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Sample records for 3c protease cleaves

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

    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.

  2. The nuclear protein Sam68 is cleaved by the FMDV 3C protease redistributing Sam68 to the cytoplasm during FMDV infection of host cells

    Lawrence, Paul; Schafer, Elizabeth A.; Rieder, Elizabeth, E-mail: elizabeth.rieder@ars.usda.gov

    2012-03-30

    Picornavirus infection can lead to disruption of nuclear pore traffic, shut-off of cell translation machinery, and cleavage of proteins involved in cellular signal transduction and the innate response to infection. Here, we demonstrated that the FMDV 3C{sup pro} induced the cleavage of nuclear RNA-binding protein Sam68 C-terminus containing the nuclear localization sequence (NLS). Consequently, it stimulated the redistribution of Sam68 to the cytoplasm. The siRNA knockdown of Sam68 resulted in a 1000-fold reduction in viral titers, which prompted us to study the effect of Sam68 on FMDV post-entry events. Interestingly, Sam68 interacts with the internal ribosomal entry site within the 5 Prime non-translated region of the FMDV genome, and Sam68 knockdown decreased FMDV IRES-driven activity in vitro suggesting that it could modulate translation of the viral genome. The results uncover a novel role for Sam68 in the context of picornaviruses and the proteolysis of a new cellular target of the FMDV 3C{sup pro}.

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

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

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

    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.

  5. In silico prediction of mutant HIV-1 proteases cleaving a target sequence

    Jensen, Jan H; Winther, Jakob R; De Vico, Luca

    2014-01-01

    HIV-1 protease represents an appealing system for directed enzyme re-design, since it has various different endogenous targets, a relatively simple structure and it is well studied. Recently Chaudhury and Gray (Structure (2009) 17: 1636 -- 1648) published a computational algorithm to discern the specificity determining residues of HIV-1 protease. In this paper we present two computational tools aimed at re-designing HIV-1 protease, derived from the algorithm of Chaudhuri and Gray. First, we present an energy-only based methodology to discriminate cleavable and non cleavable peptides for HIV-1 proteases, both wild type and mutant. Secondly, we show an algorithm we developed to predict mutant HIV-1 proteases capable of cleaving a new target substrate peptide, different from the natural targets of HIV-1 protease. The obtained in silico mutant enzymes were analyzed in terms of cleavability and specificity towards the target peptide using the energy-only methodology. We found two mutant proteases as best candidate...

  6. Rhinovirus 3C protease facilitates specific nucleoporin cleavage and mislocalisation of nuclear proteins in infected host cells.

    Erin J Walker

    Full Text Available Human Rhinovirus (HRV infection results in shut down of essential cellular processes, in part through disruption of nucleocytoplasmic transport by cleavage of the nucleoporin proteins (Nups that make up the host cell nuclear pore. Although the HRV genome encodes two proteases (2A and 3C able to cleave host proteins such as Nup62, little is known regarding the specific contribution of each. Here we use transfected as well as HRV-infected cells to establish for the first time that 3C protease is most likely the mediator of cleavage of Nup153 during HRV infection, while Nup62 and Nup98 are likely to be targets of HRV2A protease. HRV16 3C protease was also able to elicit changes in the appearance and distribution of the nuclear speckle protein SC35 in transfected cells, implicating it as a key mediator of the mislocalisation of SC35 in HRV16-infected cells. In addition, 3C protease activity led to the redistribution of the nucleolin protein out of the nucleolus, but did not affect nuclear localisation of hnRNP proteins, implying that complete disruption of nucleocytoplasmic transport leading to relocalisation of hnRNP proteins from the nucleus to the cytoplasm in HRV-infected cells almost certainly requires 2A in addition to 3C protease. Thus, a specific role for HRV 3C protease in cleavage and mislocalisation of host cell nuclear proteins, in concert with 2A, is implicated for the first time in HRV pathogenesis.

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

    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.

  8. Cleaving for growth: threonine aspartase 1-a protease relevant for development and disease.

    Stauber, Roland H; Hahlbrock, Angelina; Knauer, Shirley K; Wünsch, Désirée

    2016-03-01

    From the beginning of life, proteases are key to organismal development comprising morphogenesis, cellular differentiation, and cell growth. Regulated proteolytic activity is essential for the orchestration of multiple developmental pathways, and defects in protease activity can account for multiple disease patterns. The highly conserved protease threonine aspartase 1 is a member of such developmental proteases and critically involved in the regulation of complex processes, including segmental identity, head morphogenesis, spermatogenesis, and proliferation. Additionally, threonine aspartase 1 is overexpressed in numerous liquid as well as in solid malignancies. Although threonine aspartase 1 is able to cleave the master regulator mixed lineage leukemia protein as well as other regulatory proteins in humans, our knowledge of its detailed pathobiological function and the underlying molecular mechanisms contributing to development and disease is still incomplete. Moreover, neither effective genetic nor chemical inhibitors for this enzyme are available so far precluding the detailed dissection of the pathobiological functions of threonine aspartase 1. Here, we review the current knowledge of the structure-function relationship of threonine aspartase 1 and its mechanistic impact on substrate-mediated coordination of the cell cycle and development. We discuss threonine aspartase 1-mediated effects on cellular transformation and conclude by presenting a short overview of recent interference strategies.-Stauber, R. H., Hahlbrock, A., Knauer, S. K., Wünsch, D. Cleaving for growth: threonine aspartase 1-a protease relevant for development and disease. PMID:26578689

  9. Mutants of complement component C3 cleaved by the C4-specific C1-s protease.

    Mathias, P; Carrillo, C J; Zepf, N E; Cooper, N R; Ogata, R T

    1992-01-01

    To identify some of the structural features determining specific protease recognition of complement components C3 and C4, we used site-specific mutagenesis to construct mutants of murine C3 that are cleaved by the C4-specific C1-s protease. Insertion of three amino acid residues corresponding to residues at the C1-s cleavage site of human C4 into murine C3 at the analogous C3 convertase cleavage site was adequate to render the mutant protein susceptible to C1-s cleavage. In addition, insertio...

  10. Drosophila insulin degrading enzyme and rat skeletal muscle insulin protease cleave insulin at similar sites

    Insulin degradation is an integral part of the cellular action of insulin. Recent evidence suggests that the enzyme insulin protease is involved in the degradation of insulin in mammalian tissues. Drosophila, which has insulin-like hormones and insulin receptor homologues, also expresses an insulin degrading enzyme with properties that are very similar to those of mammalian insulin protease. In the present study, the insulin cleavage products generated by the Drosophila insulin degrading enzyme were identified and compared with the products generated by the mammalian insulin protease. Both purified enzymes were incubated with porcine insulin specifically labeled with 125I on either the A19 or B26 position, and the degradation products were analyzed by HPLC before and after sulfitolysis. Isolation and sequencing of the cleavage products indicated that both enzymes cleave the A chain of intact insulin at identical sites between residues A13 and A14 and A14 and A15. These results demonstrate that all the insulin cleavage sites generated by the Drosopohila insulin degrading enzyme are shared in common with the mammalian insulin protease. These data support the hypothesis that there is evolutionary conservation of the insulin degrading enzyme and further suggest that this enzyme plays an important role in cellular function

  11. Unc93b Induces Apoptotic Cell Death and Is Cleaved by Host and Enteroviral Proteases.

    Katharine G Harris

    Full Text Available Unc93b is an endoplasmic reticulum (ER-resident transmembrane protein that serves to bind and traffic toll-like receptors (TLRs from the ER to their appropriate subcellular locations for ligand sensing. Because of its role in TLR trafficking, Unc93b is necessary for an effective innate immune response to coxsackievirus B3 (CVB, a positive-sense single stranded RNA virus belonging to the enterovirus family. Here, we show that Unc93b is cleaved by a CVB-encoded cysteine protease (3Cpro during viral replication. Further, we define a role for Unc93b in the induction of apoptotic cell death and show that expression of wild-type Unc93b, but not a mutant incapable of binding TLRs or exiting the ER (H412R, induces apoptosis. Furthermore, we show that cellular caspases activated during apoptosis directly cleave Unc93b. Interestingly, we show that the 3Cpro- and caspase-mediated cleavage of Unc93b both occur within ten amino acids in the distal N-terminus of Unc93b. Mechanistically, neither caspase-mediated nor 3Cpro-mediated cleavage of Unc93b altered its trafficking function, inhibited its role in facilitating TLR3 or TLR8 signaling, or altered its apoptosis-inducing effects. Taken together, our studies show that Unc93b is targeted by both viral- and host cell-specific proteases and identify a function of Unc93b in the induction of apoptotic cell death.

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

    Gullberg, Maria; Polacek, Charlotta; Belsham, Graham

    The foot-and-mouth disease virus (FMDV) capsid protein precursor P1-2A is cleaved by the 3C protease to produce VP0, VP3, VP1 and 2A. It was shown previously that modification of a single amino acid residue (K210) within the VP1 protein, close to the VP1/2A cleavage site, inhibited cleavage...

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

    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...

  14. Serum protease cleaves proANF into a 14-kilodalton peptide and ANF

    Proatrial natriuretic factor (proANF), the 126-amino acid precursor of ANF, is the major storage form in mammalian atria. In contrast, two ANF peptides containing the 28- and 24-carboxyterminal residues of proANF have been isolated from rat plasma. Whether the cleavage of proANF in vivo to these ANF peptides occurs during or after its release into the circulation has not been determined. The latter possibility was suggested by a previous study where, by using a cultured rat cardiocyte preparation, the authors demonstrated that proANF is secreted intact into the culture medium. They now report that serum, but not plasma, contains a protease that specifically cleaves the 17-kdalton proANF to a 14-kdalton amino-terminal peptide and the carboxyterminal 3-kdalton circulating forms of ANF. The role of this proANF-cleaving enzyme in the generation of the biologically active ANF peptides remains to be defined. Its isolation and characterization should provide insights into its site of production and whether in vivo it is involved in the processing of circulating proANF. Radiolabeled proANF is used in these studies

  15. Canine hepacivirus NS3 serine protease can cleave the human adaptor proteins MAVS and TRIF.

    Mariona Parera

    Full Text Available Canine hepacivirus (CHV was recently identified in domestic dogs and horses. The finding that CHV is genetically the virus most closely related to hepatitis C virus (HCV has raised the question of whether HCV might have evolved as the result of close contact between dogs and/or horses and humans. The aim of this study was to investigate whether the NS3/4A serine protease of CHV specifically cleaves human mitochondrial antiviral signaling protein (MAVS and Toll-IL-1 receptor domain-containing adaptor inducing interferon-beta (TRIF. The proteolytic activity of CHV NS3/4A was evaluated using a bacteriophage lambda genetic screen. Human MAVS- and TRIF-specific cleavage sites were engineered into the lambda cI repressor. Upon infection of Escherichia coli cells coexpressing these repressors and a CHV NS3/4A construct, lambda phage replicated up to 2000-fold more efficiently than in cells expressing a CHV protease variant carrying the inactivating substitution S139A. Comparable results were obtained when several HCV NS3/4A constructs of genotype 1b were assayed. This indicates that CHV can disrupt the human innate antiviral defense signaling pathway and suggests a possible evolutionary relationship between CHV and HCV.

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

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

    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 ...

  17. Cleavage of Maize chlorotic dwarf virus R78 protein by the viral 3C protease

    Maize chlorotic dwarf virus (MCDV) is a member of the genus Waikavirus and encodes a 389 kDa polyprotein from its 11784 nt genomic RNA. Like many polyprotein-encoding viruses, MCDV contains a 3C-like virus protease that is presumably responsible for maturation cleavages of the polyprotein. However,...

  18. [Stable expression and characterization of the von Willebrand factor cleaving protease].

    Ma, Zhenni; Dong, Ningzheng; Zhang, Jingyu; Su, Jian; Wang, Anyou; Ruan, Changgeng

    2010-02-01

    This study was to acquire recombinant protein of von Willebrand factor cleaving protease (ADAMTS13, a disintegrin and metalloprotease with a thromboSpondin type 1 motifs 13), for further studies on its biological function in thrombosis and hemostasis. We transfected the Hela cells with the plasmid pSecTag-ADAMTS13 by lipofectamine. A positive cell cloning was selected by hygromycin-B. The recombinant protein was purified with Ni-NTA agarose column by gradient imidazole. The purity and immune activity of purified products were identified with SDS-PAGE and Western blotting respectively. We also measured the enzymatic activity of recombinant protein (rADAMTS13) by GST-His two-site ELISA assay. The results showed that we successfully constructed Hela cells ADAMTS2-4 which expressed high level of rADAMTS13. We received about 5.8 mg recombinant protein in culture supernantants per liter purified with Ni-NTA column. The protein formed a main lane at the position of 190 kDa with SDS-PAGE and reacted with polyclonal antibody against ADAMTS13 by Western blotting. The amount of rADAMTS13 activity was 6.4 U/mL, according to the normal plasma defined as 1 U/mL. In conclusion, rADAMTS13 protein had high purity, immune activity and good enzymatic activity, which could establish the experimental foundation for further research on biological function and mechanism of this unique metalloprotease. PMID:20432945

  19. Luteoloside Acts as 3C Protease Inhibitor of Enterovirus 71 In Vitro.

    Cao, Zeyu; Ding, Yue; Ke, Zhipeng; Cao, Liang; Li, Na; Ding, Gang; Wang, Zhenzhong; Xiao, Wei

    2016-01-01

    Luteoloside is a member of the flavonoids family that exhibits several bioactivities including anti-microbial and anti-cancer activities. However, the antiviral activity of luteoloside against enterovirus 71 (EV71) and the potential mechanism(s) responsible for this effect remain unknown. In this study, the antiviral potency of luteoloside against EV71 and its inhibitory effects on 3C protease activity were evaluated. First, we investigated the cytotoxicity of luteoloside against rhabdomyosarcoma (RD) cells, which was the cell line selected for an in vitro infection model. In a subsequent antiviral assay, the cytopathic effect of EV71 was significantly and dose-dependently relieved by the administration of luteoloside (EC50 = 0.43 mM, selection index = 5.3). Using a plaque reduction assay, we administered luteoloside at various time points and found that the compound reduced EV71 viability in RD cells rather than increasing defensive mobilization or viral absorption. Moreover, biochemical studies focused on VP1 (a key structural protein of EV71) mRNA transcript and protein levels also revealed the inhibitory effects of luteoloside on the EV71 viral yield. Finally, we performed inhibition assays using luteoloside to evaluate its effect on recombinant 3C protease activity. Our results demonstrated that luteoloside blocked 3C protease enzymatic activity in a dose-dependent manner (IC50 = 0.36 mM) that was similar to the effect of rutin, which is a well-known C3 protease inhibitor. Collectively, the results from this study indicate that luteoloside can block 3C protease activity and subsequently inhibit EV71 production in vitro. PMID:26870944

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

    Seiichi Mawatari

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

  1. Characterization and purification of a protease in serum that cleaves proatrial natriuretic factor (ProANF) to its circulating forms

    Atrial natriuretic factor (ANF) is synthesized and stored in atrial cardiocytes as a 17-kilodalton (kDa), 126 amino acid polypeptide, proANF, but circulates as smaller, 24 and 28 amino acid peptide fragments of the carboxy terminus of proANF. This reports describes the purification and characterization of this proANF-cleaving protease from rat serum. The cleavages both of 35S-labeled proANF derived from rat atrial cell cultures, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/autoradiography, and of a synthetic p-nitroanilide-containing substrate were used as assays for the detection of enzyme activity. ProANF-cleaving activity was found in rat serum, with no such activity detectable in rat plasma. Fractionation of either whole serum or the purified enzyme by gel filtration chromatography revealed a single peak of activity corresponding to a protein with a Stokes radius of 45 A. Incubation of the purified enzyme with [3H]DFP followed by SDS-PAGE and autoradiography revealed a specifically labeled 38-kDa peptide, the substrate binding subunit. Analysis by high-performance liquid chromatography of the 3-kDa products resulting from the cleavage of 35S-labeled proANF by the purified enzyme revealed, as previously described with whole serum, two radiolabeled peptides which coeluted with the 28 and 24 amino acid C-terminal peptides. These observations imply a precursor-product relationship, with the initial cleavage of proANF to the 28 amino acid peptide, which is then cleaved to the 24 amino acid peptide. These studies indicate that the majority of proANF cleavage activity found in rat serum is represented by that of a distinct serine protease whose properties are different from a variety of well-characterized proteases. The role of this protease in the in vivo processing of proANF remains to be defined

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

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

    2016-01-01

    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...... 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...

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

    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. PMID:27093053

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

    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.

  5. A spatially localized rhomboid protease cleaves cell surface adhesins essential for invasion by Toxoplasma

    Brossier, Fabien; Jewett, Travis J.; Sibley, L. David; Urban, Sinisa

    2005-01-01

    Apicomplexan parasites cause serious human and animal diseases, the treatment of which requires identification of new therapeutic targets. Host-cell invasion culminates in the essential cleavage of parasite adhesins, and although the cleavage site for several adhesins maps within their transmembrane domains, the protease responsible for this processing has not been discovered. We have identified, cloned, and characterized the five nonmitochondrial rhomboid intramembrane proteases encoded in t...

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

    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. PMID:27265257

  7. Peptidomimetic ethyl propenoate covalent inhibitors of the enterovirus 71 3C protease: a P2-P4 study.

    Ang, Melgious J Y; Lau, Qiu Ying; Ng, Fui Mee; Then, Siew Wen; Poulsen, Anders; Cheong, Yuen Kuen; Ngoh, Zi Xian; Tan, Yong Wah; Peng, Jianhe; Keller, Thomas H; Hill, Jeffrey; Chu, Justin J H; Chia, C S Brian

    2016-01-01

    Enterovirus 71 (EV71) is a highly infectious pathogen primarily responsible for Hand, Foot, and Mouth Disease, particularly among children. Currently, no approved antiviral drug has been developed against this disease. The EV71 3C protease is deemed an attractive drug target due to its crucial role in viral polyprotein processing. Rupintrivir, a peptide-based inhibitor originally developed to target the human rhinovirus 3C protease, was found to inhibit the EV71 3C protease. In this communication, we report the inhibitory activities of 30 Rupintrivir analogs against the EV71 3C protease. The most potent inhibitor, containing a P2 ring-constrained phenylalanine analog (compound 9), was found to be two-fold more potent than Rupintrivir (IC50 value 3.4 ± 0.4 versus 7.3 ± 0.8 μM). Our findings suggest that employing geometrically constrained residues in peptide-based protease inhibitors can potentially enhance their inhibitory activities. PMID:25792507

  8. Characterization of the protease activity that cleaves the extracellular domain of β-dystroglycan

    Dystroglycan (DG) complex, composed of αDG and βDG, provides a link between the extracellular matrix (ECM) and cortical cytoskeleton. Although the proteolytic processing of βDG was reported in various physiological and pathological conditions, its exact mechanism remains unknown. In this study, we addressed this issue using the cell culture system of rat schwannoma cell line RT4. We found that the culture medium of RT4 cells was enriched with the protease activity that degrades the fusion protein construct of the extracellular domain of βDG specifically. This activity was suppressed by the inhibitor of matrix metalloproteinase-2 (MMP-2) and MMP-9, but not by the inhibitors of MMP-1, MMP-3, MMP-8, and MMP-13. Zymography and RT-PCR analysis showed that RT4 cells secreted MMP-2 and MMP-9 into the culture medium. Finally, active MMP-2 and MMP-9 enzymes degraded the fusion protein construct of the extracellular domain of βDG. These results indicate (1) that RT4 cells secrete the protease activity that degrades the extracellular domain of βDG specifically and (2) that MMP-2 and MMP-9 may be involved in this process

  9. Cysteine protease antigens cleave CD123, the α subunit of murine IL-3 receptor, on basophils and suppress IL-3-mediated basophil expansion

    Nishikado, Hideto [Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, Tokyo (Japan); Fujimura, Tsutomu; Taka, Hikari; Mineki, Reiko [Laboratory of Proteomics and Biomolecular Science, BioMedical Research Center, Juntendo University Graduate School of Medicine, Tokyo (Japan); Ogawa, Hideoki; Okumura, Ko [Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, Tokyo (Japan); Takai, Toshiro, E-mail: t-takai@juntendo.ac.jp [Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, Tokyo (Japan)

    2015-05-01

    Th2 type immune responses are essential for protective immunity against parasites and play crucial roles in allergic disorders. Helminth parasites secrete a variety of proteases for their infectious cycles including for host entry, tissue migration, and suppression of host immune effector cell function. Furthermore, a number of pathogen-derived antigens, as well as allergens such as papain, belong to the family of cysteine proteases. Although the link between protease activity and Th2 type immunity is well documented, the mechanisms by which proteases regulate host immune responses are largely unknown. Here, we demonstrate that the cysteine proteases papain and bromelain selectively cleave the α subunit of the IL-3 receptor (IL-3Rα/CD123) on the surface of murine basophils. The decrease in CD123 expression on the cell surface, and the degradation of the extracellular domain of recombinant CD123 were dependent on the protease activity of papain and bromelain. Pre-treatment of murine basophils with papain resulted in inhibition of IL-3-IL-3R signaling and suppressed IL-3- but not thymic stromal lymphopoietin-induced expansion of basophils in vitro. Our unexpected findings illuminate a novel mechanism for the regulation of basophil functions by protease antigens. Because IL-3 plays pivotal roles in the activation and proliferation of basophils and in protective immunity against helminth parasites, pathogen-derived proteases might contribute to the pathogenesis of infections by regulating IL-3-mediated functions in basophils. - Highlights: • We identified the murine IL3R as a novel target of papain-family cysteine proteases. • Papain-family cysteine proteases cleaved IL3Rα/CD123 on murine basophils. • Papain suppressed IL3- but not TSLP-induced expansion of murine basophils. • The inactivation of IL3R might be a strategy for pathogens to suppress host immunity.

  10. Cysteine protease antigens cleave CD123, the α subunit of murine IL-3 receptor, on basophils and suppress IL-3-mediated basophil expansion

    Th2 type immune responses are essential for protective immunity against parasites and play crucial roles in allergic disorders. Helminth parasites secrete a variety of proteases for their infectious cycles including for host entry, tissue migration, and suppression of host immune effector cell function. Furthermore, a number of pathogen-derived antigens, as well as allergens such as papain, belong to the family of cysteine proteases. Although the link between protease activity and Th2 type immunity is well documented, the mechanisms by which proteases regulate host immune responses are largely unknown. Here, we demonstrate that the cysteine proteases papain and bromelain selectively cleave the α subunit of the IL-3 receptor (IL-3Rα/CD123) on the surface of murine basophils. The decrease in CD123 expression on the cell surface, and the degradation of the extracellular domain of recombinant CD123 were dependent on the protease activity of papain and bromelain. Pre-treatment of murine basophils with papain resulted in inhibition of IL-3-IL-3R signaling and suppressed IL-3- but not thymic stromal lymphopoietin-induced expansion of basophils in vitro. Our unexpected findings illuminate a novel mechanism for the regulation of basophil functions by protease antigens. Because IL-3 plays pivotal roles in the activation and proliferation of basophils and in protective immunity against helminth parasites, pathogen-derived proteases might contribute to the pathogenesis of infections by regulating IL-3-mediated functions in basophils. - Highlights: • We identified the murine IL3R as a novel target of papain-family cysteine proteases. • Papain-family cysteine proteases cleaved IL3Rα/CD123 on murine basophils. • Papain suppressed IL3- but not TSLP-induced expansion of murine basophils. • The inactivation of IL3R might be a strategy for pathogens to suppress host immunity

  11. Crystal structure of the 3C protease from Southern African Territories type 2 foot-and-mouth disease virus.

    Yang, Jingjie; Leen, Eoin N; Maree, Francois F; Curry, Stephen

    2016-01-01

    The replication of foot-and-mouth disease virus (FMDV) is dependent on the virus-encoded 3C protease (3C(pro)). As in other picornaviruses, 3C(pro) performs most of the proteolytic processing of the polyprotein expressed from the large open reading frame in the RNA genome of the virus. Previous work revealed that the 3C(pro) from serotype A-one of the seven serotypes of FMDV-adopts a trypsin-like fold. On the basis of capsid sequence comparisons the FMDV serotypes are grouped into two phylogenetic clusters, with O, A, C, and Asia 1 in one, and the three Southern African Territories serotypes, (SAT-1, SAT-2 and SAT-3) in another, a grouping pattern that is broadly, but not rigidly, reflected in 3C(pro) amino acid sequences. We report here the cloning, expression and purification of 3C proteases from four SAT serotype viruses (SAT2/GHA/8/91, SAT1/NIG/5/81, SAT1/UGA/1/97, and SAT2/ZIM/7/83) and the crystal structure at 3.2 Å resolution of 3C(pro) from SAT2/GHA/8/91. PMID:27168976

  12. Cysteine protease antigens cleave CD123, the α subunit of murine IL-3 receptor, on basophils and suppress IL-3-mediated basophil expansion.

    Nishikado, Hideto; Fujimura, Tsutomu; Taka, Hikari; Mineki, Reiko; Ogawa, Hideoki; Okumura, Ko; Takai, Toshiro

    2015-05-01

    Th2 type immune responses are essential for protective immunity against parasites and play crucial roles in allergic disorders. Helminth parasites secrete a variety of proteases for their infectious cycles including for host entry, tissue migration, and suppression of host immune effector cell function. Furthermore, a number of pathogen-derived antigens, as well as allergens such as papain, belong to the family of cysteine proteases. Although the link between protease activity and Th2 type immunity is well documented, the mechanisms by which proteases regulate host immune responses are largely unknown. Here, we demonstrate that the cysteine proteases papain and bromelain selectively cleave the α subunit of the IL-3 receptor (IL-3Rα/CD123) on the surface of murine basophils. The decrease in CD123 expression on the cell surface, and the degradation of the extracellular domain of recombinant CD123 were dependent on the protease activity of papain and bromelain. Pre-treatment of murine basophils with papain resulted in inhibition of IL-3-IL-3R signaling and suppressed IL-3- but not thymic stromal lymphopoietin-induced expansion of basophils in vitro. Our unexpected findings illuminate a novel mechanism for the regulation of basophil functions by protease antigens. Because IL-3 plays pivotal roles in the activation and proliferation of basophils and in protective immunity against helminth parasites, pathogen-derived proteases might contribute to the pathogenesis of infections by regulating IL-3-mediated functions in basophils. PMID:25778870

  13. Degradation of the encephalomyocarditis virus and hepatitis A virus 3C proteases by the ubiquitin/26S proteasome system in vivo

    We have isolated stably transfected mouse embryonic fibroblast cell lines that inducibly express either the mature encephalomyocarditis virus (EMCV) or hepatitis A virus (HAV) 3C protease and have used these cells to demonstrate that both proteins are subject to degradation in vivo by the ubiquitin/26S proteasome system. The detection of 3C protease expression in these cells requires inducing conditions and the presence of one of several proteasome inhibitors. Both 3C proteases are incorporated into conjugates with ubiquitin in vivo. HAV 3C protease expression has deleterious effects on cell viability, as determined by observation and counting of cells cultured in the absence or presence of inducing conditions. The EMCV 3C protease was found to be preferentially localized to the nucleus of induced cells, while the HAV 3C protease remains in the cytoplasm. The absence of polyubiquitinated EMCV 3C protease conjugates in nuclear fraction preparations suggests that localization to the nucleus can protect this protein from ubiquitination

  14. Synthesis and structure-activity relationship of α-keto amides as enterovirus 71 3C protease inhibitors.

    Zeng, Debin; Ma, Yuying; Zhang, Rui; Nie, Quandeng; Cui, Zhengjie; Wang, Yaxin; Shang, Luqing; Yin, Zheng

    2016-04-01

    α-Keto amide derivatives as enterovirus 71 (EV71) 3C protease (3C(pro)) inhibitors have been synthesized and assayed for their biochemical and antiviral activities. structure-activity relationship (SAR) study indicated that small moieties were primarily tolerated at P1' and the introduction of para-fluoro benzyl at P2 notably improved the potency of inhibitor. Inhibitors 8v, 8w and 8x exhibited satisfactory activity (IC50=1.32±0.26μM, 1.88±0.35μM and 1.52±0.31μM, respectively) and favorable CC50 values (CC50>100μM). α-Keto amide may represent a good choice as a warhead for EV71 3C(pro) inhibitor. PMID:26916437

  15. IRES mediated expression of viral 3C protease for enhancing the yield of FMDV empty capsids using baculovirus system.

    Vivek Srinivas, V M; Basagoudanavar, Suresh H; Hosamani, Madhusudan

    2016-03-01

    For expression of FMDV empty capsids, high protease activity associated with 3C co-expressed with P1 polyprotein has been reported to adversely affect the yields of capsids. Limiting the levels of 3Cpro relative to P1-2A polypeptide is thus critical to enhance the yields. In this study, FMDV internal ribosome entry site (IRES) sequence which serves as an alternative to the CAP-dependent translation initiation mechanism, was used for controlled translation of 3C protease. Baculovirus expressing bicistronic cDNA cassette containing two open reading frames-FMDV capsid gene (P1-2A) and 3Cpro intervened by IRES was prepared. Analysis of the expression in insect cells infected with baculovirus showed increased accumulation of processed capsids. Recombinant capsids showed higher immunoreactivity similar to the whole virus antigen, when reacted with polyclonal antibodies against the purified whole virus 146S particles. Thus, inclusion of the IRES upstream of 3Cpro facilitated reduced expression of the protease in baculovirus expression system, without causing significant proteolysis, thereby contributing to improved yields of the processed capsid antigens. PMID:26775685

  16. Crystal structure of the 3C protease from Southern African Territories type 2 foot-and-mouth disease virus

    Yang, Jingjie; Leen, Eoin N.; Maree, Francois F.

    2016-01-01

    The replication of foot-and-mouth disease virus (FMDV) is dependent on the virus-encoded 3C protease (3Cpro). As in other picornaviruses, 3Cpro performs most of the proteolytic processing of the polyprotein expressed from the large open reading frame in the RNA genome of the virus. Previous work revealed that the 3Cpro from serotype A—one of the seven serotypes of FMDV—adopts a trypsin-like fold. On the basis of capsid sequence comparisons the FMDV serotypes are grouped into two phylogenetic clusters, with O, A, C, and Asia 1 in one, and the three Southern African Territories serotypes, (SAT-1, SAT-2 and SAT-3) in another, a grouping pattern that is broadly, but not rigidly, reflected in 3Cpro amino acid sequences. We report here the cloning, expression and purification of 3C proteases from four SAT serotype viruses (SAT2/GHA/8/91, SAT1/NIG/5/81, SAT1/UGA/1/97, and SAT2/ZIM/7/83) and the crystal structure at 3.2 Å resolution of 3Cpro from SAT2/GHA/8/91. PMID:27168976

  17. 2,3,4-Trihydroxybenzyl-hydrazide analogues as novel potent coxsackievirus B3 3C protease inhibitors.

    Kim, Bo-Kyoung; Ko, Hyojin; Jeon, Eun-Seok; Ju, Eun-Seon; Jeong, Lak Shin; Kim, Yong-Chul

    2016-09-14

    Human coxsackievirus B3 (CVB3) 3C protease plays an essential role in the viral replication of CVB3, which is a non-enveloped and positive single-stranded RNA virus belonging to Picornaviridae family, causing acute viral myocarditis mainly in children. During optimization based on SAR studies of benserazide (3), which was reported as a novel anti-CVB3 3C(pro) agent from a screening of compound libraries, the 2,3,4-trihydroxybenzyl moiety of 3 was identified as a key pharmacophore for inhibitory activity against CVB3 3C(pro). Further optimization was performed by the introduction of various aryl-alkyl substituted hydrazide moieties instead of the serine moiety of 3. Among the optimized compounds, 11Q, a 4-hydroxyphenylpentanehydrazide derivative, showed the most potent inhibitory activity (IC50 = 0.07 μM). Enzyme kinetics studies indicated that 11Q exhibited a mixed inhibitory mechanism of action. The antiviral activity against CVB3 was confirmed using the further derived analogue (14b) with more cell permeable valeryl ester group at the 2,3,4-trihydroxy moiety. PMID:27191615

  18. A mammalian cell-based reverse two-hybrid system for functional analysis of 3C viral protease of human enterovirus 71.

    Lee, Jin-Ching; Shih, Shin-Ru; Chang, Ten-Yuan; Tseng, Huan-Yi; Shih, Ya-Feng; Yen, Kuei-Jung; Chen, Wei-Chun; Shie, Jiun-Jie; Fang, Jim-Min; Liang, Po-Huang; Chao, Yu-Sheng; Hsu, John T-A

    2008-04-01

    Although several cell-based reporter assays have been developed for screening of viral protease inhibitors, most of these assays have a significant limitation in that numerous false positives can be generated for the compounds that are interfering with reporter gene detection due to the cellular viability. To improve, we developed a mammalian cell-based assay based on the reverse two-hybrid system to monitor the proteolytic activity of human enterovirus 71 (EV71) 3C protease and to validate the cytotoxicity of compounds at the same time. In this system, the GAL4 DNA binding domain (M3) and transactivation domain (VP16) were fused, in-frame, with 3C or 3C(mut). The 3C(mut) was an inactivated protease with mutations at the predicted catalytic triad. The reporter plasmid contains a secreted alkaline phosphatase (SEAP) gene under the control of GAL4 activating sequences. We demonstrated that M3-3C-VP16 failed to turn on the expression of SEAP due to the separation of M3 and the VP16 domains by self-cleavage of 3C. In contrast, SEAP expression was induced by the M3-3C(mut)-VP16 fusion protein or the M3-3C-VP16 in cells treated with AG7088, a potent inhibitor of human rhinoviruses (HRVs) 3C protease. Potentially, this protease detection system should greatly facilitate anti-EV71 drug discovery through a high-throughput screening. PMID:18190777

  19. Escherichia coli DegP Protease Cleaves between Paired Hydrophobic Residues in a Natural Substrate: the PapA Pilin

    Jones, C. Hal; Dexter, Paul; Evans, Amy K.; Liu, Christopher; Hultgren, Scott J.; Hruby, Dennis E.

    2002-01-01

    The DegP protein, a multifunctional chaperone and protease, is essential for clearance of denatured or aggregated proteins from the inner-membrane and periplasmic space in Escherichia coli. To date, four natural targets for DegP have been described: colicin A lysis protein, pilin subunits and MalS from E. coli, and high-molecular-weight adherence proteins from Haemophilus influenzae. In vitro, DegP has shown weak protease activity with casein and several other nonnative substrates. We report ...

  20. Secreted aspartic proteases of pathogenic Candida spp. are temporarily retained in the cell wall and cleave the extracellular substrates

    Pichová, Iva; Vinterová, Zuzana; Šanda, Miloslav; Dostál, Jiří; Hrušková-Heidingsfeldová, Olga

    2012-01-01

    Roč. 21, S1 (2012), s. 206-206. ISSN 0961-8368. [Annual Symposium of the Protein-Society /26./. 05.08.2012-08.08.2012, San Diego] R&D Projects: GA ČR GA310/09/1945 Institutional research plan: CEZ:AV0Z40550506 Keywords : aspartic proteases * Candida spp. * cell wall Subject RIV: CE - Biochemistry

  1. Distinct roles of secreted HtrA proteases from gram-negative pathogens in cleaving the junctional protein and tumor suppressor E-cadherin.

    Hoy, Benjamin; Geppert, Tim; Boehm, Manja; Reisen, Felix; Plattner, Patrick; Gadermaier, Gabriele; Sewald, Norbert; Ferreira, Fatima; Briza, Peter; Schneider, Gisbert; Backert, Steffen; Wessler, Silja

    2012-03-23

    The periplasmic chaperone and serine protease HtrA is important for bacterial stress responses and protein quality control. Recently, we discovered that HtrA from Helicobacter pylori is secreted and cleaves E-cadherin to disrupt the epithelial barrier, but it remained unknown whether this maybe a general virulence mechanism. Here, we show that important other pathogens including enteropathogenic Escherichia coli, Shigella flexneri, and Campylobacter jejuni, but not Neisseria gonorrhoeae, cleaved E-cadherin on host cells. HtrA deletion in C. jejuni led to severe defects in E-cadherin cleavage, loss of cell adherence, paracellular transmigration, and basolateral invasion. Computational modeling of HtrAs revealed a conserved pocket in the active center exhibiting pronounced proteolytic activity. Differential E-cadherin cleavage was determined by an alanine-to-glutamine exchange in the active center of neisserial HtrA. These data suggest that HtrA-mediated E-cadherin cleavage is a prevalent pathogenic mechanism of multiple gram-negative bacteria representing an attractive novel target for therapeutic intervention to combat bacterial infections. PMID:22337879

  2. X-Ray Structure and Inhibition of 3C-like Protease from Porcine Epidemic Diarrhea Virus.

    St John, Sarah E; Anson, Brandon J; Mesecar, Andrew D

    2016-01-01

    Porcine epidemic diarrhea virus (PEDV) is a coronavirus that infects pigs and can have mortality rates approaching 100% in piglets, causing serious economic impact. The 3C-like protease (3CL(pro)) is essential for the coronaviral life cycle and is an appealing target for the development of therapeutics. We report the expression, purification, crystallization and 2.10 Å X-ray structure of 3CL(pro) from PEDV. Analysis of the PEDV 3CL(pro) structure and comparison to other coronaviral 3CL(pro)'s from the same alpha-coronavirus phylogeny shows that the overall structures and active site architectures across 3CL(pro)'s are conserved, with the exception of a loop that comprises the protease S2 pocket. We found a known inhibitor of severe acute respiratory syndrome coronavirus (SARS-CoV) 3CL(pro), (R)-16, to have inhibitor activity against PEDV 3CL(pro), despite that SARS-3CL(pro) and PEDV 3CL(pro) share only 45.4% sequence identity. Structural comparison reveals that the majority of residues involved in (R)-16 binding to SARS-3CL(pro) are conserved in PEDV-3CL(pro); however, the sequence variation and positional difference in the loop forming the S2 pocket may account for large observed difference in IC50 values. This work advances our understanding of the subtle, but important, differences in coronaviral 3CL(pro) architecture and contributes to the broader structural knowledge of coronaviral 3CL(pro)'s. PMID:27173881

  3. THAP5 is a human cardiac-specific inhibitor of cell cycle that is cleaved by the proapoptotic Omi/HtrA2 protease during cell death.

    Balakrishnan, Meenakshi P; Cilenti, Lucia; Mashak, Zineb; Popat, Paiyal; Alnemri, Emad S; Zervos, Antonis S

    2009-08-01

    Omi/HtrA2 is a mitochondrial serine protease that has a dual function: while confined in the mitochondria, it promotes cell survival, but when released into the cytoplasm, it participates in caspase-dependent as well as caspase-independent cell death. To investigate the mechanism of Omi/HtrA2's function, we set out to isolate and characterize novel substrates for this protease. We have identified Thanatos-associated protein 5 (THAP5) as a specific interactor and substrate of Omi/HtrA2 in cells undergoing apoptosis. This protein is an uncharacterized member of the THAP family of proteins. THAP5 has a unique pattern of expression and is found predominantly in the human heart, although a very low expression is also seen in the human brain and muscle. THAP5 protein is localized in the nucleus and, when ectopically expressed, induces cell cycle arrest. During apoptosis, THAP5 protein is degraded, and this process can be blocked using a specific Omi/HtrA2 inhibitor, leading to reduced cell death. In patients with coronary artery disease, THAP5 protein levels substantially decrease in the myocardial infarction area, suggesting a potential role of this protein in human heart disease. This work identifies human THAP5 as a cardiac-specific nuclear protein that controls cell cycle progression. Furthermore, during apoptosis, THAP5 is cleaved and removed by the proapoptotic Omi/HtrA2 protease. Taken together, we provide evidence to support that THAP5 and its regulation by Omi/HtrA2 provide a new link between cell cycle control and apoptosis in cardiomyocytes. PMID:19502560

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

    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.

  5. The nuclear inclusion a (NIa protease of turnip mosaic virus (TuMV cleaves amyloid-β.

    Hye-Eun Han

    Full Text Available BACKGROUND: The nuclear inclusion a (NIa protease of turnip mosaic virus (TuMV is responsible for the processing of the viral polyprotein into functional proteins. NIa was previously shown to possess a relatively strict substrate specificity with a preference for Val-Xaa-His-Gln↓, with the scissile bond located after Gln. The presence of the same consensus sequence, Val(12-His-His-Gln(15, near the presumptive α-secretase cleavage site of the amyloid-β (Aβ peptide led us to hypothesize that NIa could possess activity against Aβ. METHODOLOGY/PRINCIPAL FINDINGS: Western blotting results showed that oligomeric as well as monomeric forms of Aβ can be degraded by NIa in vitro. The specific cleavage of Aβ was further confirmed by mass spectrometry analysis. NIa was shown to exist predominantly in the cytoplasm as observed by immunofluorescence microscopy. The overexpression of NIa in B103 neuroblastoma cells resulted in a significant reduction in cell death caused by both intracellularly generated and exogenously added Aβ. Moreover, lentiviral-mediated expression of NIa in APP(sw/PS1 transgenic mice significantly reduced the levels of Aβ and plaques in the brain. CONCLUSIONS/SIGNIFICANCE: These results indicate that the degradation of Aβ in the cytoplasm could be a novel strategy to control the levels of Aβ, plaque formation, and the associated cell death.

  6. Significance of plasma von Willebrand factor level and von Willebrand factor-cleaving protease activity in patients with chronic renal diseases

    LU Guo-yuan; SHEN Lei; WANG Zhao-yue; GUO Xiao-fang; BAI Xia; SU Jian; RUAN Chang-geng

    2008-01-01

    Background yon Willebrand factor(vWF)mediates the initial capture of platelets to vascular subendothelium and is essential for platelet aggregation under high fluid shear stress as in arteriaI stenosis.On release frOm endothelial cells,vWF is rapidly cleaved by ADAMTSl 3/vWF-cleaving protease (vWF-CP).We investigated the clinical significance of changes in plasma vWF and vWF-CP activities in chronic renal disease.Methods Plasma vWF and vWF-CP activities were measured using enzyme-linked immunosorbent assay(ELISA)and residual collagen binding assay respectively in patients with lupus nephritis(n=31),primary nephritic syndrome(n=25),diabetic nephropathy(n=45),chronic glomerulonephritis(n=38)and 40 normal controls.The reIation of their levels with pathological and renal status was analyzed.Results In all diseased patients the levels of vWF were significantly higher and vWF-CP activity significantly lower than the controls(both P<0.01).vWF in the four subgroups did not correlate with the stage of disease but correlated negatively with vWF-CP activity.vWF-CP activity was not changed two weeks after renal transplantation.Renal biopsy demonstrated that the vWF level in stage Ⅳ was higher than in stages Ⅱ and Ⅲ while vWF-CP activity was lower in patients with lupus nephritis.After eight-week treatment,the vWF level significantly decreased and the vWF-CP activity significantly increased in systemic lupus erythema,disease activity index<9,but not with index≥9.Even though the vWF-CP activity was significantly lower in membranous nephropathy than in minimal change disease,mesangial proliferative glomerulonephritis or IgA glomerulonephritis,the vWF level was not significantly different.Conclusions The alterations of plasma vWF and vWF-CP activities were associated with different renal pathologies.Injury to endothelial cells and autoantibodies against vWF-CP activity may result in higher vWF Ievel and Iower vWF-CP activity in chronic renaI disease and thus a

  7. The nuclear protein Sam68 is cleaved by the FMDV 3C protease redistributing Sam68 to the cytoplasm during FMDV infection of host cells

    Picornavirus infection can lead to disruption of nuclear pore traffic, shut-off of cell translation machinery, and cleavage of proteins involved in cellular signal transduction and the innate response to infection. Here, we demonstrated that the FMDV 3Cpro induced the cleavage of nuclear RNA-binding protein Sam68 C-terminus containing the nuclear localization sequence (NLS). Consequently, it stimulated the redistribution of Sam68 to the cytoplasm. The siRNA knockdown of Sam68 resulted in a 1000-fold reduction in viral titers, which prompted us to study the effect of Sam68 on FMDV post-entry events. Interestingly, Sam68 interacts with the internal ribosomal entry site within the 5′ non-translated region of the FMDV genome, and Sam68 knockdown decreased FMDV IRES-driven activity in vitro suggesting that it could modulate translation of the viral genome. The results uncover a novel role for Sam68 in the context of picornaviruses and the proteolysis of a new cellular target of the FMDV 3Cpro.

  8. Efficient production of foot-and-mouth disease virus empty capsids in insect cells following down regulation of 3C protease activity

    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...... precursor P1-2A by the action of the virus-encoded 3C protease. To date recombinant empty capsid assembly has been limited by poor expression levels, restricting the development of empty capsids as a viable vaccine. Here expression of the FMDV structural protein precursor P1-2A in insect cells is shown...... assembled into empty capsids. Expression was independent of the insect host cell background and leads to capsids that are recognised as authentic by a range of anti-FMDV bovine sera suggesting their feasibility as an alternate vaccine....

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

    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...... with that achieved with a single P1-2A-3C polyprotein. Expression of the FMDV 3Cpro is poorly tolerated by mammalian cells and higher levels of the 3Cpro greatly inhibit protein expression. In addition, it is demonstrated that both the intact P1-2A precursor and the processed capsid proteins can be efficiently...... detected by FMDV antigen detection assays. Furthermore, the P1-2A and the processed forms each bind to the integrin αvβ6, the major FMDV receptor. These results contribute to the development of systems which efficiently express the components of empty capsid particles and may represent the basis for safer...

  10. Entamoeba histolytica cysteine proteases cleave the MUC2 mucin in its C-terminal domain and dissolve the protective colonic mucus gel

    Lidell, Martin E.; Moncada, Darcy M.; Chadee, Kris; Hansson, Gunnar C.

    2006-01-01

    In order for the protozoan parasite Entamoeba histolytica (E.h.) to cause invasive intestinal and extraintestinal infection, which leads to significant morbidity and mortality, it must disrupt the protective mucus layer by a previously unknown mechanism. We hypothesized that cysteine proteases secreted from the amoeba disrupt the mucin polymeric network, thereby overcoming the protective mucus barrier. The MUC2 mucin is the major structural component of the colonic mucus gel. Heavily O-glycos...

  11. Cleaving DNA with DNA

    Carmi, Nir; Balkhi, Shameelah R.; Breaker, Ronald R.

    1998-03-01

    A DNA structure is described that can cleave single-stranded DNA oligonucleotides in the presence of ionic copper. This ``deoxyribozyme'' can self-cleave or can operate as a bimolecular complex that simultaneously makes use of duplex and triplex interactions to bind and cleave separate DNA substrates. Bimolecular deoxyribozyme-mediated strand scission proceeds with a kobs of 0.2 min-1, whereas the corresponding uncatalyzed reaction could not be detected. The duplex and triplex recognition domains can be altered, making possible the targeted cleavage of single-stranded DNAs with different nucleotide sequences. Several small synthetic DNAs were made to function as simple ``restriction enzymes'' for the site-specific cleavage of single-stranded DNA.

  12. Enteroviral proteases: structure, host interactions and pathogenicity.

    Laitinen, Olli H; Svedin, Emma; Kapell, Sebastian; Nurminen, Anssi; Hytönen, Vesa P; Flodström-Tullberg, Malin

    2016-07-01

    Enteroviruses are common human pathogens, and infections are particularly frequent in children. Severe infections can lead to a variety of diseases, including poliomyelitis, aseptic meningitis, myocarditis and neonatal sepsis. Enterovirus infections have also been implicated in asthmatic exacerbations and type 1 diabetes. The large disease spectrum of the closely related enteroviruses may be partially, but not fully, explained by differences in tissue tropism. The molecular mechanisms by which enteroviruses cause disease are poorly understood, but there is increasing evidence that the two enteroviral proteases, 2A(pro) and 3C(pro) , are important mediators of pathology. These proteases perform the post-translational proteolytic processing of the viral polyprotein, but they also cleave several host-cell proteins in order to promote the production of new virus particles, as well as to evade the cellular antiviral immune responses. Enterovirus-associated processing of cellular proteins may also contribute to pathology, as elegantly demonstrated by the 2A(pro) -mediated cleavage of dystrophin in cardiomyocytes contributing to Coxsackievirus-induced cardiomyopathy. It is likely that improved tools to identify targets for these proteases will reveal additional host protein substrates that can be linked to specific enterovirus-associated diseases. Here, we discuss the function of the enteroviral proteases in the virus replication cycle and review the current knowledge regarding how these proteases modulate the infected cell in order to favour virus replication, including ways to avoid detection by the immune system. We also highlight new possibilities for the identification of protease-specific cellular targets and thereby a way to discover novel mechanisms contributing to disease. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27145174

  13. Functional study of elafin cleaved by Pseudomonas aeruginosa metalloproteinases.

    Guyot, Nicolas

    2010-06-01

    Elafin is a 6-kDa innate immune protein present at several epithelial surfaces including the pulmonary epithelium. It is a canonical protease inhibitor of two neutrophil serine proteases [neutrophil elastase (NE) and proteinase 3] with the capacity to covalently bind extracellular matrix proteins by transglutamination. In addition to these properties, elafin also possesses antimicrobial and immunomodulatory activities. The aim of the present study was to investigate the effect of Pseudomonas aeruginosa proteases on elafin function. We found that P. aeruginosa PAO1-conditioned medium and two purified Pseudomonas metalloproteases, pseudolysin (elastase) and aeruginolysin (alkaline protease), are able to cleave recombinant elafin. Pseudolysin was shown to inactivate the anti-NE activity of elafin by cleaving its protease-binding loop. Interestingly, antibacterial properties of elafin against PAO1 were found to be unaffected after pseudolysin treatment. In contrast to pseudolysin, aeruginolysin failed to inactivate the inhibitory properties of elafin against NE. Aeruginolysin cleaves elafin at the amino-terminal Lys6-Gly7 peptide bond, resulting in a decreased ability to covalently bind purified fibronectin following transglutaminase activity. In conclusion, this study provides evidence that elafin is susceptible to proteolytic cleavage at alternative sites by P. aeruginosa metalloproteinases, which can affect different biological functions of elafin.

  14. Cleaving DNA with DNA

    Carmi, Nir; Balkhi, Shameelah R.; Breaker, Ronald R.

    1998-01-01

    A DNA structure is described that can cleave single-stranded DNA oligonucleotides in the presence of ionic copper. This “deoxyribozyme” can self-cleave or can operate as a bimolecular complex that simultaneously makes use of duplex and triplex interactions to bind and cleave separate DNA substrates. Bimolecular deoxyribozyme-mediated strand scission proceeds with a kobs of 0.2 min−1, whereas the corresponding uncatalyzed reaction could not be detected. The duplex and triplex recognition domai...

  15. Autoprocessing: an essential step for expression and purification of enterovirus 71 3C(pro) in Escherichia coli.

    Huang, Shuqiong; Lyu, Yanning; Qing, Xianyun; Wang, Weiwei; Tang, Liang; Cheng, Kedi; Wang, Wei

    2013-11-01

    A gene encoding the 3BC of human enterovirus 71 (EV71) was cloned and inserted into a derivative of plasmid pET-32a(+) driven by T7 promoter. The expressed 3C protease (3C(pro)) autocatalytically cleaved itself from the recombinant protein Trx-3BC and the mature 3C(pro) partitioned in the soluble fraction of bacterial lysate. The 13-amino-acid peptide substrates with the junction of 3B/3C were used to verify the proteolysis activity of the purified 3C(pro). The EV71 3C(pro) had a Km value of 63 μM (measured by a continuous fluorescence assay). The other solid-phase activity assay of the EV71 3C(pro) was developed using HPLC to analyze the proteolytic products. The combination of two activity assays contributes to promote the identification of the specific inhibitors targeted to the EV71 3C(pro). PMID:23881322

  16. Biased Signaling of Protease-activated Receptors

    PeishenZhao; NigelWilliamBunnett

    2014-01-01

    In addition to their role in protein degradation and digestion, proteases can also function as hormone-like signaling molecules that regulate vital patho-physiological processes, including inflammation, hemostasis, pain and repair mechanisms. Certain proteases can signal to cells by cleaving protease-activated receptors (PARs), a family of four G protein-coupled receptors. PARs are expressed by almost all cell types, control important physiological and disease-relevant processes, and are an e...

  17. Intramembrane Proteolysis by Signal Peptide Peptidases: A Comparative Discussion of GXGD-type Aspartyl Proteases*

    Fluhrer, Regina; Steiner, Harald; Haass, Christian

    2009-01-01

    Intramembrane-cleaving proteases are required for reverse signaling and membrane protein degradation. A major class of these proteases is represented by the GXGD-type aspartyl proteases. GXGD describes a novel signature sequence that distinguishes these proteases from conventional aspartyl proteases. Members of the family of the GXGD-type aspartyl proteases are the Alzheimer disease-related γ-secretase, the signal peptide peptidases and their homologs, and the bacteria...

  18. Cysteine Proteases: Modes of Activation and Future Prospects as Pharmacological Targets

    Verma, Sonia; Dixit, Rajnikant; Pandey, Kailash C.

    2016-01-01

    Proteolytic enzymes are crucial for a variety of biological processes in organisms ranging from lower (virus, bacteria, and parasite) to the higher organisms (mammals). Proteases cleave proteins into smaller fragments by catalyzing peptide bonds hydrolysis. Proteases are classified according to their catalytic site, and distributed into four major classes: cysteine proteases, serine proteases, aspartic proteases, and metalloproteases. This review will cover only cysteine proteases, papain fam...

  19. Protease-mediated drug delivery

    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.

  20. The Role of Protease Activity in ErbB Biology

    Blobel, Carl P; Carpenter, Graham; Freeman, Matthew

    2008-01-01

    Proteases are now recognized as having an active role in a variety of processes aside from their recognized metabolic role in protein degradation. Within the ErbB system of ligands and receptors proteases are known to be necessary for the generation of soluble ligands from transmembrane precursers and for the processing of the ErbB4 receptor, such that its intracellular domain is translocated to the nucleus. There are two protease activities involved in the events: proteases that cleave withi...

  1. Effects of TSP2-8 and CUB1+2 Domains on Secretion Direction of Von Willebrand Factor-cleaving Protease%TSP2-8和CUB1+2片段对血管性血友病因子裂解酶分泌方向的影响

    高单萍; 刘琼; 陈素华; 艾继辉

    2011-01-01

    本研究旨在探讨羧基端TSP2-8和CUB1+2片段是否决定血管性血友病因子裂解酶(ADAMTS13)合成后细胞内运输的方向,以了解金属蛋白酶ADAMTS13羧基端结构与功能的关系.利用脂质体转染技术将重组质粒pcDNA3.1-ADAMTS13及peDNA3.1-de1TSP2-8CUB1+2ADAMTS13分别转染犬肾上皮极性细胞MDCK,筛选出阳性细胞克隆后传代铺到中间有特殊分子筛膜的双池培养皿内培养,待细胞生长到无空隙后收集上、下池培养液;通过Western blot检测上、下池培养液中ADAMTS13蛋白表达水平,以对比分析ADAMTS13蛋白在极性细胞内的分泌方向.结果表明,德定表达野生型ADAMTS13的MDCK细胞组,在分子筛膜的上池培养液中检测到ADAMTS13蛋白,而表达缺失TSP2-8CUB1 +2区域ADAMTS13的细胞组,在分子膜的上、下池培养液中均检测到ADAMTS13重组蛋白.结论:金属蛋白酶ADAMTS13的分泌是有极性的,且羧基端TSP2-8和CUB1+2结构域与ADAMTS13合成后细胞内的运输方向密切相关.%This study was aimed to explore if the intracellular transportation direction of von Willebrand factor-cleaving protease (ADAMTS13, vWF-CP) after synthesis is determined by the carboxyl terminal TSP2-8CUB1 + 2 domains of ADAMTS13 and to decipher the relationship between the structure and function of ADAMTS13. The recombinant plasmids pcDNA3.1-ADAMTS13 and pcDNA3. 1-delTSP2-8CUB1 + 2 ADAMTS13 were introduced into Madin-Darby canine kidney cells (MDCK) by lipofectamine-mediated DNA transfection. Positive cell clones gained after antibioticscreening were grown on 6-well transwell filter units with a zeolite membrane in the middle layer. The conditioned culture media in both apical and basolateral wells were collected when cells reached confluency and the tight cell monolayer formed. ADAMTS13 proteases in the conditioned media were determined by Western blot, and the direction of ADAMTS13 secretion in polarized cells was comparatively analyzed. The results

  2. Self and non-self discrimination by "restriction proteases".

    Lefkovits, I

    1986-01-01

    I propose that an organism possesses a set of specific enzymes ("restriction proteases") that cleave self proteins at defined amino acid sequences unless these sequences are rendered inaccessible by glycosylation. Intracellular proteins are degraded by restriction proteases when cells die. In this way, intracellular proteins remain undetected by the immune system. I propose that some autoimmune diseases are caused by the absence of a specific restriction protease.

  3. Protease inhibitor

    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...

  4. Processing Proteases

    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-terminal of...... the scissile bond, leaving C-terminal fusions to have non-native C-termini after processing. A solution yielding native C-termini would allow novel expression and purification systems for therapeutic proteins and peptides.The peptidyl-Lys metallopeptidase (LysN) of the fungus Armillaria mellea (Am) is...

  5. Purification and characterization of an immunoglobulin A1 protease from Bacteroides melaninogenicus.

    Mortensen, S B; Kilian, M

    1984-01-01

    Attention has recently been focused on bacterial proteases with the capacity to cleave immunoglobulin A (IgA proteases) as possible pathogenic factors in bacterial meningitis, gonorrhoea, and destructive periodontal disease. Here, we describe a method for the rapid purification of a specific IgA1 protease from Bacteroides melaninogenicus. The IgA1 protease was purified 6,172-fold with a yield of 9% by ammonium sulfate precipitation, DEAE-ion exchange chromatography, and separation on a prepar...

  6. Supermarket Proteases.

    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)

  7. Earthworm Protease

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

  8. ADAM 12-S cleaves IGFBP-3 and IGFBP-5 and is inhibited by TIMP-3

    Loechel, F; Fox, J W; Murphy, G;

    2000-01-01

    that it cleaves insulin-like growth factor binding protein-3 (IGFBP-3). This result supports a role for ADAM 12-S in the degradation of IGFBP-3 in the blood of pregnant women. Furthermore, we tested for proteolysis of other members of the IGF binding protein family and found that ADAM 12-S cleaves...... IGFBP-5 in addition to IGFBP-3, but does not cleave IGFBP-1, -2, -4, or -6. ADAM 12-S may therefore be the IGFBP-5 protease that is secreted by osteoblasts and other cells. Cleavage of both IGFBP-3 and -5 by ADAM 12-S was inhibited by TIMP-3, raising the possibility that TIMP-3 is a physiological...

  9. Cleavage Luminescence from Cleaved Indium Phosphide

    We outline the experiments performed to gain further information about the structure and properties of cleaved InP surfaces. The experiments involved detecting the luminescence produced after cleaving thin InP plates within a high vacuum, by a process of converting the luminescence to an electrical signal which could be amplified and measured accurately. The experimental results show that the detected luminescence durations from cleaved InP are usually only about 10μs. It is believed that this time represents the time of travel of the crack with the actual recombination time being much shorter. Strong signals could also be picked up from cleaved InP in air

  10. Fabrication of Graphene by Cleaving Graphite Chemically

    ZHAO Shu-hua; ZHAO Xiao-ting; FAN Hou-gang; YANG Li-li; ZHANG Yong-jun; YANG Jing-hai

    2011-01-01

    Graphite was chemically cleaved to graphene by Billups Reaction,and the morphologies and microstructures of graphene were characterized by SEM,Raman and AFM.The results show that the graphite was first functionalized by l-iodododecane,which led to the cleavage of the graphene layer in the graphite.The second decoration cleaved the graphite further and graphene was obtained.The heights of the graphene layer were larger than 1 nm due to the organic decoration.

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

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

    2008-06-27

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

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

    Kousted, Tina M; Skjødt, Karsten; Petersen, Steen V;

    2014-01-01

    , conversion to an inactive conformation or induction of serpin substrate behaviour. Until now, no inhibitory antibodies against PN-1 have been thoroughly characterised. Here we report the development of three monoclonal antibodies binding specifically and with high affinity to human PN-1. The antibodies all...... serpin presenting its so-called reactive centre loop as a substrate to its target protease, resulting in a covalent complex with the inactivated enzyme. Previously, three mechanisms have been proposed for the inactivation of serpins by monoclonal antibodies: steric blockage of protease recognition...... abolish the protease inhibitory activity of PN-1. In the presence of the antibodies, PN-1 does not form a complex with its target proteases, but is recovered in a reactive centre cleaved form. Using site-directed mutagenesis, we mapped the three overlapping epitopes to an area spanning the gap between the...

  13. Evaluation on Potential Contributions of Protease Activated Receptors Related Mediators in Allergic Inflammation

    Huiyun Zhang; Xiaoning Zeng; Shaoheng He

    2014-01-01

    Protease activated receptors (PARs) have been recognized as a distinctive four-member family of seven transmembrane G protein-coupled receptors (GPCRs) that can be cleaved by certain serine proteases. In recent years, there has been considerable interest in the role of PARs in allergic inflammation, the fundamental pathologic changes of allergy, but the potential roles of PARs in allergy remain obscure. Since many of these proteases are produced and actively involved in the pathologic process...

  14. Caught in the act: the crystal structure of cleaved cathepsin L bound to the active site of Cathepsin L.

    Sosnowski, Piotr; Turk, Dušan

    2016-04-01

    Cathepsin L is a ubiquitously expressed papain-like cysteine protease involved in the endosomal degradation of proteins and has numerous roles in physiological and pathological processes, such as arthritis, osteoporosis, and cancer. Insight into the specificity of cathepsin L is important for elucidating its physiological roles and drug discovery. To study interactions with synthetic ligands, we prepared a presumably inactive mutant and crystallized it. Unexpectedly, the crystal structure determined at 1.4 Å revealed that the cathepsin L molecule is cleaved, with the cleaved region trapped in the active site cleft of the neighboring molecule. Hence, the catalytic mutant demonstrated low levels of catalytic activity. PMID:26992470

  15. Mast cell proteases as pharmacological targets.

    Caughey, George H

    2016-05-01

    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

  16. INTEGRATED POLARIZATION PROPERTIES OF 3C48, 3C138, 3C147, AND 3C286

    Perley, R. A.; Butler, B. J., E-mail: RPerley@nrao.edu, E-mail: BButler@nrao.edu [National Radio Astronomy Observatory, P.O. Box O, Socorro, NM 87801 (United States)

    2013-06-01

    We present the integrated polarization properties of the four compact radio sources 3C48, 3C138, 3C147, and 3C286, from 1 to 50 GHz, over a 30 yr time frame spanning 1982-2012. Using the polarized emission of Mars, we have determined that the position angle of the linearly polarized emission of 3C286 rises from 33 Degree-Sign at 8 GHz to 36 Degree-Sign at 45 GHz. There is no evidence for a change in the position angle over time. Using these values, the position angles of the integrated polarized emission from the other three sources are determined as a function of frequency and time. The fractional polarization of 3C286 is found to be slowly rising, at all frequencies, at a rate of {approx}0.015% yr{sup -1}. The fractional polarizations of 3C48, 3C138, and 3C147 are all slowly variable, with the variations correlated with changes in the total flux densities of these sources.

  17. Characterization of Fibrinolytic Proteases from Gloydius blomhoffii siniticus Venom

    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.

  18. Kallikrein Promotes Inflammation in Human Dental Pulp Cells Via Protease-Activated Receptor-1.

    Hayama, Tomomi; Kamio, Naoto; Okabe, Tatsu; Muromachi, Koichiro; Matsushima, Kiyoshi

    2016-07-01

    Plasma kallikrein (KLKB1), a serine protease, cleaves high-molecular weight kininogen to produce bradykinin, a potent vasodilator and pro-inflammatory peptide. In addition, KLKB1 activates plasminogen and other leukocyte and blood coagulation factors and processes pro-enkephalin, prorenin, and C3. KLKB1 has also been shown to cleave protease-activated receptors in vascular smooth muscle cells to regulate the expression of epidermal growth factor receptor. In this study, we investigated KLKB1-dependent inflammation and activation of protease-activated receptor-1 in human dental pulp cells. These cells responded to KLKB1 stimulation by increasing intracellular Ca(2+) , upregulating cyclooxygenase-2, and secreting prostaglandin E2 . Remarkably, SCH79797, an antagonist of protease-activated receptor-1, blocked these effects. Thus, these data indicate that KLKB1 induces inflammatory reactions in human dental tissues via protease-activated receptor 1. J. Cell. Biochem. 117: 1522-1528, 2016. © 2015 Wiley Periodicals, Inc. PMID:26566265

  19. The surface layer of cleaved bilayer manganites

    Recently, several informative reports have been published on spectroscopy experiments performed on cleaved surfaces of the bilayered colossal magnetoresistive manganite La2-2xSr1+2xMn2O7 (Konoto et al 2004 Phys. Rev. Lett. 93 107201, Freeland et al 2005 Nat. Mater. 4 62, Mannella et al 2005 Nature 438 474, Roennow et al 2006 Nature 440 1025). For the detailed interpretation of these results, it is of importance to know exactly which layer within the crystal structure is exposed to the surface upon cleavage. Here we combine crystal structure arguments, scanning tunnelling microscopy and x-ray photoelectron spectroscopy measurements to demonstrate that the crystals cleave between the rare-earth rock-salt oxide layers, leaving one outermost rare-earth oxide layer before the first electronically active MnO bilayer

  20. Development of a protease-sensitive molecular imaging agent for optoacoustic tomography

    La Rivière, Patrick J.; Green, Anthony; Norris, James R.

    2007-02-01

    We are working to develop a molecular imaging agent that will allow for in vivo imaging of proteases by use of optoacoustic tomography. Proteases are protein-cleaving proteins known to be overactive in a number of pathologies, including cancers and vascular disease. Protease-sensitive "smart probes" have previously been developed in the context of pure optical imaging. These involve pairs of mutually quenching fluorophores attached to a backbone by protease-cleavable peptide side chains; cleaving of the side chains liberates the fluorophores and leads to increase in fluorescence. Optoacoustic imaging is sensitive not to fluorescence but to optical absorption and so a smart imaging probe for protease imaging would need to shift its absorption peak upon cleavage. Naturally, the absorption peaks of the cleaved (and, ideally, uncleaved) molecules should be in the near infrared for maximum tissue penetration. We have designed a molecule that should achieve these specifications. It comprises two active sites, derivatives of natural photosynthetic bacteriochlorophylls that absorb in the near IR, conjugated to a lysine backbone by peptide spacers specific to the protease being imaged. When these bacteriochlorophylls dimerize and stack in the uncleaved molecule, their absorption peak shifts about 20-30 nm. When they are cleaved from the molecule the absorption peak shifts back to that of bacteriochlorophyll monomers. We have performed a preliminary synthesis of the molecule and confirmed by use of a spectrometer that the pairing of the bacteriochlorophylls leads to the expected absorption shift.

  1. Proteases as Insecticidal Agents

    Harrison, Robert L.; 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...

  2. Characterization and inhibition of norovirus proteases of genogroups I and II using a fluorescence resonance energy transfer assay

    Noroviruses are the major cause of food- or water-borne gastroenteritis outbreaks in humans. The norovirus protease that cleaves a large viral polyprotein to nonstructural proteins is essential for virus replication and an attractive target for antiviral drug development. Noroviruses show high genetic diversity with at least five genogroups, GI–GV, of which GI and GII are responsible for the majority of norovirus infections in humans. We cloned and expressed proteases of Norwalk virus (GI) and MD145 virus (GII) and characterized the enzymatic activities with fluorescence resonance energy transfer substrates. We demonstrated that the GI and GII proteases cleaved the substrates derived from the naturally occurring cleavage site in the open reading frame (ORF) 1 of G1 norovirus with similar efficiency, and that enzymatic activity of both proteases was inhibited by commercial protease inhibitors including chymostatin. The interaction of chymostatin to Norwalk virus protease was validated by nuclear magnetic resonance (NMR) spectroscopy.

  3. Characterization and inhibition of norovirus proteases of genogroups I and II using a fluorescence resonance energy transfer assay

    Chang, Kyeong-Ok [Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, KS 66506 (United States); Takahashi, Daisuke; Prakash, Om [Department of Biochemistry, Kansas State University, Manhattan, KS 66506 (United States); Kim, Yunjeong, E-mail: ykim@vet.ksu.edu [Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, KS 66506 (United States)

    2012-02-20

    Noroviruses are the major cause of food- or water-borne gastroenteritis outbreaks in humans. The norovirus protease that cleaves a large viral polyprotein to nonstructural proteins is essential for virus replication and an attractive target for antiviral drug development. Noroviruses show high genetic diversity with at least five genogroups, GI-GV, of which GI and GII are responsible for the majority of norovirus infections in humans. We cloned and expressed proteases of Norwalk virus (GI) and MD145 virus (GII) and characterized the enzymatic activities with fluorescence resonance energy transfer substrates. We demonstrated that the GI and GII proteases cleaved the substrates derived from the naturally occurring cleavage site in the open reading frame (ORF) 1 of G1 norovirus with similar efficiency, and that enzymatic activity of both proteases was inhibited by commercial protease inhibitors including chymostatin. The interaction of chymostatin to Norwalk virus protease was validated by nuclear magnetic resonance (NMR) spectroscopy.

  4. Insights into the serine protease mechanism based on structural observations of the conversion of a peptidyl serine protease inhibitor to a substrate

    Jiang, Longguang; Andersen, Lisbeth Moreau; Andreasen, Peter A; Chen, Liqing; Huang, Mingdong

    2016-01-01

    BACKGROUND: Serine proteases are one of the most studied group of enzymes. Despite the extensive mechanistic studies, some crucial details remain controversial, for example, how the cleaved product is released in the catalysis reaction. A cyclic peptidyl inhibitor (CSWRGLENHRMC, upain-1) of a...... serine protease, urokinase-type plasminogen activator (uPA), was found to become a slow substrate and cleaved slowly upon the replacement of single residue (W3A). METHODS: By taking advantage of the unique property of this peptide, we report the high-resolution structures of uPA in complex with upain-1-W...

  5. Functional imaging of proteases: recent advances in the design and application of substrate- and activity- based probes

    Edgington, Laura E.; Verdoes, Martijn; Bogyo, Matthew

    2011-01-01

    Proteases are enzymes that cleave peptide bonds in protein substrates. This process can be important for regulated turnover of a target protein but it can also produce protein fragments that then perform other functions. Because the last few decades of protease research have confirmed that proteolysis is an essential regulatory process in both normal physiology and in multiple disease-associated conditions, there has been an increasing interest in developing methods to image protease activity...

  6. MMP-15 is upregulated in preeclampsia, but does not cleave endoglin to produce soluble endoglin.

    Tu'uhevaha J Kaitu'u-Lino

    Full Text Available Preeclampsia is a major pregnancy complication, characterized by severe endothelial dysfunction, hypertension and maternal end-organ damage. Soluble endoglin is an anti-angiogenic protein released from placenta and thought to play a central role in causing the endothelial dysfunction and maternal organ injury seen in severe preeclampsia. We recently reported MMP-14 was the protease producing placentally-derived soluble endoglin by cleaving full-length endoglin present on the syncytiotrophoblast surface. This find identifies a specific drug target for severe preeclampsia; interfering with MMP-14 mediated cleavage of endoglin could decrease soluble endoglin production, ameliorating clinical disease. However, experimental MMP-14 inhibition alone only partially repressed soluble endoglin production, implying other proteases might have a role in producing soluble endoglin. Here we investigated whether MMP-15--phylogenetically the closest MMP relative to MMP-14 with 66% sequence similarity--also cleaves endoglin to produce soluble endoglin. MMP-15 was localized to the syncytiotrophoblast layer of the placenta, the same site where endoglin was localized. Interestingly, it was significantly (p = 0.03 up-regulated in placentas from severe early-onset preeclamptic pregnancies (n = 8 compared to gestationally matched preterm controls (n = 8. However, siRNA knockdown of MMP-15 yielded no significant decrease of soluble endoglin production from either HUVECs or syncytialised BeWo cells in vitro. Importantly, concurrent siRNA knockdown of both MMP-14 and MMP-15 in HUVECS did not yield further decrease in soluble endoglin production compared to MMP-14 siRNA alone. We conclude MMP-15 is up-regulated in preeclampsia, but does not cleave endoglin to produce soluble endoglin.

  7. Complement component 3 (C3)

    ... page: //medlineplus.gov/ency/article/003539.htm Complement component 3 (C3) To use the sharing features on this page, ... be some throbbing. Why the Test is Performed C3 and C4 are the most commonly measured complement components. A complement test may be used to monitor ...

  8. CLIP proteases and Plasmodium melanization in Anopheles gambiae.

    Barillas-Mury, Carolina

    2007-07-01

    Melanization is a potent immune response mediated by phenoloxidase (PO). Multiple Clip-domain serine proteases (CLIP) regulate PO activation as part of a complex cascade of proteases that are cleaved sequentially. The role of several CLIP as key activators or suppressors of the melanization responses of Anopheles gambiae to Plasmodium berghei (murine malaria) has been established recently using a genome-wide reverse genetics approach. Important differences in regulation of PO activation between An. gambiae strains were also identified. This review summarizes these findings and discusses our current understanding of the An. gambiae melanization responses to Plasmodium. PMID:17512801

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

    Budatha, Madhusudhan; Silva, Simone; Montoya, Teodoro Ignacio; Suzuki, Ayako; Shah-Simpson, Sheena; Wieslander, Cecilia Karin; Yanagisawa, Masashi; Word, Ruth Ann; Yanagisawa, Hiromi

    2013-01-01

    Mice deficient for the fibulin-5 gene (Fbln5(-/-)) develop pelvic organ prolapse (POP) due to compromised elastic fibers and upregulation of matrix metalloprotease (MMP)-9. Here, we used casein zymography, inhibitor profiling, affinity pull-down, and mass spectrometry to discover additional protease upregulated in the vaginal wall of Fbln5(-/-) mice, herein named V1 (25 kDa). V1 was a serine protease with trypsin-like activity similar to protease, serine (PRSS) 3, a major extrapancreatic trypsinogen, was optimum at pH 8.0, and predominantly detected in estrogenized vaginal epithelium of Fbln5(-/-) mice. PRSS3 was (a) localized in epithelial secretions, (b) detected in media of vaginal organ culture from both Fbln5(-/-) and wild type mice, and (c) cleaved fibulin-5 in vitro. Expression of two serine protease inhibitors [Serpina1a (α1-antitrypsin) and Elafin] was dysregulated in Fbln5(-/-) epithelium. Finally, we confirmed that PRSS3 was expressed in human vaginal epithelium and that SERPINA1 and Elafin were downregulated in vaginal tissues from women with POP. These data collectively suggest that the balance between proteases and their inhibitors contributes to support of the pelvic organs in humans and mice. PMID:23437119

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

    Madhusudhan Budatha

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

  11. Proteases of neutrophilic granulocytes

    Wiesława Roszkowska-Jakimiec

    2002-06-01

    Full Text Available The literature referring to proteolytic enzymes of neutrophilic granulocytes was surveyed. Biosynthesis, subcellular distribution, division according to the catalytic site structure, inhibitors and methods used to determine the activity of these enzymes were discussed. The survey included metaloproteases (granulocytic collagenase, gelatinase B, serine proteases (granulocytic elastase, cathepsin G, protease 3, membraneous proteases (aminopeptidase N, aminopeptidase P, neprilisine, cysteine and aspartic cathepsins. The role of these proteases in the pathology and diagnostics of certain diseases was considered.

  12. Endonuclease V cleaves at inosines in RNA.

    Vik, Erik Sebastian; Nawaz, Meh Sameen; Strøm Andersen, Pernille; Fladeby, Cathrine; Bjørås, Magnar; Dalhus, Bjørn; Alseth, Ingrun

    2013-01-01

    Endonuclease V orthologues are highly conserved proteins found in all kingdoms of life. While the prokaryotic enzymes are DNA repair proteins for removal of deaminated adenosine (inosine) from the genome, no clear role for the eukaryotic counterparts has hitherto been described. Here we report that human endonuclease V (ENDOV) and also Escherichia coli endonuclease V are highly active ribonucleases specific for inosine in RNA. Inosines are normal residues in certain RNAs introduced by specific deaminases. Adenosine-to-inosine editing is essential for proper function of these transcripts and defects are linked to various human disease. Here we show that human ENDOV cleaves an RNA substrate containing inosine in a position corresponding to a biologically important site for deamination in the Gabra-3 transcript of the GABA(A) neurotransmitter. Further, human ENDOV specifically incises transfer RNAs with inosine in the wobble position. This previously unknown RNA incision activity may suggest a role for endonuclease V in normal RNA metabolism. PMID:23912683

  13. Proteases of neutrophilic granulocytes

    Wiesława Roszkowska-Jakimiec; Anna Worowska; Marek Gacko; Tomasz Maksimowicz

    2002-01-01

    The literature referring to proteolytic enzymes of neutrophilic granulocytes was surveyed. Biosynthesis, subcellular distribution, division according to the catalytic site structure, inhibitors and methods used to determine the activity of these enzymes were discussed. The survey included metaloproteases (granulocytic collagenase, gelatinase B), serine proteases (granulocytic elastase, cathepsin G, protease 3), membraneous proteases (aminopeptidase N, aminopeptidase P, neprilisine), cysteine ...

  14. MMP-9 cleaves SP-D and abrogates its innate immune functions in vitro.

    Preston E Bratcher

    Full Text Available Possession of a properly functioning innate immune system in the lung is vital to prevent infections due to the ongoing exposure of the lung to pathogens. While mechanisms of pulmonary innate immunity have been well studied, our knowledge of how these systems are altered in disease states, leading to increased susceptibility to infections, is limited. One innate immune protein in the lung, the pulmonary collectin SP-D, has been shown to be important in innate immune defense, as well as clearance of allergens and apoptotic cells. MMP-9 is a protease with a wide variety of substrates, and has been found to be dysregulated in a myriad of lung diseases ranging from asthma to cystic fibrosis; in many of these conditions, there are decreased levels of SP-D. Our results indicate that MMP-9 is able to cleave SP-D in vitro and this cleavage leads to loss of its innate immune functions, including its abilities to aggregate bacteria and increase phagocytosis by mouse alveolar macrophages. However, MMP-9-cleaved SP-D was still detected in a solid-phase E. coli LPS-binding assay, while NE-cleaved SP-D was not. In addition, MMP-9 seems to cleave SP-D much more efficiently than NE at physiological levels of calcium. Previous studies have shown that in several diseases, including cystic fibrosis and asthma, patients have increased expression of MMP-9 in the lungs as well as decreased levels of intact SP-D. As patients suffering from many of the diseases in which MMP-9 is over-expressed can be more susceptible to pulmonary infections, it is possible that MMP-9 cleavage of SP-D may contribute to this phenotype.

  15. Scintillation observations of the 3C48, 3C273 and 3C295 at 25 MHz

    Interplanetary scintillations of 3C 48, 3C 273, and 3C 295 have been observed at 25 MHz. 3C 48 is found to possess a halo. 3C 295 at the decametric waves becomes an one -component source. 3C 273 has the same angular structure as in the meter - wavelength range. The models of the sources corresponding to the present observations are proposed

  16. Simple Room Temperature Method for Polymer Optical Fibre Cleaving

    Saez-Rodriguez, David; Nielsen, Kristian; Bang, Ole;

    2015-01-01

    . In this paper, we make use of the temperature-time equivalence in polymers to replace the use of heating by an increase of the cleaving time and use a sawing motion to reduce fibre end face damage. In this way, the polymer fibre can be cleaved at room temperature in seconds with the resulting end...

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

    Sonia eVerma

    2016-04-01

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

  18. Beta-secretase-cleaved amyloid precursor protein in Alzheimer brain: a morphologic study

    Sennvik, Kristina; Bogdanovic, N; Volkmann, Inga; Fastbom, J; Benedikz, Eirikur

    2004-01-01

    (beta-sAPP) in brain tissue sections from the frontal, temporal and occipital lobe. Strong granular beta-sAPP staining was found throughout the gray matter of all three areas, while white matter staining was considerably weaker. beta-sAPP was found to be localized in astrocytes and in axons. We found...... the beta-sAPP immunostaining to be stronger and more extensive in gray matter in Alzheimer disease (AD) cases than controls. The axonal beta-sAPP staining was patchy and unevenly distributed for the AD cases, indicating impaired axonal transport. beta-sAPP was also found surrounding senile plaques and......beta-amyloid (Abeta) is the main constituent of senile plaques seen in Alzheimer's disease. Abeta is derived from the amyloid precursor protein (APP) via proteolytic cleavage by proteases beta- and gamma-secretase. In this study, we examined content and localization of beta-secretase-cleaved APP...

  19. Sam68 is cleaved by caspases under apoptotic cell death induced by ionizing radiation

    The RNA-binding protein Sam68, a mitotic substrate of tyrosine kinases, has been reported to participate in the cell cycle, apoptosis, and signaling. In particular, overexpression of Sam68 protein is known to suppress cell growth and cell cycle progression in NIH3T3 cells. Although Sam68 is involved in many cellular activities, the function of Sam68, especially in response to apoptotic stimulation, is not well understood. In this study, we found that Sam68 protein is cleaved in immune cells undergoing apoptosis induced by γ-radiation. Moreover, we found that Sam68 cleavage was induced by apoptotic stimuli containing γ-radiation in a caspase-dependent manner. In particular, we showed that activated casepase-3, 7, 8 and 9 can directly cleave Sam68 protein through in vitro protease cleavage assay. Finally, we found that the knockdown of Sam68 attenuated γ-radiation-induced cell death and growth suppression. Conclusively, the cleavage of Sam68 is a new indicator for the cell damaging effects of ionizing radiation. (author)

  20. Structural, kinetic, and thermodynamic studies of specificity designed HIV-1 protease

    Alvizo, Oscar; Mittal, Seema; Mayo, Stephen L.; Schiffer, Celia A. (CIT); (UMASS, MED)

    2012-10-23

    HIV-1 protease recognizes and cleaves more than 12 different substrates leading to viral maturation. While these substrates share no conserved motif, they are specifically selected for and cleaved by protease during viral life cycle. Drug resistant mutations evolve within the protease that compromise inhibitor binding but allow the continued recognition of all these substrates. While the substrate envelope defines a general shape for substrate recognition, successfully predicting the determinants of substrate binding specificity would provide additional insights into the mechanism of altered molecular recognition in resistant proteases. We designed a variant of HIV protease with altered specificity using positive computational design methods and validated the design using X-ray crystallography and enzyme biochemistry. The engineered variant, Pr3 (A28S/D30F/G48R), was designed to preferentially bind to one out of three of HIV protease's natural substrates; RT-RH over p2-NC and CA-p2. In kinetic assays, RT-RH binding specificity for Pr3 increased threefold compared to the wild-type (WT), which was further confirmed by isothermal titration calorimetry. Crystal structures of WT protease and the designed variant in complex with RT-RH, CA-p2, and p2-NC were determined. Structural analysis of the designed complexes revealed that one of the engineered substitutions (G48R) potentially stabilized heterogeneous flap conformations, thereby facilitating alternate modes of substrate binding. Our results demonstrate that while substrate specificity could be engineered in HIV protease, the structural pliability of protease restricted the propagation of interactions as predicted. These results offer new insights into the plasticity and structural determinants of substrate binding specificity of the HIV-1 protease.

  1. Cysteine Proteases: Modes of Activation and Future Prospects as Pharmacological Targets.

    Verma, Sonia; Dixit, Rajnikant; Pandey, Kailash C

    2016-01-01

    Proteolytic enzymes are crucial for a variety of biological processes in organisms ranging from lower (virus, bacteria, and parasite) to the higher organisms (mammals). Proteases cleave proteins into smaller fragments by catalyzing peptide bonds hydrolysis. Proteases are classified according to their catalytic site, and distributed into four major classes: cysteine proteases, serine proteases, aspartic proteases, and metalloproteases. This review will cover only cysteine proteases, papain family enzymes which are involved in multiple functions such as extracellular matrix turnover, antigen presentation, processing events, digestion, immune invasion, hemoglobin hydrolysis, parasite invasion, parasite egress, and processing surface proteins. Therefore, they are promising drug targets for various diseases. For preventing unwanted digestion, cysteine proteases are synthesized as zymogens, and contain a prodomain (regulatory) and a mature domain (catalytic). The prodomain acts as an endogenous inhibitor of the mature enzyme. For activation of the mature enzyme, removal of the prodomain is necessary and achieved by different modes. The pro-mature domain interaction can be categorized as protein-protein interactions (PPIs) and may be targeted in a range of diseases. Cysteine protease inhibitors are available that can block the active site but no such inhibitor available yet that can be targeted to block the pro-mature domain interactions and prevent it activation. This review specifically highlights the modes of activation (processing) of papain family enzymes, which involve auto-activation, trans-activation and also clarifies the future aspects of targeting PPIs to prevent the activation of cysteine proteases. PMID:27199750

  2. Cleavage of DAP5 by coxsackievirus B3 2A protease facilitates viral replication and enhances apoptosis by altering translation of IRES-containing genes.

    Hanson, P J; Ye, X; Qiu, Y; Zhang, H M; Hemida, M G; Wang, F; Lim, T; Gu, A; Cho, B; Kim, H; Fung, G; Granville, D J; Yang, D

    2016-05-01

    Cleavage of eukaryotic translation initiation factor 4G (eIF4G) by enterovirus proteases during infection leads to the shutoff of cellular cap-dependent translation, but does not affect the initiation of cap-independent translation of mRNAs containing an internal ribosome entry site (IRES). Death-associated protein 5 (DAP5), a structural homolog of eIF4G, is a translation initiation factor specific for IRES-containing mRNAs. Coxsackievirus B3 (CVB3) is a positive single-stranded RNA virus and a primary causal agent of human myocarditis. Its RNA genome harbors an IRES within the 5'-untranslated region and is translated by a cap-independent, IRES-driven mechanism. Previously, we have shown that DAP5 is cleaved during CVB3 infection. However, the protease responsible for cleavage, cleavage site and effects on the translation of target genes during CVB3 infection have not been investigated. In the present study, we demonstrated that viral protease 2A but not 3C is responsible for DAP5 cleavage, generating 45- and 52-kDa N- (DAP5-N) and C-terminal (DAP5-C) fragments, respectively. By site-directed mutagenesis, we found that DAP5 is cleaved at amino acid G434. Upon cleavage, DAP5-N largely translocated to the nucleus at the later time points of infection, whereas the DAP5-C largely remained in the cytoplasm. Overexpression of these DAP5 truncates demonstrated that DAP5-N retained the capability of initiating IRES-driven translation of apoptosis-associated p53, but not the prosurvival Bcl-2 (B-cell lymphoma 2) when compared with the full-length DAP5. Similarly, DAP5-N expression promoted CVB3 replication and progeny release; on the other hand, DAP5-C exerted a dominant-negative effect on cap-dependent translation. Taken together, viral protease 2A-mediated cleavage of DAP5 results in the production of two truncates that exert differential effects on protein translation of the IRES-containing genes, leading to enhanced host cell death. PMID:26586572

  3. Unproductive cleavage and the inactivation of protease-activated receptor-1 by trypsin in vascular endothelial cells

    Nakayama, Tetsuzo; Hirano, Katsuya; Shintani, Yoshinobu; Nishimura, Junji; Nakatsuka, Akio; Kuga, Hirotaka; Takahashi, Shosuke; Kanaide, Hideo

    2003-01-01

    Using fura-2 fluorometry of [Ca2+]i in response to thrombin, trypsin and protease-activated receptor activating peptides (PAR-APs), we determined whether trypsin cleaves protease-activated receptor 1 (PAR1) and activates it in the endothelial cells of the porcine aortic valves and human umbilical vein.Once stimulated with thrombin, the subsequent application of trypsin induced a [Ca2+]i elevation similar to that obtained without the preceding stimulation with thrombin in the valvular endothel...

  4. MIB-MIP is a mycoplasma system that captures and cleaves immunoglobulin G.

    Arfi, Yonathan; Minder, Laetitia; Di Primo, Carmelo; Le Roy, Aline; Ebel, Christine; Coquet, Laurent; Claverol, Stephane; Vashee, Sanjay; Jores, Joerg; Blanchard, Alain; Sirand-Pugnet, Pascal

    2016-05-10

    Mycoplasmas are "minimal" bacteria able to infect humans, wildlife, and a large number of economically important livestock species. Mycoplasma infections include a spectrum of clinical manifestations ranging from simple fever to fulminant inflammatory diseases with high mortality rates. These infections are mostly chronic, suggesting that mycoplasmas have developed means to evade the host immune response. Here we present and functionally characterize a two-protein system from Mycoplasma mycoides subspecies capri that is involved in the capture and cleavage of IgG. The first component, Mycoplasma Ig binding protein (MIB), is an 83-kDa protein that is able to tightly bind to the Fv region of a wide range of IgG. The second component, Mycoplasma Ig protease (MIP), is a 97-kDa serine protease that is able to cleave off the VH domain of IgG. We demonstrate that MIB is necessary for the proteolytic activity of MIP. Cleavage of IgG requires a sequential interaction of the different partners of the system: first MIB captures the IgG, and then MIP is recruited to the MIB-IgG complex, enabling protease activity. MIB and MIP are encoded by two genes organized in tandem, with homologs found in the majority of pathogenic mycoplasmas and often in multiple copies. Phylogenetic studies suggest that genes encoding the MIB-MIP system are specific to mycoplasmas and have been disseminated by horizontal gene transfer. These results highlight an original and complex system targeting the host immunoglobulins, playing a potentially key role in the immunity evasion by mycoplasmas. PMID:27114507

  5. An Efficient Catalytic DNA that Cleaves L-RNA.

    Kha Tram

    Full Text Available Many DNAzymes have been isolated from synthetic DNA pools to cleave natural RNA (D-RNA substrates and some have been utilized for the design of aptazyme biosensors for bioanalytical applications. Even though these biosensors perform well in simple sample matrices, they do not function effectively in complex biological samples due to ubiquitous RNases that can efficiently cleave D-RNA substrates. To overcome this issue, we set out to develop DNAzymes that cleave L-RNA, the enantiomer of D-RNA, which is known to be completely resistant to RNases. Through in vitro selection we isolated three L-RNA-cleaving DNAzymes from a random-sequence DNA pool. The most active DNAzyme exhibits a catalytic rate constant ~3 min-1 and has a structure that contains a kissing loop, a structural motif that has never been observed with D-RNA-cleaving DNAzymes. Furthermore we have used this DNAzyme and a well-known ATP-binding DNA aptamer to construct an aptazyme sensor and demonstrated that this biosensor can achieve ATP detection in biological samples that contain RNases. The current work lays the foundation for exploring RNA-cleaving DNAzymes for engineering biosensors that are compatible with complex biological samples.

  6. Commercial proteases: present and future.

    Li, Qing; Yi, Li; Marek, Peter; Iverson, Brent L

    2013-04-17

    This review presents a brief overview of the general categories of commercially used proteases, and critically surveys the successful strategies currently being used to improve the properties of proteases for various commercial purposes. We describe the broad application of proteases in laundry detergents, food processing, and the leather industry. The review also introduces the expanding development of proteases as a class of therapeutic agents, as well as highlighting recent progress in the field of protease engineering. The potential commercial applications of proteases are rapidly growing as recent technological advances are producing proteases with novel properties and substrate specificities. PMID:23318711

  7. Tomato ringspot nepovirus protease: characterization and cleavage site specificity

    Hans, F.; Sanfacon, H.

    1995-01-01

    We have cloned the region of tomato ringspot nepovirus (TomRSV) RNA-1 coding for the putative TomRSV 3C-related protease (amino acids 1213 to 1508) in a transcription vector and in a transient expression vector. Using cell-free transcription and translation systems and plant protoplasts, we have dem

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

    Catherine S. Adamson

    2012-01-01

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

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

    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.

  10. Camelid single domain antibodies (VHHs) as neuronal cell intrabody binding agents and inhibitors of Clostridium botulinum neurotoxin (BoNT) proteases

    Tremblay, Jacqueline M.; Kuo, Chueh-Ling; Abeijon, Claudia; Sepulveda, Jorge; Oyler, George; Hu, Xuebo; Jin, Moonsoo M.; Shoemaker, Charles B.

    2010-01-01

    Botulinum neurotoxins (BoNTs) function by delivering a protease to neuronal cells that cleave SNARE proteins and inactivate neurotransmitter exocytosis. Small (14 kDa) binding domains specific for the protease of BoNT serotypes A or B were selected from libraries of heavy chain only antibody domains (VHHs or nanobodies) cloned from immunized alpacas. Several VHHs bind the BoNT proteases with high affinity (KD near 1 nM) and include potent inhibitors of BoNT/A protease activity (Ki near 1 nM)....

  11. Proteases involved in generation of beta- and alpha-amylases from a large amylase precursor in Bacillus polymyxa.

    S. Takekawa; Uozumi, N; Tsukagoshi, N; Udaka, S

    1991-01-01

    The genes for extracellular neutral protease (Npr) and intracellular serine protease (Isp) were cloned from Bacillus polymyxa in order to elucidate the process involved in the generation of multiple beta-amylases and an alpha-amylase from a large amylase precursor. The npr gene was composed of 1,770 bp and 570 amino acids, while the isp gene was composed of 978 bp and 326 amino acids. Both proteases produced by E. coli cleaved the amylase precursor to generate beta- and alpha-amylases. Furthe...

  12. BACE2, a β-secretase homolog, cleaves at the β site and within the amyloid-β region of the amyloid-β precursor protein

    Farzan, Michael; Schnitzler, Christine E.; Vasilieva, Natalya; Leung, Doris; Choe, Hyeryun

    2000-01-01

    Production of amyloid-β protein (Aβ) is initiated by a β-secretase that cleaves the Aβ precursor protein (APP) at the N terminus of Aβ (the β site). A recently identified aspartyl protease, BACE, cleaves the β site and at residue 11 within the Aβ region of APP. Here we show that BACE2, a BACE homolog, cleaves at the β site and more efficiently at a different site within Aβ. The Flemish missense mutation of APP, implicated in a form of familial Alzheimer's disease, is adjacent to this latter site and markedly increases Aβ production by BACE2 but not by BACE. BACE and BACE2 respond identically to conservative β-site mutations, and alteration of a common active site Arg inhibits β-site cleavage but not cleavage within Aβ by both enzymes. These data suggest that BACE2 contributes to Aβ production in individuals bearing the Flemish mutation, and that selective inhibition of these highly similar proteases may be feasible and therapeutically advantageous. PMID:10931940

  13. 3C 273 - half a century later

    Slavcheva-Mihova, L.; Mihov, B.; I. Iliev

    2013-01-01

    We have presented an optical monitoring of 3C 273, the first quasar discovered fifty years ago. It does not show variability both on intra-night and long-term time scales. To facilitate the further monitoring of 3C 273, we compiled the available calibrations of the comparison stars in its field into a mean sequence.

  14. A Genomic Analysis of Rat Proteases and Protease Inhibitors

    Puente, Xose S.; López-Otín, Carlos

    2004-01-01

    Proteases perform important roles in multiple biological and pathological processes. The availability of the rat genome sequence has facilitated the analysis of the complete protease repertoire or degradome of this model organism. The rat degradome consists of at least 626 proteases and homologs, which are distributed into 24 aspartic, 160 cysteine, 192 metallo, 221 serine, and 29 threonine proteases. This distribution is similar to that of the mouse degradome but is more complex than that of...

  15. Bacterial proteases and virulence

    Frees, Dorte; Brøndsted, Lone; Ingmer, Hanne

    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...... tolerance to adverse conditions such as those experienced in the host. In the membrane, HtrA performs similar functions whereas the extracellular proteases, in close contact with host components, pave the way for spreading infections by degrading host matrix components or interfering with host cell...... cell. 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....

  16. Protease inhibitors decrease IgG shedding from Staphylococcus aureus, increasing complement activation and phagocytosis efficiency.

    Fernandez Falcon, Maria F; Echague, Charlene G; Hair, Pamela S; Nyalwidhe, Julius O; Cunnion, Kenji M

    2011-10-01

    Staphylococcus aureus is a major pathogen for immunologically intact humans and its pathogenesis is a model system for evasion of host defences. Antibodies and complement are essential elements of the humoral immune system for prevention and control of S. aureus infections. The specific hypothesis for the proposed research is that S. aureus modifies humoral host defences by cleaving IgG that has bound to the bacterial surface, thereby inhibiting opsonophagocytosis. S. aureus was coated with pooled, purified human IgG and assayed for the shedding of cleaved IgG fragments using ELISA and Western blot analysis. Surface-bound IgG was shed efficiently from S. aureus in the absence of host blood proteins. Broad-spectrum protease inhibitors prevented cleavage of IgG from the S. aureus surface, suggesting that staphylococcal proteases are responsible for IgG cleavage. Serine protease inhibitors and cysteine protease inhibitors decreased the cleavage of surface-bound IgG; however, a metalloprotease inhibitor had no effect. Using protease inhibitors to prevent the cleavage of surface-bound IgG increased the binding of complement C3 fragments on the surface of S. aureus, increased the association with human neutrophils and increased phagocytosis by human neutrophils. PMID:21636671

  17. RNA-Cleaving DNA Enzymes with Altered Regio- or Enantioselectivity

    Ordoukhanian, Phillip; Joyce, Gerald F.

    2002-01-01

    In vitro evolution methods were used to obtain DNA enzymes that cleave either a 2',5' - phosphodiester following a wibonucleotide or a 3',5' -phosphodiester following an L-ribonucleotide. Both enzymes can operate in an intermolecular reaction format with multiple turnover. The DNA enzyme that cleaves a 2',5' -phosphodiester exhibits a k(sub cat) of approx. 0.01/ min and catalytic efficiency, k(sub cat)/k(sub m) of approx. 10(exp 5)/ M min. The enzyme that cleaves an L-ribonudeotide is about 10-fold slower and has a catalytic efficiency of approx. 4 x 10(exp 5)/ M min. Both enzymes require a divalent metal cation for their activity and have optimal catalytic rate at pH 7-8 and 35-50 C. In a comparison of each enzyme s activity with either its corresponding substrate that contains an unnatural ribonudeotide or a substrate that instead contains a standard ribonucleotide, the 2',5' -phosphodiester-deaving DNA enzyme exhibited a regioselectivity of 6000- fold, while the L-ribonucleotide-cleaving DNA enzyme exhibited an enantioselectivity of 50-fold. These molecules demonstrate how in vitro evolution can be used to obtain regio- and enantioselective catalysts that exhibit specificities for nonnatural analogues of biological compounds.

  18. Protease determination using an optimized alcohol enzyme electrode.

    Bardeletti, G; Carillon, C

    1993-12-01

    A new method for the determination of protease activities is described. In this large family, trypsin is used as a protease model that cleaves the ethyl or methyl ester of artificial substrates producing ethanol or methanol. Alcohol is detected using an alcohol oxidase enzyme electrode. The H2O2 production that occurs is measured amperometrically. At 30 degrees C, in a 0.1M phosphate buffer, pH 7.5, the enzyme electrode response for ethanol was calibrated at 3.10(-6)-3.10(-3)M and for methanol from 3.10(-7) to 4.10(-4)M in the cell measurement. Trypsin levels as determined by the proposed method and by a conventional spectrophotometric method are in good agreement when using the same measurement conditions. A detection limit of 10 U.L-1 and a linear calibration curve of 10-100,000 U.L-1 in the sample were obtained. Measuring time for the required trypsin solution concentration was from 4 min (for the most dilute samples) to 1 min (for the most concentrate samples). In a typical experiment, protease measurements did not inactivate the alcohol oxidase on the probe, nor did a more classical use for alcohol detection. The procedure developed could permit any protease estimation on the condition that they hydrolyze ester bonds from synthetic substrate. PMID:8109959

  19. Trichuris suis: thiol protease activity from adult worms.

    Hill, D E; Sakanari, J A

    1997-01-01

    Trichuris suis, the whipworm of swine, causes anemia, weight loss, anorexia, mucohemorrhagic diarrhea, and death in heavy infections. A zinc metalloprotease has been suggested to play a role in the severe enteric pathology associated with infection and the infiltration of opportunistic bacteria into deeper tissues in the swine colon. In this study, a thiol protease from gut extracts of adult T. suis and from excretory/secretory components (E/S) of adult worms was characterized using fluorogenic peptide substrates and protein substrate gels. The protease cleaved the fluorogenic substrate Z-Phe-Arg-AMC, and this cleavage was completely inhibited by the thiol protease inhibitors E-64, leupeptin, Z-Phe-Ala-CH2F, and Z-Phe-Arg-CH2F. Gelatin substrate gels and fluorescence assays using both the gut and the stichosome extracts and E/S revealed enhanced activity when 2 mM dithiothreitol or 5 mM cysteine was included in the incubation buffer, and optimal activity was seen over a pH range of 5.5 to 8.5. Incubation of gut extracts or E/S material with inhibitors of aspartic, serine, or metalloproteases had no effect on the cleavage of Z-Phe-Arg-AMC. Thiol protease activity was found in extracts of gut tissue but not in the extracts of stichocytes of adult worms. N-terminal amino acid sequencing of the protease revealed sequence homologies with cathepsin B-like thiol protease identified from parasitic and free-living nematodes. PMID:9024202

  20. Nucleic Acid Aptamers Against Proteases

    Dupont, D M; Andersen, L M; Bøtkjær, Kenneth Alrø; Andreasen, P A

    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 small molecule protease inhibitors of sufficient specificity has proved a daunting task. Aptamers seem to represent a promising alternative. In our review, we concentrate on biochemical mechanisms of...

  1. Death proteases come alive

    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

  2. Proteases in Periodontal Disease

    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.

  3. Structure of the catalytic domain of the hepatitis C virus NS2-3 protease

    Lorenz,I.; Marcotrigiano, J.; Dentzer, T.; Rice, C.

    2006-01-01

    Hepatitis C virus is a major global health problem affecting an estimated 170 million people worldwide. Chronic infection is common and can lead to cirrhosis and liver cancer. There is no vaccine available and current therapies have met with limited success. The viral RNA genome encodes a polyprotein that includes two proteases essential for virus replication. The NS2-3 protease mediates a single cleavage at the NS2/NS3 junction, whereas the NS3-4A protease cleaves at four downstream sites in the polyprotein. NS3-4A is characterized as a serine protease with a chymotrypsin-like fold, but the enzymatic mechanism of the NS2-3 protease remains unresolved. Here we report the crystal structure of the catalytic domain of the NS2-3 protease at 2.3 Angstroms resolution. The structure reveals a dimeric cysteine protease with two composite active sites. For each active site, the catalytic histidine and glutamate residues are contributed by one monomer, and the nucleophilic cysteine by the other. The carboxy-terminal residues remain coordinated in the two active sites, predicting an inactive post-cleavage form. Proteolysis through formation of a composite active site occurs in the context of the viral polyprotein expressed in mammalian cells. These features offer unexpected insights into polyprotein processing by hepatitis C virus and new opportunities for antiviral drug design.

  4. Antimicrobial proteins and peptides in human lung diseases: A friend and foe partnership with host proteases.

    Lecaille, Fabien; Lalmanach, Gilles; Andrault, Pierre-Marie

    2016-03-01

    Lung antimicrobial proteins and peptides (AMPs) are major sentinels of innate immunity by preventing microbial colonization and infection. Nevertheless bactericidal activity of AMPs against Gram-positive and Gram-negative bacteria is compromised in patients with chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF) and asthma. Evidence is accumulating that expression of harmful human serine proteases, matrix metalloproteases and cysteine cathepsins is markedely increased in these chronic lung diseases. The local imbalance between proteases and protease inhibitors compromises lung tissue integrity and function, by not only degrading extracellular matrix components, but also non-matrix proteins. Despite the fact that AMPs are somewhat resistant to proteolytic degradation, some human proteases cleave them efficiently and impair their antimicrobial potency. By contrast, certain AMPs may be effective as antiproteases. Host proteases participate in concert with bacterial proteases in the degradation of key innate immunity peptides/proteins and thus may play immunomodulatory activities during chronic lung diseases. In this context, the present review highlights the current knowledge and recent discoveries on the ability of host enzymes to interact with AMPs, providing a better understanding of the role of human proteases in innate host defense. PMID:26341472

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

    Palarasah, Yaseelan; Skjødt, 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. The C3c mAb was confirmed to be C3c specific, as it showed no cross-reactivity with native (un-cleaved) C3, with C3b, iC3b, or with C3d. Also, no significant reaction was observed with C3 fragments in factor I deficient sera or plasma. This antibody forms the basis for the generation of a robust...

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

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

    2014-01-01

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

  7. Substrate-Induced Conformational Changes Occur in All Cleaved Forms of Caspase-6

    S Vaidya; E Velazquez-Delgado; G Abbruzzese; J Hardy

    2011-12-31

    Caspase-6 is an apoptotic cysteine protease that also governs disease progression in Huntington's and Alzheimer's diseases. Caspase-6 is of great interest as a target for treatment of these neurodegenerative diseases; however, the molecular basis of caspase-6 function and regulation remains poorly understood. In the recently reported structure of caspase-6, the 60's and 130's helices at the base of the substrate-binding groove extend upward, in a conformation entirely different from that of any other caspase. Presently, the central question about caspase-6 structure and function is whether the extended conformation is the catalytically competent conformation or whether the extended helices must undergo a large conformational rearrangement in order to bind substrate. We have generated a series of caspase-6 cleavage variants, including a novel constitutively two-chain form, and determined crystal structures of caspase-6 with and without the intersubunit linker. This series allows evaluation of the role of the prodomain and intersubunit linker on caspase-6 structure and function before and after substrate binding. Caspase-6 is inherently more stable than closely related caspases. Cleaved caspase-6 with both the prodomain and the linker present is the most stable, indicating that these two regions act in concert to increase stability, but maintain the extended conformation in the unliganded state. Moreover, these data suggest that caspase-6 undergoes a significant conformational change upon substrate binding, adopting a structure that is more like canonical caspases.

  8. Structural and functional characterization of cleavage and inactivation of human serine protease inhibitors by the bacterial SPATE protease EspPα from enterohemorrhagic E. coli.

    André Weiss

    Full Text Available EspPα and EspI are serine protease autotransporters found in enterohemorrhagic Escherichia coli. They both belong to the SPATE autotransporter family and are believed to contribute to pathogenicity via proteolytic cleavage and inactivation of different key host proteins during infection. Here, we describe the specific cleavage and functional inactivation of serine protease inhibitors (serpins by EspPα and compare this activity with the related SPATE EspI. Serpins are structurally related proteins that regulate vital protease cascades, such as blood coagulation and inflammatory host response. For the rapid determination of serpin cleavage sites, we applied direct MALDI-TOF-MS or ESI-FTMS analysis of coincubations of serpins and SPATE proteases and confirmed observed cleavage positions using in-gel-digest of SDS-PAGE-separated degradation products. Activities of both serpin and SPATE protease were assessed in a newly developed photometrical assay using chromogenic peptide substrates. EspPα cleaved the serpins α1-protease inhibitor (α1-PI, α1-antichymotrypsin, angiotensinogen, and α2-antiplasmin. Serpin cleavage led to loss of inhibitory function as demonstrated for α1-PI while EspPα activity was not affected. Notably, EspPα showed pronounced specificity and cleaved procoagulatory serpins such as α2-antiplasmin while the anticoagulatory antithrombin III was not affected. Together with recently published research, this underlines the interference of EspPα with hemostasis or inflammatory responses during infection, while the observed interaction of EspI with serpins is likely to be not physiologically relevant. EspPα-mediated serpin cleavage occurred always in flexible loops, indicating that this structural motif might be required for substrate recognition.

  9. Neisseria meningitidis NalP cleaves human complement C3, facilitating degradation of C3b and survival in human serum

    Del Tordello, Elena; Vacca, Irene; Ram, Sanjay; Rappuoli, Rino; Serruto, Davide

    2013-01-01

    The complement system is a crucial component of the innate immune response in humans. In this study, we report the characterization of an autotransporter protease of Neisseria meningitidis named NalP. We show that NalP is able to cleave the α-chain of the human complement factor C3 in a species-specific manner. As a consequence, the deposition of C3b on the bacterial surface is reduced and, in human serum, the NalP-generated C3b fragment is further degraded by host factors. Our results signif...

  10. Effect of mutations in the human immunoglobulin A1 (IgA1) hinge on its susceptibility to cleavage by diverse bacterial IgA1 proteases.

    Senior, Bernard W; Woof, Jenny M

    2005-03-01

    Components of the human immunoglobulin A1 (IgA1) hinge governing sensitivity to cleavage by bacterial IgA1 proteases were investigated. Recombinant antibodies with distinct hinge mutations were constructed from a hybrid comprised of human IgA2 bearing half of the human IgA1 hinge region. This hybrid antibody and all the mutant antibodies derived from it were resistant to cleavage by the IgA1 proteases from Streptococcus oralis and Streptococcus mitis biovar 1 strains but were cleaved to various degrees by those of Streptococcus pneumoniae, some Streptococcus sanguis strains, and the type 1 and 2 IgA1 proteases of Haemophilus influenzae, Neisseria meningitidis, and Neisseria gonorrhoeae. Remarkably, those proteases that cleave a Pro-Ser peptide bond in the wild-type IgA1 hinge were able to cleave mutant antibodies lacking a Pro-Ser peptide bond in the hinge, and those that cleave a Pro-Thr peptide bond in the wild-type IgA1 hinge were able to cleave mutant antibodies devoid of a Pro-Thr peptide bond in the hinge. Thus, the enzymes can cleave alternatives to their preferred postproline peptide bond when such a bond is unavailable. Peptide sequence analysis of a representative antibody digestion product confirmed this conclusion. The presence of a cleavable peptide bond near the CH2 end of the hinge appeared to result in greater cleavage than if the scissile bond was at the CH1 end of the hinge. Proline-to-serine substitution at residue 230 in a hinge containing potentially cleavable Pro-Ser and Pro-Thr peptide bonds increased the resistance of the antibody to cleavage by many IgA1 proteases. PMID:15731049

  11. NATO-3C/Delta launch

    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.

  12. BATSE observations of 3C273

    Paciesas, W.S.; Mallozzi, R. S.; Pendleton, G. N.; Harmon, B. A.; Wilson, C. A.; Zhang, S. N.; Fishman, G. J.

    1994-01-01

    The quasar 3C273 has been detected by all instruments on CGRO. The emission from this source is monitored continuously by BATSE using Earth occultation. We present results of a preliminary analysis of BATSE data, including light curves of the 3C273 flux covering approximately 150 days in the interval April-August 1991 and approximately 350 days in the interval July 1992-April 1993. The source intensity in the energy range 50-300 keV is typically approx. 0.002 ph cm(exp -2)s(exp -1). We find weak evidence for variations of as much as a factor of 3 in the intensity. We derive spectral parameters of 3C273 during the intervals TJD 8422-8435 (15-28 June 1991) and TJD 8532-8546 (3-17 October 1991) for comparison with other CGRO instruments.

  13. Analyzing polarization swings in 3C 279

    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.

  14. Analyzing polarization swings in 3C 279

    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.

  15. The spectrum of 3C 58

    Digital spectra of three faint knots in the SNR 3C 58 have been obtained in the wavelength range lambdalambda4700--7300 with the intensified Reticon spectrometer at the 1.3 m telescope of McGraw-Hill Observatory. Emission lines of [S II], [N II], Hα, and [O III] were detected with radial velocities less than 100 km s-1. Although 3C 58 resembles the Crab Nebula in its radio properties and is thought to be the remnant of the supernova observed in A.D. 1181, the relative line intensities and radial velocities reported here more nearly resemble those of the Cygnus Loop and Kepler's SNR

  16. The Bright Quasar 3C 273

    Courvoisier, Thierry J. -L.

    1998-01-01

    We review the observed properties of the bright quasar 3C~273 and discuss the implications of these observations for the emission processes and in view of gaining a more global understanding of the object. Continuum and line emission are discussed. The emission from the radio domain to gamma rays are reviewed. Emphasis is given to variability studies across the spectrum as a means to gain some understanding on the relationships between the emission components. 3C~273 has a small scale jet and...

  17. Broad Spectrum Activity of a Lectin-Like Bacterial Serine Protease Family on Human Leukocytes

    Ayala-Lujan, Jorge Luis; Vijayakumar, Vidhya; Gong, Mei; Smith, Rachel; Santiago, Araceli E.; Ruiz-Perez, Fernando

    2014-01-01

    The serine protease autotransporter from Enterobacteriaceae (SPATE) family, which number more than 25 proteases with apparent diverse functions, have been phylogenetically divided into two distinct classes, designated 1 and 2. We recently demonstrated that Pic and Tsh, two members of the class-2 SPATE family produced by intestinal and extraintestinal pathogenic E. coli, were able to cleave a number of O-glycosylated proteins on neutrophils and lymphocytes resulting in impaired leukocyte functions. Here we show that most members of the class-2 SPATE family have lectin-like properties and exhibit differential protease activity reliant on glycoprotein type and cell lineage. Protease activity was seen in virtually all tested O-glycosylated proteins including CD34, CD55, CD164, TIM1, TIM3, TIM4 and C1-INH. We also show that although SPATE proteins bound and cleaved glycoproteins more efficiently on granulocytes and monocytes, they also targeted glycoproteins on B, T and natural killer lymphocytes. Finally, we found that the characteristic domain-2 of class-2 SPATEs is not required for glycoprotease activity, but single amino acid mutations in Pic domain-1 to those residues naturally occurring in domain-1 of SepA, were sufficient to hamper Pic glycoprotease activity. This study shows that most class-2 SPATEs have redundant activities and suggest that they may function as immunomodulators at several levels of the immune system. PMID:25251283

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

    Jorge Luis Ayala-Lujan

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

  19. L3+C air shower array

    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.

  20. Radio jet of 3C273

    Radio observation at 408 MHz of 3C273 are reported which show that the brightness of the postulated counter-jet is <1/100 of the brightness of the visible jet. Possible explanations of these observations are discussed. (U.K.)

  1. IRIS photometry of 3C273

    Twenty-seven blue plates of 3C 273 originally measured by Zwicky, Karpowicz, and Rudnicki using the Argelander method and measurement of image diameters were remeasured on an iris photometer to improve their precision. These measures, including two later observations, have an accuracy approximately +-0.1m. Some errors in the Julian day numbers given in the earlier study are corrected

  2. IRIS photometry of 3C273

    Lanning, H.H.

    1976-04-01

    Twenty-seven blue plates of 3C 273 originally measured by Zwicky, Karpowicz, and Rudnicki using the Argelander method and measurement of image diameters were remeasured on an iris photometer to improve their precision. These measures, including two later observations, have an accuracy approximately +-0.1m. Some errors in the Julian day numbers given in the earlier study are corrected.

  3. PROSPER: an integrated feature-based tool for predicting protease substrate cleavage sites.

    Jiangning Song

    Full Text Available The ability to catalytically cleave protein substrates after synthesis is fundamental for all forms of life. Accordingly, site-specific proteolysis is one of the most important post-translational modifications. The key to understanding the physiological role of a protease is to identify its natural substrate(s. Knowledge of the substrate specificity of a protease can dramatically improve our ability to predict its target protein substrates, but this information must be utilized in an effective manner in order to efficiently identify protein substrates by in silico approaches. To address this problem, we present PROSPER, an integrated feature-based server for in silico identification of protease substrates and their cleavage sites for twenty-four different proteases. PROSPER utilizes established specificity information for these proteases (derived from the MEROPS database with a machine learning approach to predict protease cleavage sites by using different, but complementary sequence and structure characteristics. Features used by PROSPER include local amino acid sequence profile, predicted secondary structure, solvent accessibility and predicted native disorder. Thus, for proteases with known amino acid specificity, PROSPER provides a convenient, pre-prepared tool for use in identifying protein substrates for the enzymes. Systematic prediction analysis for the twenty-four proteases thus far included in the database revealed that the features we have included in the tool strongly improve performance in terms of cleavage site prediction, as evidenced by their contribution to performance improvement in terms of identifying known cleavage sites in substrates for these enzymes. In comparison with two state-of-the-art prediction tools, PoPS and SitePrediction, PROSPER achieves greater accuracy and coverage. To our knowledge, PROSPER is the first comprehensive server capable of predicting cleavage sites of multiple proteases within a single substrate

  4. Studies on the gonococcal IgA1 protease II. Improved methods of enzyme purification and production of monoclonal antibodies to the enzyme.

    Blake, M S; Eastby, C

    1991-11-22

    Two types of extremely active proteases that cleave human IgA1 are produced by pathogenic Neisseria in minute concentrations. To study the antigenicity of these enzymes, a simplified method is described to purify these enzymes from large batch cultures to obtain a sufficient quantity of these IgA1 proteases to study these characteristics. In addition, we describe the production of both rabbit polyclonal and mouse monoclonal antibodies to one of these enzymes. One such monoclonal antibody seemed directed toward the active site of the IgA1 protease and inhibited its enzymatic activity. PMID:1960418

  5. Cathepsin proteases in Toxoplasma gondii

    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...

  6. Nucleic Acid Aptamers Against Proteases

    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...

  7. Substrate specificity of rhomboid intramembrane proteases is governed by helix-breaking residues in the substrate transmembrane domain.

    Urban, Sinisa; Freeman, Matthew

    2003-06-01

    Rhomboid intramembrane proteases initiate cell signaling during Drosophila development and Providencia bacterial growth by cleaving transmembrane ligand precursors. We have determined how specificity is achieved: Drosophila Rhomboid-1 is a site-specific protease that recognizes its substrate Spitz by a small region of the Spitz transmembrane domain (TMD). This substrate motif is necessary and sufficient for cleavage and is composed of residues known to disrupt helices. Rhomboids from diverse organisms including bacteria and vertebrates recognize the same substrate motif, suggesting that they use a universal targeting strategy. We used this information to search for other rhomboid substrates and identified a family of adhesion proteins from the human parasite Toxoplasma gondii, the TMDs of which were efficient substrates for rhomboid proteases. Intramembrane cleavage of these proteins is required for host cell invasion. These results provide an explanation of how rhomboid proteases achieve specificity, and allow some rhomboid substrates to be predicted from sequence information. PMID:12820957

  8. Optical fiber alignment using cleaved-edge diffracted light

    Brun, Louis C.; Bergeron, Patrick; Duguay, Michel A.; Ouellette, Francois; Tetu, Michel

    1993-08-01

    We describe a simple technique for aligning optical fibers prior to fusion splicing. The technique relies on the fact that well-cleaved fiber ends have extremely sharp edges. By making the narrow pencil of light emerging from one fiber scan laterally over the entrance face of a second fiber, and by monitoring the light diffracted past its sharp edges, we can locate precisely the geometric center of the output fiber. With this technique, we have aligned fiber cores with a mean lateral offset of 0.81 micrometers , the major part of this offset caused by the eccentricity of the core relative to the cladding's circular perimeter.

  9. STM observation of twin microlayers on cleaved bismuth surfaces

    Edel'man, V. S.

    1996-02-01

    Scanning tunneling microscopy of cleaved bismuth surfaces at low temperatures revealed the presence of thin twin microlayers, whose width ∼70 Å is determined by the fact that the height gained in crossing such a microlayer due to its tilt with respect to the surface of the undisturbed crystal equals the interlunar distance in the [0001] direction. The microlayers were strictly aligned with the surface atomic rows. Their lengths were macroscopically large, being no smaller than a few fractions of a micron. It was found that, near the microlayer boundaries, the electron properties were significantly different from those at other points on the surface.

  10. Superluminal expansion of quasar 3C273

    Using the very long baseline interferometry technique observations of the radio structure of the quasar 3C273 have been obtained from mid-1977 to mid-1980 at 10.65 and 5.0 GHz. Maps based on the 10.65 GHz results are presented which provide unambiguous evidence of superluminal expansion. It is argued that the apparent constant velocity of 9.6c observed in this period is an important constraint on superluminal expansion theories. (U.K.)

  11. Fabrication of HgI2 nuclear radiation detectors by machine cleaving

    A new device has been designed to facilitate the cleaving of thin sections from bulk crystals of mercuric iodide. Crystallographic perfection of the machine-cleaved sections was established by gamma-ray diffraction rocking curves and was found to be higher than the perfection of hand-cleaved sections or of as-grown thin platelets, approaching the perfection of string-sawn sections of HgI2. A correlation was found between the perfection and thickness of the machine-cleaved sections, i.e., the thicker the section the more perfect it is. Reproducibility of the fabrication was significantly improved by using machine cleaving in the fabrication process

  12. Observations with a VLB array. III. The sources 3C 120, 3C 273B, 2134+004, and 3C 84

    The sources 3C 120, 3C 273B, 2134+004, and 3C 84 have been observed at several epochs at 2.8 cm using a multielement very-long-baseline interferometer (VLBI). The resulting visibility data show that the brightness distributions of each source are variable. There are large changes in the visibilities of 3C 120 and 3C 273B on a time scale of months. For 3C 120, and perhaps 3C 273B, the changes can be explained by source distributions in which the components are separating at high (probably relativistic) speeds

  13. The nature of the air-cleaved mica surface

    Christenson, Hugo K.; Thomson, Neil H.

    2016-06-01

    The accepted image of muscovite mica is that of an inert and atomically smooth surface, easily prepared by cleavage in an ambient atmosphere. Consequently, mica is extensively used a model substrate in many fundamental studies of surface phenomena and as a substrate for AFM imaging of biomolecules. In this review we present evidence from the literature that the above picture is not quite correct. The mica used in experimental work is almost invariably cleaved in laboratory air, where a reaction between the mica surface, atmospheric CO2 and water occurs immediately after cleavage. The evidence suggests very strongly that as a result the mica surface becomes covered by up to one formula unit of K2CO3 per nm2, which is mobile under humid conditions, and crystallises under drier conditions. The properties of mica in air or water vapour cannot be fully understood without reference to the surface K2CO3, and many studies of the structure of adsorbed water on mica surfaces may need to be revisited. With this new insight, however, the air-cleaved mica should provide exciting opportunities to study phenomena such as two-dimensional ion diffusion, electrolyte effects on surface conductivity, and two-dimensional crystal nucleation.

  14. The relationship between the spectrum and flux density of 3C273 and 3C446

    Yuan, Yuhai; Fan, Junhui

    2015-06-01

    In the radio band, the relationship between the emission spectrum ( α) and flux density ( F) can demonstrate the emission theory and process. In this paper, we used the radio data of 3C273 and 3C446 from UMRAO (the University of Michigan Radio Observatory) to calculate the spectral indices ( α), and analyzed the relationship between spectral indices ( α) and flux densities ( F). We obtained the following results. (1) There were anti-correlations between α and F, for 3C273, α=-0.024 F 14.5+0.91, with the correlation coefficient r=-0.92, the chance p3C273, the time spans of two α- F circles were 8.43 years and 7.79 years; for 3C446, the time spans of two α- F circles were 5.66 years and 6.64 years. Not only for 3C273, but also for 3C446, the time spans were consistent with the quasi-periodicities calculated from the lightcurve or spectral variance.

  15. Plasmodium falciparum signal peptide peptidase cleaves malaria heat shock protein 101 (HSP101). Implications for gametocytogenesis

    Highlights: • PfSPP is an ER resident protease. • PfSPP is expressed both as a monomer and dimer. • The signal peptide of HSP101 is the first known substrate of PfSPP. • Reduced PfSPP activity may significantly affect ER homeostasis. - Abstract: Previously we described the identification of a Plasmodium falciparum signal peptide peptidase (PfSPP) functioning at the blood stage of malaria infection. Our studies also demonstrated that mammalian SPP inhibitors prevent malaria parasite growth at the late-ring/early trophozoite stage of intra-erythrocytic development. Consistent with its role in development, we tested the hypothesis that PfSPP functions at the endoplasmic reticulum of P.falciparum where it cleaves membrane-bound signal peptides generated following the enzyme activity of signal peptidase. The localization of PfSPP to the endoplasmic reticulum was confirmed by immunofluorescence microscopy and immunogold electron microscopy. Biochemical analysis indicated the existence of monomer and dimer forms of PfSPP in the parasite lysate. A comprehensive bioinformatics screen identified several candidate PfSPP substrates in the parasite genome. Using an established transfection based in vivo luminescence assay, malaria heat shock protein 101 (HSP101) was identified as a substrate of PfSPP, and partial inhibition of PfSPP correlated with the emergence of gametocytes. This finding unveils the first known substrate of PfSPP, and provides new perspectives for the function of intra-membrane proteolysis at the erythrocyte stage of malaria parasite life cycle

  16. Plasmodium falciparum signal peptide peptidase cleaves malaria heat shock protein 101 (HSP101). Implications for gametocytogenesis

    Baldwin, Michael; Russo, Crystal; Li, Xuerong [Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States); Chishti, Athar H., E-mail: athar.chishti@tufts.edu [Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States); Sackler School of Graduate Biomedical Sciences, Programs in Physiology, Pharmacology, and Microbiology, Tufts University School of Medicine, Boston, MA 02111 (United States)

    2014-08-08

    Highlights: • PfSPP is an ER resident protease. • PfSPP is expressed both as a monomer and dimer. • The signal peptide of HSP101 is the first known substrate of PfSPP. • Reduced PfSPP activity may significantly affect ER homeostasis. - Abstract: Previously we described the identification of a Plasmodium falciparum signal peptide peptidase (PfSPP) functioning at the blood stage of malaria infection. Our studies also demonstrated that mammalian SPP inhibitors prevent malaria parasite growth at the late-ring/early trophozoite stage of intra-erythrocytic development. Consistent with its role in development, we tested the hypothesis that PfSPP functions at the endoplasmic reticulum of P.falciparum where it cleaves membrane-bound signal peptides generated following the enzyme activity of signal peptidase. The localization of PfSPP to the endoplasmic reticulum was confirmed by immunofluorescence microscopy and immunogold electron microscopy. Biochemical analysis indicated the existence of monomer and dimer forms of PfSPP in the parasite lysate. A comprehensive bioinformatics screen identified several candidate PfSPP substrates in the parasite genome. Using an established transfection based in vivo luminescence assay, malaria heat shock protein 101 (HSP101) was identified as a substrate of PfSPP, and partial inhibition of PfSPP correlated with the emergence of gametocytes. This finding unveils the first known substrate of PfSPP, and provides new perspectives for the function of intra-membrane proteolysis at the erythrocyte stage of malaria parasite life cycle.

  17. The radio jet of 3C273

    Most radio sources are two-sided and a minority are one-sided. The first-known and brightest example is 3C273, a high-luminosity QSO, showing 'super-luminal' proper motions in the core. The explanation of such one-sided sources may follow one of two lines: on the one hand, the ejection of material from the central object may truly be one-sided, while on the other hand the ejection may be two-sided but at a relativistic speed, so that the receding half is hidden by Doppler beaming. (Auth.)

  18. Rapid paracellular transmigration of Campylobacter jejuni across polarized epithelial cells without affecting TER: role of proteolytic-active HtrA cleaving E-cadherin but not fibronectin

    Boehm, Manja

    2012-04-25

    AbstractBackgroundCampylobacter jejuni is one of the most important bacterial pathogens causing food-borne illness worldwide. Crossing the intestinal epithelial barrier and host cell entry by C. jejuni is considered the primary reason of damage to the intestinal tissue, but the molecular mechanisms as well as major bacterial and host cell factors involved in this process are still widely unclear.ResultsIn the present study, we characterized the serine protease HtrA (high-temperature requirement A) of C. jejuni as a secreted virulence factor with important proteolytic functions. Infection studies and in vitro cleavage assays showed that C. jejuni’s HtrA triggers shedding of the extracellular E-cadherin NTF domain (90 kDa) of non-polarised INT-407 and polarized MKN-28 epithelial cells, but fibronectin was not cleaved as seen for H. pylori’s HtrA. Deletion of the htrA gene in C. jejuni or expression of a protease-deficient S197A point mutant did not lead to loss of flagella or reduced bacterial motility, but led to severe defects in E-cadherin cleavage and transmigration of the bacteria across polarized MKN-28 cell layers. Unlike other highly invasive pathogens, transmigration across polarized cells by wild-type C. jejuni is highly efficient and is achieved within a few minutes of infection. Interestingly, E-cadherin cleavage by C. jejuni occurs in a limited fashion and transmigration required the intact flagella as well as HtrA protease activity, but does not reduce transepithelial electrical resistance (TER) as seen with Salmonella, Shigella, Listeria or Neisseria.ConclusionThese results suggest that HtrA-mediated E-cadherin cleavage is involved in rapid crossing of the epithelial barrier by C. jejuni via a very specific mechanism using the paracellular route to reach basolateral surfaces, but does not cleave the fibronectin receptor which is necessary for cell entry.

  19. Rapid paracellular transmigration of Campylobacter jejuni across polarized epithelial cells without affecting TER: role of proteolytic-active HtrA cleaving E-cadherin but not fibronectin

    Boehm Manja

    2012-04-01

    Full Text Available Abstract Background Campylobacter jejuni is one of the most important bacterial pathogens causing food-borne illness worldwide. Crossing the intestinal epithelial barrier and host cell entry by C. jejuni is considered the primary reason of damage to the intestinal tissue, but the molecular mechanisms as well as major bacterial and host cell factors involved in this process are still widely unclear. Results In the present study, we characterized the serine protease HtrA (high-temperature requirement A of C. jejuni as a secreted virulence factor with important proteolytic functions. Infection studies and in vitro cleavage assays showed that C. jejuni’s HtrA triggers shedding of the extracellular E-cadherin NTF domain (90 kDa of non-polarised INT-407 and polarized MKN-28 epithelial cells, but fibronectin was not cleaved as seen for H. pylori’s HtrA. Deletion of the htrA gene in C. jejuni or expression of a protease-deficient S197A point mutant did not lead to loss of flagella or reduced bacterial motility, but led to severe defects in E-cadherin cleavage and transmigration of the bacteria across polarized MKN-28 cell layers. Unlike other highly invasive pathogens, transmigration across polarized cells by wild-type C. jejuni is highly efficient and is achieved within a few minutes of infection. Interestingly, E-cadherin cleavage by C. jejuni occurs in a limited fashion and transmigration required the intact flagella as well as HtrA protease activity, but does not reduce transepithelial electrical resistance (TER as seen with Salmonella, Shigella, Listeria or Neisseria. Conclusion These results suggest that HtrA-mediated E-cadherin cleavage is involved in rapid crossing of the epithelial barrier by C. jejuni via a very specific mechanism using the paracellular route to reach basolateral surfaces, but does not cleave the fibronectin receptor which is necessary for cell entry.

  20. The peculiar radio galaxy 3C 433

    Van Breugel, W.; Helfand, D.; Balick, B.; Heckman, T.; Miley, G.

    1983-01-01

    Radio, optical and X-ray observations are presented of the peculiar radio galaxy 3C 433, a Seyfert 2 object with luminosity an order of magnitude greater than that expected from its complex, shell-type morphology. Observations conducted at 6 and 12 cm with the VLA and at 21 cm with the Westerbork telescope show a striking asymmetry between the northern and southern radio emissions, and an overall X-shaped morphology. Optical observations using the Video Camera and High Gain Video Spectrometer on the 4-m telescope and the Intensified Image Dissector Scanner on the 2.1-m telescope at Kitt Peak confirm the identification of the source with a pair of bright galaxies. Observations in the X-ray from the Einstein Observatory IPC reveal an unresolved source at the position of 3C 433, as well as two serendipitous X-ray sources. The observations may be used to explain the overall structure of the source either in terms of tidal torquing or precessing models of double galaxies; however, it is argued that the tidal torquing model requires fewer assumptions to account for the brightness asymmetry.

  1. Chemistry and Biology of Self-Cleaving Ribozymes.

    Jimenez, Randi M; Polanco, Julio A; Lupták, Andrej

    2015-11-01

    Self-cleaving ribozymes were discovered 30 years ago, but their biological distribution and catalytic mechanisms are only beginning to be defined. Each ribozyme family is defined by a distinct structure, with unique active sites accelerating the same transesterification reaction across the families. Biochemical studies show that general acid-base catalysis is the most common mechanism of self-cleavage, but metal ions and metabolites can be used as cofactors. Ribozymes have been discovered in highly diverse genomic contexts throughout nature, from viroids to vertebrates. Their biological roles include self-scission during rolling-circle replication of RNA genomes, co-transcriptional processing of retrotransposons, and metabolite-dependent gene expression regulation in bacteria. Other examples, including highly conserved mammalian ribozymes, suggest that many new biological roles are yet to be discovered. PMID:26481500

  2. Protease-Sensitive Synthetic Prions

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

    2010-01-01

    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, ...

  3. Juggling jobs: roles and mechanisms of multifunctional protease inhibitors in plants.

    Grosse-Holz, Friederike M; van der Hoorn, Renier A L

    2016-05-01

    Multifunctional protease inhibitors juggle jobs by targeting different enzymes and thereby often controlling more than one biological process. Here, we discuss the biological functions, mechanisms and evolution of three types of multifunctional protease inhibitors in plants. The first type is double-headed inhibitors, which feature two inhibitory sites targeting proteases with different specificities (e.g. Bowman-Birk inhibitors) or even different hydrolases (e.g. α-amylase/protease inhibitors preventing both early germination and seed predation). The second type consists of multidomain inhibitors which evolved by intragenic duplication and are released by processing (e.g. multicystatins and potato inhibitor II, implicated in tuber dormancy and defence, respectively). The third type consists of promiscuous inhibitory folds which resemble mouse traps that can inhibit different proteases cleaving the bait they offer (e.g. serpins, regulating cell death, and α-macroglobulins). Understanding how multifunctional inhibitors juggle biological jobs increases our knowledge of the connections between the networks they regulate. These examples show that multifunctionality evolved independently from a remarkable diversity of molecular mechanisms that can be exploited for crop improvement and provide concepts for protein design. PMID:26800491

  4. Structural Evidence for Regulation and Specificity of Flaviviral Proteases and Evolution of the Flaviviridae Fold

    Aleshin,A.; Shiryaev, S.; Strongin, A.; Liddington, R.

    2007-01-01

    Pathogenic members of the flavivirus family, including West Nile Virus (WNV) and Dengue Virus (DV), are growing global threats for which there are no specific treatments. The two-component flaviviral enzyme NS2B-NS3 cleaves the viral polyprotein precursor within the host cell, a process that is required for viral replication. Here, we report the crystal structure of WNV NS2B-NS3pro both in a substrate-free form and in complex with the trypsin inhibitor aprotinin/BPTI. We show that aprotinin binds in a substrate-mimetic fashion in which the productive conformation of the protease is fully formed, providing evidence for an 'induced fit' mechanism of catalysis and allowing us to rationalize the distinct substrate specificities of WNV and DV proteases. We also show that the NS2B cofactor of WNV can adopt two very distinct conformations and that this is likely to be a general feature of flaviviral proteases, providing further opportunities for regulation. Finally, by comparing the flaviviral proteases with the more distantly related Hepatitis C virus, we provide insights into the evolution of the Flaviviridae fold. Our work should expedite the design of protease inhibitors to treat a range of flaviviral infections.

  5. Expedient screening for HIV-1 protease inhibitors using a simplified immunochromatographic assay.

    Kitidee, Kuntida; Khamaikawin, Wannisa; Thongkum, Weeraya; Tawon, Yardpiroon; Cressey, Tim R; Jevprasesphant, Rachaneekorn; Kasinrerk, Watchara; Tayapiwatana, Chatchai

    2016-05-15

    A colloidal gold-based immunochromatographic (IC) strip test was developed and validated for the detection of HIV-1 protease (HIV-PR) activity and inhibitory effect of HIV-PR inhibitors (PIs). It is a unique 'two-step' process requiring the combination of proteolysis of HIV-PR and an immunochromatographic reaction. Monoclonal antibodies to the free C-terminus of HIV matrix protein (HIV-MA) conjugated to gold particles and a monoclonal antibody against intact and cleaved forms of the HIV-MA are immobilized on the 'Test'-line of the IC strip. Using lopinavir, a potent HIV protease inhibitor, the IC-strip was optimized to detect inhibitory activity against HIV-protease. At a lopinavir concentration of 1000ng/mL (its suggested minimum effective concentration), a HIV-PRH6 concentration of 6mg/mL and incubation period of 60min were the optimal conditions. A preliminary comparison between a validated high-performance liquid chromatography assay and the IC-strip to semi-quantify HIV protease inhibitor concentrations (lopinavir and atazanavir) demonstrated good agreement. This simplified method is suitable for the rapid screening of novel protease inhibitors for future therapeutic use. Moreover, the IC strip could also be optimized to semi-quantify PIs concentrations in plasma samples. PMID:26490422

  6. Inner radio jet of 3C273

    Zensus, J.A.; Cohen, M.H.; Baaaath, L.B.; Nicolson, G.D.

    1988-08-04

    Radio maps of 3C273 obtained with very long baseline interferometry (VLBI) have been limited by low dynamic range and poor north-south resolution resulting from the low declination of this quasar. Dramatic improvement can now be achieved using larger arrays and antennas in the Southern Hemisphere. A new VLBI map, made at 5 GHz with angular resolution and dynamic range unsurpassed at this frequency for this source, shows a narrow jet extending to a projected distance lsub(proj) approx. 125 h/sup -1/ parsecs from the core. Superluminal motion exists out to at least lsub(proj) ''approx ='' 46 h/sup -1/ parsecs. Successive superluminal components emerge from the core and appear to move on a fixed curved path with similar speeds of about 1 milliarcseconds per year.

  7. Double Faraday rotation toward 3C 27

    Goldstein, S. J., Jr.; Reed, J. A.

    1984-08-01

    From observations of the integrated flux of 3C 27 with the NRAO 140 foot (43 m) telescope at 40 frequencies between 1250 and 1445 MHz, the authors deduce rotation measures of 165±15 and -104±4 rad m-2. Since the source (assumed to be a radio galaxy) has components 45arcsec apart, it is concluded that the net magnetic field reverses between these directions. One explanation is that a large magnetic field surrounding the central galaxy of the distant source covers one component but not the other. Another explanation is that our Galaxy contains a dipole field with a scale of order 1 pc. One component of the distant source is seen inside the current loop associated with the dipole field, while the other is seen outside the loop.

  8. Solution Conformations of the substrates and Inhibitor of Hepatitis C Virus NS3 Protease

    Hepatitis C virus (HCV) has been known to be an enveloped virus with a positive strand RNA genome and the major agent of the vast majority of transfusion associated cases of hepatitis. For viral replication, HCV structural proteins are first processed by host cell signal peptidases and NS2/NS3 site of the nonstructural protein is cleaved by a zinc-dependent protease NS2 with N-terminal NS3. The four remaining junctions are cleaved by a separate NS3 protease. The solution conformations of NS4B/5A, NS5A/5B substrates and NS5A/5B inhibitor have been determined by two-dimensional nuclear magnetic resonance (NMR) spectroscopy. NMR data suggested that the both NS5A/5B substrate and inhibitor appeared to have a folded turn-like conformation not only between P1 and P6 position but also C-terminal region, whereas the NS4B/5A substrate exhibited mostly extended conformation. In addition, we have found that the conformation of the NS5A/5B inhibitor slightly differs from that of NS5A/5B substrate peptide, suggesting different binding mode for NS3 protease. These findings will be of importance for designing efficient inhibitor to suppress HCV processing

  9. Amino acid sequence requirements in the hinge of human immunoglobulin A1 (IgA1) for cleavage by streptococcal IgA1 proteases.

    Batten, Margaret R; Senior, Bernard W; Kilian, Mogens; Woof, Jenny M

    2003-03-01

    The amino acid sequence requirements in the hinge of human immunoglobulin A1 (IgA1) for cleavage by IgA1 proteases of different species of Streptococcus were investigated. Recombinant IgA1 antibodies were generated with point mutations at proline 227 and threonine 228, the residues lying on either side of the peptide bond at which all streptococcal IgA1 proteases cleave wild-type human IgA1. The amino acid substitutions produced no major effect upon the structure of the mutant IgA1 antibodies or their functional ability to bind to Fcalpha receptors. However, the substitutions had a substantial effect upon sensitivity to cleavage with some streptococcal IgA1 proteases, with, in some cases, a single point mutation rendering the antibody resistant to a particular IgA1 protease. This effect was least marked with the IgA1 protease from Streptococcus pneumoniae, which showed no absolute requirement for either proline or threonine at residues 227 to 228. By contrast, the IgA1 proteases of Streptococcus oralis, Streptococcus sanguis, and Streptococcus mitis had an absolute requirement for proline at 227 but not for threonine at 228, which could be replaced by valine. There was evidence in S. mitis that proteases from different strains may have different amino acid requirements for cleavage. Remarkably, some streptococcal proteases appeared able to cleave the hinge at a distant alternative site if substitution prevented efficient cleavage of the original site. Hence, this study has identified key residues required for the recognition of the IgA1 hinge as a substrate by streptococcal IgA1 proteases, and it marks a preliminary step towards development of specific enzyme inhibitors. PMID:12595464

  10. Serine proteases of parasitic helminths.

    Yang, Yong; Wen, Yun jun; Cai, Ya Nan; Vallée, Isabelle; Boireau, Pascal; Liu, Ming Yuan; Cheng, Shi Peng

    2015-02-01

    Serine proteases form one of the most important families of enzymes and perform significant functions in a broad range of biological processes, such as intra- and extracellular protein metabolism, digestion, blood coagulation, regulation of development, and fertilization. A number of serine proteases have been identified in parasitic helminths that have putative roles in parasite development and nutrition, host tissues and cell invasion, anticoagulation, and immune evasion. In this review, we described the serine proteases that have been identified in parasitic helminths, including nematodes (Trichinella spiralis, T. pseudospiralis, Trichuris muris, Anisakis simplex, Ascaris suum, Onchocerca volvulus, O. lienalis, Brugia malayi, Ancylostoma caninum, and Steinernema carpocapsae), cestodes (Spirometra mansoni, Echinococcus granulosus, and Schistocephalus solidus), and trematodes (Fasciola hepatica, F. gigantica, and Schistosoma mansoni). Moreover, the possible biological functions of these serine proteases in the endogenous biological phenomena of these parasites and in the host-parasite interaction were also discussed. PMID:25748703

  11. Formation of a high quality two-dimensional electron gas on cleaved GaAs

    Pfeiffer, Loren; West, K.W.; Stormer, H. L.; Eisenstein, J. P.; Baldwin, K. W.; Gershoni, D.; Spector, J

    1990-01-01

    We have succeeded in fabricating a two-dimensional electron gas (2DEG) on the cleaved (110) edge of a GaAs wafer by molecular beam epitaxy (MBE). A (100) wafer previously prepared by MBE growth is reinstalled in the MBE chamber so that an in situ cleave exposes a fresh (110) GaAs edge for further MBE overgrowth. A sequence of Si-doped AlGaAs layers completes the modulation-doped structure at the cleaved edge. Mobilities as high as 6.1×10^5 cm^2/V s are measured in the 2DEG at the cleaved inte...

  12. Serine Proteases of Parasitic Helminths

    Yong YANG; Wen, Yun jun; Cai, Ya Nan; Vallée, Isabelle; Boireau, Pascal; Liu, Ming Yuan; Cheng, Shi Peng

    2015-01-01

    Serine proteases form one of the most important families of enzymes and perform significant functions in a broad range of biological processes, such as intra- and extracellular protein metabolism, digestion, blood coagulation, regulation of development, and fertilization. A number of serine proteases have been identified in parasitic helminths that have putative roles in parasite development and nutrition, host tissues and cell invasion, anticoagulation, and immune evasion. In this review, we...

  13. The parsec-scale jets in 3C 273 and 3C 345

    Unwin, Stephen C.; Wehrle, Ann E.; Davis, Richard J.; Muxlow, Thomas W. B.

    1992-01-01

    We present recent centimeter-wavelength 'global-array' VLBI images of the quasars 3C 273 and 3C 345 with dynamic ranges in excess of 3000:1. They trace the jet emission on scales from r about 5 parsecs out to about 200 parsecs. The maps of 3C273 at 18 cm wavelength show a well-collimated one-sided jet with a wavy ridge line; these wiggles exist on size scales ranging from about 1 pc to over 10 kpc. We show that the well-known superluminal flow extends to r about 120 pc. Images of 3C 345 at 6 cm wavelength from data taken in 1989 and 1990 show surprising features not seen in lower dynamic-range maps of this otherwise well-studied quasar: the inner part of the jet shows edge-brightening, which is important for modeling of jet confinement. The jet fades out very abruptly at r about 40 pc, then reappears at about 70 pc; beyond 70 pc, the resumed jet flares and is more diffuse than an extrapolation of the inner jet would predict. This morphology is reminiscent of M 87, and is suggestive of a shock wave.

  14. 3C236 Radio Source, Interrupted?

    O'Dea, C P; Baum, S A; Sparks, W B; Martel, A R; Allen, M G; Macchetto, F D; Miley, G K; Dea, Christopher P. O'; Koekemoer, Anton M.; Baum, Stefi A.; Sparks, William B.; Martel, Andre R.; Allen, Mark G.; Macchetto, Ferdinando D.; Miley, George K.

    2001-01-01

    We present new HST STIS/MAMA near-UV images and archival WFPC2 V and R band images which reveal the presence of four star forming regions in an arc along the edge of the dust lane in the giant (4 Mpc) radio galaxy 3C236. Two of the star forming regions are relatively young with ages of order 1E7 yr, while the other two are older with ages of order 1E8 - 1E9 yr which is comparable to the estimated age of the giant radio source. Based on dynamical and spectral aging arguments, we suggest that the fuel supply to the AGN was interrupted for 1E7 yr and has now been restored, resulting in the formation of the inner 2 kpc scale radio source. This time scale is similar to that of the age of the youngest of the star forming regions. We suggest that the transport of gas in the disk is non-steady and that this produces both the multiple episodes of star formation in the disk as well as the multiple epochs of radio source activity. If the inner radio source and the youngest star forming region are related by the same eve...

  15. Atomically precise, coupled quantum dots fabricated by cleaved edge overgrowth

    Wegscheider, W.; Schedelbeck, G.; Bichler, M.; Abstreiter, G.

    Recent progress in the fabrication of quantum dots by molecular beam epitaxy along three directions in space is reviewed. The optical properties of different sample structures consisting of individual quantum dots, pairs of coupled dots as well as of linear arrays of dots are studied by microscopic photoluminescence spectroscopy. The high degree of control over shape, composition and position of the 7×7×7 nm3 size GaAs quantum dots, which form at the intesection of three orthogonal quantum wells, allows a detailed investigation of the influence of coupling between almost identical zero-dimensional objects. In contrast to the inhomogeneously broadened quantum well and quantum wire signals originating from the complex twofold cleaved edge overgrowth structure, the photoluminescence spetrum of an individual quantum dot exhibits a single sharp line (full width at half maximum denomination "artificial atoms" for the quantum dots. It is further demonstrated that an "artifical molecule", characterized by the existence of bonding and antibonding states can be assembled from two of such "artificial atoms". The coupling strength between the "artificial atoms" is adjusted by the "interatomic" distance and is reflected in the energetic separation of the bonding and antibonding levels and the linewidths of the corresponding interband transitions.

  16. Universal binding energy relation for cleaved and structurally relaxed surfaces

    The universal binding energy relation (UBER), derived earlier to describe the cohesion between two rigid atomic planes, does not accurately capture the cohesive properties when the cleaved surfaces are allowed to relax. We suggest a modified functional form of UBER that is analytical and at the same time accurately models the properties of surfaces relaxed during cleavage. We demonstrate the generality as well as the validity of this modified UBER through first-principles density functional theory calculations of cleavage in a number of crystal systems. Our results show that the total energies of all the relaxed surfaces lie on a single (universal) energy surface, that is given by the proposed functional form which contains an additional length-scale associated with structural relaxation. This functional form could be used in modelling the cohesive zones in crack growth simulation studies. We find that the cohesive law (stress–displacement relation) differs significantly in the case where cracked surfaces are allowed to relax, with lower peak stresses occurring at higher displacements. (paper)

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

    The crystallization of the recombinant protease from Southampton norovirus is described. Whilst the native crystals were found to diffract only to medium resolution (2.9 Å), cocrystals with a designed covalently bound inhibitor diffracted X-rays to 1.7 Å resolution. 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. Whilst the native crystals were found to diffract only to medium resolution (2.9 Å), cocrystals of an inhibitor complex diffracted X-rays to 1.7 Å 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

  18. Simultaneous selection and counter-selection for the directed evolution of proteases in E. coli using a cytoplasmic anchoring strategy.

    Carrico, Zachary M; Strobel, Kathryn L; Atreya, Meera E; Clark, Douglas S; Francis, Matthew B

    2016-06-01

    With the goal of generating new enzymes that can cleave custom sequences, this article describes a selection strategy for evolving proteases with desirable characteristics. Positive selection and counter-selection are combined to select for and against specified cleavage sequences simultaneously. Cleavage of the positive selection sequence permits E. coli growth, and cleavage of the counter-selection sequence slows growth. Growth occurs when cleavage of the positive selection sequence releases β-lactamase into the periplasm where it can confer antibiotic resistance. The counter-selection traps β-lactamase in the cytoplasm, preventing antibiotic resistance and growth. Thus, proteases with a preference for the positive selection sequence relative to the counter-selection sequence grow more rapidly. This system was used to select a tobacco etch virus (TEV) protease mutant with new substrate compatibility. Biotechnol. Bioeng. 2016;113: 1187-1193. © 2015 Wiley Periodicals, Inc. PMID:26666461

  19. Diversity and transcription of proteases involved in the maturation of hydrogenases in Nostoc punctiforme ATCC 29133 and Nostoc sp. strain PCC 7120

    Lindblad Peter

    2009-03-01

    Full Text Available Abstract Background The last step in the maturation process of the large subunit of [NiFe]-hydrogenases is a proteolytic cleavage of the C-terminal by a hydrogenase specific protease. Contrary to other accessory proteins these hydrogenase proteases are believed to be specific whereby one type of hydrogenases specific protease only cleaves one type of hydrogenase. In cyanobacteria this is achieved by the gene product of either hupW or hoxW, specific for the uptake or the bidirectional hydrogenase respectively. The filamentous cyanobacteria Nostoc punctiforme ATCC 29133 and Nostoc sp strain PCC 7120 may contain a single uptake hydrogenase or both an uptake and a bidirectional hydrogenase respectively. Results In order to examine these proteases in cyanobacteria, transcriptional analyses were performed of hupW in Nostoc punctiforme ATCC 29133 and hupW and hoxW in Nostoc sp. strain PCC 7120. These studies revealed numerous transcriptional start points together with putative binding sites for NtcA (hupW and LexA (hoxW. In order to investigate the diversity and specificity among hydrogeanse specific proteases we constructed a phylogenetic tree which revealed several subgroups that showed a striking resemblance to the subgroups previously described for [NiFe]-hydrogenases. Additionally the proteases specificity was also addressed by amino acid sequence analysis and protein-protein docking experiments with 3D-models derived from bioinformatic studies. These studies revealed a so called "HOXBOX"; an amino acid sequence specific for protease of Hox-type which might be involved in docking with the large subunit of the hydrogenase. Conclusion Our findings suggest that the hydrogenase specific proteases are under similar regulatory control as the hydrogenases they cleave. The result from the phylogenetic study also indicates that the hydrogenase and the protease have co-evolved since ancient time and suggests that at least one major horizontal gene transfer

  20. The Trypanosoma cruzi protease cruzain mediates immune evasion.

    Patricia S Doyle

    2011-09-01

    Full Text Available Trypanosoma cruzi is the causative agent of Chagas' disease. Novel chemotherapy with the drug K11777 targets the major cysteine protease cruzain and disrupts amastigote intracellular development. Nevertheless, the biological role of the protease in infection and pathogenesis remains unclear as cruzain gene knockout failed due to genetic redundancy. A role for the T. cruzi cysteine protease cruzain in immune evasion was elucidated in a comparative study of parental wild type- and cruzain-deficient parasites. Wild type T. cruzi did not activate host macrophages during early infection (<60 min and no increase in ∼P iκB was detected. The signaling factor NF-κB P65 colocalized with cruzain on the cell surface of intracellular wild type parasites, and was proteolytically cleaved. No significant IL-12 expression occurred in macrophages infected with wild type T. cruzi and treated with LPS and BFA, confirming impairment of macrophage activation pathways. In contrast, cruzain-deficient parasites induced macrophage activation, detectable iκB phosphorylation, and nuclear NF-κB P65 localization. These parasites were unable to develop intracellularly and survive within macrophages. IL 12 expression levels in macrophages infected with cruzain-deficient T. cruzi were comparable to LPS activated controls. Thus cruzain hinders macrophage activation during the early (<60 min stages of infection, by interruption of the NF-κB P65 mediated signaling pathway. These early events allow T. cruzi survival and replication, and may lead to the spread of infection in acute Chagas' disease.

  1. Microbial inhibitors of cysteine proteases.

    Kędzior, Mateusz; Seredyński, Rafał; Gutowicz, Jan

    2016-08-01

    Cysteine proteases are one of the major classes of proteolytic enzymes involved in a number of physiological and pathological processes in plants, animals and microorganisms. When their synthesis, activity and localization in mammalian cells are altered, they may contribute to the development of many diseases, including rheumatoid arthritis, osteoporosis and cancer. Therefore, cysteine proteases have become promising drug targets for the medical treatment of these disorders. Inhibitors of cysteine proteases are also produced by almost every group of living organisms, being responsible for the control of intracellular proteolytic activity. Microorganisms synthesize cysteine protease inhibitors not only to regulate the activity of endogenous, often virulent enzymes, but also to hinder the host's proteolytic defense system and evade its immune responses against infections. Present work describes known to date microbial inhibitors of cysteine proteases in terms of their structure, enzyme binding mechanism, specificity and pathophysiological roles. The overview of both proteinaceous and small-molecule inhibitors produced by all groups of microorganisms (bacteria, archaea, fungi, protists) and viruses is provided. Subsequently, possible applications of microbial inhibitors in science, medicine and biotechnology are also highlighted. PMID:27048482

  2. Investigations of radio jets in M87, 3C273, and 3C345

    Biretta, J.A.

    1986-01-01

    Observational studies of extra-galactic radio jets in M87, 3C273, and 3C345 are presented. Observations of the M87 jet were made at 15 GHz with 0.12'' resolution. All of the knots are clearly resolved both along and across the jet. Most of the knots are found to be smooth in appearance with no evidence of shocklike discontinuities. The brightest knot and the innermost knot are exceptions to this. The brightest knot (knot A) seems consistent with a shock caused by unsteady flow in the jet. Models for this feature are discussed. Combining these data with x-ray data suggests that the jet is neither freely expanding, thermally confined, nor ram-pressure confined. The jet may, however, be magnetically confined. The author presents 10.7 GHz VLBI observations of 3C273 with high north-south resolution. A strong, nonmonotonic curvature is found in the jet at projected radii less than or equal to 5 pc. It is unlikely that this curvature can be caused by precession. Measurements of the core size show that bulk relativistic motion in the core is not required for consistency with the observed x-ray flux.

  3. Investigations of radio jets in M87, 3C273, and 3C345

    Observational studies of extra-galactic radio jets in M87, 3C273, and 3C345 are presented. Observations of the M87 jet were made at 15 GHz with 0.12'' resolution. All of the knots are clearly resolved both along and across the jet. Most of the knots are found to be smooth in appearance with no evidence of shocklike discontinuities. The brightest knot and the innermost knot are exceptions to this. The brightest knot (knot A) seems consistent with a shock caused by unsteady flow in the jet. Models for this feature are discussed. Combining these data with x-ray data suggests that the jet is neither freely expanding, thermally confined, nor ram-pressure confined. The jet may, however, be magnetically confined. The author presents 10.7 GHz VLBI observations of 3C273 with high north-south resolution. A strong, nonmonotonic curvature is found in the jet at projected radii less than or equal to 5 pc. It is unlikely that this curvature can be caused by precession. Measurements of the core size show that bulk relativistic motion in the core is not required for consistency with the observed x-ray flux

  4. Type II transmembrane serine proteases as potential targets for cancer therapy

    Murray, Andrew S.; Varela, Fausto A.

    2016-01-01

    Carcinogenesis is accompanied by increased protein and activity levels of extracellular cell-surface proteases that are capable of modifying the tumor micro-environment by directly cleaving the extracellular matrix, as well as activating growth factors and proinflammatory mediators involved in proliferation and invasion of cancer cells, and recruitment of inflammatory cells. These complex processes ultimately potentiate neoplastic progression leading to local tumor cell invasion, entry into the vasculature, and metastasis to distal sites. Several members of the type II transmembrane serine protease (TTSP) family have been shown to play critical roles in cancer progression. In this review the knowledge collected over the past two decades about the molecular mechanisms underlying the pro-cancerous properties of selected TTSPs will be summarized. Furthermore, we will discuss how these insights may facilitate the translation into clinical settings in the future by specifically targeting TTSPs as part of novel cancer treatment regimens. PMID:27078673

  5. The Resolved Outflow from 3C 48

    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

  6. Modulation of the Bacillus anthracis secretome by the immune inhibitor A1 protease.

    Pflughoeft, Kathryn J; Swick, Michelle C; Engler, David A; Yeo, Hye-Jeong; Koehler, Theresa M

    2014-01-01

    The Bacillus anthracis secretome includes protective antigen, lethal factor, and edema factor, which are the components of anthrax toxin, and other proteins with known or potential roles in anthrax disease. Immune inhibitor A1 (InhA1) is a secreted metalloprotease that is unique to pathogenic members of the Bacillus genus and has been associated with cleavage of host proteins during infection. Here, we report the effect of InhA1 on the B. anthracis secretome. Differential in-gel electrophoresis of proteins present in culture supernatants from a parent strain and an isogenic inhA1-null mutant revealed multiple differences. Of the 1,340 protein spots observed, approximately one-third were less abundant and one-third were more abundant in the inhA1 secretome than in the parent strain secretome. Proteases were strongly represented among those proteins exhibiting a 9-fold or greater change. InhA1 purified from a B. anthracis culture supernatant directly cleaved each of the anthrax toxin proteins as well as an additional secreted protease, Npr599. The conserved zinc binding motif HEXXH of InhA1 (HEYGH) was critical for its proteolytic activity. Our data reveal that InhA1 directly and indirectly modulates the form and/or abundance of over half of all the secreted proteins of B. anthracis. The proteolytic activity of InhA1 on established secreted virulence factors, additional proteases, and other secreted proteins suggests that this major protease plays an important role in virulence not only by cleaving mammalian substrates but also by modulating the B. anthracis secretome itself. PMID:24214942

  7. Identification of BACE2 as an avid ß-amyloid-degrading protease

    Abdul-Hay Samer O

    2012-09-01

    Full Text Available Abstract Background Proteases that degrade the amyloid ß-protein (Aß have emerged as key players in the etiology and potential treatment of Alzheimer’s disease (AD, but it is unlikely that all such proteases have been identified. To discover new Aß-degrading proteases (AßDPs, we conducted an unbiased, genome-scale, functional cDNA screen designed to identify proteases capable of lowering net Aß levels produced by cells, which were subsequently characterized for Aß-degrading activity using an array of downstream assays. Results The top hit emerging from the screen was ß-site amyloid precursor protein-cleaving enzyme 2 (BACE2, a rather unexpected finding given the well-established role of its close homolog, BACE1, in the production of Aß. BACE2 is known to be capable of lowering Aß levels via non-amyloidogenic processing of APP. However, in vitro, BACE2 was also found to be a particularly avid AßDP, with a catalytic efficiency exceeding all known AßDPs except insulin-degrading enzyme (IDE. BACE1 was also found to degrade Aß, albeit ~150-fold less efficiently than BACE2. Aß is cleaved by BACE2 at three peptide bonds—Phe19-Phe20, Phe20-Ala21, and Leu34-Met35—with the latter cleavage site being the initial and principal one. BACE2 overexpression in cultured cells was found to lower net Aß levels to a greater extent than multiple, well-established AßDPs, including neprilysin (NEP and endothelin-converting enzyme-1 (ECE1, while showing comparable effectiveness to IDE. Conclusions This study identifies a new functional role for BACE2 as a potent AßDP. Based on its high catalytic efficiency, its ability to degrade Aß intracellularly, and other characteristics, BACE2 represents a particulary strong therapeutic candidate for the treatment or prevention of AD.

  8. Cytomegalovirus protease targeted prodrug development.

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

    2013-04-01

    Human cytomegalovirus (HCMV) is a prevalent virus that infects up to 90% of the population. The goal of this research is to determine if small molecular prodrug substrates can be developed for a specific HCMV encoded protease and thus achieve site-specific activation. HCMV encodes a 256 amino acid serine protease that is responsible for capsid assembly, an essential process for herpes virus production. The esterase activity of the more stable HCMV A143T/A144T protease mutant was evaluated with model p-nitrophenol (ONp) esters, Boc-Xaa-ONp (Ala, Leu, Ile, Val, Gln, Phe at the Xaa position). We demonstrate that the A143T/A144T mutant has esterase activity toward specific small ester compounds, e.g., Boc-L-Ala-ONp. Mono amino acid and dipeptide prodrugs of ganciclovir (GCV) were also synthesized and evaluated for hydrolysis by the A143T/A144T protease mutant in solution. Hydrolysis of these prodrugs was also evaluated in Caco-2 cell homogenates, human liver microsomes (HLMs), and rat and human plasma. For the selectivity potential of the prodrugs, the hydrolysis ratio was evaluated as a percentage of prodrug hydrolyzed by the HCMV protease over the percentages of prodrug hydrolyses by Caco-2 cell homogenates, HLMs, and human/rat plasma. A dipeptide prodrug of ganciclovir, Ac-l-Gln-l-Ala-GCV, emerged as a potential selective prodrug candidate. The results of this research demonstrate that targeting prodrugs for activation by a specific protease encoded by the infectious HCMV pathogen may be achievable. PMID:23485093

  9. PROTEOLYTIC PROCESSING OF VON WILLEBRAND FACTOR BY ADAMTS13 AND LEUKOCYTE PROTEASES

    Stefano Lancellotti

    2013-09-01

    Full Text Available ADAMTS13 is a 190 kDa zinc protease encoded by a gene located on chromosome 9q34.   This protease specifically hydrolyzes von Willebrand factor (VWF multimers, thus causing VWF size reduction. ADAMTS13 belongs to the A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats (ADAMTS family, involved in proteolytic processing of many matrix proteins. ADAMTS13 consists of numerous domains including a metalloprotease domain, a disintegrin domain, several thrombospondin type 1 (TSP1 repeats, a cysteine-rich domain, a spacer domain and 2 CUB (Complement c1r/c1s, sea Urchin epidermal growth factor, and Bone morphogenetic protein domains. ADAMTS13 cleaves a single peptide bond (Tyr1605-Met1606 in the central A2 domain of the VWF molecule. This proteolytic cleavage is essential to reduce the size of ultra-large VWF polymers, which, when exposed to high shear stress in the microcirculation, are prone to form with platelets clumps, which cause severe syndromes called thrombotic microangiopathies (TMAs. In this review, we a discuss the current knowledge of structure-function aspects of ADAMTS13 and its involvement in the pathogenesis of TMAs, b address the recent findings concerning proteolytic processing of VWF multimers by different proteases, such as the leukocyte-derived serine and metallo-proteases and c indicate the direction of future investigations

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

    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.

  11. Amino acid sequence requirements in the human IgA1 hinge for cleavage by streptococcal IgA1 proteases

    Senior, BW; Batten, MR; Kilian, Mogens;

    2002-01-01

    All the IgA1 proteases of the different pathogenic species of Streptococcus cleave the hinge of the alpha chain of human IgA1 only at one proline-threonine peptide bond. In order to study the importance of these amino acids for cleavage, several hinge mutant recombinant IgA1 antibodies were...... constructed. The mutations were found to be without major effect upon the structure or functional abilities of the antibodies. However, they had a major effect upon their sensitivity to cleavage by some of the IgA1 proteases....

  12. Amino acid sequence requirements in the human IgA1 hinge for cleavage by streptococcal IgA1 proteases.

    Senior, B W; Batten, M R; Kilian, M; Woof, J M

    2002-08-01

    All the IgA1 proteases of the different pathogenic species of Streptococcus cleave the hinge of the alpha chain of human IgA1 only at one proline-threonine peptide bond. In order to study the importance of these amino acids for cleavage, several hinge mutant recombinant IgA1 antibodies were constructed. The mutations were found to be without major effect upon the structure or functional abilities of the antibodies. However, they had a major effect upon their sensitivity to cleavage by some of the IgA1 proteases. PMID:12196126

  13. Purification of two high molecular weight proteases from rabbit reticulocyte lysate

    The authors have purified two large proteases from rabbit reticulocyte lysate. The enzymes are so similar in their chromatographic behavior that each is the only significant contaminant of the other during the final stages of purification. At pH 7.8, both hydrolyze 125I-α-casein and 4-methylcoumaryl-7-amide (MCA) derivatives with tyrosine, phenylalanine or arginine at the P1 position. The larger, ATP-dependent enzyme degrades ubiquitin-lysozyme conjugates, but it does not degrade unmodified lysozyme. Hydrolysis of Suc-Leu-Leu-Val-Tyr-MCA by this enzyme is also stimulated two-fold in the presence of ATP. The protease has a molecular weight of 950,000 based on sedimentation, gel filtration and non-denaturing PAGE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the protease is composed of a number of subunits with molecular masses between 32 and 110 kDa. Densitometric analysis showed equivalent amounts of the two larger chains, and the presence of one copy of each in the native enzyme would be consistent with an M/sub r/ of 950,000. The smaller protease has a molecular weight of 700,000 and is composed of 8 to 10 subunits ranging from 21,000 to 32,000. It cleaves ubiquitin-lysozyme conjugates only slightly, and hydrolysis of conjugates or fluorogenic peptide substrates is not stimulated by ATP. This protease appears similar, if not identical, to the multicatalytic protease complex first purified by Wilk and Orlowski

  14. Enzymatic Degradation of Ovalbumin by Various Proteases

    Matsumoto, Kiyoshi; Yoshimaru, Tetsuro; Matsui, Toshiro; Osajima, Yutaka

    1997-01-01

    An investigation was made of the enzymatic hydrolysis of ovalbumin (OVA), a major allergen in egg white, by various acid and alkaline proteases. Protease YP-SS (acid protease) from Aspergillus niger and alcalase (alkaline protease) from BacilLus licheniformis were found to be useful for the degradation of OVA, respectively. OVA was almost totally hydrolyzed within 15 hr at 37℃ by alcalase. Alcalase acted rapidly to hydrolyze OVA, with about 90% of OVA being hydrolyzed within 30min., the react...

  15. Enterovirus 71 contains a type I IRES element that functions when eukaryotic initiation factor eIF4G is cleaved

    Human enterovirus 71 (EV71) is a member of the Enterovirus genus of the Picornaviridae family. Other members of this family utilize an unusual mechanism of translation initiation whereby ribosomes are recruited internally to the viral RNA by an internal ribosome site (IRES) located in their 5' noncoding regions (5' NCR). Using dicistronic reporter constructs, we demonstrate that the 5' NCRs of the 7423/MS/87 and BrCr strains of EV71 function as an IRES both in extracts and in cultured cells. Preincubation of translation extracts with purified coxsackievirus 2A protease cleaved elF4G, a component of the cap binding complex, resulting in a significant decrease in translation of capped mRNAs. In contrast, the translational efficiency of the EV71 IRES was enhanced under this condition, demonstrating that the EV71 IRES functions similar to other enterovirus IRES elements when components of the cap binding protein complex are cleaved. Finally, insertion of an upstream, out-of-frame start codon in the 5' NCR of the EV71 genome inhibited IRES activity, suggesting that EV71 can be classified as a type I IRES, in which ribosomes first bind upstream of the initiation codon and then scan the mRNA until an appropriate downstream AUG start codon is encountered and protein synthesis commences

  16. Elafin, an elastase-specific inhibitor, is cleaved by its cognate enzyme neutrophil elastase in sputum from individuals with cystic fibrosis.

    Guyot, Nicolas

    2008-11-21

    Elafin is a neutrophil serine protease inhibitor expressed in lung and displaying anti-inflammatory and anti-bacterial properties. Previous studies demonstrated that some innate host defense molecules of the cystic fibrosis (CF) and chronic obstructive pulmonary disease airways are impaired due to increased proteolytic degradation observed during lung inflammation. In light of these findings, we thus focused on the status of elafin in CF lung. We showed in the present study that elafin is cleaved in sputum from individuals with CF. Pseudomonas aeruginosa-positive CF sputum, which was found to contain lower elafin levels and higher neutrophil elastase (NE) activity compared with P. aeruginosa-negative samples, was particularly effective in cleaving recombinant elafin. NE plays a pivotal role in the process as only NE inhibitors are able to inhibit elafin degradation. Further in vitro studies demonstrated that incubation of recombinant elafin with excess of NE leads to the rapid cleavage of the inhibitor. Two cleavage sites were identified at the N-terminal extremity of elafin (Val-5-Lys-6 and Val-9-Ser-10). Interestingly, purified fragments of the inhibitor (Lys-6-Gln-57 and Ser-10-Gln-57) were shown to still be active for inhibiting NE. However, NE in excess was shown to strongly diminish the ability of elafin to bind lipopolysaccharide (LPS) and its capacity to be immobilized by transglutamination. In conclusion, this study provides evidence that elafin is cleaved by its cognate enzyme NE present at excessive concentration in CF sputum and that P. aeruginosa infection promotes this effect. Such cleavage may have repercussions on the innate immune function of elafin.

  17. Curcumin derivatives as HIV-1 protease inhibitors

    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.

  18. Candidate prognostic markers in breast cancer: focus on extracellular proteases and their inhibitors

    Roy DM

    2014-07-01

    Full Text Available David M Roy,1 Logan A Walsh21Weill Cornell/Rockefeller/Sloan-Kettering Tri-Institutional MD-PhD Program, New York, NY, USA; 2Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USAAbstract: The extracellular matrix (ECM is the complex network of proteins that surrounds cells in multicellular organisms. Due to its diverse nature and composition, the ECM has a multifaceted role in both normal tissue homeostasis and pathophysiology. It provides structural support, segregates tissues from one another, and regulates intercellular communication. Furthermore, the ECM sequesters a wide range of growth factors and cytokines that may be released upon specific and well-coordinated cues. Regulation of the ECM is performed by the extracellular proteases, which are tasked with cleaving and remodeling this intricate and diverse protein matrix. Accordingly, extracellular proteases are differentially expressed in various tissue types and in many diseases such as cancer. In fact, metastatic dissemination of tumor cells requires degradation of extracellular matrices by several families of proteases, including metalloproteinases and serine proteases, among others. Extracellular proteases are emerging as strong candidate cancer biomarkers for aiding and predicting patient outcome. Not surprisingly, inhibition of these protumorigenic enzymes in animal models of metastasis has shown impressive therapeutic effects. As such, many of these proteolytic inhibitors are currently in various phases of clinical investigation. In addition to direct approaches, aberrant expression of extracellular proteases in disease states may also facilitate the selective delivery of other therapeutic or imaging agents. Herein, we outline extracellular proteases that are either bona fide or probable prognostic markers in breast cancer. Furthermore, using existing patient data and multiple robust statistical analyses, we highlight several

  19. Exogenous proteases for meat tenderization.

    Bekhit, Alaa A; Hopkins, David L; Geesink, Geert; Bekhit, Adnan A; Franks, Philip

    2014-01-01

    The use of exogenous proteases to improve meat tenderness has attracted much interest recently, with a view to consistent production of tender meat and added value to lower grade meat cuts. This review discusses the sources, characteristics, and use of exogenous proteases in meat tenderization to highlight the specificity of the proteases toward meat proteins and their impact on meat quality. Plant enzymes (such as papain, bromelain, and ficin) have been extensively investigated as meat tenderizers. New plant proteases (actinidin and zingibain) and microbial enzyme preparations have been of recent interest due to controlled meat tenderization and other advantages. Successful use of these enzymes in fresh meat requires their enzymatic kinetics and characteristics to be determined, together with an understanding of the impact of the surrounding environmental conditions of the meat (pH, temperature) on enzyme function. This enables the optimal conditions for tenderizing fresh meat to be established, and the elimination or reduction of any negative impacts on other quality attributes. PMID:24499119

  20. Comparative Studies on Retroviral Proteases: Substrate Specificity

    József Tözsér

    2010-01-01

    Full Text Available Exogenous retroviruses are subclassified into seven genera and include viruses that cause diseases in humans. The viral Gag and Gag-Pro-Pol polyproteins are processed by the retroviral protease in the last stage of replication and inhibitors of the HIV-1 protease are widely used in AIDS therapy. Resistant mutations occur in response to the drug therapy introducing residues that are frequently found in the equivalent position of other retroviral proteases. Therefore, besides helping to understand the general and specific features of these enzymes, comparative studies of retroviral proteases may help to understand the mutational capacity of the HIV-1 protease.

  1. Partial genetic suppression of a loss-of-function mutant of the neuronal ceroid lipofuscinosis-associated protease TPP1 in Dictyostelium discoideum

    Phillips, Jonathan E.; Gomer, Richard H.

    2014-01-01

    Neuronal ceroid lipofuscinosis (NCL) is the most common childhood-onset neurodegenerative disease. NCL is inevitably fatal, and there is currently no treatment available. Children with NCL show a progressive decline in movement, vision and mental abilities, and an accumulation of autofluorescent deposits in neurons and other cell types. Late-infantile NCL is caused by mutations in the lysosomal protease tripeptidyl peptidase 1 (TPP1). TPP1 cleaves tripeptides from the N-terminus of proteins i...

  2. Biotechnology of Cold-Active Proteases

    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.

  3. ATP-dependent protease in maize mitochondria

    ATP-dependent protease was identified in the matrix of Zea mays L. Sachara mitochondria. 14C-methylated casein has been used as a substrate, and the matrix ATP-dependent protease exhibited similar sensitivity towards specific inhibitors as the Lon protease from E. coli nd analogues from rat liver and yeast mitochondria. Here we report the existence of Lon like ATP-dependent protease in intact mitochondria prepared from 4-days-old epicotyls of Zea mays L. seedling. Enzyme has been purified from Lubrol treated mitochondria using ion exchange chromatography and gel filtration. The enzyme activity has been estimated using 14C-methylated casein as a substrate and sensitivity of the protease towards the specific inhibitors has been tested. ATP-dependent protease from the mitochondrial matrix of maize exhibit similar sensitivity to the above mentioned inhibitors like Lon protease from yeast and rat liver mitochondria as well as from E. coli. (authors)

  4. Glycine decarboxylase in C3, C4 and C3-C4 intermediate species.

    Schulze, Stefanie; Westhoff, Peter; Gowik, Udo

    2016-06-01

    The glycine decarboxylase complex (GDC) plays a central role in photorespiration. GDC is localized in the mitochondria and together with serine hydroxymethyltransferase it converts two molecules of glycine to one molecule of serine, CO2 and NH3. Overexpression of GDC subunits in the C3 species Arabidopsis thaliana can increase the metabolic flux through the photorespiratory pathway leading to enhanced photosynthetic efficiency and consequently to an enhanced biomass production of the transgenic plants. Changing the spatial expression patterns of GDC subunits was an important step during the evolution of C3-C4 intermediate and likely also C4 plants. Restriction of the GDC activity to the bundle sheath cells led to the establishment of a photorespiratory CO2 pump. PMID:27038285

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

    Nicole Hofmann

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

  6. A Cell-Based Assay Reveals Nuclear Translocation of Intracellular Domains Released by SPPL Proteases.

    Mentrup, Torben; Häsler, Robert; Fluhrer, Regina; Saftig, Paul; Schröder, Bernd

    2015-08-01

    During regulated intramembrane proteolysis (RIP) a membrane-spanning substrate protein is cleaved by an ectodomain sheddase and an intramembrane cleaving protease. A cytoplasmic intracellular domain (ICD) is liberated, which can migrate to the nucleus thereby influencing transcriptional regulation. Signal peptide peptidase-like (SPPL) 2a and 2b have been implicated in RIP of type II transmembrane proteins. Even though SPPL2a might represent a potential pharmacological target for treatment of B-cell-mediated autoimmunity, no specific and potent inhibitors for this enzyme are currently available. We report here on the first quantitative cell-based assay for measurement of SPPL2a/b activity. Demonstrating the failure of standard Gal4/VP16 reporter assays for SPPL2a/b analysis, we have devised a novel system employing β-galactosidase (βGal) complementation. This is based on detecting nuclear translocation of the proteolytically released substrate ICDs, which results in specific restoration of βGal activity. Utilizing this potentially high-throughput compatible new setup, we demonstrate nuclear translocation of the ICDs from integral membrane protein 2B (ITM2B), tumor necrosis factor (TNF) and CD74 and identify secreted frizzled-related protein 2 (SFRP2) as potential transcriptional downstream target of the CD74 ICD. We show that the presented assay is easily adaptable to other intramembrane proteases and therefore represents a valuable tool for the functional analysis and development of new inhibitors of this class of enzymes. PMID:25824657

  7. Effect of ionizing radiation on the ability of PvuII enzyme to cleave plasmid DNA

    Restriction endonucleases type II are enzymes which recognize palindromic DNA base sequences and specifically cleave the double-stranded DNA at the recognition sites. The ability of PvuII enzyme irradiated by gamma radiation to cleave DNA plasmid pcDNA3 was examined by the agarose electrophoresis method. Doses above 200 Gy reduced the enzyme's recognition efficiency for specific DNA base sequences and doses above 500 Gy inactivated the enzyme completely. (orig.)

  8. Improved fabrication of HgI2 nuclear radiation detectors by machine-cleaving

    The perfection of machine-cleaved sections from HgI2 bulk crystals was examined. The perfection of the machine-cleaved sections as established by gamma diffraction rocking curves was found to be much better than the perfection of hand-cleaved sections or as grown thin platelets, reaching a perfection similar to that of the wire-sawn sections of HgI2. A correlation between the perfection and the thickness of the machine-cleaved section was also found, i.e., the thicker the cleaved-section the more perfect it is. The reproducibility of the fabrication was significantly improved by using machine cleaving in the process of fabrication. Large single crystals of HgI2 weighing 20 to 200 g, can be grown from the vapor phase using the TOM Technique. In order to fabricate nuclear radiation detectors from these single crystals, thin sections of about 0.4 to 0.8 mm thickness have to be prepared. Up till now, the state-of-the-art of fabricating HgI2 nuclear radiation detectors involved two methods to get thin sections from the large single crystals: (1) hand-cleaving using a razor-blade and (2) solution wire sawing. The chemical wire sawing method involves a loss of about 50% of the crystal volume and is usually followed by a chemical polishing process which involves a significant loss of volume of the original volume. This procedure is complicated and wasteful. The traditional fabrication method, i.e., hand-cleaving followed by rapid nonselective chemical etching, is simpler and less wasteful

  9. The cleavage specificity of the aspartic protease of cocoa beans involved in the generation of the cocoa-specific aroma precursors.

    Janek, Katharina; Niewienda, Agathe; Wöstemeyer, Johannes; Voigt, Jürgen

    2016-11-15

    Particular peptides generated from the vicilin-class(7S) globulin of the cocoa beans by acid-induced proteolysis during cocoa fermentation are essential precursors of the cocoa-specific aroma notes. As revealed by in vitro studies, the formation of the cocoa-specific aroma precursors depends on the particular cleavage specificity of the cocoa aspartic protease, which cannot be substituted by pepsin. Therefore, we have investigated the effects of aspartic protease inhibitors on both enzymes and comparatively studied their cleavage specificities using different protein substrates and MALDI-TOF mass spectrometric analyses of the generated oligopeptides. Three classes of cleavage sites have been identified and characterized: (I) sequences exclusively cleaved by the cocoa enzyme, (II) sequences cleaved by both pepsin and the cocoa enzyme, and (III) those cleaved exclusively by pepsin. In contrast to most aspartic proteases from other origins, basic amino acid residues, particularly lysine, were found to be abundant in the specific cleavage sites of the cocoa enzyme. PMID:27283639

  10. On the Y-chromosome haplogroup C3c classification.

    Malyarchuk, Boris A; Derenko, Miroslava; Denisova, Galina

    2012-10-01

    As there are ambiguities in classification of the Y-chromosome haplogroup C3c, relatively frequent in populations of Northern Asia, we analyzed all three haplogroup-defining markers M48, M77 and M86 in C3-M217-individuals from Siberia, Eastern Asia and Eastern Europe. We have found that haplogroup C3c is characterized by the derived state at M48, whereas mutations at both M77 and M86 define subhaplogroup C3c1. The branch defined by M48 alone would belong to subhaplogroup C3c*, characteristic for some populations of Central and Eastern Siberia, such as Koryaks, Evens, Evenks and Yukaghirs. Subhaplogroup C3c* individuals could be considered as remnants of the Neolithic population of Siberia, based on the age of C3c*-short tandem repeat variation amounting to 4.5 ± 2.4 thousand years. PMID:22810113

  11. Identification, synthesis and evaluation of SARS-CoV and MERS-CoV 3C-like protease inhibitors.

    Kumar, Vathan; Tan, Kian-Pin; Wang, Ying-Ming; Lin, Sheng-Wei; Liang, Po-Huang

    2016-07-01

    Severe acute respiratory syndrome (SARS) led to a life-threatening form of atypical pneumonia in late 2002. Following that, Middle East Respiratory Syndrome (MERS-CoV) has recently emerged, killing about 36% of patients infected globally, mainly in Saudi Arabia and South Korea. Based on a scaffold we reported for inhibiting neuraminidase (NA), we synthesized the analogues and identified compounds with low micromolar inhibitory activity against 3CL(pro) of SARS-CoV and MERS-CoV. Docking studies show that a carboxylate present at either R(1) or R(4) destabilizes the oxyanion hole in the 3CL(pro). Interestingly, 3f, 3g and 3m could inhibit both NA and 3CL(pro) and serve as a starting point to develop broad-spectrum antiviral agents. PMID:27240464

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

    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.

  13. 86 GHz Very Long Baseline Polarimetry of 3C273 and 3C279 with the Coordinated Millimeter VLBI Array

    Attridge, J M

    2001-01-01

    (86) GHz Very Long Baseline Polarimetry probes magnetic field structures within the cores of Active Galactic Nuclei at higher angular resolutions and a spectral octave higher than previously achievable. Observations of 3C273 and 3C279 taken in April 2000 with the Coordinated Millimeter VLBI Array have resulted in the first total intensity (Stokes I) and linear polarization VLBI images reported of any source at 86 GHz. These results reveal the 86 GHz electric vector position angles within the jets of 3C273 and 3C279 to be orthogonal to each other, and the core of 3C273 to be unpolarized. If this lack of polarization is due to Faraday depolarization alone, the dispersion in rotation measure is >=90000 rad/m^2 for the core of 3C273.

  14. Protease gene families in Populus and Arabidopsis

    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.

  15. Inhibitors of lysosomal cysteine proteases

    Lyanna O. L.; Chorna V. I.

    2011-01-01

    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 anal...

  16. Inhibitors of lysosomal cysteine proteases

    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.

  17. Protease-sensitive synthetic prions.

    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.

  18. Multifrequency Observations of the Virgo Blazars 3C 273 and 3C 279 in CGRO Cycle 8

    Collmar, W; Grove, J E; Hartman, R C; Heindl, W A; Kraus, A L; Teraesranta, H; Villata, M; Bennett, K; Blömen, H; Johnson, W N; Krichbaum, T P; Raiteri, C M; Ryan, J; Sobrito, G; Schönfelder, V; Williams, O R; Wilms, J

    2000-01-01

    We report first observational results of multifrequency campaigns on the prominent Virgo blazars 3C 273 and 3C 279 which were carried out in January and February 1999. Both blazars are detected from radio to gamma-ray energies. We present the measured X- to gamma-ray spectra of both sources, and for 3C 279 we compare the 1999 broad-band (radio to gamma-ray) spectrum to measured previous ones.

  19. Role and efforts of T3C in corrosion economics

    The basic purpose of T3C activity is to show how to acquire specific corrosion cost information so that overall costs for doing business can be reduced. The scope of T3C is to accumulate data, appraise methods, develop recommended practices, promote knowledge and communicate relative to the economic evaluation of corrosion and counter corrosion techniques

  20. 27 CFR 21.37 - Formula No. 3-C.

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Formula No. 3-C. 21.37... OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.37 Formula No. 3-C. (a) Formula. To every 100 gallons of alcohol add:...

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

    Liu, Lei

    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 similarity between them suggests that there is a low-dimensional chaotic attractor in the light curve of 3C 273.

  2. Characterization of cysteine proteases in Malian medicinal plants.

    Bah, Sékou; Paulsen, Berit S; Diallo, Drissa; Johansen, Harald T

    2006-09-19

    Extracts form 10 different Malian medicinal plants with a traditional use against schistosomiasis were investigated for their possible content of proteolytic activity. The proteolytic activity was studied by measuring the hydrolysis of two synthetic peptide substrates Z-Ala-Ala-Asn-NHMec and Z-Phe-Arg-NHMec. Legumain- and papain-like activities were found in all tested crude extracts except those from Entada africana, with the papain-like activity being the strongest. Cissus quadrangularis, Securidaca longepedunculata and Stylosanthes erecta extracts showed high proteolytic activities towards both substrates. After gel filtration the proteolytic activity towards the substrate Z-Ala-Ala-Asn-NHMec in root extract of Securidaca longepedunculata appeared to have Mr of 30 and 97kDa, while the activity in extracts from Cissus quadrangularis was at 39kDa. Enzymatic activity cleaving the substrate Z-Phe-Arg-NHMec showed apparent Mr of 97 and 26kDa in extracts from roots and leaves of Securidaca longepedunculata, while in Cissus quadrangularis extracts the activity eluted at 39 and 20kDa, with the highest activity in the latter. All Z-Phe-Arg-NHMec activities were inhibited by E-64 but unaffected by PMSF. The legumain activity was unaffected by E-64 and PMSF. The SDS-PAGE analysis exhibited five distinct gelatinolytic bands for Cissus quadrangularis extracts (115, 59, 31, 22 and 20kDa), while two bands (59 and 30kDa) were detected in Securidaca longepedunculata extracts. The inhibition profile of the gelatinolytic bands and that of the hydrolysis of the synthetic substrates indicate the cysteine protease class of the proteolytic activities. Several cysteine protease activities with different molecular weights along with a strong variability of these activities between species as well as between plant parts from the same species were observed. PMID:16621376

  3. Quantitative Characterization of the Activation Steps of Mannan-binding Lectin (MBL)-associated Serine Proteases (MASPs) Points to the Central Role of MASP-1 in the Initiation of the Complement Lectin Pathway*

    Megyeri, Márton; Harmat, Veronika; Major, Balázs; Végh, Ádám; Balczer, Júlia; Héja, Dávid; Szilágyi, Katalin; Datz, Dániel; Pál, Gábor; Závodszky, Péter; Gál, Péter; Dobó, József

    2013-01-01

    Mannan-binding lectin (MBL)-associated serine proteases, MASP-1 and MASP-2, have been thought to autoactivate when MBL/ficolin·MASP complexes bind to pathogens triggering the complement lectin pathway. Autoactivation of MASPs occurs in two steps: 1) zymogen autoactivation, when one proenzyme cleaves another proenzyme molecule of the same protease, and 2) autocatalytic activation, when the activated protease cleaves its own zymogen. Using recombinant catalytic fragments, we demonstrated that a stable proenzyme MASP-1 variant (R448Q) cleaved the inactive, catalytic site Ser-to-Ala variant (S646A). The autoactivation steps of MASP-1 were separately quantified using these mutants and the wild type enzyme. Analogous mutants were made for MASP-2, and rate constants of the autoactivation steps as well as the possible cross-activation steps between MASP-1 and MASP-2 were determined. Based on the rate constants, a kinetic model of lectin pathway activation was outlined. The zymogen autoactivation rate of MASP-1 is ∼3000-fold higher, and the autocatalytic activation of MASP-1 is about 140-fold faster than those of MASP-2. Moreover, both activated and proenzyme MASP-1 can effectively cleave proenzyme MASP-2. MASP-3, which does not autoactivate, is also cleaved by MASP-1 quite efficiently. The structure of the catalytic region of proenzyme MASP-1 R448Q was solved at 2.5 Å. Proenzyme MASP-1 R448Q readily cleaves synthetic substrates, and it is inhibited by a specific canonical inhibitor developed against active MASP-1, indicating that zymogen MASP-1 fluctuates between an inactive and an active-like conformation. The determined structure provides a feasible explanation for this phenomenon. In summary, autoactivation of MASP-1 is crucial for the activation of MBL/ficolin·MASP complexes, and in the proenzymic phase zymogen MASP-1 controls the process. PMID:23386610

  4. Quantitative characterization of the activation steps of mannan-binding lectin (MBL)-associated serine proteases (MASPs) points to the central role of MASP-1 in the initiation of the complement lectin pathway.

    Megyeri, Márton; Harmat, Veronika; Major, Balázs; Végh, Ádám; Balczer, Júlia; Héja, Dávid; Szilágyi, Katalin; Datz, Dániel; Pál, Gábor; Závodszky, Péter; Gál, Péter; Dobó, József

    2013-03-29

    Mannan-binding lectin (MBL)-associated serine proteases, MASP-1 and MASP-2, have been thought to autoactivate when MBL/ficolin·MASP complexes bind to pathogens triggering the complement lectin pathway. Autoactivation of MASPs occurs in two steps: 1) zymogen autoactivation, when one proenzyme cleaves another proenzyme molecule of the same protease, and 2) autocatalytic activation, when the activated protease cleaves its own zymogen. Using recombinant catalytic fragments, we demonstrated that a stable proenzyme MASP-1 variant (R448Q) cleaved the inactive, catalytic site Ser-to-Ala variant (S646A). The autoactivation steps of MASP-1 were separately quantified using these mutants and the wild type enzyme. Analogous mutants were made for MASP-2, and rate constants of the autoactivation steps as well as the possible cross-activation steps between MASP-1 and MASP-2 were determined. Based on the rate constants, a kinetic model of lectin pathway activation was outlined. The zymogen autoactivation rate of MASP-1 is ∼3000-fold higher, and the autocatalytic activation of MASP-1 is about 140-fold faster than those of MASP-2. Moreover, both activated and proenzyme MASP-1 can effectively cleave proenzyme MASP-2. MASP-3, which does not autoactivate, is also cleaved by MASP-1 quite efficiently. The structure of the catalytic region of proenzyme MASP-1 R448Q was solved at 2.5 Å. Proenzyme MASP-1 R448Q readily cleaves synthetic substrates, and it is inhibited by a specific canonical inhibitor developed against active MASP-1, indicating that zymogen MASP-1 fluctuates between an inactive and an active-like conformation. The determined structure provides a feasible explanation for this phenomenon. In summary, autoactivation of MASP-1 is crucial for the activation of MBL/ficolin·MASP complexes, and in the proenzymic phase zymogen MASP-1 controls the process. PMID:23386610

  5. Advances in protease engineering for laundry detergents.

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

    2015-12-25

    Proteases are essential ingredients in modern laundry detergents. Over the past 30 years, subtilisin proteases employed in the laundry detergent industry have been engineered by directed evolution and rational design to tailor their properties towards industrial demands. This comprehensive review discusses recent success stories in subtilisin protease engineering. Advances in protease engineering for laundry detergents comprise simultaneous improvement of thermal resistance and activity at low temperatures, a rational strategy to modulate pH profiles, and a general hypothesis for how to increase promiscuous activity towards the production of peroxycarboxylic acids as mild bleaching agents. The three protease engineering campaigns presented provide in-depth analysis of protease properties and have identified principles that can be applied to improve or generate enzyme variants for industrial applications beyond laundry detergents. PMID:25579194

  6. Extracellular proteases as targets for drug development.

    Cudic, Mare; Fields, Gregg B

    2009-08-01

    Proteases constitute one of the primary targets in drug discovery. In the present review, we focus on extracellular proteases (ECPs) because of their differential expression in many pathophysiological processes, including cancer, cardiovascular conditions, and inflammatory, pulmonary, and periodontal diseases. Many new ECP inhibitors are currently under clinical investigation and a significant increase in new therapies based on protease inhibition can be expected in the coming years. In addition to directly blocking the activity of a targeted protease, one can take advantage of differential expression in disease states to selectively deliver therapeutic or imaging agents. Recent studies in targeted drug development for the metalloproteases (matrix metalloproteinases, adamalysins, pappalysins, neprilysin, angiotensin-converting enzyme, metallocarboxypeptidases, and glutamate carboxypeptidase II), serine proteases (elastase, coagulation factors, tissue/urokinase plasminogen activator system, kallikreins, tryptase, dipeptidyl peptidase IV) and cysteine proteases (cathepsin B) are discussed herein. PMID:19689354

  7. Protein targeting to ATP-dependent proteases

    Inobe, Tomonao; Matouschek, Andreas

    2008-01-01

    ATP-dependent proteases control diverse cellular processes by degrading specific regulatory proteins. Understanding how these regulatory proteins are targeted to ATP-dependent proteases is of central importance to understanding their biological role as regulators. Recent work has shown that protein substrates are specifically transferred to ATP-dependent proteases through different routes. These routes can function in parallel or independently. In all of these targeting mechanisms it can be u...

  8. Extracellular proteases as targets for drug development

    Cudic, Mare; Fields, Gregg B.

    2009-01-01

    Proteases constitute one of the primary targets in drug discovery. In the present review, we focus on extracellular proteases (ECPs) because of their differential expression in many pathophysiological processes, including cancer, cardiovascular conditions, and inflammatory, pulmonary, and periodontal diseases. Many new ECP inhibitors are currently under clinical investigation and a significant increase in new therapies based on protease inhibition can be expected in the coming years. In addit...

  9. Synthesis of macrocyclic trypanosomal cysteine protease inhibitors

    Chen, Yen Ting; Lira, Ricardo; Hansell, Elizabeth; McKerrow, James H.; Roush, William R.

    2008-01-01

    The importance of cysteine proteases in parasites, compounded with the lack of redundancy compared to their mammalian hosts makes proteases attractive targets for the development of new therapeutic agents. The binding mode of K11002 to cruzain, the major cysteine protease of Trypanosoma cruzi was used in the design of conformationally constrained inhibitors. Vinyl sulfone-containing macrocycles were synthesized via olefin ring-closing metathesis and evaluated against cruzain and the closely r...

  10. A biotechnology perspective of fungal proteases

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

    2015-01-01

    Proteases hydrolyze the peptide bonds of proteins into peptides and amino acids, being found in all living organisms, and are essential for cell growth and differentiation. Proteolytic enzymes have potential application in a wide number of industrial processes such as food, laundry detergent and pharmaceutical. Proteases from microbial sources have dominated applications in industrial sectors. Fungal proteases are used for hydrolyzing protein and other components of soy beans and wheat in soy...

  11. Thioamide-Based Fluorescent Protease Sensors

    Goldberg, Jacob M.; Chen, Xing; Meinhardt, Nataline; Greenbaum, Doron C.; Petersson, E. James

    2014-01-01

    Thioamide quenchers can be paired with compact fluorophores to design “turn-on” fluorescent protease substrates. We have used this method to study a variety of serine-, cysteine-, carboxyl-, and metallo-proteases, including trypsin, chymotrypsin, pepsin, thermolysin, papain, and calpain. Since thioamides quench some fluorophores red-shifted from those naturally occurring in proteins, this technique can be used for real time monitoring of protease activity in crude preparations of virtually an...

  12. THE ESCHERICHIA COLI SIGNAL PEPTIDE PEPTIDASE A IS A SERINE-LYSINE PROTEASE WITH A LYSINE RECRUITED TO THE NON-CONSERVED AMINO-TERMINAL DOMAIN IN THE S49 PROTEASE FAMILY

    Wang, Peng; Shim, Eunjung; Cravatt, Benjamin; Jacobsen, Richard; Schoeniger, Joe; Kim, Apollos C.; Paetzel, Mark; Dalbey, Ross E.

    2008-01-01

    The E. coli signal peptide peptidase A (SppA) is a serine protease which cleaves signal peptides after they have been proteolytically removed from exported proteins by signal peptidase processing. We present here results of site-directed mutagenesis studies of all the conserved serines of SppA in the carboxyl-terminal domain showing that only Ser 409 is essential for enzymatic activity. Also, we show that the serine hydrolase inhibitor FP-biotin inhibits SppA and modifies the protein, but doe...

  13. Proteolytic crosstalk in multi-protease networks

    Ogle, Curtis T.; Mather, William H.

    2016-04-01

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

  14. Site-specific cleavage of the host poly(A) binding protein by the encephalomyocarditis virus 3C proteinase stimulates viral replication.

    Kobayashi, Mariko; Arias, Carolina; Garabedian, Alexandra; Palmenberg, Ann C; Mohr, Ian

    2012-10-01

    Although picornavirus RNA genomes contain a 3'-terminal poly(A) tract that is critical for their replication, the impact of encephalomyocarditis virus (EMCV) infection on the host poly(A)-binding protein (PABP) remains unknown. Here, we establish that EMCV infection stimulates site-specific PABP proteolysis, resulting in accumulation of a 45-kDa N-terminal PABP fragment in virus-infected cells. Expression of a functional EMCV 3C proteinase was necessary and sufficient to stimulate PABP cleavage in uninfected cells, and bacterially expressed 3C cleaved recombinant PABP in vitro in the absence of any virus-encoded or eukaryotic cellular cofactors. N-terminal sequencing of the resulting C-terminal PABP fragment identified a 3C(pro) cleavage site on PABP between amino acids Q437 and G438, severing the C-terminal protein-interacting domain from the N-terminal RNA binding fragment. Single amino acid substitution mutants with changes at Q437 were resistant to 3C(pro) cleavage in vitro and in vivo, validating that this is the sole detectable PABP cleavage site. Finally, while ongoing protein synthesis was not detectably altered in EMCV-infected cells expressing a cleavage-resistant PABP variant, viral RNA synthesis and infectious virus production were both reduced. Together, these results establish that the EMCV 3C proteinase mediates site-specific PABP cleavage and demonstrate that PABP cleavage by 3C regulates EMCV replication. PMID:22837200

  15. Expression and partial biochemical characterization of a recombinant serine protease from Bothrops pauloensis snake venom.

    Isabel, Thais F; Costa, Guilherme Nunes Moreira; Pacheco, Isabela B; Barbosa, Luana G; Santos-Junior, Célio D; Fonseca, Fernando P P; Boldrini França, Johara; Henrique-Silva, Flávio; Yoneyama, Kelly A G; Rodrigues, Renata S; Rodrigues, Veridiana de Melo

    2016-06-01

    Snake venom serine proteases (SVSPs) are enzymes capable of interfering at several points of hemostasis. Some serine proteases present thrombin-like activity, which makes them targets for the development of therapeutics agents in the treatment of many hemostatic disorders. In this study, a recombinant thrombin-like serine protease, denominated rBpSP-II, was obtained from cDNA of the Bothrops pauloensis venom gland and was characterized enzymatically and biochemically. The enzyme rBpSP-II showed clotting activity on bovine plasma and proteolytic activity on fibrinogen, cleaving exclusively the Aα chain. The evaluation of rBpSP-II activity on chromogenic substrates demonstrated thrombin-like activity of the enzyme due to its capacity to hydrolyze the thrombin substrate. These characteristics make rBpSP-II an attractive molecule for additional studies. Further research is needed to verify whether rBpSP-II can serve as a template for the synthesis of therapeutic agents to treat hemostatic disorders. PMID:26965926

  16. A noncovalent class of papain-like protease/deubiquitinase inhibitors blocks SARS virus replication

    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.

  17. Cell Surface Human Airway Trypsin-Like Protease Is Lost During Squamous Cell Carcinogenesis.

    Duhaime, Michael J; Page, Khaliph O; Varela, Fausto A; Murray, Andrew S; Silverman, Michael E; Zoratti, Gina L; List, Karin

    2016-07-01

    Cancer progression is accompanied by increased levels of extracellular proteases that are capable of remodeling the extracellular matrix, as well as cleaving and activating growth factors and receptors that are involved in pro-cancerous signaling pathways. Several members of the type II transmembrane serine protease (TTSP) family have been shown to play critical roles in cancer progression, however, the expression or function of the TTSP Human Airway Trypsin-like protease (HAT) in carcinogenesis has not been examined. In the present study we aimed to determine the expression of HAT during squamous cell carcinogenesis. HAT transcript is present in several tissues containing stratified squamous epithelium and decreased expression is observed in carcinomas. We determined that HAT protein is consistently expressed on the cell surface in suprabasal/apical layers of squamous cells in healthy cervical and esophageal epithelia. To assess whether HAT protein is differentially expressed in normal tissue versus tissue in different stages of carcinogenesis, we performed a comprehensive immunohistochemical analysis of HAT protein expression levels and localization in arrays of paraffin embedded human cervical and esophageal carcinomas compared to the corresponding normal tissue. We found that HAT protein is expressed in the non-proliferating, differentiated cellular strata and is lost during the dedifferentiation of epithelial cells, a hallmark of squamous cell carcinogenesis. Thus, HAT expression may potentially be useful as a marker for clinical grading and assessment of patient prognosis in squamous cell carcinomas. J. Cell. Physiol. 231: 1476-1483, 2016. © 2015 Wiley Periodicals, Inc. PMID:26297835

  18. Optical nuclear activity in the radio galaxy 3C 465

    De Robertis, M.M.; Yee, H.K.C. (York Univ., North York (Canada) Toronto Univ. (Canada))

    1990-07-01

    The presently discussed discovery of weak, high-ionization emission lines in the nuclei of radio galaxies which had been classified as quiescent absorption-line systems demonstrates that AGN-like activity does occur in the central galaxies of rich clusters. 3C 465-like objects can be considered the extreme low-luminosity end of active nuclei in the centers of rich clusters; the estimated magnitude of 3C 465's nuclear component, at -15.7, is consistent with the precipitous drop of the luminosities of quasars in clusters. 3C 465 appears to represent a new class of optically active objects. 48 refs.

  19. Optical nuclear activity in the radio galaxy 3C 465

    The presently discussed discovery of weak, high-ionization emission lines in the nuclei of radio galaxies which had been classified as quiescent absorption-line systems demonstrates that AGN-like activity does occur in the central galaxies of rich clusters. 3C 465-like objects can be considered the extreme low-luminosity end of active nuclei in the centers of rich clusters; the estimated magnitude of 3C 465's nuclear component, at -15.7, is consistent with the precipitous drop of the luminosities of quasars in clusters. 3C 465 appears to represent a new class of optically active objects. 48 refs

  20. Privacy Issues of the W3C Geolocation API

    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.

  1. Privacy Issues of the W3C Geolocation API

    Doty, Nick; Mulligan, Deirdre K.; 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 s...

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

    Whitcomb, David C

    2014-01-01

    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 associate...

  3. The Caspase-8 Homolog Dredd Cleaves Imd and Relish but Is Not Inhibited by p35*

    Kim, Chan-Hee; Paik, Donggi; Rus, Florentina; Silverman, Neal

    2014-01-01

    In Drosophila, the Imd pathway is activated by diaminopimelic acid-type peptidoglycan and triggers the humoral innate immune response, including the robust induction of antimicrobial peptide gene expression. Imd and Relish, two essential components of this pathway, are both endoproteolytically cleaved upon immune stimulation. Genetic analyses have shown that these cleavage events are dependent on the caspase-8 like Dredd, suggesting that Imd and Relish are direct substrates of Dredd. Among the seven Drosophila caspases, we find that Dredd uniquely promotes Imd and Relish processing, and purified recombinant Dredd cleaves Imd and Relish in vitro. In addition, interdomain cleavage of Dredd is not required for Imd or Relish processing and is not observed during immune stimulation. Baculovirus p35, a suicide substrate of executioner caspases, is not cleaved by purified Dredd in vitro. Consistent with this biochemistry but contrary to earlier reports, p35 does not interfere with Imd signaling in S2* cells or in vivo. PMID:24891502

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

    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...

  5. Cauliflower mosaic virus produces an aspartic proteinase to cleave its polyproteins.

    Torruella, M; Gordon, K; Hohn, T

    1989-01-01

    Cauliflower mosaic virus (CaMV), a plant pararetrovirus, produces polyproteins from its adjacent genes for the coat protein (ORF IV) and for enzymatic functions (ORF V). The N-terminal domain of the latter gene includes a sequence showing homology to the active site of other retroviral and acid proteases. We have now shown that this domain does indeed produce a functional aspartic protease that can process both the polyproteins. Mutations in the putative active site abolished virus infectivit...

  6. Extracellular and membrane-bound proteases from Bacillus subtilis.

    Mäntsälä, P; Zalkin, H

    1980-01-01

    Bacillus subtilis YY88 synthesizes increased amounts of extracellular and membrane-bound proteases. More than 99% of the extracellular protease activity is accounted for by an alkaline serine protease and a neutral metalloprotease. An esterase having low protease activity accounts for less than 1% of the secreted protease. These enzymes were purified to homogeneity. Molecular weights of approximately 28,500 and 39,500 were determined for the alkaline and neutral proteases, respectively. The e...

  7. Optical Periodicity Analysis of 3C 446 using Period04

    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.

  8. Radio jet of the quasar 3C273

    Flatters, C.; Conway, R.G.

    1985-04-04

    Although 3C273 was one of the first quasars to be identified, the extended feature 3C273A, which can be detected at radio, optical and X-ray wavelengths, remains an enigma. The source is an extreme example of a one-sided radio source (3C273A has no detectable counter component) and this fact, coupled with the presence of the optical emission, makes it unlikely that 3C273A is a normal (slow-moving) radio lobe. Superluminal transverse motion at milliarc second scales shows that relativistic velocities occur within the quasar itself, 3C273B; it is an open question whether these velocities persist out to 3C273A. It has been widely suggested that Doppler beaming causes the one-sidedness of this and similar sources by suppressing the receding half of the source, but there are no spectral lines by which the Doppler shift of 3C273A could be directly measured. Thus, any (indirect) indication of the velocity is of interest. Here new MERLIN observations of the brightness and polarization of the radio jet of 3C273 at a resolution of 0.35 arc s are presented. One of the most marked features of the new map, the high polarization found within the head of the source, is hard to explain. If the motion is indeed fast, then relativistic aberration should be taken into account; it suggests that this leads to a natural explanation of the high observed polarization. 18 references, 1 figure, 1 table.

  9. A Modified Stratified Model for 3C 273 Jet

    Liu, Wen-Po; Shen, Zhi-Qiang

    2008-01-01

    We present a modified stratified jet model to interpret the observed spectral energy distributions of knots in 3C 273 jet. Based on the hypothesis of the single index of the particle energy spectrum at injection and identical emission processes among all the knots, the observed difference of spectral shape among different 3C273 knots can be understood as a manifestation of deviation of the equivalent Doppler factor of stratified emission regions in individual knot from a characteristic one. T...

  10. Immobilization to prevent enzyme incompatibility with proteases

    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 s