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Sample records for 3clpro proteinase cleavage

  1. Characterization of trans- and cis-cleavage activity of the SARS coronavirus 3CLpro protease: basis for the in vitro screening of anti-SARS drugs.

    PubMed

    Lin, Cheng-Wen; Tsai, Chang-Hai; Tsai, Fuu-Jen; Chen, Pei-Jer; Lai, Chien-Chen; Wan, Lei; Chiu, Hua-Hao; Lin, Kuan-Hsun

    2004-09-10

    Severe acute respiratory syndrome (SARS) has been globally reported. A novel coronavirus (CoV), SARS-CoV, was identified as the etiological agent of the disease. SARS-CoV 3C-like protease (3CLpro) mediates the proteolytic processing of replicase polypeptides 1a and 1ab into functional proteins, playing an important role in viral replication. In this study, we demonstrated the expression of the SARS-CoV 3CLpro in Escherichia coli and Vero cells, and then characterized the in vitro trans-cleavage and the cell-based cis-cleavage by the 3CLpro. Mutational analysis of the 3CLpro demonstrated the importance of His41, Cys145, and Glu166 in the substrate-binding subsite S1 for keeping the proteolytic activity. In addition, alanine substitution of the cleavage substrates indicated that Gln-(P1) in the substrates mainly determined the cleavage efficiency. Therefore, this study not only established the quantifiable and reliable assay for the in vitro and cell-based measurement of the 3CLpro activity, but also characterized the molecular interaction of the SARS-CoV 3CLpro with the substrates. The results will be useful for the rational development of the anti-SARS drugs.

  2. Effect of retroviral proteinase inhibitors on Mason-Pfizer monkey virus maturation and transmembrane glycoprotein cleavage.

    PubMed Central

    Sommerfelt, M A; Petteway, S R; Dreyer, G B; Hunter, E

    1992-01-01

    Mason-Pfizer monkey virus (M-PMV) is the prototype type D retrovirus which preassembles immature intracytoplasmic type A particles within the infected cell cytoplasm. Intracytoplasmic type A particles are composed of uncleaved polyprotein precursors which upon release are cleaved by the viral proteinase to their constituent mature proteins. This results in a morphological change in the virion described as maturation. We have investigated the role of the viral proteinase in virus maturation and infectivity by inhibiting the function of the enzyme through mutagenesis of the proteinase gene and by using peptide inhibitors originally designed to block human immunodeficiency virus type 1 proteinase activity. Mutation of the active-site aspartic acid, Asp-26, to asparagine abrogated the activity of the M-PMV proteinase but did not affect the assembly of noninfectious, immature virus particles. In mutant virions, the transmembrane glycoprotein (TM) of M-PMV, initially synthesized as a cell-associated gp22, is not cleaved to gp20, as is observed with wild-type virions. This demonstrates that the viral proteinase is responsible for this cleavage event. Hydroxyethylene isostere human immunodeficiency virus type 1 proteinase inhibitors were shown to block M-PMV proteinase cleavage of the TM glycoprotein and Gag-containing precursors in a dose-dependent manner. The TM cleavage event was more sensitive than cleavage of the Gag precursors to inhibition. The infectivity of treated particles was reduced significantly, but experiments showed that inhibition of precursor and TM cleavage may be at least partially reversible. These results demonstrate that the M-PMV aspartyl proteinase is activated in released virions and that the hydroxyethylene isostere proteinase inhibitors used in this study exhibit a broad spectrum of antiretroviral activity. Images PMID:1602542

  3. Cleavage of Grb2-Associated Binding Protein 2 by Viral Proteinase 2A during Coxsackievirus Infection

    PubMed Central

    Deng, Haoyu; Fung, Gabriel; Qiu, Ye; Wang, Chen; Zhang, Jingchun; Jin, Zheng-Gen; Luo, Honglin

    2017-01-01

    Coxsackievirus type B3 (CV-B3), an enterovirus associated with the pathogenesis of several human diseases, subverts, or employs the host intracellular signaling pathways to support effective viral infection. We have previously demonstrated that Grb2-associated binding protein 1 (GAB1), a signaling adaptor protein that serves as a platform for intracellular signaling assembly and transduction, is cleaved upon CV-B3 infection, resulting in a gain-of-pro-viral-function via the modification of GAB1-mediated ERK1/2 pathway. GAB2 is a mammalian homolog of GAB1. In this study, we aim to address whether GAB2 plays a synergistic role with GAB1 in the regulation of CV-B3 replication. Here, we reported that GAB2 is also a target of CV-B3-encoded viral proteinase. We showed that GAB2 is cleaved at G238 during CV-B3 infection by viral proteinase 2A, generating two cleaved fragments of GAB2-N1−237 and GAB2-C238−676. Moreover, knockdown of GAB2 significantly inhibits the synthesis of viral protein and subsequent viral progeny production, accompanied by reduced levels of phosphorylated p38, suggesting a pro-viral function for GAB2 linked to p38 activation. Finally, we examined whether the cleavage of GAB2 can promote viral replication as observed for GAB1 cleavage. We showed that expression of neither GAB2-N1−237 nor GAB2-C238−676 results in enhanced viral infectivity, indicating a loss-of-function, rather than a gain-of-function of GAB2 cleavage in mediating virus replication. Taken together, our findings in this study suggest a novel host defense machinery through which CV-B3 infection is limited by the cleavage of a pro-viral protein. PMID:28361043

  4. Conformational Flexibility of a Short Loop near the Active Site of the SARS-3CLpro is Essential to Maintain Catalytic Activity

    PubMed Central

    Li, Chunmei; Teng, Xin; Qi, Yifei; Tang, Bo; Shi, Hailing; Ma, Xiaomin; Lai, Luhua

    2016-01-01

    The SARS 3C-like proteinase (SARS-3CLpro), which is the main proteinase of the SARS coronavirus, is essential to the virus life cycle. This enzyme has been shown to be active as a dimer in which only one protomer is active. However, it remains unknown how the dimer structure maintains an active monomer conformation. It has been observed that the Ser139-Leu141 loop forms a short 310-helix that disrupts the catalytic machinery in the inactive monomer structure. We have tried to disrupt this helical conformation by mutating L141 to T in the stable inactive monomer G11A/R298A/Q299A. The resulting tetra-mutant G11A/L141T/R298A/Q299A is indeed enzymatically active as a monomer. Molecular dynamics simulations revealed that the L141T mutation disrupts the 310-helix and helps to stabilize the active conformation. The coil-310-helix conformational transition of the Ser139-Leu141 loop serves as an enzyme activity switch. Our study therefore indicates that the dimer structure can stabilize the active conformation but is not a required structure in the evolution of the active enzyme, which can also arise through simple mutations. PMID:26879383

  5. Conformational Flexibility of a Short Loop near the Active Site of the SARS-3CLpro is Essential to Maintain Catalytic Activity

    NASA Astrophysics Data System (ADS)

    Li, Chunmei; Teng, Xin; Qi, Yifei; Tang, Bo; Shi, Hailing; Ma, Xiaomin; Lai, Luhua

    2016-02-01

    The SARS 3C-like proteinase (SARS-3CLpro), which is the main proteinase of the SARS coronavirus, is essential to the virus life cycle. This enzyme has been shown to be active as a dimer in which only one protomer is active. However, it remains unknown how the dimer structure maintains an active monomer conformation. It has been observed that the Ser139-Leu141 loop forms a short 310-helix that disrupts the catalytic machinery in the inactive monomer structure. We have tried to disrupt this helical conformation by mutating L141 to T in the stable inactive monomer G11A/R298A/Q299A. The resulting tetra-mutant G11A/L141T/R298A/Q299A is indeed enzymatically active as a monomer. Molecular dynamics simulations revealed that the L141T mutation disrupts the 310-helix and helps to stabilize the active conformation. The coil-310-helix conformational transition of the Ser139-Leu141 loop serves as an enzyme activity switch. Our study therefore indicates that the dimer structure can stabilize the active conformation but is not a required structure in the evolution of the active enzyme, which can also arise through simple mutations.

  6. In vitro molecular genetics as a tool for determining the differential cleavage specificities of the poliovirus 3C proteinase.

    PubMed Central

    Ypma-Wong, M F; Semler, B L

    1987-01-01

    We describe a completely in vitro system for generating defined poliovirus proteinase mutations and subsequently assaying the phenotypic expression of such mutations. A complete cDNA copy of the entire poliovirus genome has been inserted into a bacteriophage T7 transcription vector. We have introduced proteinase and/or cleavage site mutations into this cDNA. Mutant RNA is transcribed from the altered cDNA template and is subsequently translated in vitro. Employing such a system, we provide direct evidence for the bimolecular cleavage events carried out by the 3C proteinase. We show that specific genetically-altered precursor polypeptides containing authentic Q-G cleavage sites will not act as substrates for 3C either in cis or in trans. We also provide evidence that almost the entire P3 region is required to generate 3C proteinase activity capable of cleaving the P1 precursor to capsid proteins. However, only the 3C portion of P3 is required to generate 3C proteinase activity capable of cleaving P2 and its processing products. Images PMID:3031587

  7. Foot-and-mouth disease virus leader proteinase: structural insights into the mechanism of intermolecular cleavage.

    PubMed

    Steinberger, Jutta; Grishkovskaya, Irina; Cencic, Regina; Juliano, Luiz; Juliano, Maria A; Skern, Tim

    2014-11-01

    Translation of foot-and-mouth disease virus RNA initiates at one of two start codons leading to the synthesis of two forms of leader proteinase L(pro) (Lab(pro) and Lb(pro)). These forms free themselves from the viral polyprotein by intra- and intermolecular self-processing and subsequently cleave the cellular eukaryotic initiation factor (eIF) 4 G. During infection, Lb(pro) removes six residues from its own C-terminus, generating sLb(pro). We present the structure of sLb(pro) bound to the inhibitor E64-R-P-NH2, illustrating how sLb(pro) can cleave between Lys/Gly and Gly/Arg pairs. In intermolecular cleavage on polyprotein substrates, Lb(pro) was unaffected by P1 or P1' substitutions and processed a substrate containing nine eIF4GI cleavage site residues whereas sLb(pro) failed to cleave the eIF4GI containing substrate and cleaved appreciably more slowly on mutated substrates. Introduction of 70 eIF4GI residues bearing the Lb(pro) binding site restored cleavage. These data imply that Lb(pro) and sLb(pro) may have different functions in infected cells.

  8. Human cytomegalovirus maturational proteinase: expression in Escherichia coli, purification, and enzymatic characterization by using peptide substrate mimics of natural cleavage sites.

    PubMed Central

    Burck, P J; Berg, D H; Luk, T P; Sassmannshausen, L M; Wakulchik, M; Smith, D P; Hsiung, H M; Becker, G W; Gibson, W; Villarreal, E C

    1994-01-01

    The proteolytic processing of the human cytomegalovirus (HCMV) assembly protein, resulting in truncation of its C terminus, is an essential step in virion maturation. The proteinase responsible for this cleavage is the amino-terminal half of the protein encoded by the UL80a open reading fame. We have obtained high expression levels of this 256-amino-acid HCMV proteinase, assemblin, in Escherichia coli. In addition to the 28-kDa proteinase, a 15-kDa protein comprising the first 143 amino acids and a 13-kDa protein comprising the last 113 amino acids of the 28-kDa HCMV proteinase were present. Both the 28-kDa proteinase and the 15-kDa protein were purified by a two-step chromatographic procedure utilizing anion exchange in urea and dithiothreitol and size exclusion in NaSCN and dithiothreitol. Activation of the purified 28-kDa proteinase required denaturation in urea as well as complete reduction of all five cysteine residues in the molecule. Removal of the urea by dialysis with retention of the reducing agent yielded an active proteinase. Addition of glycerol to 50% enhanced the activity. The HCMV proteinase cleaved the peptides RGVVNASSRLAK and SYVKASVSPE, which are mimics of the maturational (M)- and release (R)-site sequences, respectively, in the UL80a-encoded protein. The cleavage site in the peptides was at the same Ala-Ser scissile bond as observed in the UL80a protein. The Km value for the cleavage of RGVVNASSRLAK (M-site mimic) by the proteinase was similar to that for SYVKASVSPE (R-site mimic), but the turnover (kcat) of the M-site peptide mimic substrate by the proteinase was six to eight times faster. The peptide homologs of the herpes simplex virus type 1 M- and R-site sequences in the UL26-encoded protein were also cleaved by the HCMV proteinase, although at rates slower than those for the HCMV substrates. The HCMV proteinase was inhibited by Zn2+ and by alkylating agents, but only at very high inhibitor concentrations. The purified 15-kDa protein

  9. Potential Broad Spectrum Inhibitors of the Coronavirus 3CLpro: A Virtual Screening and Structure-Based Drug Design Study

    PubMed Central

    Berry, Michael; Fielding, Burtram C.; Gamieldien, Junaid

    2015-01-01

    Human coronaviruses represent a significant disease burden; however, there is currently no antiviral strategy to combat infection. The outbreak of severe acute respiratory syndrome (SARS) in 2003 and Middle East respiratory syndrome (MERS) less than 10 years later demonstrates the potential of coronaviruses to cross species boundaries and further highlights the importance of identifying novel lead compounds with broad spectrum activity. The coronavirus 3CLpro provides a highly validated drug target and as there is a high degree of sequence homology and conservation in main chain architecture the design of broad spectrum inhibitors is viable. The ZINC drugs-now library was screened in a consensus high-throughput pharmacophore modeling and molecular docking approach by Vina, Glide, GOLD and MM-GBSA. Molecular dynamics further confirmed results obtained from structure-based techniques. A highly defined hit-list of 19 compounds was identified by the structure-based drug design methodologies. As these compounds were extensively validated by a consensus approach and by molecular dynamics, the likelihood that at least one of these compounds is bioactive is excellent. Additionally, the compounds segregate into 15 significantly dissimilar (p < 0.05) clusters based on shape and features, which represent valuable scaffolds that can be used as a basis for future anti-coronaviral inhibitor discovery experiments. Importantly though, the enriched subset of 19 compounds identified from the larger library has to be validated experimentally. PMID:26694449

  10. The eIF4G-eIF4E complex is the target for direct cleavage by the rhinovirus 2A proteinase.

    PubMed Central

    Haghighat, A; Svitkin, Y; Novoa, I; Kuechler, E; Skern, T; Sonenberg, N

    1996-01-01

    The 2A proteinases (2Apro) of certain picornaviruses induce the cleavage of the eIF4G subunit of the cap-binding protein complex, eIF4F. Several reports have demonstrated that 2Apro of rhinovirus and coxsackievirus B4 cleave eIF4G directly. However, it was suggested that in poliovirus infection, the 2Apro induces the activation of a cellular proteinase which in turn cleaves eIF4G. Furthermore, it is not clear whether eIF4G is cleaved as part of the eIF4F complex or as an individual polypeptide. To address these issues, recombinant eIF4G was purified from Sf9 insect cells and tested for cleavage by purified rhinovirus 2Apro. Here we report that eIF4G alone is a relatively poor substrate for cleavage by the rhinovirus 2Apro. However, an eIF4G-eIF4E complex is cleaved efficiently by the 2Apro, suggesting that eIF4F is a preferred substrate for cleavage by rhinovirus 2Apro. Furthermore, 2Apr drastically reduced the translation of a capped mRNA. An eIF4G-eIF4E complex, but not eIF4G alone, was required to restore translation. PMID:8970966

  11. Discovery of N-(benzo[1,2,3]triazol-1-yl)-N-(benzyl)acetamido)phenyl) carboxamides as severe acute respiratory syndrome coronavirus (SARS-CoV) 3CLpro inhibitors: identification of ML300 and non-covalent nanomolar inhibitors with an induced-fit binding

    PubMed Central

    Turlington, Mark; Chun, Aspen; Tomar, Sakshi; Eggler, Aimee; Grum-Tokars, Valerie; Jacobs, Jon; Daniels, J. Scott; Dawson, Eric; Saldanha, Adrian; Chase, Peter; Baez-Santos, Yahira M.; Lindsley, Craig W.; Hodder, Peter; Mesecar, Andrew; Stauffer, Shaun R.

    2013-01-01

    Herein we report the discovery and SAR of a novel series of SARS-CoV 3CLpro inhibitors identified through the NIH Molecular Libraries Probe Production Centers Network (MLPCN). In addition to ML188, ML300 represents the second probe declared for 3CLpro from this collaborative effort. The X-ray structure of SARS-CoV 3CLpro bound with a ML300 analog highlights a unique induced-fit reorganization of the S2-S4 binding pockets leading to the first sub-micromolar non-covalent 3CLpro inhibitors retaining a single amide bond. PMID:24080461

  12. The serine proteinase chain of human complement component C1s. Cyanogen bromide cleavage and N-terminal sequences of the fragments.

    PubMed Central

    Carter, P E; Dunbar, B; Fothergill, J E

    1983-01-01

    Human complement component C1s was purified from fresh blood by conventional methods of precipitation and chromatography. The single-chain zymogen form was activated by treatment with C1r. Reduction and carboxymethylation then allowed the light chain and heavy chain to be separated on DEAE-Sepharose CL-6B in 8 M-urea. Liquid-phase sequencing of the light chain determined 50 residues from the N-terminus. CNBr-cleavage fragments of the light chain were separated by high-pressure liquid chromatography on gel-permeation and reverse-phase columns. N-Terminal sequencing of these fragments determined the order of a further 138 residues, giving a total of 188 residues or about 75% of the light chain. Seven of these eight sequences could be readily aligned with the amino acid sequences of other serine proteinases. The typical serine proteinase active-site residues are clearly conserved in C1s, and the specificity-related side chain of the substrate-binding pocket is aspartic acid, as in trypsin, consistent with the proteolytic action of C1s on C4 at an arginine residue. Somewhat surprisingly, when the C1s sequence is compared with that of complement subcomponent C1r, the percentage difference (59%) is approximately the same as that found between the other mammalian serine proteinases (56-71%). PMID:6362661

  13. Cleavage at a novel site in the NS4A region by the yellow fever virus NS2B-3 proteinase is a prerequisite for processing at the downstream 4A/4B signalase site.

    PubMed Central

    Lin, C; Amberg, S M; Chambers, T J; Rice, C M

    1993-01-01

    Flavivirus proteins are produced by co- and posttranslational proteolytic processing of a large polyprotein by both host- and virus-encoded proteinases. The viral serine proteinase, which consists of NS2B and NS3, is responsible for cleavage of at least four dibasic sites (2A/2B, 2B/3, 3/4A, and 4B/5) in the nonstructural region. Since the amino acid sequence preceding NS4B shares characteristics with signal peptides used for translocation of nascent polypeptides into the lumen of the endoplasmic reticulum, it has been proposed that cleavage at the 4A/4B site is mediated by a cellular signal peptidase. In this report, cell-free translation and in vivo transient expression assays were used to study processing in the NS4 region of the yellow fever virus polyprotein. With a construct which contained NS4B preceded by 17 residues constituting the putative signal peptide (sig4B), membrane-dependent cleavage at the 4A/4B site was demonstrated in vitro. Surprisingly, processing of NS4A-4B was not observed in cell-free translation studies, and in vivo expression of several yellow fever virus polyproteins revealed that the 4A/4B cleavage occurred only during coexpression of NS2B and the proteinase domain of NS3. Examination of mutant derivatives of the NS3 proteinase domain demonstrated that cleavage at the 4A/4B site correlated with expression of an active NS2B-3 proteinase. From these results, we propose a model in which the signalase cleavage generating the N terminus of NS4B requires a prior NS2B-3 proteinase-mediated cleavage at a novel site (called the 4A/2K site) which is conserved among flaviviruses and located 23 residues upstream of the signalase site. In support of this model, mutations at the 4A/4B signalase site did not eliminate processing in the NS4 region. In contrast, substitutions at the 4A/2K site, which were engineered to block NS2B-3 proteinase-mediated cleavage, eliminated signalase cleavage at the 4A/4B site. In addition, the size of the 3(502)-4A

  14. Steady-state and pre-steady-state kinetic evaluation of severe acute respiratory syndrome coronavirus (SARS-CoV) 3CLpro cysteine protease: development of an ion-pair model for catalysis.

    PubMed

    Solowiej, James; Thomson, James A; Ryan, Kevin; Luo, Chun; He, Mingying; Lou, Jihong; Murray, Brion W

    2008-02-26

    Severe acute respiratory syndrome (SARS) was a worldwide epidemic caused by a coronavirus that has a cysteine protease (3CLpro) essential to its life cycle. Steady-state and pre-steady-state kinetic methods were used with highly active 3CLpro to characterize the reaction mechanism. We show that 3CLpro has mechanistic features common and disparate to the archetypical proteases papain and chymotrypsin. The kinetic mechanism for 3CLpro-mediated ester hydrolysis, including the individual rate constants, is consistent with a simple double displacement mechanism. The pre-steady-state burst rate was independent of ester substrate concentration indicating a high commitment to catalysis. When homologous peptidic amide and ester substrates were compared, a series of interesting observations emerged. Despite a 2000-fold difference in nonenzymatic reactivity, highly related amide and ester substrates were found to have similar kinetic parameters in both the steady-state and pre-steady-state. Steady-state solvent isotope effect (SIE) studies showed an inverse SIE for the amide but not ester substrates. Evaluation of the SIE in the pre-steady-state revealed normal SIEs for both amide and ester burst rates. Proton inventory (PI) studies on amide peptide hydrolysis were consistent with two proton-transfer reactions in the transition state while the ester data was consistent with a single proton-transfer reaction. Finally, the pH-inactivation profile of 3CLpro with iodoacetamide is indicative of an ion-pair mechanism. Taken together, the data are consistent with a 3CLpro mechanism that utilizes an "electrostatic" trigger to initiate the acylation reaction, a cysteine-histidine catalytic dyad ion pair, an enzyme-facilitated release of P1, and a general base-catalyzed deacylation reaction.

  15. Neutrophil proteinase cathepsin G is proteolytically active on the human platelet glycoprotein Ib-IX receptor: characterization of the cleavage sites within the glycoprotein Ib alpha subunit.

    PubMed Central

    Pidard, D; Renesto, P; Berndt, M C; Rabhi, S; Clemetson, K J; Chignard, M

    1994-01-01

    The proteolytic activity of the neutrophil serine-proteinase cathepsin G (CG) on platelet adherence receptors, the glycoprotein (GP) Ib-IX complex and the integrin alpha IIb beta 3, has been investigated. In the range 50 to 200 nmol/l, CG is a potent platelet agonist which induces shape change, granule exocytosis and aggregation. Investigation of the proteolysis of the receptors' subunits during the course of platelet activation by CG was performed by immunoblot analysis of platelet proteins using a panel of specific antibodies. Exposure of platelets for 3 min at 37 degrees C to CG at a concentration that induces full cell activation resulted in an extensive cleavage of the N-terminal region of the extracellular domain of GPIb alpha, the largest (relative molecular mass, M(r), 143,000) of the three subunits constituting the GPIb-IX complex. In contrast, no detectable proteolytic modification of the two other subunits, GPIb beta and GPIX, was detected. Similarly, we observed that neither of the two subunits of the alpha IIb beta 3 receptor were proteolytically modified by CG. Cleavage of GPIb alpha by CG leaves a remnant of the polypeptide chain with M(r) approx. 106,000 in the plasma membrane, while releasing into the extracellular milieu the N-terminal domain with M(r) in the range 40,000 to 46,000. N-terminal sequencing of the CG-derived fragments of GPIb alpha indicated that the Leu275-Tyr276 peptide bond was the primary cleavage site for this proteinase. Proteolysis of GPIb alpha was already detectable at concentrations of CG as low as 25 nmol/l, while with 200 nmol/l the cleavage was detected as soon as 10 s after exposure of platelets to the proteinase. Comparison of the kinetics and concentration dependency for the proteolysis of GPIb alpha and for the activation of platelets by CG showed that cleavage of the GPIb-IX receptor is an early event that accompanies exocytosis and aggregation. Quantitative evaluation of the conversion of GPIb alpha into its

  16. Cinanserin is an inhibitor of the 3C-like proteinase of severe acute respiratory syndrome coronavirus and strongly reduces virus replication in vitro.

    PubMed

    Chen, Lili; Gui, Chunshan; Luo, Xiaomin; Yang, Qingang; Günther, Stephan; Scandella, Elke; Drosten, Christian; Bai, Donglu; He, Xichang; Ludewig, Burkhard; Chen, Jing; Luo, Haibin; Yang, Yiming; Yang, Yifu; Zou, Jianping; Thiel, Volker; Chen, Kaixian; Shen, Jianhua; Shen, Xu; Jiang, Hualiang

    2005-06-01

    The 3C-like proteinase (3CLpro) of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is one of the most promising targets for anti-SARS-CoV drugs due to its crucial role in the viral life cycle. In this study, a database containing structural information of more than 8,000 existing drugs was virtually screened by a docking approach to identify potential binding molecules of SARS-CoV 3CLpro. As a target for screening, both a homology model and the crystallographic structure of the binding pocket of the enzyme were used. Cinanserin (SQ 10,643), a well-characterized serotonin antagonist that has undergone preliminary clinical testing in humans in the 1960s, showed a high score in the screening and was chosen for further experimental evaluation. Binding of both cinanserin and its hydrochloride to bacterially expressed 3CLpro of SARS-CoV and the related human coronavirus 229E (HCoV-229E) was demonstrated by surface plasmon resonance technology. The catalytic activity of both enzymes was inhibited with 50% inhibitory concentration (IC50) values of 5 microM, as tested with a fluorogenic substrate. The antiviral activity of cinanserin was further evaluated in tissue culture assays, namely, a replicon system based on HCoV-229E and quantitative test assays with infectious SARS-CoV and HCoV-229E. All assays revealed a strong inhibition of coronavirus replication at nontoxic drug concentrations. The level of virus RNA and infectious particles was reduced by up to 4 log units, with IC50 values ranging from 19 to 34 microM. These findings demonstrate that the old drug cinanserin is an inhibitor of SARS-CoV replication, acting most likely via inhibition of the 3CL proteinase.

  17. Engineering the substrate specificity of rhizopuspepsin: the role of Asp 77 of fungal aspartic proteinases in facilitating the cleavage of oligopeptide substrates with lysine in P1.

    PubMed Central

    Lowther, W. T.; Majer, P.; Dunn, B. M.

    1995-01-01

    Rhizopuspepsin and other fungal aspartic proteinases are distinct from the mammalian enzymes in that they are able to cleave substrates with lysine in the P1 position. Sequence and structural comparisons suggest that two aspartic acid residues, Asp 30 and Asp 77 (pig pepsin numbering), may be responsible for generating this unique specificity. Asp 30 and Asp 77 were changed to the corresponding residues in porcine pepsin, Ile 30 and Thr 77, to create single and double mutants. The zymogen forms of the wild-type and mutant enzymes were overexpressed in Escherichia coli as inclusion bodies. Following solubilization, denaturation, refolding, activation, and purification to homogeneity, structural and kinetic comparisons were made. The mutant enzymes exhibited a high degree of structural similarity to the wild-type recombinant protein and a native isozyme. The catalytic activities of the recombinant proteins were analyzed with chromogenic substrates containing lysine in the P1, P2, or P3 positions. Mutation of Asp 77 resulted in a loss of 7 kcal mol-1 of transition-state stabilization energy in the hydrolysis of the substrate containing lysine in P1. An inhibitor containing the positively charged P1-lysine side chain inhibited only the enzymes containing Asp 77. Inhibition of the Asp 77 mutants of rhizopuspepsin and several mammalian enzymes was restored upon acetylation of the lysine side chain. These results suggest that an exploitation of the specific electrostatic interaction of Asp 77 in the active site of fungal enzymes may lead to the design of compounds that preferentially inhibit a variety of related Candida proteinases in immunocompromised patients. PMID:7613467

  18. A second hepatitis C virus-encoded proteinase.

    PubMed Central

    Grakoui, A; McCourt, D W; Wychowski, C; Feinstone, S M; Rice, C M

    1993-01-01

    Host and viral proteinases are believed to be required for the production of at least nine hepatitis C virus (HCV)-specific polyprotein cleavage products. Although several cleavages appear to be catalyzed by host signal peptidase or the HCV NS3 serine proteinase, the enzyme responsible for cleavage at the 2/3 site has not been identified. In this report, we have defined the 2/3 cleavage site and obtained evidence which suggests that this cleavage is mediated by a second HCV-encoded proteinase, located between aa 827 and 1207. This region encompasses the C-terminal portion of the 23-kDa NS2 protein, the 2/3 cleavage site, and the serine proteinase domain of NS3. Efficient processing at the 2/3 site was observed in mammalian cells, Escherichia coli, and in plant or animal cell-free translation systems in the absence of microsomal membranes. Cleavage at the 2/3 site was abolished by alanine substitutions for NS2 residues His-952 or Cys-993 but was unaffected by several other substitution mutations, including those that inactivate NS3 serine proteinase function. Mutations abolishing cleavage at the 2/3 site did not block cleavage at other sites in the HCV polyprotein. Cotransfection experiments indicate that the 2/3 site can be cleaved in trans, which should facilitate purification and further characterization of this enzyme. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:8248148

  19. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  20. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  1. Manduca sexta hemolymph proteinase 21 activates prophenoloxidase-activating proteinase 3 in an insect innate immune response proteinase cascade.

    PubMed

    Gorman, Maureen J; Wang, Yang; Jiang, Haobo; Kanost, Michael R

    2007-04-20

    Melanization, an insect immune response, requires a set of hemolymph proteins including pathogen recognition proteins that initiate the response, a cascade of mostly unknown serine proteinases, and phenoloxidase. Until now, only initial and final proteinases in the pathways have been conclusively identified. Four such proteinases have been purified from the larval hemolymph of Manduca sexta: hemolymph proteinase 14 (HP14), which autoactivates in the presence of microbial surface components, and three prophenoloxidase-activating proteinases (PAP1-3). In this study, we have used two complementary approaches to identify a serine proteinase that activates proPAP3. Partial purification from hemolymph of an activator of proPAP3 resulted in an active fraction with two abundant polypeptides of approximately 32 and approximately 37 kDa. Labeling of these polypeptides with a serine proteinase inhibitor, diisopropyl fluorophosphate, indicated that they were active serine proteinases. N-terminal sequencing revealed that both were cleaved forms of the previously identified hemolymph serine proteinase, HP21. Surprisingly, cleavage of proHP21 had occurred not at the predicted activation site but more N-terminal to it. In vitro reactions carried out with purified HP14 (which activates proHP21), proHP21, proPAP3, and site-directed mutant forms of the latter two proteinases confirmed that HP21 activates proPAP3 by limited proteolysis. Like the HP21 products purified from hemolymph, HP21 that was activated by HP14 in the in vitro reactions was not cleaved at its predicted activation site.

  2. The reaction of serpins with proteinases involves important enthalpy changes.

    PubMed

    Boudier, C; Bieth, J G

    2001-08-21

    When active serpins are proteolytically inactivated in a substrate-like reaction, they undergo an important structural transition with a resultant increase in their conformational stability. We have used microcalorimetry to show that this conformational alteration is accompanied by an important enthalpy change. For instance, the cleavage of alpha(1)-proteinase inhibitor by Pseudomonas aeruginosa elastase, Staphylococcus aureus V8 proteinase, or papain and that of antithrombin by leukocyte elastase are characterized by large enthalpy changes (DeltaH = -53 to -63 kcal mol(-1)). The former reaction also has a large and negative heat capacity (DeltaC(p)() = -566 cal K(-1) mol(-1)). In contrast, serpins release significantly less heat when they act as proteinase inhibitors. For example, the inhibition of pancreatic elastase, leukocyte elastase, and pancreatic chymotrypsin by alpha(1)-proteinase inhibitor and that of pancreatic trypsin and coagulation factor Xa by antithrombin are accompanied by a DeltaH of -20 to -31 kcal mol(-1). We observe no heat release upon proteolytic cleavage of inactive serpins or following inhibition of serine proteinases by canonical inhibitors or upon acylation of chymotrypsin by N-trans-cinnamoylimidazole. We suggest that part of the large enthalpy change that occurs during the structural transition of serpins is used to stabilize the proteinase in its inactive state.

  3. Picornaviral 3C cysteine proteinases have a fold similar to the chymotrypsin-like serine proteinases

    SciTech Connect

    Allaire,M.; Chernaia, M.; Malcolm, B.; James, M.

    1994-01-01

    The picornavirus family includes several pathogens such as poliovirus, rhinovirus (the major cause of the common cold), hepatitis A virus and the foot-and-mouth disease virus. Picornaviral proteins are expressed by direct translation of the genomic RNA into a single, large polyprotein precursor. Proteolysis of the viral polyprotein into the mature proteins is assured by the viral 3C enzymes, which are cysteine proteinases. Here we report the X-ray crystal structure at 2.3 {angstrom} resolution of the 3C proteinase from hepatitis A virus (HAV-3C). The overall architecture of HAV-3C reveals a fold resembling that of the chymotrypsin family of serine proteinases, which is consistent with earlier predictions. Catalytic residues include Cys 172 as nucleophile and His 44 as general base. The 3C cleavage specificity for glutamine residues is defined primarily by His 191. The overall structure suggests that an inter-molecular (trans) cleavage releases 3C and that there is an active proteinase in the polyprotein.

  4. Isolation and characterization of recombinant Drosophila Copia aspartic proteinase

    PubMed Central

    Athauda, Senarath B. P.; Yoshioka, Katsuji; Shiba, Tadayoshi; Takahashi, Kenji

    2006-01-01

    The wild type Copia Gag precursor protein of Drosophila melanogaster expressed in Escherichia coli was shown to be processed autocatalytically to generate two daughter proteins with molecular masses of 33 and 23 kDa on SDS/PAGE. The active-site motif of aspartic proteinases, Asp-Ser-Gly, was present in the 23 kDa protein corresponding to the C-terminal half of the precursor protein. The coding region of this daughter protein (152 residues) in the copia gag gene was expressed in E. coli to produce the recombinant enzyme protein as inclusion bodies, which was then purified and refolded to create the active enzyme. Using the peptide substrate His-Gly-Ile-Ala-Phe-Met-Val-Lys-Glu-Val-Asn (cleavage site: Phe–Met) designed on the basis of the sequence of the cleavage-site region of the precursor protein, the enzymatic properties of the proteinase were investigated. The optimum pH and temperature of the proteinase toward the synthetic peptide were 4.0 and 70 °C respectively. The proteolytic activity was increased with increasing NaCl concentration in the reaction mixture, the optimum concentration being 2 M. Pepstatin A strongly inhibited the enzyme, with a Ki value of 15 nM at pH 4.0. On the other hand, the active-site residue mutant, in which the putative catalytic aspartic acid residue was mutated to an alanine residue, had no activity. These results show that the Copia proteinase belongs to the family of aspartic proteinases including HIV proteinase. The B-chain of oxidized bovine insulin was hydrolysed at the Leu15−–Tyr16 bond fairly selectively. Thus the recombinant Copia proteinase partially resembles HIV proteinase, but is significantly different from it in certain aspects. PMID:16813567

  5. Human immunodeficiency virus type 1 proteinase is rapidly and efficiently inactivated in human plasma by alpha 2-macroglobulin.

    PubMed

    Kisselev, A F; von der Helm, K

    1994-10-01

    Human plasma impairs the activity of the human immunodeficiency virus (HIV-1) proteinase to cleave the HIV-1 gag-polyprotein precursor. The inhibition is due to the entrapment of the proteinase by plasma alpha 2-macroglobulin (alpha 2M). In methylamine-treated plasma, where alpha 2M is inactivated, HIV proteinase is not blocked. The interaction of alpha 2M and HIV-1 proteinase resulting in covalent complexes of proteinase and alpha 2M was demonstrated by immunoblotting with antiserum either to alpha 2M or to the HIV proteinase. We suggest if HIV-1 proteinase would be released in vivo from infected patients' cells, alpha 2M entrapment may prevent or minimize a conceivable cleavage of extracellular matrix or plasma proteins by the HIV-1 enzyme.

  6. Sensitive method to identify and characterize proteinases in situ after SDS-PAGE.

    PubMed

    Williams, J; McGrath, W J; Mangel, W F

    2000-11-01

    Cells and body fluids contain numerous, different proteinases; to identify and characterize them are both important and difficult tasks. Especially difficult to identify and characterize are highly specific proteinases. Here, we present an extremely sensitive and quantitative method to characterize proteinases fractionated by SDS-PAGE that cleave specific rhodamine-based fluorogenic substrates. To test the sensitivity of the technique, we used trypsin as our model system. Filter paper impregnated with rhodamine-based fluorogenic substrates was placed on a gel, and bands of fluorescence originating from specific proteinases were visualized in real time. The method is very sensitive; picogram amounts of trypsin can be detected. The method should be very general, in that even proteinases whose substrates require amino acids C-terminal to the cleavage site may be identified and characterized. The results allow one to obtain not only information on the substrate specificity of a specific enzyme but also information about its molecular weight.

  7. Co-factor activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying the peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  8. Co-factor activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  9. Class specific inhibition of house dust mite proteinases which cleave cell adhesion, induce cell death and which increase the permeability of lung epithelium

    PubMed Central

    Winton, Helen L; Wan, Hong; Cannell, Mark B; Thompson, Philip J; Garrod, David R; Stewart, Geoffrey A; Robinson, Clive

    1998-01-01

    House dust mite (HDM) allergens with cysteine and serine proteinase activity are risk factors for allergic sensitization and asthma. A simple method to fractionate proteinase activity from HDM faecal pellets into cysteine and serine class activity is described. Both proteinase fractions increased the permeability of epithelial cell monolayers. The effects of the serine proteinase fraction were inhibited by 4-(2-aminoethyl)-benzenesulphonyl fluoride hydrochloride (AEBSF) and soybean trypsin inhibitor (SBTI). The effects of the cysteine proteinase fraction could be inhibited by E-64. No reciprocity of action was found. Treatment of epithelial monolayers with either proteinase fraction caused breakdown of tight junctions (TJs). AEBSF inhibited TJ breakdown caused by the serine proteinase fraction, whereas E-64 inhibited the cysteine proteinase fraction. Agarose gel electrophoresis revealed that the proteinases induced DNA cleavage which was inhibited by the matrix metalloproteinase inhibitor BB-250. Compound E-64 inhibited DNA fragmentation caused by the cysteine proteinase fraction, but was without effect on the serine proteinase fraction. Staining of proteinase-treated cells with annexin V (AV) and propidium iodide (PI) revealed a diversity of cellular responses. Some cells stained only with AV indicating early apoptosis, whilst others were dead and stained with both AV and PI. HDM proteinases exert profound effects on epithelial cells which will promote allergic sensitization; namely disruption of intercellular adhesion, increased paracellular permeability and initiation of cell death. Attenuation of these actions by proteinase inhibitors leads to the conclusion that compounds designed to be selective for the HDM enzymes may represent a novel therapy for asthma. PMID:9720772

  10. Identification of a novel proteinase (ameloprotease-I) responsible for the complete degradation of amelogenin during enamel maturation.

    PubMed Central

    Moradian-Oldak, J; Leung, W; Simmer, J P; Zeichner-David, M; Fincham, A G

    1996-01-01

    During enamel formation the proteins of the extracellular matrix, particularly amelogenins, are removed prior to maturation. In order to investigate this process and to improve our understanding of the function of proteinases during enamel maturation, proteinase fractions were isolated from developing pig enamel and assayed for proteolytic activity in vitro. A recombinant murine amelogenin, M179, was used as a substrate. Two major groups of enamel proteinases were defined as high-molecular-mass ['high-molecular-weight' in Moradian-Oldak, Simmer, Sarte, Zeichner-David and Fincham (1994) Arch. Oral Biol.39, 647-656] and low-molecular-mass proteinases. Here we report the characterization of one of the proteinases present in the low-molecular-mass group. We demonstrate that this proteinase is a serine proteinase capable of degradation of M179 following cleavage of the tyrosine-rich amelogenin polypeptide from the N-terminal region. A partial N-terminal sequence of the proteinase was obtained (LPHVPHRIPPGYGRPXTXNEEGXNPYFXFFXXHG). An anti-peptide antibody directed against a synthetic peptide corresponding to the first 14 amino acids of the above sequence was produced. The presence of the proteinase in the acetic acid extract was confirmed by Western blotting. Searching using the amino acid sequence determined in this study showed it to be also present in the 32 kDa and 89 kDa enamelin proteins reported by Fukae, Tanabe, Murakami and Tohi [(1996) Adv. Dent. Res., in the press]. We therefore identify the 32 kDa enamelin as an enamel proteinase ('ameloprotease-I') which is responsible for amelogenin degradation in maturing enamel. We propose that the 89 kDa enamelin is a precursor of ameloprotease-I, the first enamel protein for which a function has been defined. PMID:8836151

  11. Characterization of the Proteinase that Initiates the Degradation of the Trypsin Inhibitor in Germinating Mung Beans (Vigna radiata).

    PubMed

    Wilson, K A; Tan-Wilson, A L

    1987-05-01

    The proteinase (proteinase F) responsible for the initial proteolysis of the mung bean (Vigna radiata) trypsin inhibitor (MBTI) during germination has been purified 1400-fold from dry beans. The enzyme acts as an endopeptidase, cleaving the native inhibitor, MBTI-F, to produce the first modified inhibitor form, MBTI-E. The cleavage of the Asp76-Lys77 peptide bond of MBTI-F occurs at a pH optimum of 4.5, with the tetrapeptide Lys-Asp-Asp-Asp being released. Proteinase F exhibited no activity against the modified inhibitor forms MBTI-E and MBTI-C. Vicilin, the major storage protein of the mung bean, does not serve as a substrate for proteinase F between pH 4 and 7. Proteinase F is inhibited by phenylmethylsulfonyl fluoride, chymostatin, p-hydroxymercuribenzoate, and p-chlorophenylsulfonate, but not by iodoacetate and CuCl(2). It is not activated by dithiothreitol, and is stable for extended periods of time (10 months, 4 degrees C, pH 4.0) in the absence of reducing agents. An apparent molecular weight of 65,000 was found for proteinase F by gel filtration. Subcellular fractionation in glycerol suggests that greater than 85% of the proteinase F activity is found in the protein bodies of the ungerminated mung bean. The same studies indicate that at least 56% of the MBTI of the seed is also localized in the protein bodies.

  12. A pepstatin-insensitive aspartic proteinase from a thermophilic Bacillus sp.

    PubMed Central

    Toogood, H S; Prescott, M; Daniel, R M

    1995-01-01

    Bacillus sp. strain Wp22.A1 produced a cell-associated aspartic proteinase which was purified to homogeneity using phenyl-Sepharose (hydrophobic and affinity chromatography) and Mono Q. The proteinase has a molecular mass of 45 kDa by SDS/PAGE and a pI of 3.8. It is insensitive to pepstatin, but is sensitive to the other aspartic proteinase-specific inhibitors diazoacetyl-DL-norleucine methyl ester (DAN) and 1,2-epoxy-3-(p-nitrophenoxy)propane. Inactivation by DAN was only partial, suggesting that it had non-specifically modified an aspartate residue at a site other than the active site. The enzyme was not inhibited by any of the serine or cysteine proteinase inhibitors tested. Maximum proteolytic activity was observed at pH 3.5. The proteinase had a higher activity with haemoglobin, but was more specific (Vmax./Km) for cytochrome c. Substrate inhibition was observed with both these substrates. The cleavage of oxidized insulin B chain tended to occur at sites where the P1 amino acid was bulky and non-polar, and the P1' amino acid was bulky and polar, such as its primary cleavage site of Val2-Asn3. The proteinase was stable in the pH range 2.5-5.5. Thermostability was increased in the presence of Ca2+, although to a lesser extent at higher temperatures. The thermostabilities at 60, 70, 80 and 90 degrees C were 45 h, 102, 21 and 3 min respectively in the presence of Ca2+. Images Figure 1 PMID:7741709

  13. Poliovirus polypeptide precursors: expression in vitro and processing by exogenous 3C and 2A proteinases.

    PubMed Central

    Nicklin, M J; Kräusslich, H G; Toyoda, H; Dunn, J J; Wimmer, E

    1987-01-01

    Plasmids have been constructed to generate substrates for the study of proteinases 2A and 3C of poliovirus. They contain the P1 (capsomer precursor) region of the poliovirus genome or P1 and part of P2 (a nonstructural precursor), which can be transcribed and translated in vitro. A transcript containing the entire 5' nontranslated region and the P1 region of the viral RNA gave poor translation in a reticulocyte translation system. Truncation of the 5' nontranslated region to its 3'-most segment gave acceptably good yields of radiolabeled P1. P1 was specifically processed to yield capsomer proteins by enzymes supplied in a postmitochondrial supernatant from poliovirus-infected cells. Thus, proteinase 3C can be supplied exogenously (in trans) and effect processing. This system may be used to provide P1 for the assay of proteinase 3C. Precursors that lacked either the 1A or 1D regions were poor substrates for proteinase 3C--observations that demonstrated a stringent structural requirement in processing by 3C. The translation product of a transcript encoding P1 and part of P2 was rapidly cleaved at the P1-P2 site in the absence of infected-cell extract. A transcript that contained a mutated 2A region gave a stable P1-P2 precursor that could be processed specifically by exogenous proteinase from infected-cell fractions. Processing of P1 appeared to require cleavage of the P1-P2 bond. These results support our previous data that 2A is the second polioviral proteinase and also provides a means of assaying proteinase 2A in vitro. Images PMID:3035560

  14. Simple Bond Cleavage

    SciTech Connect

    Gary S. Groenewold

    2005-08-01

    Simple bond cleavage is a class of fragmentation reactions in which a single bond is broken, without formation of new bonds between previously unconnected atoms. Because no bond making is involved, simple bond cleavages are endothermic, and activation energies are generally higher than for rearrangement eliminations. The rate of simple bond cleavage reactions is a strong function of the internal energy of the molecular ion, which reflects a loose transition state that resembles reaction products, and has a high density of accessible states. For this reason, simple bond cleavages tend to dominate fragmentation reactions for highly energized molecular ions. Simple bond cleavages have negligible reverse activation energy, and hence they are used as valuable probes of ion thermochemistry, since the energy dependence of the reactions can be related to the bond energy. In organic mass spectrometry, simple bond cleavages of odd electron ions can be either homolytic or heterolytic, depending on whether the fragmentation is driven by the radical site or the charge site. Simple bond cleavages of even electron ions tend to be heterolytic, producing even electron product ions and neutrals.

  15. Cleavage of nucleic acids

    DOEpatents

    Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow; Mary Ann D.; Dahlberg, James E.

    2010-11-09

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

  16. Cleavage of nucleic acids

    DOEpatents

    Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor L.; Brow, Mary Ann D.; Dahlberg, James E.

    2007-12-11

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

  17. The cysteine proteinases of the pineapple plant.

    PubMed

    Rowan, A D; Buttle, D J; Barrett, A J

    1990-03-15

    The pineapple plant (Ananas comosus) was shown to contain at least four distinct cysteine proteinases, which were purified by a procedure involving active-site-directed affinity chromatography. The major proteinase present in extracts of plant stem was stem bromelain, whilst fruit bromelain was the major proteinase in the fruit. Two additional cysteine proteinases were detected only in the stem: these were ananain and a previously undescribed enzyme that we have called comosain. Stem bromelain, fruit bromelain and ananain were shown to be immunologically distinct. Enzymic characterization revealed differences in both substrate-specificities and inhibition profiles. A study of the cysteine proteinase derived from the related bromeliad Bromelia pinguin (pinguinain) indicated that in many respects it was similar to fruit bromelain, although it was found to be immunologically distinct.

  18. The cysteine proteinases of the pineapple plant.

    PubMed Central

    Rowan, A D; Buttle, D J; Barrett, A J

    1990-01-01

    The pineapple plant (Ananas comosus) was shown to contain at least four distinct cysteine proteinases, which were purified by a procedure involving active-site-directed affinity chromatography. The major proteinase present in extracts of plant stem was stem bromelain, whilst fruit bromelain was the major proteinase in the fruit. Two additional cysteine proteinases were detected only in the stem: these were ananain and a previously undescribed enzyme that we have called comosain. Stem bromelain, fruit bromelain and ananain were shown to be immunologically distinct. Enzymic characterization revealed differences in both substrate-specificities and inhibition profiles. A study of the cysteine proteinase derived from the related bromeliad Bromelia pinguin (pinguinain) indicated that in many respects it was similar to fruit bromelain, although it was found to be immunologically distinct. Images Fig. 4. Fig. 5. PMID:2327970

  19. Activity dependent CAM cleavage and neurotransmission

    PubMed Central

    Conant, Katherine; Allen, Megan; Lim, Seung T.

    2015-01-01

    Spatially localized proteolysis represents an elegant means by which neuronal activity dependent changes in synaptic structure, and thus experience dependent learning and memory, can be achieved. In vitro and in vivo studies suggest that matrix metalloproteinase and adamalysin activity is concentrated at the cell surface, and emerging evidence suggests that increased peri-synaptic expression, release and/or activation of these proteinases occurs with enhanced excitatory neurotransmission. Synaptically expressed cell adhesion molecules (CAMs) could therefore represent important targets for neuronal activity-dependent proteolysis. Several CAM subtypes are expressed at the synapse, and their cleavage can influence the efficacy of synaptic transmission through a variety of non-mutually exclusive mechanisms. In the following review, we discuss mechanisms that regulate neuronal activity-dependent synaptic CAM shedding, including those that may be calcium dependent. We also highlight CAM targets of activity-dependent proteolysis including neuroligin and intercellular adhesion molecule-5 (ICAM-5). We include discussion focused on potential consequences of synaptic CAM shedding, with an emphasis on interactions between soluble CAM cleavage products and specific pre- and post-synaptic receptors. PMID:26321910

  20. Inactivation of key factors of the plasma proteinase cascade systems by Bacteroides gingivalis.

    PubMed Central

    Nilsson, T; Carlsson, J; Sundqvist, G

    1985-01-01

    The effect of Bacteroides gingivalis W83 on various key components of the human plasma proteinase cascade systems was studied. When purified C1-inhibitor was incubated with the bacterium, the inhibitor was rapidly inactivated by limited proteolytic cleavage. In citrated whole plasma, C1-inhibitor, antithrombin, plasminogen, prekallikrein, prothrombinase complex, the clotting factor X, and most of the alpha 2-antiplasmin were functionally eliminated after 30 min of incubation with the bacterium. Fibrinogen disappeared from the plasma almost immediately upon mixing with the bacterial suspension. In contrast, there was no appreciable decrease in the bulk of other plasma proteins, such as various transport proteins (albumin, prealbumin, transferrin) and immunoglobulins, during 4 h of incubation with the bacterium. Most of the observed effects can be assigned to the proteolytic activity of the bacterium itself, since there was little evidence for generation of intrinsic plasma proteinase activity, despite the loss of proteinase inhibitory activities. B. gingivalis W83 thus seems to be equipped with proteolytic enzyme systems which selectively recognize and rapidly inactivate the most important proteinase inhibitors and proenzymes present in human plasma. This bacterium therefore seems to be able to efficiently paralyze the host's various defenses against invading microorganisms. Images PMID:3902645

  1. Evolutionary mechanisms acting on proteinase inhibitor variability.

    PubMed

    Christeller, John T

    2005-11-01

    The interaction of proteinase inhibitors produced, in most cases, by host organisms and the invasive proteinases of pathogens or parasites or the dietary proteinases of predators, results in an evolutionary 'arms race' of rapid and ongoing change in both interacting proteins. The importance of these interactions in pathogenicity and predation is indicated by the high level and diversity of observable evolutionary activity that has been found. At the initial level of evolutionary change, recruitment of other functional protein-folding families has occurred, with the more recent evolution of one class of proteinase inhibitor from another, using the same mechanism and proteinase contact residues. The combination of different inhibitor domains into a single molecule is also observed. The basis from which variation is possible is shown by the high rate of retention of gene duplication events and by the associated process of inhibitory domain multiplication. At this level of reorganization, mutually exclusive splicing is also observed. Finally, the major mechanism by which variation is achieved rapidly is hypervariation of contact residues, an almost ubiquitous feature of proteinase inhibitors. The diversity of evolutionary mechanisms in a single class of proteins is unlikely to be common, because few systems are under similar pressure to create variation. Proteinase inhibitors are therefore a potential model system in which to study basic evolutionary process such as functional diversification.

  2. [Extracellular proteinases of filamentous fungi as potential markers of phytopathogenesis].

    PubMed

    Dunaevskiĭ, Ia E; Gruban', T N; Beliakova, G A; Belozerskiĭ, M A

    2006-01-01

    The presence of proteins in the culture liquid of filamentous fungi under study was found to induce the secretion of proteinases. The inhibitory analysis of the major extracellular proteinases of the saprotrophic fungus Trichoderma harzianum and the phytopathogenic fungus Alternaria alternata showed that they both belong to the group of serine proteinases. The substrate specificity of these proteinases and their sensitivity to inhibitors suggest that the enzyme of T. harzianum is a subtilisin-like proteinase and the enzyme of A. alternata is a trypsin-like proteinase. This difference between the proteinases may reflect the physiological difference between their producers (saprotroph and phytopathogen).

  3. Mutational analysis of human immunodeficiency virus type 1 protease suggests functional homology with aspartic proteinases.

    PubMed Central

    Loeb, D D; Hutchison, C A; Edgell, M H; Farmerie, W G; Swanstrom, R

    1989-01-01

    Processing of the retroviral gag and pol gene products is mediated by a viral protease. Bacterial expression systems have been developed which permit genetic analysis of the human immunodeficiency virus type 1 protease as measured by cleavage of the pol protein precursor. Deletion analysis of the pol reading frame locates the sequences required to encode a protein with appropriate proteolytic activity near the left end of the pol reading frame but largely outside the gag-pol overlap region, which is at the extreme left end of pol. Most missense mutations within an 11-amino-acid domain highly conserved among retroviral proteases and with sequence similarity to the active site of aspartic proteinases abolish appropriate processing, suggesting that the retrovirus proteases share a catalytic mechanism with aspartic proteinases. Substitution of the amino acids flanking the scissile bond at three of the processing sites encoded by pol demonstrates distinct sequence requirements for cleavage at these different sites. The inclusion of a charged amino acid at the processing site blocks cleavage. A subset of these substitutions also inhibits processing at the nonmutated sites. Images PMID:2642305

  4. In vitro and ex vivo inhibition of hepatitis A virus 3C proteinase by a peptidyl monofluoromethyl ketone.

    PubMed

    Morris, T S; Frormann, S; Shechosky, S; Lowe, C; Lall, M S; Gauss-Müller, V; Purcell, R H; Emerson, S U; Vederas, J C; Malcolm, B A

    1997-05-01

    Hepatitis A virus (HAV) 3C proteinase is the enzyme responsible for the processing of the viral polyprotein. Although a cysteine proteinase, it displays an active site configuration like those of the mammalian serine proteinases (Malcolm, B. A. Protein Science 1995, 4, 1439). A peptidyl monofluoromethyl ketone (peptidyl-FMK) based on the preferred peptide substrates for HAV 3C proteinase was generated by first coupling the precursor, N,N-dimethylglutamine fluoromethylalcohol, to the tripeptide, Ac-Leu-Ala-Ala-OH, and then oxidizing the product to the corresponding peptidyl-FMK (Ac-LAAQ'-FMK). This molecule was found to be an irreversible inactivator of HAV 3C with a second-order rate constant of 3.3 x 10(2) M-1 s-1. 19F NMR spectroscopy indicates the displacement of fluoride on inactivation of the enzyme by the fluoromethyl ketone. NMR spectroscopy of the complex between the 13C-labeled inhibitor and the HAV 3C proteinase indicates that an (alkylthio)methyl ketone is formed. Studies of polyprotein processing, using various substrates generated by in vitro transcription/translation, demonstrated efficient blocking of even the most rapid proteolytic events such as cleavage of the 2A-2B and 2C-3A junctions. Subsequent ex vivo studies, to test for antiviral activity, show a 25-fold reduction in progeny virus production as the result of treatment with 5 microM inhibitor 24 h post-infection.

  5. Extracellular alkaline proteinase of Colletotrichum gloeosporioides.

    PubMed

    Dunaevsky, Ya E; Matveeva, A R; Beliakova, G A; Domash, V I; Belozersky, M A

    2007-03-01

    The main proteinase of the filamentous fungus Colletotrichum gloeosporioides causing anthracnoses and serious problems for production and storage of agricultural products has molecular mass of 57 kD and was purified more than 200-fold to homogeneity with the yield of 5%. Maximal activity of the proteinase is at pH 9.0-10.0, and the enzyme is stable at pH 6.0-11.5 (residual activity not less than 70%). The studied enzyme completely kept its activity to 55 degrees C, with a temperature optimum of 45 degrees C. The purified C. gloeosporioides proteinase is stable at alkaline pH values, but rapidly loses its activity at pH values lower than 5.0. Addition of bovine serum albumin stabilizes the enzyme under acidic conditions. Data on inhibitor analysis and substrate specificity of the enzyme allow its classification as a serine proteinase of subtilisin family. It is demonstrated that the extracellular proteinase of C. gloeosporioides specifically effects plant cell wall proteins. It is proposed that the studied proteinase--via hydrolysis of cell wall--provides for penetration of the fungus into the tissues of the host plant.

  6. Solution structure of the squash aspartic acid proteinase inhibitor (SQAPI) and mutational analysis of pepsin inhibition.

    PubMed

    Headey, Stephen J; Macaskill, Ursula K; Wright, Michele A; Claridge, Jolyon K; Edwards, Patrick J B; Farley, Peter C; Christeller, John T; Laing, William A; Pascal, Steven M

    2010-08-27

    The squash aspartic acid proteinase inhibitor (SQAPI), a proteinaceous proteinase inhibitor from squash, is an effective inhibitor of a range of aspartic proteinases. Proteinaceous aspartic proteinase inhibitors are rare in nature. The only other example in plants probably evolved from a precursor serine proteinase inhibitor. Earlier work based on sequence homology modeling suggested SQAPI evolved from an ancestral cystatin. In this work, we determined the solution structure of SQAPI using NMR and show that SQAPI shares the same fold as a plant cystatin. The structure is characterized by a four-strand anti-parallel beta-sheet gripping an alpha-helix in an analogous manner to fingers of a hand gripping a tennis racquet. Truncation and site-specific mutagenesis revealed that the unstructured N terminus and the loop connecting beta-strands 1 and 2 are important for pepsin inhibition, but the loop connecting strands 3 and 4 is not. Using ambiguous restraints based on the mutagenesis results, SQAPI was then docked computationally to pepsin. The resulting model places the N-terminal strand of SQAPI in the S' side of the substrate binding cleft, whereas the first SQAPI loop binds on the S side of the cleft. The backbone of SQAPI does not interact with the pepsin catalytic Asp(32)-Asp(215) diad, thus avoiding cleavage. The data show that SQAPI does share homologous structural elements with cystatin and appears to retain a similar protease inhibitory mechanism despite its different target. This strongly supports our hypothesis that SQAPI evolved from an ancestral cystatin.

  7. Solution Structure of the Squash Aspartic Acid Proteinase Inhibitor (SQAPI) and Mutational Analysis of Pepsin Inhibition

    PubMed Central

    Headey, Stephen J.; MacAskill, Ursula K.; Wright, Michele A.; Claridge, Jolyon K.; Edwards, Patrick J. B.; Farley, Peter C.; Christeller, John T.; Laing, William A.; Pascal, Steven M.

    2010-01-01

    The squash aspartic acid proteinase inhibitor (SQAPI), a proteinaceous proteinase inhibitor from squash, is an effective inhibitor of a range of aspartic proteinases. Proteinaceous aspartic proteinase inhibitors are rare in nature. The only other example in plants probably evolved from a precursor serine proteinase inhibitor. Earlier work based on sequence homology modeling suggested SQAPI evolved from an ancestral cystatin. In this work, we determined the solution structure of SQAPI using NMR and show that SQAPI shares the same fold as a plant cystatin. The structure is characterized by a four-strand anti-parallel β-sheet gripping an α-helix in an analogous manner to fingers of a hand gripping a tennis racquet. Truncation and site-specific mutagenesis revealed that the unstructured N terminus and the loop connecting β-strands 1 and 2 are important for pepsin inhibition, but the loop connecting strands 3 and 4 is not. Using ambiguous restraints based on the mutagenesis results, SQAPI was then docked computationally to pepsin. The resulting model places the N-terminal strand of SQAPI in the S′ side of the substrate binding cleft, whereas the first SQAPI loop binds on the S side of the cleft. The backbone of SQAPI does not interact with the pepsin catalytic Asp32–Asp215 diad, thus avoiding cleavage. The data show that SQAPI does share homologous structural elements with cystatin and appears to retain a similar protease inhibitory mechanism despite its different target. This strongly supports our hypothesis that SQAPI evolved from an ancestral cystatin. PMID:20538608

  8. Purification, cDNA cloning and characterization of proteinase B, an asparagine-specific endopeptidase from germinating vetch (Vicia sativa L.) seeds.

    PubMed

    Becker, C; Shutov, A D; Nong, V H; Senyuk, V I; Jung, R; Horstmann, C; Fischer, J; Nielsen, N C; Müntz, K

    1995-03-01

    Proteinase B, an asparagine-specific endopeptidase, has been purified from germinating vetch (Vicia sativa L.) seeds. The final preparation consists of two enzymically active proteins with molecular masses of approximately 39 kDa and 37 kDa. Synthetic substrates were used to confirm cleavage specificity of the proteinase B preparation. As expected, the enzyme cleaves the substrates at the C-terminal side of Asn residues. The octapeptide ETRNGVEE was digested most efficiently. When Gly was replaced by Ile or Glu, cleavage took place with lower efficiency. Polyclonal antibodies displayed both proteins in cotyledon extracts of germinated vetch seeds. In addition, a strong cross-reacting protein band was found in cotyledon extracts of developing seeds, indicating the presence of a very similar enzyme during seed development. cDNA clones encoding proteinase B precursor have been obtained on the basis of the N-terminal amino acid sequence DDDFEGTRWAILLAGS, by means of the polymerase chain reaction. The cDNA clones contain an open reading frame of 1479 bp encoding a polypeptide of 493 amino acids. The precursor displayed 59% sequence identity to the cDNA-derived amino acid sequence of a vacuolar Asn-specific enzyme from the developing castor beam endosperm which is thought to catalyze the post-translational processing of pro-proteins into the mature forms. Proteinase B is synthesized de novo during seed germination. The results of Southern-blot analyses suggested that there are at least two genes for proteinase B.

  9. The picornaviral 3C proteinases: cysteine nucleophiles in serine proteinase folds.

    PubMed

    Malcolm, B A

    1995-08-01

    The 3C proteinases are a novel group of cysteine proteinases with a serine proteinase-like fold that are responsible for the bulk of polyprotein processing in the Picornaviridae. Because members of this viral family are to blame for several ongoing global pandemic problems (rhinovirus, hepatitis A virus) as well as sporadic outbreaks of more serious pathologies (poliovirus), there has been continuing interest over the last two decades in the development of antiviral therapies. The recent determination of the structure of two of the 3C proteinases by X-ray crystallography opens the door for the application of the latest advances in computer-assisted identification and design of anti-proteinase therapeutic/chemoprophylactic agents.

  10. Roles of the Picornaviral 3C Proteinase in the Viral Life Cycle and Host Cells

    PubMed Central

    Sun, Di; Chen, Shun; Cheng, Anchun; Wang, Mingshu

    2016-01-01

    The Picornaviridae family comprises a large group of non-enveloped viruses that have a major impact on human and veterinary health. The viral genome contains one open reading frame encoding a single polyprotein that can be processed by viral proteinases. The crucial 3C proteinases (3Cpros) of picornaviruses share similar spatial structures and it is becoming apparent that 3Cpro plays a significant role in the viral life cycle and virus host interaction. Importantly, the proteinase and RNA-binding activity of 3Cpro are involved in viral polyprotein processing and the initiation of viral RNA synthesis. In addition, 3Cpro can induce the cleavage of certain cellular factors required for transcription, translation and nucleocytoplasmic trafficking to modulate cell physiology for viral replication. Due to interactions between 3Cpro and these essential factors, 3Cpro is also involved in viral pathogenesis to support efficient infection. Furthermore, based on the structural conservation, the development of irreversible inhibitors and discovery of non-covalent inhibitors for 3Cpro are ongoing and a better understanding of the roles played by 3Cpro may provide insights into the development of potential antiviral treatments. In this review, the current knowledge regarding the structural features, multiple functions in the viral life cycle, pathogen host interaction, and development of antiviral compounds for 3Cpro is summarized. PMID:26999188

  11. Roles of the Picornaviral 3C Proteinase in the Viral Life Cycle and Host Cells.

    PubMed

    Sun, Di; Chen, Shun; Cheng, Anchun; Wang, Mingshu

    2016-03-17

    The Picornaviridae family comprises a large group of non-enveloped viruses that have a major impact on human and veterinary health. The viral genome contains one open reading frame encoding a single polyprotein that can be processed by viral proteinases. The crucial 3C proteinases (3C(pro)s) of picornaviruses share similar spatial structures and it is becoming apparent that 3C(pro) plays a significant role in the viral life cycle and virus host interaction. Importantly, the proteinase and RNA-binding activity of 3C(pro) are involved in viral polyprotein processing and the initiation of viral RNA synthesis. In addition, 3C(pro) can induce the cleavage of certain cellular factors required for transcription, translation and nucleocytoplasmic trafficking to modulate cell physiology for viral replication. Due to interactions between 3C(pro) and these essential factors, 3C(pro) is also involved in viral pathogenesis to support efficient infection. Furthermore, based on the structural conservation, the development of irreversible inhibitors and discovery of non-covalent inhibitors for 3C(pro) are ongoing and a better understanding of the roles played by 3C(pro) may provide insights into the development of potential antiviral treatments. In this review, the current knowledge regarding the structural features, multiple functions in the viral life cycle, pathogen host interaction, and development of antiviral compounds for 3C(pro) is summarized.

  12. Inhibition of serine proteinases from human blood clotting system by squash inhibitor mutants.

    PubMed

    Grzesiak, A; Buczek, O; Petry, I; Szewczuk, Z; Otlewski, J

    2000-05-23

    A series of six CMTI I variants mutated in the P(2)-P(4)' region of the canonical binding loop were used to probe the role of single amino acid substitutions on binding to the following human proteinases involved in blood clotting: plasmin, plasma kallikrein, factors X(a) and XII(a). The mutants were expressed as fusion proteins with the LE1413 hydrophobic polypeptide in Escherichia coli, purified from inclusion bodies, followed by cyanobromide cleavage and refolding. The mutants inhibited the proteinases with the association constants in the range 10(3)-10(9) M(-1). Inhibition of plasma kallikrein and factors X(a) and XII(a) could be improved up to 30-fold by single mutations. In contrast, neither of the introduced mutations increased inhibitory properties of CMTI I against plasmin. Additionally, using two inhibitors of natural origin, CMTI I (P(1) Arg) and CPTI II (P(1) Lys), we determined the effect of Lys-->Arg on binding to four proteinases. With the exception of plasmin (no effect), P(1) Arg resulted in up to 30-fold stronger binding than P(1) Lys.

  13. Molecular dynamic and docking interaction study of Heterodera glycines serine proteinase with Vigna mungo proteinase inhibitor.

    PubMed

    Prasad, C V S Siva; Gupta, Saurabh; Gaponenko, Alex; Tiwari, Murlidhar

    2013-08-01

    Many plants do produce various defense proteins like proteinase inhibitors (PIs) to protect them against various pests. PIs function as pseudosubstrates of digestive proteinase, which inhibits proteolysis in pests and leads to amino acid deficiency-based mortality. This work reports the structural interaction studies of serine proteinase of Heterodera glycines (SPHG) with Vigna mungo proteinase inhibitor (VMPI). 3D protein structure modeling, validation of SPHG and VMPI, and their putative protein-protein binding sites were predicted. Protein-protein docking followed by molecular dynamic simulation was performed to find the reliable confirmation of SPHG-VMPI complex. Trajectory analysis of each successive conformation concludes better interaction of first loop in comparison with second loop. Lysine residues of first loop were actively participating in complex formation. Overall, this study discloses the structural aspects and interaction mechanisms of VMPI with SPHG, and it would be helpful in the development of pest-resistant genetically modified crops.

  14. Novel proteinase inhibitor promotes resistance to insects

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A novel Beta vulgaris serine proteinase inhibitor gene (BvSTI) and its protein are identified in response to insect feeding on B. vulgaris seedlings. BvSTI is cloned into an expression vector with constitutive promoter and transformed into Nicotiana benthamiana plants to assess BvSTI’s ability to ...

  15. Proteinase activity in human and murine saliva as a biomarker for proteinase inhibitor efficacy.

    PubMed

    Fingleton, Barbara; Menon, Ramkumar; Carter, Kathy J; Overstreet, P Dawn; Hachey, David L; Matrisian, Lynn M; McIntyre, J Oliver

    2004-12-01

    As molecularly targeted agents reach the clinic, there is a need for assays to detect their presence and effectiveness against target molecules in vivo. Proteinase inhibitors are one example of a class of therapeutic agent for which satisfactory methods of identifying successful target modulation in vivo are lacking. This is of particular importance while these drugs are in clinical trials because standard maximum-tolerated dose-finding studies often are not suitable due to lack of toxicity. Saliva represents a readily accessible bodily fluid that can be repeatedly sampled and used for assaying in vivo effects of systemic drugs. Here we show the development of a simple assay that can be used to measure proteinase activity in saliva and proteinase inhibition after systemic treatment with three different proteinase inhibitors. A variety of gelatinolytic activities present in human and murine saliva have been assayed with a fluorescent dye-labeled substrate and assigned to different proteinase categories by inclusion of specific classes of inhibitors. Treatment of mice with either matrix metalloproteinase inhibitors or a urokinase inhibitor for a period as short as 48 hours results in levels of the drugs that can be detected in saliva by mass spectrometry and concomitant decreases in salivary proteinase activity, thus demonstrating that these inhibitors successfully modulate their targets in vivo.

  16. Inhibition of a Plasmodium vinckei cysteine proteinase cures murine malaria.

    PubMed Central

    Rosenthal, P J; Lee, G K; Smith, R E

    1993-01-01

    Intraerythrocytic malaria parasites degrade hemoglobin as a principal source of amino acids for parasite protein synthesis. We have previously identified a Plasmodium falciparum trophozoite cysteine proteinase as a putative hemoglobinase and shown that specific inhibitors of this proteinase block the hydrolysis of globin and the development of cultured parasites. We now show that the murine malaria parasite Plasmodium vinckei has an analogous cysteine proteinase with similar biochemical properties to the P. falciparum proteinase, including an acid pH optimum, a preference for the peptide proteolytic substrate benzyloxycarbonyl (Z)-Phe-Arg-7-amino-4-methylcoumarin, and nonomolar inhibition by seven peptide fluoromethyl ketone proteinase inhibitors. Thus, P. vinckei offers a model system for the in vivo testing of the antimalarial properties of cysteine proteinase inhibitors. One of the proteinase inhibitors studied, morpholine urea (Mu)-Phe-Homophenylalanine (HPhe)-CH2F strongly inhibited the P. vinckei cysteine proteinase in vitro and rapidly blocked parasite cysteine proteinase activity in vivo. When administered four times a day for 4 d to P. vinckei-infected mice, Mu-Phe-HPhe-CH2F elicited long-term cures in 80% of the treated animals. These results show that peptide proteinase inhibitors can be effective antimalarial compounds in vivo and suggest that the P. falciparum cysteine proteinase is a promising target for chemotherapy. Images PMID:8450035

  17. Silk gland-specific proteinase inhibitor serpin16 from the Bombyx mori shows cysteine proteinase inhibitory activity.

    PubMed

    Guo, Peng-Chao; Dong, Zhaoming; Xiao, Li; Li, Tao; Zhang, Yan; He, Huawei; Xia, Qingyou; Zhao, Ping

    2015-01-30

    Serpins (serine proteinase inhibitors) are widely distributed in different species and are well known for their inhibitory activities towards serine proteinases. Here, we report the functional characterization of Bombyx mori serpin16. Expression analysis showed that serpin16 was specifically expressed at high levels in the silk gland at both the transcriptional and translational levels. Moreover, homology modeling and multi-sequence alignment suggested that serpin16 had a canonical serpin fold, but it contained a unique reactive center loop, which was obviously shorter than that of typical serpins. Inhibitory activity analyses revealed that the target proteinase of serpin18 is a cysteine proteinase, rather than a serine proteinase. Furthermore, a Michaelis complex model of serpin16 with its target proteinase was constructed to explain the structural basis of how serpin16 recognizes the cysteine proteinase and its target specificity.

  18. Matrix metalloproteinases - From the cleavage data to the prediction tools and beyond.

    PubMed

    Cieplak, Piotr; Strongin, Alex Y

    2017-03-24

    Understanding the physiological role of any protease requires identification of both its cleavage substrates and their relative cleavage efficacy as compared with other substrates and other proteinases. Our review manuscript is focused on the cleavage preferences of the individual matrix metalloproteinases (MMPs) and the cleavage similarity and distinction that exist in the human MMP family. The recent in-depth analysis of MMPs by us and many others greatly increased knowledge of the MMP biology and structural-functional relationships among this protease family members. A better knowledge of cleavage preferences of MMPs has led us to the development of the prediction tools that are now capable of the high throughput reliable prediction and ranking the MMP cleavage sites in the peptide sequences in silico. Our software unifies and consolidates volumes of the pre-existing data. Now this prediction-ranking in silico tool is ready to be used by others. The software we developed may facilitate both the identification of the novel proteolytic regulatory pathways and the discovery of the previously uncharacterized substrates of the individual MMPs. Because now the MMP research may be based on the mathematical probability parameters rather than on either random luck or common sense alone, the researchers armed with this novel in silico tool will be better equipped to fine-tune or, at least, to sharply focus their wet chemistry experiments. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman.

  19. Inhibition of the 20S proteosome by a protein proteinase inhibitor: evidence that a natural serine proteinase inhibitor can inhibit a threonine proteinase.

    PubMed

    Yabe, Kimihiko; Koide, Takehiko

    2009-02-01

    The 20S proteasome (20S) is an intracellular threonine proteinase (Mr 750,000) that plays important roles in many cellular regulations. Several synthetic peptide inhibitors and bacteria-derived inhibitors such as lactacystin and epoxomicin have been identified as potent proteasome inhibitors. However, essentially no protein proteinase inhibitor has been characterized. By examining several small size protein proteinase inhibitors, we found that a well-known serine proteinase inhibitor from bovine pancreas, basic pancreatic trypsin inhibitor (BPTI), inhibits the 20S in vitro and ex vivo. Inhibition of the 20S by BPTI was time- and concentration-dependent, and stoichiometric. To inhibit the 20S activity, BPTI needs to enter into the interior of the 20S molecule. The molar ratio of BPTI to the 20S in the complex was estimated as approximately six BPTI to one 20S, thereby two sets of three peptidase activities (trypsin-like, chymotrypsin-like and caspase-like) of the 20S were all inhibited. These results indicate that an entrance hole to the 20S formed by seven alpha-subunits is sufficiently large for BPTI to enter. This report is essentially the initial description of the inhibition of a threonine proteinase by a protein serine proteinase inhibitor, suggesting a common mechanism of inhibition between serine and threonine proteinases by a natural protein proteinase inhibitor.

  20. Predicting proteinase specificities from free energy calculations.

    PubMed

    Mekonnen, Seble Merid; Olufsen, Magne; Smalås, Arne O; Brandsdal, Bjørn O

    2006-10-01

    The role of the primary binding residue (P1) in complexes between three different subtilases (subtilisin Carlsberg, thermitase and proteinase K) and their canonical protein inhibitor eglin c have been studied by free energy calculations. Based on the crystal structures of eglin c in complex with subtilisin Carlsberg and thermitase, and a homology model of the eglin c-proteinase K complex, a total of 57 mutants have been constructed and docked into their host proteins. The binding free energy was then calculated using molecular dynamics (MD) simulations combined with the linear interaction energy (LIE) method for all complexes differing only in the nature of the amino acid at the P1 position. LIE calculations for 19 different complexes for each subtilase were thus carried out excluding proline. The effects of substitutions at the P1 position on the binding free energies are found to be very large, and positively charged residues (Arg, Lys and His) are particularly deleterious for all three enzymes. The charged variants of the acidic side chains are found to bind more favorably as compared to their protonated states in all three subtilases. Furthermore, hydrophobic amino acids are accommodated most favorably at the S1-site in all three enzymes. Comparison of the three series of binding free energies shows only minor differences in the 19 computed relative binding free energies among these subtilases. This is further reflected in the correlation coefficient between the 23 relative binding free energies obtained, including the possible protonation states of ionizable side chains, but excluding the P1 Pro, for subtilisin Carlsberg versus thermitase (0.95), subtilisin versus proteinase K (0.94) and thermitase versus proteinase K (0.96).

  1. Specificity of a wheat gluten aspartic proteinase.

    PubMed

    Bleukx, W; Brijs, K; Torrekens, S; Van Leuven, F; Delcour, J A

    1998-09-08

    The substrate and peptide bond specificities of a purified wheat gluten aspartic proteinase (GlAP) are studied. GlAP shows maximum gluten hydrolysing activity at pH 3.0. At this pH, especially the wheat high molecular weight glutenin subunits (HMW-GS) and to a lesser extent the low molecular weight glutenin subunits and gliadins are hydrolysed. GlAP has no obvious effect on albumins and globulins. In its action on oxidised insulin B-chain, GlAP forms eight peptides and has high specificity for peptide bonds located between amino acid residues with large hydrophobic side chains (Leu, Phe, Tyr) but the peptide bond Glu13-Ala14 is also hydrolysed. Although structurally quite similar to a barley aspartic proteinase, the peptide bond specificity of GlAP towards oxidised insulin B-chain resembles slightly more that of a cardoon aspartic proteinase, cardosin B. HMW-GS 7, purified from cultivar Galahad-77, is rapidly hydrolysed by GlAP. N-Terminal amino acid sequence data show that GlAP cleaves at least one Met-Ile peptide bond at the end of the N-terminal domain and two Val-Leu peptide bonds in the repetitive domain of HMW-GS 7.

  2. A Compact Viral Processing Proteinase/Ubiquitin Hydrolase from the OTU Family

    PubMed Central

    Chenon, Mélanie; Andreani, Jessica; Guerois, Raphaël; Jupin, Isabelle; Bressanelli, Stéphane

    2013-01-01

    Turnip yellow mosaic virus (TYMV) - a member of the alphavirus-like supergroup of viruses - serves as a model system for positive-stranded RNA virus membrane-bound replication. TYMV encodes a precursor replication polyprotein that is processed by the endoproteolytic activity of its internal cysteine proteinase domain (PRO). We recently reported that PRO is actually a multifunctional enzyme with a specific ubiquitin hydrolase (DUB) activity that contributes to viral infectivity. Here, we report the crystal structure of the 150-residue PRO. Strikingly, PRO displays no homology to other processing proteinases from positive-stranded RNA viruses, including that of alphaviruses. Instead, the closest structural homologs of PRO are DUBs from the Ovarian tumor (OTU) family. In the crystal, one molecule's C-terminus inserts into the catalytic cleft of the next, providing a view of the N-terminal product complex in replication polyprotein processing. This allows us to locate the specificity determinants of PRO for its proteinase substrates. In addition to the catalytic cleft, at the exit of which the active site is unusually pared down and solvent-exposed, a key element in molecular recognition by PRO is a lobe N-terminal to the catalytic domain. Docking models and the activities of PRO and PRO mutants in a deubiquitylating assay suggest that this N-terminal lobe is also likely involved in PRO's DUB function. Our data thus establish that DUBs can evolve to specifically hydrolyze both iso- and endopeptide bonds with different sequences. This is achieved by the use of multiple specificity determinants, as recognition of substrate patches distant from the cleavage sites allows a relaxed specificity of PRO at the sites themselves. Our results thus shed light on how such a compact protein achieves a diversity of key functions in viral genome replication and host-pathogen interaction. PMID:23966860

  3. A compact viral processing proteinase/ubiquitin hydrolase from the OTU family.

    PubMed

    Lombardi, Charlotte; Ayach, Maya; Beaurepaire, Lionel; Chenon, Mélanie; Andreani, Jessica; Guerois, Raphaël; Jupin, Isabelle; Bressanelli, Stéphane

    2013-08-01

    Turnip yellow mosaic virus (TYMV)--a member of the alphavirus-like supergroup of viruses--serves as a model system for positive-stranded RNA virus membrane-bound replication. TYMV encodes a precursor replication polyprotein that is processed by the endoproteolytic activity of its internal cysteine proteinase domain (PRO). We recently reported that PRO is actually a multifunctional enzyme with a specific ubiquitin hydrolase (DUB) activity that contributes to viral infectivity. Here, we report the crystal structure of the 150-residue PRO. Strikingly, PRO displays no homology to other processing proteinases from positive-stranded RNA viruses, including that of alphaviruses. Instead, the closest structural homologs of PRO are DUBs from the Ovarian tumor (OTU) family. In the crystal, one molecule's C-terminus inserts into the catalytic cleft of the next, providing a view of the N-terminal product complex in replication polyprotein processing. This allows us to locate the specificity determinants of PRO for its proteinase substrates. In addition to the catalytic cleft, at the exit of which the active site is unusually pared down and solvent-exposed, a key element in molecular recognition by PRO is a lobe N-terminal to the catalytic domain. Docking models and the activities of PRO and PRO mutants in a deubiquitylating assay suggest that this N-terminal lobe is also likely involved in PRO's DUB function. Our data thus establish that DUBs can evolve to specifically hydrolyze both iso- and endopeptide bonds with different sequences. This is achieved by the use of multiple specificity determinants, as recognition of substrate patches distant from the cleavage sites allows a relaxed specificity of PRO at the sites themselves. Our results thus shed light on how such a compact protein achieves a diversity of key functions in viral genome replication and host-pathogen interaction.

  4. Manduca sexta proprophenoloxidase activating proteinase-3 (PAP3) stimulates melanization by activating proPAP3, proSPHs, and proPOs

    PubMed Central

    Wang, Yang; Lu, Zhiqiang; Jiang, Haobo

    2014-01-01

    Melanization participates in various insect physiological processes including antimicrobial immune responses. Phenoloxidase (PO), a critical component of the enzyme system catalyzing melanin formation, is produced as an inactive precursor prophenoloxidase (proPO) and becomes active via specific proteolytic cleavage by proPO activating proteinase (PAP). In Manduca sexta, three PAPs can activate proPOs in the presence of two serine proteinase homologs (SPH1 and SPH2). While the hemolymph proteinases (HPs) that generate the active PAPs are known, it is unclear how the proSPHs (especially proSPH1) are activated. In this study, we isolated from plasma of bar-stage M. sexta larvae an Ile-Glu-Ala-Arg-p-nitroanilide hydrolyzing enzyme that cleaved the proSPHs. This proteinase, PAP3, generated active SPH1 and SPH2, which function as cofactors for PAP3 in proPO activation. Cleavage of the purified recombinant proSPHs by PAP3 yielded 38 kDa bands similar in mobility to the SPHs formed in vivo. Surprisingly, PAP3 also can activate proPAP3 to stimulate melanization in a direct positive feedback loop. The enhanced proPO activation concurred with the cleavage activation of proHP6, proHP8, proPAP1, proPAP3, proSPH1, proSPH2, proPOs, but not proHP14 or proHP21. These results indicate that PAP3, like PAP1, is a key factor of the self-reinforcing mechanism in the proPO activation system, which is linked to other immune responses in M. sexta. PMID:24768974

  5. The insect immune protein scolexin is a novel serine proteinase homolog.

    PubMed Central

    Finnerty, C. M.; Karplus, P. A.; Granados, R. R.

    1999-01-01

    Scolexin is a coagulation-provoking plasma protein induced in response to bacterial or viral infection of larval Manduca sexta, a large lepidopterous insect. Here we report the isolation and sequencing of two cDNA clones that code for scolexin isoforms sharing 80% sequence identity. The scolexin sequences have low but recognizable sequence similarity to members of the chymotrypsin family and represent a new subfamily of chymotrypsin-like serine proteinases. Comparison with known structures reveals the conservation of key catalytic residues and a possible specificity for small nonpolar residues. Most remarkable is the absence of a canonical activation peptide cleavage site. This suggests that the regulation of scolexin activity will involve a novel activation mechanism. PMID:10210202

  6. Specificity of an extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine alpha s1-casein.

    PubMed Central

    Rattray, F P; Fox, P F; Healy, A

    1996-01-01

    The specificity of the extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine alpha s1-casein was studied. Hydrolysis was monitored over time by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and urea-PAGE. The major pH 4.6-soluble peptides were isolated by high-performance liquid chromatography and identified by N-terminal amino acid sequencing and mass spectrometry. The time course of peptide formation indicated that His-8-Gln-9, Ser-161-Gly-162, and either Gln-172-Tyr-173 or Phe-23-Phe-24 were the first, second, and third bonds cleaved, respectively. Other cleavage sites included Asn-19-Leu-20, Phe-32-Gly-33, Tyr-104-Lys-105, Leu-142-Ala-143, Phe-150-Arg-151, Gln-152-Phe-153, Leu-169-Gly-170, and Thr-171-Gln-172. The proteinase had a broad specificity for the amino acid residues at the P1 and P'1 positions but showed a preference for hydrophobic residues at the P2, P3, P4, P'2, P'3, and P'4 positions. PMID:8593051

  7. Specificity of an extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine alpha s1-casein.

    PubMed

    Rattray, F P; Fox, P F; Healy, A

    1996-02-01

    The specificity of the extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine alpha s1-casein was studied. Hydrolysis was monitored over time by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and urea-PAGE. The major pH 4.6-soluble peptides were isolated by high-performance liquid chromatography and identified by N-terminal amino acid sequencing and mass spectrometry. The time course of peptide formation indicated that His-8-Gln-9, Ser-161-Gly-162, and either Gln-172-Tyr-173 or Phe-23-Phe-24 were the first, second, and third bonds cleaved, respectively. Other cleavage sites included Asn-19-Leu-20, Phe-32-Gly-33, Tyr-104-Lys-105, Leu-142-Ala-143, Phe-150-Arg-151, Gln-152-Phe-153, Leu-169-Gly-170, and Thr-171-Gln-172. The proteinase had a broad specificity for the amino acid residues at the P1 and P'1 positions but showed a preference for hydrophobic residues at the P2, P3, P4, P'2, P'3, and P'4 positions.

  8. [Suppression of activity of Candida albicans proteinases by cobalt chloride].

    PubMed

    Kutyreva, M P; Mukhametzianova, A R; Ulakhovich, N A

    2012-01-01

    Influence of cobalt (II) chloride on the system of Candida albicans proteinase (SAP C. alb.) (both in solution and immobilized on a surface of nitrocellulose membranes) has been investigated. In solution cobalt chloride inactivated inducible but not constitute enzyme. In the heterogenous sytem proteolitical effect of the cobalt ion on inductible proteinase was also observed.

  9. Some aspects of structural studies on aspartic proteinases.

    PubMed

    Andreeva, N S

    1992-01-01

    This paper gives a brief overview over the differences and similarities in the structure of aspartic proteinases presently available. Comparison of the three-dimentional structure of different aspartic proteinases by a common intramolecular coordinate system have been performed. The intramolecular movable subdomains have been localized and the role of motion in substrate binding and zymogen activation is discussed.

  10. The N-terminal octapeptide acts as a dimerization inhibitor of SARS coronavirus 3C-like proteinase.

    PubMed

    Wei, Ping; Fan, Keqiang; Chen, Hao; Ma, Liang; Huang, Changkang; Tan, Lei; Xi, Dong; Li, Chunmei; Liu, Ying; Cao, Aoneng; Lai, Luhua

    2006-01-20

    The 3C-like proteinase of severe acute respiratory syndrome (SARS) coronavirus has been proposed to be a key target for structural-based drug design against SARS. Accurate determination of the dimer dissociation constant and the role of the N-finger (residues 1-7) will provide more insights into the enzyme catalytic mechanism of SARS 3CL proteinase. The dimer dissociation constant of the wild-type protein was determined to be 14.0microM by analytical ultracentrifugation method. The N-finger fragment of the enzyme plays an important role in enzyme dimerization as shown in the crystal structure. Key residues in the N-finger have been studied by site-directed mutagenesis, enzyme assay, and analytical ultracentrifugation. A single mutation of M6A was found to be critical to maintain the dimer structure of the enzyme. The N-terminal octapeptide N8 and its mutants were also synthesized and tested for their potency as dimerization inhibitors. Peptide cleavage assay confirms that peptide N8 is a dimerization inhibitor with a K(i) of 2.20mM. The comparison of the inhibitory activities of N8 and its mutants indicates that the hydrophobic interaction of Met-6 and the electrostatic interaction of Arg-4 contribute most for inhibitor binding. This study describes the first example of inhibitors targeting the dimeric interface of SARS 3CL proteinase, providing a novel strategy for drug design against SARS and other coronaviruses.

  11. Differential cleavage of IRES trans-acting factors (ITAFs) in cells infected by human rhinovirus.

    PubMed

    Chase, Amanda J; Semler, Bert L

    2014-01-20

    Human rhinovirus (HRV) is a major causative agent of the common cold, and thus has several important health implications. As a member of the picornavirus family, HRV has a small genomic RNA that utilizes several host cell proteins for RNA replication. Host proteins poly(rC) binding protein 2 (PCBP2) and polypyrimidine tract binding protein (PTB) are cleaved by a viral proteinase during the course of infection by the related picornavirus, poliovirus. The cleavage of PCBP2 and PTB inhibits poliovirus translation and has been proposed to mediate a switch in poliovirus template usage from translation to RNA replication. HRV RNA replication also requires a switch in template usage from translation to RNA replication; however, the mechanism is not yet known. We demonstrate that PCBP2 and PTB are differentially cleaved during HRV infection in different cell lines, suggesting that HRV utilizes a mechanism distinct from PCBP2 or PTB cleavage to mediate a switch in template usage.

  12. Cassava Brown Streak Virus (Potyviridae) Encodes a Putative Maf/HAM1 Pyrophosphatase Implicated in Reduction of Mutations and a P1 Proteinase That Suppresses RNA Silencing but Contains No HC-Pro ▿

    PubMed Central

    Mbanzibwa, Deusdedith R.; Tian, Yanping; Mukasa, Settumba B.; Valkonen, Jari P. T.

    2009-01-01

    The complete positive-sense single-stranded RNA genome of Cassava brown streak virus (CBSV; genus Ipomovirus; Potyviridae) was found to consist of 9,069 nucleotides and predicted to produce a polyprotein of 2,902 amino acids. It was lacking helper-component proteinase but contained a single P1 serine proteinase that strongly suppressed RNA silencing. Besides the exceptional structure of the 5′-proximal part of the genome, CBSV also contained a Maf/HAM1-like sequence (678 nucleotides, 226 amino acids) recombined between the replicase and coat protein domains in the 3′-proximal part of the genome, which is highly conserved in Potyviridae. HAM1 was flanked by consensus proteolytic cleavage sites for ipomovirus NIaPro cysteine proteinase. Homology of CBSV HAM1 with cellular Maf/HAM1 pyrophosphatases suggests that it may intercept noncanonical nucleoside triphosphates to reduce mutagenesis of viral RNA. PMID:19386713

  13. Leukocyte cell surface proteinases: regulation of expression, functions, and mechanisms of surface localization.

    PubMed

    Owen, Caroline A

    2008-01-01

    A number of proteinases are expressed on the surface of leukocytes including members of the serine, metallo-, and cysteine proteinase superfamilies. Some proteinases are anchored to the plasma membrane of leukocytes by a transmembrane domain or a glycosyl phosphatidyl inositol (GPI) anchor. Other proteinases bind with high affinity to classical receptors, or with lower affinity to integrins, proteoglycans, or other leukocyte surface molecules. Leukocyte surface levels of proteinases are regulated by: (1) cytokines, chemokines, bacterial products, and growth factors which stimulate synthesis and/or release of proteinases by cells; (2) the availability of surface binding sites for proteinases; and/or (3) internalization or shedding of surface-bound proteinases. The binding of proteinases to leukocyte surfaces serves many functions including: (1) concentrating the activity of proteinases to the immediate pericellular environment; (2) facilitating pro-enzyme activation; (3) increasing proteinase stability and retention in the extracellular space; (4) regulating leukocyte function by proteinases signaling through cell surface binding sites or other surface proteins; and (5) protecting proteinases from inhibition by extracellular proteinase inhibitors. There is strong evidence that membrane-associated proteinases on leukocytes play critical roles in wound healing, inflammation, extracellular matrix remodeling, fibrinolysis, and coagulation. This review will outline the biology of membrane-associated proteinases expressed by leukocytes and their roles in physiologic and pathologic processes.

  14. Peptide aldehyde inhibitors of hepatitis A virus 3C proteinase.

    PubMed

    Malcolm, B A; Lowe, C; Shechosky, S; McKay, R T; Yang, C C; Shah, V J; Simon, R J; Vederas, J C; Santi, D V

    1995-06-27

    Picornaviral 3C proteinases are a group of closely related thiol proteinases responsible for processing of the viral polyprotein into its component proteins. These proteinases adopt a chymotrypsin-like fold [Allaire et al. (1994) Nature 369, 72-77; Matthews et al. (1994) Cell 77, 761-771] and a display an active-site configuration like those of the serine proteinases. Peptide-aldehydes based on the preferred peptide substrates for hepatitis A virus (HAV) 3C proteinase were synthesized by reduction of a thioester precursor. Acetyl-Leu-Ala-Ala-(N,N'-dimethylglutaminal) was found to be a reversible, slow-binding inhibitor for HAV 3C with a Ki* of (4.2 +/- 0.8) x 10(-8) M. This inhibitor showed 50-fold less activity against the highly homologous human rhinovirus (strain 14) 3C proteinase, whose peptide substrate specificity is slightly different, suggesting a high degree of selectivity. NMR spectrometry of the adduct of the 13C-labeled inhibitor with the HAV-3C proteinase indicate that a thiohemiacetal is formed between the enzyme and the aldehyde carbon as previously noted for peptide-aldehyde inhibitors of papain [Lewis & Wolfenden (1977) Biochemistry 16,4890-4894; Gamcsik et al. (1983) J. Am. Chem. Soc. 105, 6324-6325]. The adduct can also be observed by electrospray mass spectrometry.

  15. Proteinases as virulence factors in Leishmania spp. infection in mammals

    PubMed Central

    2012-01-01

    Leishmania parasites cause human tegumentary and visceral infections that are commonly referred to as leishmaniasis. Despite the high incidence and prevalence of cases, leishmaniasis has been a neglected disease because it mainly affects developing countries. The data obtained from the analysis of patients’ biological samples and from assays with animal models confirm the involvement of an array of the parasite’s components in its survival inside the mammalian host. These components are classified as virulence factors. In this review, we focus on studies that have explored the role of proteinases as virulence factors that promote parasite survival and immune modulation in the mammalian host. Additionally, the direct involvement of proteinases from the host in lesion evolution is analyzed. The gathered data shows that both parasite and host proteinases are involved in the clinical manifestation of leishmaniasis. It is interesting to note that although the majority of the classes of proteinases are present in Leishmania spp., only cysteine-proteinases, metalloproteinases and, to a lesser scale, serine-proteinases have been adequately studied. Members from these classes have been implicated in tissue invasion, survival in macrophages and immune modulation by parasites. This review reinforces the importance of the parasite proteinases, which are interesting candidates for new chemo or immunotherapies, in the clinical manifestations of leishmaniasis. PMID:22871236

  16. Characterization and pathogenetic role of proteinase from Acanthamoeba castellanii.

    PubMed

    Na, B K; Kim, J C; Song, C Y

    2001-01-01

    A secreted proteinase was purified from the culture supernatant of Acanthamoeba castellanii with several chromatographic steps. The purified proteinase was a chymotrypsin-like serine proteinase. Its molecular weight was approximately 12 kDa on SDS-PAGE, and its native molecular weight was 12 kDa when determined by molecular sieve chromatography. It showed a broad temperature optimum ranging 30-55 degrees C with an optimal at 55 degrees C and an optimal pH of 8.5. It could degrade various protein substrates, such as collagen, fibronectin, laminin, secretory immunoglobulin A, immunoglobulin G, plasminogen, fibrinogen, haemoglobin and rabbit corneal proteins. It showed strong cytopathic effects in cultured cells, including HEp2 and HEK cells. The corneal lesions, induced by both the purified proteinase and A. castellanii, displayed similar clinical results for both cases, in which the stromal infiltration and opacity with the epithelial defect were revealed. These results suggest that the enzyme was highly associated with the pathogenesis of Acanthamoeba. The fact that cytopathic effects and development of corneal lesions caused by the proteinase of Acanthamoeba were inhibited by the proteinase inhibitor suggest that the proteinase inhibitor might be useful as a therapeutic agent.

  17. Action of plant proteinase inhibitors on enzymes of physiopathological importance.

    PubMed

    Oliva, Maria Luiza V; Sampaio, Misako U

    2009-09-01

    Obtained from leguminous seeds, various plant proteins inhibit animal proteinases, including human, and can be considered for the development of compounds with biological activity. Inhibitors from the Bowman-Birk and plant Kunitz-type family have been characterized by proteinase specificity, primary structure and reactive site. Our group mostly studies the genus Bauhinia, mainly the species bauhinioides, rufa, ungulata and variegata. In some species, more than one inhibitor was characterized, exhibiting different properties. Although proteins from this group share high structural similarity, they present differences in proteinase inhibition, explored in studies using diverse biological models.

  18. BMP1 controls TGFbeta1 activation via cleavage of latent TGFbeta-binding protein.

    PubMed

    Ge, Gaoxiang; Greenspan, Daniel S

    2006-10-09

    Transforming growth factor beta1 (TGFbeta1), an important regulator of cell behavior, is secreted as a large latent complex (LLC) in which it is bound to its cleaved prodomain (latency-associated peptide [LAP]) and, via LAP, to latent TGFbeta-binding proteins (LTBPs). The latter target LLCs to the extracellular matrix (ECM). Bone morphogenetic protein 1 (BMP1)-like metalloproteinases play key roles in ECM formation, by converting precursors into mature functional proteins, and in morphogenetic patterning, by cleaving the antagonist Chordin to activate BMP2/4. We provide in vitro and in vivo evidence that BMP1 cleaves LTBP1 at two specific sites, thus liberating LLC from ECM and resulting in consequent activation of TGFbeta1 via cleavage of LAP by non-BMP1-like proteinases. In mouse embryo fibroblasts, LAP cleavage is shown to be predominantly matrix metalloproteinase 2 dependent. TGFbeta1 is a potent inducer of ECM formation and of BMP1 expression. Thus, a role for BMP1-like proteinases in TGFbeta1 activation completes a novel fast-forward loop in vertebrate tissue remodeling.

  19. Isolation and characterization of βA3-crystallin associated proteinase from α-crystallin fraction of human lenses

    PubMed Central

    Chaves, J. M.

    2008-01-01

    -exclusion chromatography. The proteinase also exhibited proteolysis of γC- and γD- crystallins, and the cleavage of γD-crystallin at M1-G2, Q54-Y55, M70-G71, and Q103-M104 bonds. Further, the enzyme was also present in three fractions of human lenses (α-crystallin, βH-crystallin, and membrane fractions). Conclusions A serine-type βA3-crystallin proteinase existed in an inactive state in the α-crystallin fraction and was activated by detergents. The enzyme proteolyzed αA-, αB-, γC-, and γD-crystallins and was present in three fractions (α-crystallin, βH-crystallin, and membrane-fractions) of 60 to 70-year-old human lenses. PMID:18949065

  20. Protein inhibitors of serine proteinases: role of backbone structure and dynamics in controlling the hydrolysis constant.

    PubMed

    Song, Jikui; Markley, John L

    2003-05-13

    Standard mechanism protein inhibitors of serine proteinases bind as substrates and are cleaved by cognate proteinases at their reactive sites. The hydrolysis constant for this cleavage reaction at the P(1)-P(1)' peptide bond (K(hyd)) is determined by the relative concentrations at equilibrium of the "intact" (uncleaved, I) and "modified" (reactive site cleaved, I*) forms of the inhibitor. The pH dependence of K(hyd) can be explained in terms of a pH-independent term, K(hyd) degrees, plus the proton dissociation constants of the newly formed amino and carboxylate groups at the cleavage site. Two protein inhibitors that differ from one another by a single residue substitution have been found to have K(hyd) degrees values that differ by a factor of 5 [Ardelt, W., and Laskowski, M., Jr. (1991) J. Mol. Biol. 220, 1041-1052]: turkey ovomucoid third domain (OMTKY3) has K(hyd) degrees = 1.0, and Indian peafowl ovomucoid third domain (OMIPF3), which differs from OMTKY3 by the substitution P(2)'-Tyr(20)His, has K(hyd) degrees = 5.15. What mechanism is responsible for this small difference? Is it structural (enthalpic) or dynamic (entropic)? Does the mutation affect the free energy of the I state, the I* state, or both? We have addressed these questions through NMR investigations of the I and I forms of OMTKY3 and OMIPF3. Information about structure was derived from measurements of NMR chemical shift changes and trans-hydrogen-bond J-couplings; information about dynamics was obtained through measurements of (15)N relaxation rates and (1)H-(15)N heteronuclear NOEs with model-free analysis of the results. Although the I forms of each variant are more dynamic than the corresponding I forms, the study revealed no appreciable difference in the backbone dynamics of either intact inhibitor (OMIPF3 vs OMTKY3) or modified inhibitor (OMIPF3* vs OMTKY3*). Instead, changes in chemical shifts and trans-hydrogen-bond J-couplings suggested that the K(hyd) degrees difference arises from

  1. Multiple forms of calcium-dependent proteinase in crustacean muscle

    SciTech Connect

    Mykles, D.L.; Skinner, D.M.

    1986-01-01

    Four calcium-dependent proteinase (CDP) activities in lobster muscles have been resolved by high performance liquid chromatography. These activities differ in molecular weight and net charge. Though optimum activity occurred at high (5 and 10 mM) calcium at pH 6.8, the enzymes differ in activation at lower calcium concentrations. Only one of the CDPs is active at 100 ..mu..M calcium; none are active at 10 ..mu..M and below. Although all four CDPs are inhibited by the cysteine proteinase inhibitors leupeptin, E-64, and iodoacetamide, they show a differential response to the aspartic proteinase inhibitor pepstatin and the serine proteinase inhibitor PMSF. In contrast to CDPs from vertebrate tissues, crustacean muscles contain multiple forms that require calcium at millimolar levels. 17 refs., 6 figs.

  2. Aspartic proteinases from Mucor spp. in cheese manufacturing.

    PubMed

    Yegin, Sirma; Fernandez-Lahore, Marcelo; Jose Gama Salgado, Antonio; Guvenc, Ulgar; Goksungur, Yekta; Tari, Canan

    2011-02-01

    Filamentous fungi belonging to the order of Mucorales are well known as producers of aspartic proteinases depicting milk-clotting activity. The biosynthesis level, the biochemical characteristics, and the technological properties of the resulting proteinases are affected by the producer strain and the mode of cultivation. While the milk-clotting enzymes produced by the Rhizomucor spp. have been extensively studied in the past, much less is known on the properties and potential applications of the aspartic proteinases obtained for Mucor spp. Indeed, several Mucor spp. strains have been reported as a potential source of milk-clotting enzymes having unique technological properties. Both submerged fermentation and solid substrate cultivation are proven alternatives for the production of Mucor spp. aspartic proteinases. This review provides an overview on the bioprocessing routes to obtain large amounts of these enzymes, on their structural characteristics as related to their functional properties, and on their industrial applications with focus on cheese manufacturing.

  3. Feline Calicivirus Infection Disrupts Assembly of Cytoplasmic Stress Granules and Induces G3BP1 Cleavage

    PubMed Central

    Humoud, Majid N.; Doyle, Nicole; Royall, Elizabeth; Willcocks, Margaret M.; Sorgeloos, Frederic; van Kuppeveld, Frank; Roberts, Lisa O.; Goodfellow, Ian G.; Langereis, Martijn A.

    2016-01-01

    ABSTRACT In response to stress such as virus infection, cells can stall translation by storing mRNAs away in cellular compartments called stress granules (SGs). This defense mechanism favors cell survival by limiting the use of energy and nutrients until the stress is resolved. In some cases it may also block viral propagation as viruses are dependent on the host cell resources to produce viral proteins. Human norovirus is a member of the Caliciviridae family responsible for gastroenteritis outbreaks worldwide. Previous studies on caliciviruses have identified mechanisms by which they can usurp the host translational machinery, using the viral protein genome-linked VPg, or regulate host protein synthesis through the mitogen-activated protein kinase (MAPK) pathway. Here, we examined the effect of feline calicivirus (FCV) infection on SG accumulation. We show that FCV infection impairs the assembly of SGs despite an increased phosphorylation of eukaryotic initiation factor eIF2α, a hallmark of stress pathway activation. Furthermore, SGs did not accumulate in FCV-infected cells that were stressed with arsenite or hydrogen peroxide. FCV infection resulted in the cleavage of the SG-nucleating protein Ras-GTPase activating SH3 domain-binding protein (G3BP1), which is mediated by the viral 3C-like proteinase NS6Pro. Using mutational analysis, we identified the FCV-induced cleavage site within G3BP1, which differs from the poliovirus 3C proteinase cleavage site previously identified. Finally, we showed that NS6Pro-mediated G3BP1 cleavage impairs SG assembly. In contrast, murine norovirus (MNV) infection did not impact arsenite-induced SG assembly or G3BP1 integrity, suggesting that related caliciviruses have distinct effects on the stress response pathway. IMPORTANCE Human noroviruses are a major cause of viral gastroenteritis, and it is important to understand how they interact with the infected host cell. Feline calicivirus (FCV) and murine norovirus (MNV) are used as

  4. A low molecular weight proteinase inhibitor produced by T lymphocytes.

    PubMed Central

    Ganea, D; Teodorescu, M; Dray, S

    1986-01-01

    A low molecular weight (MW) proteinase inhibitor, between 6500 and 21,500 MW, appeared in the supernatant of rabbit spleen cells cultured at high density for 24 hr. The inhibitor inhibited the enzymatic activity of trypsin for both a high MW natural substrate, fibrinogen, and for a low MW artificial substrate, Chromozym TRY. The low MW proteinase inhibitor is protein in nature and is different, in terms of specificity for enzymes, MW and sensitivity to different physical or chemical treatments, from aprotinin, a low MW proteinase inhibitor (6500 MW) of bovine origin, and from the soybean trypsin inhibitor, a relatively high MW proteinase inhibitor (21,500 MW). The inhibitor was found in the supernatant of purified T cells but not B cells, and its production was increased in the presence of an optimal concentration of Con A. The possibility that this proteinase inhibitor has a role in the regulation of trypsin-like proteinases involved to the immune response remains to be investigated. Images Figure 4 PMID:2417942

  5. The induction of proteinases in corn and soybean by anoxia

    SciTech Connect

    VanToai, T.; Hwang, Shihying )

    1989-04-01

    This study characterized the anaerobic changes in proteinase activities in corn and soybean roots and to investigate the possibility that these changes might contribute to the differential anaerobiosis tolerance of the two species. After 24 h of anoxia, crude protein extracts from H60 corn and Keller soybean root tips (10cm) were assayed for proteinase activities at pH range from 4.5 to 9.5. Turnover of aberrant proteins was studied in seedlings labelled with {sup 3}H-leucine for 12 h under: (a) puromycin (0.64 mM) in air, (b) ethanol (1%) in air, (c) nitrogen and (d) air. After the treatment, the labelled proteins remaining in roots were determined every 2 h for 6 h. In both corn and soybean, activities of alkali proteinases increased, and activities of acid proteinases declined under anoxia. Neutral proteinases increase in anoxic corn roots, but decline in anoxic soybean roots. The protein turnover rate in corn treated with puromycin, ethanol and nitrogen was much higher than in control roots. The protein turnover rate in soybean roots treated with puromycin, ethanol was similar to the rate of the control. The results indicated that: (a) anoxic corn can degrade aberrant proteins, but anoxic soybean cannot, (b) the degradation of aberrant proteins in anoxic corn is accomplished by neutral proteinases, and (c) the accumulation of aberrant proteins in soybean might contribute to the susceptibility of this species to anoxia.

  6. Proteinases in the joint: clinical relevance of proteinases in joint destruction

    PubMed Central

    Rengel, Yvonne; Ospelt, Caroline; Gay, Steffen

    2007-01-01

    Proteinases are involved in essential steps in cartilage and bone homeostasis. Consequently, efforts have been made to establish their potential role in the pathology of rheumatic conditions such as rheumatoid arthritis, osteoarthritis and spondyloarthritis. Matrix metalloproteinases (MMPs) are sensitive markers of disease severity and response to treatment, and therefore they have potential in the assessment of rheumatic diseases. Despite disappointing early results with synthetic inhibitors of MMPs, there is still much scope for developing effective and safe MMPs inhibitors, and consequently to deliver new options to inhibit joint destruction. PMID:18001502

  7. Proteinases of the cornea and preocular tear film.

    PubMed

    Ollivier, F J; Gilger, B C; Barrie, K P; Kallberg, M E; Plummer, C E; O'Reilly, S; Gelatt, K N; Brooks, D E

    2007-01-01

    Maintenance and repair of corneal stromal extracellular matrix (ECM) requires a tightly coordinated balance of ECM synthesis, degradation and remodeling in which proteolytic enzymes (proteinases) perform important functions. There are natural proteinase inhibitors present in preocular tear film (PTF) and cornea simultaneously with proteinases that prevent excessive degradation of normal healthy tissue. Disorders occur when there is an imbalance between proteinases and proteinase inhibitors in favor of the proteinases, causing pathologic degradation of stromal collagen and proteoglycans in the cornea. Two matrix metalloproteinases (MMPs), MMP-2 and MMP-9, are of major importance in terms of remodeling and degradation of the corneal stromal collagen. Immunohistochemical studies have shown different origins of MMP-2 and -9. MMP-2 is synthesized by corneal keratocytes and performs a surveillance function in the normal cornea, becoming locally activated to degrade collagen molecules that occasionally become damaged. Alternatively, MMP-9 may be produced by epithelial cells and polymorphonuclear neutrophils following corneal wounding. Because the cornea is in close contact with the preocular tear film (PTF), proteinases have been evaluated in the PTF. In damaged corneas, total proteolytic activity in the tear fluid was found to be significantly increased compared to normal eyes and contralateral eyes. Studies analyzing the proteolytic activity in serial PTF samples during corneal healing led to the following conclusions: ulcerative keratitis in animals is associated with initially high levels of tear film proteolytic activity, which decrease as ulcers heal; proteinase levels in melting ulcers remain elevated leading to rapid progression of the ulcers. The success of medical and surgical treatment of the corneal ulcers is reflected by the proteolytic activity in tears. In animals, successful treatment leads to a rapid reduction in tear film proteolytic activity that

  8. Developing novel anthelmintics from plant cysteine proteinases

    PubMed Central

    Behnke, Jerzy M; Buttle, David J; Stepek, Gillian; Lowe, Ann; Duce, Ian R

    2008-01-01

    Intestinal helminth infections of livestock and humans are predominantly controlled by treatment with three classes of synthetic drugs, but some livestock nematodes have now developed resistance to all three classes and there are signs that human hookworms are becoming less responsive to the two classes (benzimidazoles and the nicotinic acetylcholine agonists) that are licensed for treatment of humans. New anthelmintics are urgently needed, and whilst development of new synthetic drugs is ongoing, it is slow and there are no signs yet that novel compounds operating through different modes of action, will be available on the market in the current decade. The development of naturally-occurring compounds as medicines for human use and for treatment of animals is fraught with problems. In this paper we review the current status of cysteine proteinases from fruits and protective plant latices as novel anthelmintics, we consider some of the problems inherent in taking laboratory findings and those derived from folk-medicine to the market and we suggest that there is a wealth of new compounds still to be discovered that could be harvested to benefit humans and livestock. PMID:18761736

  9. Compartmentalization of proteinases and amylases in Nauphoeta cinerea midgut.

    PubMed

    Elpidina, E N; Vinokurov, K S; Gromenko, V A; Rudenskaya, Y A; Dunaevsky, Y E; Zhuzhikov, D P

    2001-12-01

    Compartmentalization of proteinases, amylases, and pH in the midgut of Nauphoeta cinerea Oliv. (Blattoptera:Blaberidae) was studied in order to understand the organization of protein and starch digestion. Total proteolytic activity measured with azocasein was maximal at pH 11.5 both in anterior (AM) and posterior (PM) halves of the midgut, but the bulk of activity (67%) was found in PM. Total AM and PM preparations were fractionated on a Sephadex G-50 column and further analysed by means of activity electrophoresis and specific inhibitors and activators. The major activity in PM was classified as an unusual SH-dependent proteinase with M(r) 24,000 and pH optimum with synthetic substrate BApNA at 10.0. The enzyme was 43-fold activated in the presence of 1 mM DTT, insensitive to synthetic inhibitors of serine (PMSF, TLCK, TPCK) and cysteine (IAA, E-64) proteinases, strongly inhibited by STI, and displayed four active bands on zymograms. In PM, activities of trypsin-like, chymotrypsin-like, subtilisin-like, and cysteine proteinases were observed. Aspartic and metalloproteinases were not detected. In AM, activity of unusual SH-dependent proteinase also dominated and activity of chymotrypsin-like proteinase was observed, but their levels were much lower than in PM. Distribution of amylase activity, exhibiting an optimum at pH 6.0, was quite the opposite. The major part of it (67%) was located in AM. Treatment of amylase preparation with proteinases from AM and PM reduced amylase activity twofold. pH of the midgut contents was 6.0-7.2 in AM, 6.4-7.6 in the first and 8.8-9.3 in the second halves of PM. Thus, pH in AM is in good agreement with the optimal pH of amylase, located in this compartment, but the activity of proteinases, including the ability to degrade amylase, in such an environment is low. Active proteolysis takes place in the second half of PM, where pH of the gut is close to the optimal pH of proteinases.

  10. Candida albicans Secreted Aspartyl Proteinases in Virulence and Pathogenesis

    PubMed Central

    Naglik, Julian R.; Challacombe, Stephen J.; Hube, Bernhard

    2003-01-01

    Candida albicans is the most common fungal pathogen of humans and has developed an extensive repertoire of putative virulence mechanisms that allows successful colonization and infection of the host under suitable predisposing conditions. Extracellular proteolytic activity plays a central role in Candida pathogenicity and is produced by a family of 10 secreted aspartyl proteinases (Sap proteins). Although the consequences of proteinase secretion during human infections is not precisely known, in vitro, animal, and human studies have implicated the proteinases in C. albicans virulence in one of the following seven ways: (i) correlation between Sap production in vitro and Candida virulence, (ii) degradation of human proteins and structural analysis in determining Sap substrate specificity, (iii) association of Sap production with other virulence processes of C. albicans, (iv) Sap protein production and Sap immune responses in animal and human infections, (v) SAP gene expression during Candida infections, (vi) modulation of C. albicans virulence by aspartyl proteinase inhibitors, and (vii) the use of SAP-disrupted mutants to analyze C. albicans virulence. Sap proteins fulfill a number of specialized functions during the infective process, which include the simple role of digesting molecules for nutrient acquisition, digesting or distorting host cell membranes to facilitate adhesion and tissue invasion, and digesting cells and molecules of the host immune system to avoid or resist antimicrobial attack by the host. We have critically discussed the data relevant to each of these seven criteria, with specific emphasis on how this proteinase family could contribute to Candida virulence and pathogenesis. PMID:12966142

  11. A serine proteinase inhibitor from frog eggs with bacteriostatic activity.

    PubMed

    Han, Yaoping; Yu, Haining; Yang, Xinbo; Rees, Huw H; Liu, Jingze; Lai, Ren

    2008-01-01

    By Sephadex G-50 gel filtration, Resource Q anionic exchange and C4 reversed phase liquid high performance liquid chromatography, a proteinase inhibitor protein (Ranaserpin) was identified and purified from the eggs of the odour frog, Rana grahami. The protein displayed a single band adjacent to the molecular weight marker of 14.4 kDa analyzed by SDS-PAGE. The inhibitor protein homogeneity and its molecular weight were confirmed again by MALDI-TOF mass spectrometry analysis. The MALDI-TOF mass spectrum analysis gave this inhibitor protein an m/z of 14422.26 that was matched well with the result from SDS-PAGE. This protein is a serine proteinase inhibitor targeting multiple proteinases including trypsin, elastase, and subtilisin. Ranaserpin inhibited the proteolytic activities of trypsin, elastase, and subtilisin. It has an inhibitory constant (K(i)) of 6.2 x 10(-8) M, 2.7 x 10(-7) M and 2.2 x 10(-8) M for trypsin, elastase, and subtilisin, respectively. This serine proteinase inhibitor exhibited bacteriostatic effect on Gram-positive bacteria Bacillus subtilis (ATCC 6633). It was suggested that ranaserpin might act as a defensive role in resistance to invasion of pests or pathogens. This is the first report of serine proteinase inhibitor and its direct defensive role from amphibian eggs.

  12. Specificity of hammerhead ribozyme cleavage.

    PubMed Central

    Hertel, K J; Herschlag, D; Uhlenbeck, O C

    1996-01-01

    To be effective in gene inactivation, the hammerhead ribozyme must cleave a complementary RNA target without deleterious effects from cleaving non-target RNAs that contain mismatches and shorter stretches of complementarity. The specificity of hammerhead cleavage was evaluated using HH16, a well-characterized ribozyme designed to cleave a target of 17 residues. Under standard reaction conditions, HH16 is unable to discriminate between its full-length substrate and 3'-truncated substrates, even when six fewer base pairs are formed between HH16 and the substrate. This striking lack of specificity arises because all the substrates bind to the ribozyme with sufficient affinity so that cleavage occurs before their affinity differences are manifested. In contrast, HH16 does exhibit high specificity towards certain 3'-truncated versions of altered substrates that either also contain a single base mismatch or are shortened at the 5' end. In addition, the specificity of HH16 is improved in the presence of p7 nucleocapsid protein from human immunodeficiency virus (HIV)-1, which accelerates the association and dissociation of RNA helices. These results support the view that the hammerhead has an intrinsic ability to discriminate against incorrect bases, but emphasizes that the high specificity is only observed in a certain range of helix lengths. Images PMID:8670879

  13. Proteinase-Activated Receptor 2 Is a Novel Regulator of TGF-β Signaling in Pancreatic Cancer

    PubMed Central

    Witte, David; Zeeh, Franziska; Gädeken, Thomas; Gieseler, Frank; Rauch, Bernhard H.; Settmacher, Utz; Kaufmann, Roland; Lehnert, Hendrik; Ungefroren, Hendrik

    2016-01-01

    TGF-β has a dual role in tumorigenesis, acting as a tumor suppressor in normal cells and in the early stages of tumor development while promoting carcinogenesis and metastasis in advanced tumor stages. The final outcome of the TGF-β response is determined by cell-autonomous mechanisms and genetic alterations such as genomic instability and somatic mutations, but also by a plethora of external signals derived from the tumor microenvironment, such as cell-to-cell interactions, growth factors and extracellular matrix proteins and proteolytic enzymes. Serine proteinases mediate their cellular effects via activation of proteinase-activated receptors (PARs), a subclass of G protein-coupled receptors that are activated by proteolytic cleavage. We have recently identified PAR2 as a factor required for TGF-β1-dependent cell motility in ductal pancreatic adenocarcinoma (PDAC) cells. In this article, we review what is known on the TGF-β-PAR2 signaling crosstalk and its relevance for tumor growth and metastasis. Since PAR2 is activated through various serine proteinases, it may couple TGF-β signaling to a diverse range of other physiological processes, such as local inflammation, systemic coagulation or pathogen infection. Moreover, since PAR2 controls expression of the TGF-β type I receptor ALK5, PAR2 may also impact signaling by other TGF-β superfamily members that signal through ALK5, such as myostatin and GDF15/MIC-1. If so, PAR2 could represent a molecular linker between PDAC development and cancer-related cachexia. PMID:27916875

  14. Proteinase-Activated Receptor 2 Is a Novel Regulator of TGF-β Signaling in Pancreatic Cancer.

    PubMed

    Witte, David; Zeeh, Franziska; Gädeken, Thomas; Gieseler, Frank; Rauch, Bernhard H; Settmacher, Utz; Kaufmann, Roland; Lehnert, Hendrik; Ungefroren, Hendrik

    2016-11-30

    TGF-β has a dual role in tumorigenesis, acting as a tumor suppressor in normal cells and in the early stages of tumor development while promoting carcinogenesis and metastasis in advanced tumor stages. The final outcome of the TGF-β response is determined by cell-autonomous mechanisms and genetic alterations such as genomic instability and somatic mutations, but also by a plethora of external signals derived from the tumor microenvironment, such as cell-to-cell interactions, growth factors and extracellular matrix proteins and proteolytic enzymes. Serine proteinases mediate their cellular effects via activation of proteinase-activated receptors (PARs), a subclass of G protein-coupled receptors that are activated by proteolytic cleavage. We have recently identified PAR2 as a factor required for TGF-β1-dependent cell motility in ductal pancreatic adenocarcinoma (PDAC) cells. In this article, we review what is known on the TGF-β-PAR2 signaling crosstalk and its relevance for tumor growth and metastasis. Since PAR2 is activated through various serine proteinases, it may couple TGF-β signaling to a diverse range of other physiological processes, such as local inflammation, systemic coagulation or pathogen infection. Moreover, since PAR2 controls expression of the TGF-β type I receptor ALK5, PAR2 may also impact signaling by other TGF-β superfamily members that signal through ALK5, such as myostatin and GDF15/MIC-1. If so, PAR2 could represent a molecular linker between PDAC development and cancer-related cachexia.

  15. Systematic mutational analysis of the active-site threonine of HIV-1 proteinase: rethinking the "fireman's grip" hypothesis.

    PubMed Central

    Strisovsky, K.; Tessmer, U.; Langner, J.; Konvalinka, J.; Kräusslich, H. G.

    2000-01-01

    Aspartic proteinases share a conserved network of hydrogen bonds (termed "fireman's grip"), which involves the hydroxyl groups of two threonine residues in the active site Asp-Thr-Gly triplets (Thr26 in the case of human immunodeficiency virus type 1 (HIV-1) PR). In the case of retroviral proteinases (PRs), which are active as symmetrical homodimers, these interactions occur at the dimer interface. For a systematic analysis of the "fireman's grip," Thr26 of HIV-1 PR was changed to either Ser, Cys, or Ala. The variant enzymes were tested for cleavage of HIV-1 derived peptide and polyprotein substrates. PR(T26S) and PR(T26C) showed similar or slightly reduced activity compared to wild-type HIV-1 PR, indicating that the sulfhydryl group of cysteine can substitute for the hydroxyl of the conserved threonine in this position. PR(T26A), which lacks the "fireman's grip" interaction, was virtually inactive and was monomeric in solution at conditions where wild-type PR exhibited a monomer-dimer equilibrium. All three mutations had little effect when introduced into only one chain of a linked dimer of HIV-1 PR. In this case, even changing both Thr residues to Ala yielded residual activity suggesting that the "fireman's grip" is not essential for activity but contributes significantly to dimer formation. Taken together, these results indicate that the "fireman's grip" is crucial for stabilization of the retroviral PR dimer and for overall stability of the enzyme. PMID:11045610

  16. Purification and characterization of a halotolerant serine proteinase from thermotolerant Bacillus licheniformis RKK-04 isolated from Thai fish sauce.

    PubMed

    Toyokawa, Yoichi; Takahara, Hiroaki; Reungsang, Alissara; Fukuta, Masakazu; Hachimine, Yuki; Tachibana, Shinjiro; Yasuda, Masaaki

    2010-05-01

    A gram-positive thermotolerant bacterium, designated strain RKK-04, was isolated from a fermented Thai fish sauce broth as it demonstrated high proteolytic activity. A phylogenetic analysis based on comparisons of 16S rRNA gene sequences showed that strain RKK-04 is Bacillus licheniformis. The proteolytic enzyme, which was purified 80-fold with 18% yield, has a molecular mass of 31 kDa and an isoelectric point higher than 9.3. The optimum pH and temperature of the enzyme activity were found to be 10.0 and 50 degrees C, respectively. The addition of diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride completely inhibited enzymatic activity. These results showed that the enzyme is a subtilisin-like alkaline serine proteinase. On the other hand, the enzyme exhibited unique cleavage sites in oxidized insulin B-chain that differed from those of other subtilisin-like proteases. High enzymatic activity was also retained under high salt conditions (30% NaCl). The myosin heavy chain of fish protein was completely digested by reaction with this enzyme. Thus the halotolerant proteinase from B. licheniformis RKK-04 is a key enzyme for fish sauce fermentation.

  17. Effects of leupeptin on proteinase and germination of castor beans

    SciTech Connect

    Alpi, A.; Beevers, H.

    1981-10-01

    Leupeptin, tripeptide inhibitor of some proteinases, was shown previously to maintain the stability of several enzymes (isocitrate lyase, fumarase, and catalase) in crude extracts of castor bean endosperm. This reagent is now shown to inhibit the breakdown of water-soluble and crystalloid-storage proteins of the protein bodies isolated from castor beans by the SH-proteinase and it also inhibits the endopeptidase from mung beans. When suitably introduced into the endosperm of dry castor beans it strongly inhibits germination and seedling development. Application of leupeptin to endosperm halves removed from the seed prevents the normal development of enzymes concerned with gluconeogenesis from fat and drastically curtails sugar production. The results suggest that the SH-proteinase is intimately involved in the mobilization of storage proteins.

  18. Production of proteinase on noncarbohydrate carbon sources by Pseudomonas aeruginosa.

    PubMed

    Morihara, K

    1965-09-01

    Proteinase production by Pseudomonas aeruginosa was studied in medium containing noncarbohydrate materials, especially various hydrocarbons, as the sole carbon source. On heavy oil, kerosene, n-paraffinic hydrocarbon of C(12), C(14), or C(16), and propylene glycol, the bacteria grew well and high protinase production was observed. However, production on paraffinic hydrocarbon differed remarkably with strains of varied origins. The elastase-positive strain, IFO 3455, showed abundant growth and high proteinase production on medium containing a paraffin of C(12), C(14), or C(16), whereas the elastase-negative strain, IFO 3080, showed little growth on the same medium. Neither elastase-positive nor elastase-negative strains, however, utilized n-paraffins of C(5) to C(10), or various aromatic hydrocarbons such as benzene, naphthalene, phenanthrene, and anthracene. The proteinases produced on the noncarbohydrate medium were identical with those produced in glucose medium.

  19. [Isolation of a specific inhibitor of microbial serine proteinase from kidney bean seeds].

    PubMed

    Mosolov, V V; Malova, E L; Cheban, A N

    1983-10-01

    A protein acting as a specific inhibitor of microbial serine proteinases was isolated from kidney bean seeds. The purification procedure included complex formation between the inhibitor and Aspergillus oryzae proteinase. The protein with a Mr approximately 10 000 inhibits subtilisin and Asp. oryzae proteinase but does not affect trypsin and chymotrypsin. The inhibitor molecule contains no half-cystine residues.

  20. Phase diagram of crystallization of Aspergillus niger acid proteinase A, a non-pepsin-type acid proteinase

    NASA Astrophysics Data System (ADS)

    Kudo, Norio; Ataka, Mitsuo; Sasaki, Hiroshi; Muramatsu, Tomonari; Katsura, Tatsuo; Tanokura, Masaru

    1996-10-01

    Proteinase A from Aspergillus niger var. macrosporus is a non-pepsin-type acid proteinase with an extremely low isoelectric point (pI 3.3). The protein is crystallized from ammonium sulfate solutions of pH lower than 4. The crystallization is affected by the presence of dimethylsulfoxide (DMSO). We have studied the phase diagram of the crystallization of proteinase A in the absence and presence of DMSO, to clarify crystallization at such an extremely low pH and to study the effects of DMSO. The results indicate that the logarithm of protein solubility is a rectilinear function of ammonium sulfate concentration in both the absence and presence of DMSO. DMSO definitely lowers the solubility at relatively low concentrations of ammonium sulfate, but had little effect on protein solubility at higher concentrations of ammonium sulfate.

  1. Structure of the Autocatalytic Cysteine Protease Domain of Potyvirus Helper-component Proteinase*

    PubMed Central

    Guo, Bihong; Lin, Jinzhong; Ye, Keqiong

    2011-01-01

    The helper-component proteinase (HC-Pro) of potyvirus is involved in polyprotein processing, aphid transmission, and suppression of antiviral RNA silencing. There is no high resolution structure reported for any part of HC-Pro, hindering mechanistic understanding of its multiple functions. We have determined the crystal structure of the cysteine protease domain of HC-Pro from turnip mosaic virus at 2.0 Å resolution. As a protease, HC-Pro only cleaves a Gly-Gly dipeptide at its own C terminus. The structure represents a postcleavage state in which the cleaved C terminus remains tightly bound at the active site cleft to prevent trans activity. The structure adopts a compact α/β-fold, which differs from papain-like cysteine proteases and shows weak similarity to nsP2 protease from Venezuelan equine encephalitis alphavirus. Nevertheless, the catalytic cysteine and histidine residues constitute an active site that is highly similar to these in papain-like and nsP2 proteases. HC-Pro recognizes a consensus sequence YXVGG around the cleavage site between the two glycine residues. The structure delineates the sequence specificity at sites P1–P4. Structural modeling and covariation analysis across the Potyviridae family suggest a tryptophan residue accounting for the glycine specificity at site P1′. Moreover, a surface of the protease domain is conserved in potyvirus but not in other genera of the Potyviridae family, likely due to extra functional constrain. The structure provides insight into the catalysis mechanism, cis-acting mode, cleavage site specificity, and other functions of the HC-Pro protease domain. PMID:21543324

  2. Circulating ADAM17 Level Reflects Disease Activity in Proteinase-3 ANCA-Associated Vasculitis.

    PubMed

    Bertram, Anna; Lovric, Svjetlana; Engel, Alissa; Beese, Michaela; Wyss, Kristin; Hertel, Barbara; Park, Joon-Keun; Becker, Jan U; Kegel, Johanna; Haller, Hermann; Haubitz, Marion; Kirsch, Torsten

    2015-11-01

    ANCA-associated vasculitides are characterized by inflammatory destruction of small vessels accompanied by enhanced cleavage of membrane-bound proteins. One of the main proteases responsible for ectodomain shedding is disintegrin and metalloproteinase domain-containing protein 17 (ADAM17). Given its potential role in aggravating vascular dysfunction, we examined the role of ADAM17 in active proteinase-3 (PR3)-positive ANCA-associated vasculitis (AAV). ADAM17 concentration was significantly increased in plasma samples from patients with active PR3-AAV compared with samples from patients in remission or from other controls with renal nonvascular diseases. Comparably, plasma levels of the ADAM17 substrate syndecan-1 were significantly enhanced in active AAV. We also observed that plasma-derived ADAM17 retained its specific proteolytic activity and was partly located on extracellular microparticles. Transcript levels of ADAM17 were increased in blood samples of patients with active AAV, but those of ADAM10 or tissue inhibitor of metalloproteinases 3, which inhibits ADAMs, were not. We also performed a microRNA (miR) screen and identified miR-634 as significantly upregulated in blood samples from patients with active AAV. In vitro, miR-634 mimics induced a proinflammatory phenotype in monocyte-derived macrophages, with enhanced expression and release of ADAM17 and IL-6. These data suggest that ADAM17 has a prominent role in AAV and might account for the vascular complications associated with this disease.

  3. Circulating ADAM17 Level Reflects Disease Activity in Proteinase-3 ANCA-Associated Vasculitis

    PubMed Central

    Bertram, Anna; Lovric, Svjetlana; Engel, Alissa; Beese, Michaela; Wyss, Kristin; Hertel, Barbara; Park, Joon-Keun; Becker, Jan U.; Kegel, Johanna; Haller, Hermann; Haubitz, Marion

    2015-01-01

    ANCA-associated vasculitides are characterized by inflammatory destruction of small vessels accompanied by enhanced cleavage of membrane-bound proteins. One of the main proteases responsible for ectodomain shedding is disintegrin and metalloproteinase domain-containing protein 17 (ADAM17). Given its potential role in aggravating vascular dysfunction, we examined the role of ADAM17 in active proteinase-3 (PR3)-positive ANCA-associated vasculitis (AAV). ADAM17 concentration was significantly increased in plasma samples from patients with active PR3-AAV compared with samples from patients in remission or from other controls with renal nonvascular diseases. Comparably, plasma levels of the ADAM17 substrate syndecan-1 were significantly enhanced in active AAV. We also observed that plasma-derived ADAM17 retained its specific proteolytic activity and was partly located on extracellular microparticles. Transcript levels of ADAM17 were increased in blood samples of patients with active AAV, but those of ADAM10 or tissue inhibitor of metalloproteinases 3, which inhibits ADAMs, were not. We also performed a microRNA (miR) screen and identified miR-634 as significantly upregulated in blood samples from patients with active AAV. In vitro, miR-634 mimics induced a proinflammatory phenotype in monocyte-derived macrophages, with enhanced expression and release of ADAM17 and IL-6. These data suggest that ADAM17 has a prominent role in AAV and might account for the vascular complications associated with this disease. PMID:25788529

  4. Proteolysis of the endothelial cell protein C receptor by neutrophil proteinase 3

    PubMed Central

    VILLEGAS-MENDEZ, A; MONTES, R; AMBROSE, L R; WARRENS, A N; LAFFAN, M; LANE, D A

    2007-01-01

    Background The endothelial cell protein C receptor (EPCR) presents protein C to the thrombin:thrombomodulin complex on the endothelium of large vessels, and enhances the generation of activated protein C (APC) and activation of protease-activated receptor-1. A previous report has demonstrated binding of soluble (s) EPCR to activated neutrophils via surface proteinase 3 (PR3). Methods We now report further characterization of this interaction. Activated neutrophils and purified PR3 both decrease endothelial cell (EC) surface EPCR, suggestive of its proteolysis. Results When added to purified recombinant sEPCR, PR3 produced multiple cleavages, with early products including 20 kDa N-terminal and C-terminal (after Lys176) fragments. The binding of active site blocked PR3 to sEPCR was studied by surface plasmon resonance. Estimates of the KD of 18.5–102 nm were obtained with heterogeneous binding, suggestive of more than a single interaction site. Conclusions This work demonstrates PR3 binding to and proteolysis of EPCR and suggests a mechanism by which anticoagulant and cell protective pathways can be down-regulated during inflammation. PMID:17459006

  5. Potential Broad Spectrum Inhibitors of the Coronavirus 3CLpro: A Virtual Screening and Structure-Based Drug Design Study.

    PubMed

    Berry, Michael; Fielding, Burtram C; Gamieldien, Junaid

    2015-12-15

    Human coronaviruses represent a significant disease burden; however, there is currently no antiviral strategy to combat infection. The outbreak of severe acute respiratory syndrome (SARS) in 2003 and Middle East respiratory syndrome (MERS) less than 10 years later demonstrates the potential of coronaviruses to cross species boundaries and further highlights the importance of identifying novel lead compounds with broad spectrum activity. The coronavirus 3CL(pro) provides a highly validated drug target and as there is a high degree of sequence homology and conservation in main chain architecture the design of broad spectrum inhibitors is viable. The ZINC drugs-now library was screened in a consensus high-throughput pharmacophore modeling and molecular docking approach by Vina, Glide, GOLD and MM-GBSA. Molecular dynamics further confirmed results obtained from structure-based techniques. A highly defined hit-list of 19 compounds was identified by the structure-based drug design methodologies. As these compounds were extensively validated by a consensus approach and by molecular dynamics, the likelihood that at least one of these compounds is bioactive is excellent. Additionally, the compounds segregate into 15 significantly dissimilar (p < 0.05) clusters based on shape and features, which represent valuable scaffolds that can be used as a basis for future anti-coronaviral inhibitor discovery experiments. Importantly though, the enriched subset of 19 compounds identified from the larger library has to be validated experimentally.

  6. Microstructure and cleavage in lath martensitic steels.

    PubMed

    Morris, John W; Kinney, Chris; Pytlewski, Ken; Adachi, Y

    2013-02-01

    In this paper we discuss the microstructure of lath martensitic steels and the mechanisms by which it controls cleavage fracture. The specific experimental example is a 9Ni (9 wt% Ni) steel annealed to have a large prior austenite grain size, then examined and tested in the as-quenched condition to produce a relatively coarse lath martensite. The microstructure is shown to approximate the recently identified 'classic' lath martensite structure: prior austenite grains are divided into packets, packets are subdivided into blocks, and blocks contain interleaved laths whose variants are the two Kurjumov-Sachs relations that share the same Bain axis of the transformation. When the steel is fractured in brittle cleavage, the laths in the block share {100} cleavage planes and cleave as a unit. However, cleavage cracks deflect or blunt at the boundaries between blocks with different Bain axes. It follows that, as predicted, the block size governs the effective grain size for cleavage.

  7. Microstructure and cleavage in lath martensitic steels

    NASA Astrophysics Data System (ADS)

    Morris, John W., Jr.; Kinney, Chris; Pytlewski, Ken; Adachi, Y.

    2013-02-01

    In this paper we discuss the microstructure of lath martensitic steels and the mechanisms by which it controls cleavage fracture. The specific experimental example is a 9Ni (9 wt% Ni) steel annealed to have a large prior austenite grain size, then examined and tested in the as-quenched condition to produce a relatively coarse lath martensite. The microstructure is shown to approximate the recently identified ‘classic’ lath martensite structure: prior austenite grains are divided into packets, packets are subdivided into blocks, and blocks contain interleaved laths whose variants are the two Kurjumov-Sachs relations that share the same Bain axis of the transformation. When the steel is fractured in brittle cleavage, the laths in the block share {100} cleavage planes and cleave as a unit. However, cleavage cracks deflect or blunt at the boundaries between blocks with different Bain axes. It follows that, as predicted, the block size governs the effective grain size for cleavage.

  8. Studies on Proteinases from Some Blood-Sucking Insects,

    DTIC Science & Technology

    ovinus, and Pediculus humanus, but not in those of Cimex lectularius or Rhodnius prolixus. The trypsin and chymotrypsin have been partially... Cimex and Rhodnius appear to have a high molecular weight proteinase with optimal activity at pH 5 in their midguts. (Author)

  9. Proteinase Inhibitor I Accumulation in Tomato Suspension Cultures 1

    PubMed Central

    Walker-Simmons, Mary; Ryan, Clarence A.

    1986-01-01

    Suspension-cultured cells of tomato accumulate proteinase Inhibitor I as the sucrose is depleted from 1% to less than 0.1% in the culture medium. Inhibitor I can be prematurely induced to accumulate in the cells by the addition to the medium of the proteinase inhibitor inducing factor, trigalacturonic acid, ethylene glycol chitin, or chitosan. In cultures grown in 0.6% initial sucrose with no inducers added, a uronic acid-rich extracellular polysaccharide appears in the medium during growth of the cells. This extracellular polysaccharide apparently contains an `endogenous inducer' of Inhibitor I synthesis. When the partially purified polysaccharide is added to the culture medium, Inhibitor I accumulation is induced. Proteinase inhibitors also accumulate in tobacco and alfalfa suspension-cultured cells as the cell cultures age. As with the tomato cultures, a uronic acid-rich component(s) appears in the media prior to inhibitor accumulation. These data suggest that an endogenous inducer may be activating proteinase inhibitor genes through a similar mechanism in all three types of cells. PMID:16664609

  10. Serine proteinases from barley malt may degrade beta-amylase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Barley seed proteinases are critically important to seed germination and malting in that they generate amino acids from seed N reserves, supporting embryo growth during germination and yeast fermentation during brewing. However, relatively little is known regarding the endogenous protein substrate ...

  11. Phospholipase and proteinase activities of Candida isolates from denture wearers.

    PubMed

    Marcos-Arias, Cristina; Eraso, Elena; Madariaga, Lucila; Aguirre, Jose Manuel; Quindós, Guillermo

    2011-07-01

    The aim of the present study was to characterise phospholipase and proteinase activities of oral Candida isolates from 100 denture wearers and to study the relationship of these activities with denture stomatitis. Of 100 patients studied, 44 suffered from denture stomatitis. Specimens were collected by swabbing the denture and underlying mucosa. Isolates were previously identified by conventional mycological and genotypic methods. The phospholipase and proteinase activities were evaluated by agar plate methods. A total of 152 isolates were recovered from denture and underlying mucosa, including 101 Candida albicans, 18 Candida tropicalis, 14 Candida glabrata, 11 Candida guilliermondii, four Candida parapsilosis, two Saccharomyces cerevisiae and one isolate each of Candida dubliniensis and Candida krusei. Most C. albicans (97%) showed phospholipase activity; furthermore, the unique C. dubliniensis isolate showed a moderate phospholipase activity. The isolation of C. albicans (chi-square test, P = 0.0016) and phospholipase production by Candida spp. (chi-square test, P = 0.0213) was found to be significantly associated with denture stomatitis. Proteinase production was observed in <30% of isolates, and it was not related to the presence of denture stomatitis (P = 0.7675). Candida albicans isolates may produce both virulence factors, although the proteinase production was only observed in <30% of the isolates. Phospholipase production was exclusive of C. albicans and C. dubliniensis.

  12. Digestive proteinases of yellow mealworm (Tenebrio molitor) larvae: purification and characterization of a trypsin-like proteinase.

    PubMed

    Tsybina, T A; Dunaevsky, Y E; Belozersky, M A; Zhuzhikov, D P; Oppert, B; Elpidina, E N

    2005-03-01

    A new trypsin-like proteinase was purified to homogeneity from the posterior midgut of Tenebrio molitor larvae by ion-exchange chromatography on DEAE-Sephadex A-50 and gel filtration on Superdex-75. The isolated enzyme had molecular mass of 25.5 kD and pI 7.4. The enzyme was also characterized by temperature optimum at 55 degrees C, pH optimum at 8.5, and K(m) value of 0.04 mM (for hydrolysis of Bz-Arg-pNA). According to inhibitor analysis the enzyme is a trypsin-like serine proteinase stable within the pH range of 5.0-9.5. The enzyme hydrolyzes peptide bonds formed by Arg or Lys residues in the P1 position with a preference for relatively long peptide substrates. The N-terminal amino acid sequence, IVGGSSISISSVPXQIXLQY, shares 50-72% identity with other insect trypsin-like proteinases, and 44-50% identity to mammalian trypsins. The isolated enzyme is sensitive to inhibition by plant proteinase inhibitors and it can serve as a suitable target for control of digestion in this stored product pest.

  13. Cleavage plane determination in amphibian eggs.

    PubMed

    Sawai, T; Yomota, A

    1990-01-01

    In the present study using eggs of Cynops pyrrhogaster and Xenopus laevis, we examined (1) structural changes in the cytoplasm before the appearance of the cleavage furrow using a cytochemical method, (2) the time of cleavage plane determination depending on the mitotic apparatus (MA), by changing the shape of the eggs, and (3) the time of arrival of the "cleavage stimulus" at the cortex, by injecting colchicine solution or removing cytoplasm. Results were as follows: (1) In amphibian eggs the diastema was formed after development of the MA, appearing between the two asters after the MA had begun to degenerate. (2) The cleavage plane was preliminarily determined by the MA in the meta- to anaphase of karyokinesis. At this time, however, the egg cortex had not yet received the "cleavage stimulus" indispensable for furrow formation. (3) The egg cortex was really prepared to establish the furrow just after the edge of the diastema arrived at the cortex, when the MA had already degenerated. These results imply that the cleavage plane of the amphibian eggs is determined in two steps: the first, depending on the MA, is the determination of the direction of the growth of the diastema, and the second is the arrival of the "cleavage stimulus" at the cortex in association with the diastema.

  14. Immunodetection of thiol proteinase levels in various populations of Artemia cysts and during development.

    PubMed

    Lu, J; Warner, A H

    1991-01-01

    An immunodetection assay on Western blots has been used to determine the thiol proteinase content and composition in cysts from 12 populations of the brine shrimp Artemia. Our results showed no differences in the subunit composition of the thiol proteinase among cysts from eight bisexual strains and four parthenogenic strains, and confirmed an earlier finding that the proteinase is composed of two subunits of 25.9 and 31.5 kilodaltons. In contrast, we found that Artemia cysts from parthenogenic strains contain 17.1 ng/cyst of the thiol proteinase, while cysts from bisexual strains contain 8.2 ng/cyst of the thiol proteinase. Also, there was a good linear correlation (r = 0.863; p less than 0.001) between the thiol proteinase content and cyst mass. Embryo fractionation experiments showed that 82% of the thiol proteinase was in the cytosol, while 14 and 4%, respectively, were in the nuclei/yolk platelets and mitochondria/lysosome fractions. Measurements of the thiol proteinase content of developing Artemia embryos showed that the proteinase content was relatively constant during early development, suggesting that the activity of the thiol proteinase gene(s) may be constitutive and not developmentally regulated in Artemia embryos.

  15. Cleavage at Arg-1689 influences heavy chain cleavages during thrombin-catalyzed activation of factor VIII.

    PubMed

    Newell, Jennifer L; Fay, Philip J

    2009-04-24

    The procofactor, factor VIII, is activated by thrombin or factor Xa-catalyzed cleavage at three P1 residues: Arg-372, Arg-740, and Arg-1689. The catalytic efficiency for thrombin cleavage at Arg-740 is greater than at either Arg-1689 or Arg-372 and influences reaction rates at these sites. Because cleavage at Arg-372 appears rate-limiting and dependent upon initial cleavage at Arg-740, we investigated whether cleavage at Arg-1689 influences catalysis at this step. Recombinant B-domainless factor VIII mutants, R1689H and R1689Q were prepared and stably expressed to slow and eliminate cleavage, respectively. Specific activity values for the His and Gln mutations were approximately 50 and approximately 10%, respectively, that of wild type. Thrombin activation of the R1689H variant showed an approximately 340-fold reduction in the rate of Arg-1689 cleavage, whereas the R1689Q variant was resistant to thrombin cleavage at this site. Examination of heavy chain cleavages showed approximately 4- and 11-fold reductions in A2 subunit generation and approximately 3- and 7-fold reductions in A1 subunit generation for the R1689H and R1689Q mutants, respectively. These results suggest a linkage between light chain cleavage and cleavages in heavy chain. Results obtained evaluating proteolysis of the factor VIII mutants by factor Xa revealed modest rate reductions (<5-fold) in generating A2 and A1 subunits and in cleaving light chain at Arg-1721 from either variant, suggesting little dependence upon prior cleavage at residue 1689 as compared with thrombin. Overall, these results are consistent with a competition between heavy and light chains for thrombin exosite binding and subsequent proteolysis with binding of the former chain preferred.

  16. Ozone inactivation of human alpha 1-proteinase inhibitor

    SciTech Connect

    Johnson, D.A.

    1980-06-01

    Ozone decreased the trypsin, chymotrypsin, and elastase inhibitory activities of human alpha 1-proteinase inhibitor both in plasma and in solutions of the pure inhibitor. The total loss of porcine elastase inhibitory activity required 18 mol of ozone/mol of pure alpha 1-PI and approximately 850 mol of ozone/mol of alpha 1-PI in plasma. A corresponding loss of the ability to inhibit human leukocyte elastase was observed. Inactivated alpha 1-PI contains four residues of methionine sulfoxide, in addition to oxidized tryosine and tryptophan. Electrophoretic analysis demonstrated that the ozone-inactivated alpha 1-PI did not form normal complexes with serine proteinases. These findings suggest that the inhalation of ozone could inactivate alpha 1-PI on the airspace side of the lung to create a localized alpha 1-PI deficiency, which might contribute to the development of emphysema.

  17. Cleavage sites in the polypeptide precursors of poliovirus protein P2-X

    SciTech Connect

    Selmer, B.L.; Hanecak, R.; Anderson, C.W.; Wimmer, E.

    1981-01-01

    Partial amino-terminal sequence analysis has been performed on the three major polypeptide products (P2-3b, P2-5b, and P2-X) from the central region (P2) of the poliovirus polyprotein, and this analysis precisely locates the amino termini of these products with respect to the nucleotide sequence of the poliovirus RNA genome. Like most of the products of the replicase region (P3), the amino termini of P2-5b and P2-X are generated by cleavage between glutamine and glycine residues. Thus, P2-5b and P2-X are probably both produced by the action of a singly (virus-encoded.) proteinase. The amino terminus of P2-3b, on the other hand, is produced by a cleavage between the carboxy-terminal tyrosine of VP1 and the glycine encoded by nucleotides 3381-3383. This result may suggest that more than one proteolytic activity is required for the complete processing of the poliovirus polyprotein.

  18. CP30, a Cysteine Proteinase Involved in Trichomonas vaginalis Cytoadherence

    PubMed Central

    Mendoza-López, M. Remedios; Becerril-Garcia, Cecilia; Fattel-Facenda, Loriz V.; Avila-Gonzalez, Leticia; Ruíz-Tachiquín, Martha E.; Ortega-Lopez, Jaime; Arroyo, Rossana

    2000-01-01

    We describe here the participation of a Trichomonas vaginalis 30-kDa proteinase (CP30) with affinity to the HeLa cell surface in attachment of this parasite to host epithelial cells. The CP30 band is a cysteine proteinase because its activity was inhibited by E-64, a thiol proteinase inhibitor. In two-dimensional substrate gel electrophoresis of total extracts of the trichomonad isolate CNCD 147, three spots with proteolytic activity were detected in the 30-kDa region, in the pI range from 4.5 to 5.5. Two of the spots (pI 4.5 and 5.0) bound to the surfaces of fixed HeLa cells corresponding to the CP30 band. The immunoglobulin G fraction of the rabbit anti-CP30 antiserum that recognized a 30-kDa band by Western blotting and immunoprecipitated CP30 specifically inhibited trichomonal cytoadherence to HeLa cell monolayers in a concentration-dependent manner and reacted with CP30 at the parasite surface. CP30 degraded proteins found on the female urogenital tract, including fibronectin, collagen IV, and hemoglobin. Interestingly, CP30 digested fibronectin and collagen IV only at pH levels between 4.5 and 5.0. Moreover, trichomonosis patients whose diagnosis was confirmed by in vitro culture possessed antibody to CP30 in both sera and vaginal washes, and CP30 activity was found in vaginal washes. Our results suggest that surface CP30 is a cysteine proteinase necessary for trichomonal adherence to human epithelial cells. PMID:10948104

  19. Dental Enamel Development: Proteinases and Their Enamel Matrix Substrates

    PubMed Central

    Bartlett, John D.

    2013-01-01

    This review focuses on recent discoveries and delves in detail about what is known about each of the proteins (amelogenin, ameloblastin, and enamelin) and proteinases (matrix metalloproteinase-20 and kallikrein-related peptidase-4) that are secreted into the enamel matrix. After an overview of enamel development, this review focuses on these enamel proteins by describing their nomenclature, tissue expression, functions, proteinase activation, and proteinase substrate specificity. These proteins and their respective null mice and human mutations are also evaluated to shed light on the mechanisms that cause nonsyndromic enamel malformations termed amelogenesis imperfecta. Pertinent controversies are addressed. For example, do any of these proteins have a critical function in addition to their role in enamel development? Does amelogenin initiate crystallite growth, does it inhibit crystallite growth in width and thickness, or does it do neither? Detailed examination of the null mouse literature provides unmistakable clues and/or answers to these questions, and this data is thoroughly analyzed. Striking conclusions from this analysis reveal that widely held paradigms of enamel formation are inadequate. The final section of this review weaves the recent data into a plausible new mechanism by which these enamel matrix proteins support and promote enamel development. PMID:24159389

  20. Biochemical characterization of Acacia schweinfurthii serine proteinase inhibitor.

    PubMed

    Odei-Addo, Frank; Frost, Carminita; Smith, Nanette; Ogawa, Tomohisa; Muramoto, Koji; Oliva, Maria Luiza Vilela; Gráf, László; Naude, Ryno

    2014-10-01

    One of the many control mechanisms of serine proteinases is their specific inhibition by protein proteinase inhibitors. An extract of Acacia schweinfurthii was screened for potential serine proteinase inhibition. It was successfully purified to homogeneity by precipitating with 80% (v/v) acetone and sequential chromatographic steps, including ion-exchange, affinity purification and reversed-phase high performance liquid chromatography. Reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis conditions revealed an inhibitor (ASTI) consisting of two polypeptide chains A and B of approximate molecular weights of 16 and 10 kDa, respectively, and under non-reducing conditions, 26 kDa was observed. The inhibitor was shown to inhibit bovine trypsin (Ki of 3.45 nM) at an approximate molar ratio of inhibitor:trypsin (1:1). The A- and B-chains revealed complete sequences of 140 and 40 amino acid residues, respectively. Sequence similarity (70%) was reported between ASTI A-chain and ACTI A-chain (Acacia confusa) using ClustalW. The B-chain produced a 76% sequence similarity between ASTI and Leucaena leucocephala trypsin inhibitor.

  1. Intracellular localization and trafficking of serine proteinase AhSub and cysteine proteinase AhCP of Acanthamoeba healyi.

    PubMed

    Moon, E-K; Lee, S-T; Chung, D-I; Kong, H-H

    2006-01-01

    Proteinases have been proposed to play important roles in pathogenesis and various biologic actions in Acanthamoeba. Although genetic characteristics of several proteases of Acanthamoeba have been reported, the intracellular localization and trafficking of these enzymes has yet to be studied. In the present study, we analyzed the intracellular localization and trafficking of two proteinases, AhSub and AhCP, of Acanthamoeba healyi by transient transfection. Full-length AhSub-enhanced green fluorescent protein (EGFP) fusion protein was found in intracellular vesicle-like structures of transfected amoebae. Time-lapse photographs confirmed the secretion of the fluorescent material of the vesicle toward the extracellular space. The mutated AhSub, of which the pre or prepro region was deleted, was found to localize diffusely throughout the cytoplasm of the amoeba rather than concentrated in the secretory vesicle. Transfection of the construct containing the pre region only showed the same localization and trafficking of the full-length AhSub. A cysteine proteinase AhCP-EGFP fusion protein showed similar localization in the vesicle-like structure in the amoeba. However, using Lyso Tracker analysis, these vesicular structures of AhCP were confirmed to be lysosomes rather than secretory vesicles. The AhCP construct with a deletion of the prepro region showed a dispersed distribution of fluorescence in the cytoplasm of the cells. These results indicated that AhSub and AhCP would play different roles in Acanthameoba biology and that the pre region of AhSub and pro region of AhCP are important for proper intracellular localization and trafficking of each proteinase.

  2. Antiviral cytokines induce hepatic expression of the granzyme B inhibitors, proteinase inhibitor 9 and serine proteinase inhibitor 6.

    PubMed

    Barrie, Mahmoud B; Stout, Heather W; Abougergi, Marwan S; Miller, Bonnie C; Thiele, Dwain L

    2004-05-15

    Expression of the granzyme B inhibitors, human proteinase inhibitor 9 (PI-9), or the murine orthologue, serine proteinase inhibitor 6 (SPI-6), confers resistance to CTL or NK killing by perforin- and granzyme-dependent effector mechanisms. In light of prior studies indicating that virally infected hepatocytes are selectively resistant to this CTL effector mechanism, the present studies investigated PI-9 and SPI-6 expression in hepatocytes and hepatoma cells in response to adenoviral infection and to cytokines produced during antiviral immune responses. Neither PI-9 nor SPI-6 expression was detected by immunoblotting in uninfected murine or human hepatocytes. Similarly, human Huh-7 hepatoma cells were found to express only very low levels of PI-9 relative to levels detected in perforin- and granzyme-resistant CTL or lymphokine-activated killer cells. Following in vivo adenoviral infection or in vitro culture with IFN-alphabeta or IFN-gamma, SPI-6 expression was induced in murine hepatocytes. Similarly, after culture with IFN-alpha, induction of PI-9 mRNA and protein expression was observed in human hepatocytes and Huh-7 cells. IFN-gamma and TNF-alpha also induced 4- to 10-fold higher levels of PI-9 mRNA expression in Huh-7 cells, whereas levels of mRNA encoding a related serine proteinase inhibitor, proteinase inhibitor 8, were unaffected by culture of Huh-7 cells with IFN-alpha, IFN-gamma, or TNF-alpha. These findings indicate that cytokines that promote antiviral cytopathic responses also regulate expression of the cytoprotective molecules, PI-9 and SPI-6, in hepatocytes that are potential targets of CTL and NK effector mechanisms.

  3. Characterization of a novel Kazal-type serine proteinase inhibitor of Arabidopsis thaliana.

    PubMed

    Pariani, Sebastián; Contreras, Marisol; Rossi, Franco R; Sander, Valeria; Corigliano, Mariana G; Simón, Francisco; Busi, María V; Gomez-Casati, Diego F; Pieckenstain, Fernando L; Duschak, Vilma G; Clemente, Marina

    2016-04-01

    Many different types of serine proteinase inhibitors have been involved in several kinds of plant physiological processes, including defense mechanisms against phytopathogens. Kazal-type serine proteinase inhibitors, which are included in the serine proteinase inhibitor family, are present in several organisms. These proteins play a regulatory role in processes that involve serine proteinases like trypsin, chymotrypsin, thrombin, elastase and/or subtilisin. In the present work, we characterized two putative Kazal-type serine proteinase inhibitors from Arabidopsis thaliana, which have a single putative Kazal-type domain. The expression of these inhibitors is transiently induced in response to leaf infection by Botrytis cinerea, suggesting that they play some role in defense against pathogens. We also evaluated the inhibitory specificity of one of the Kazal-type serine proteinase inhibitors, which resulted to be induced during the local response to B. cinerea infection. The recombinant Kazal-type serine proteinase inhibitor displayed high specificity for elastase and subtilisin, but low specificity for trypsin, suggesting differences in its selectivity. In addition, this inhibitor exhibited a strong antifungal activity inhibiting the germination rate of B. cinerea conidia in vitro. Due to the important role of proteinase inhibitors in plant protection against pathogens and pests, the information about Kazal-type proteinase inhibitors described in the present work could contribute to improving current methods for plant protection against pathogens.

  4. The effect of nitrogen and carbon sources on proteinase production by Pseudomonas fluorescens.

    PubMed

    Fairbairn, D J; Law, B A

    1987-02-01

    Some factors influencing the production of an extracellular proteinase by Pseudomonas fluorescens NCDO 2085 were studied. Proteinase production was optimal at 20 degrees C and pH 6.9 in static culture when calcium was included in the medium. Proteinase was not detectable in basal medium but could be induced by organic nitrogen compounds. The proteinase was produced in the exponential phase of growth on protein substrates but not until early stationary phase during growth on amino acids. The organism did not utilize lactose, the most abundant carbohydrate in milk. Citrate was readily utilized as an energy source but had a strong repressive effect on proteinase production. A medium containing sodium caseinate and pyruvate supported good growth and enzyme production. All the amino acids utilized as a sole carbon source, with the exception of serine, could induce proteinase production. Asparagine was the most effective amino acid inducer. Particular combinations of amino acids could induce or repress proteinase production. The regulation of proteinase production by Ps. fluorescens NCDO 2085 appears to be based on a balance between induction by low concentrations of low molecular weight degradation products and sensitivity to end product catabolite repression. The results suggest that the function of the proteinase is to ensure a supply of carbon rather than amino acids for protein synthesis.

  5. Disruption of the endothelial barrier by proteases from the bacterial pathogen Pseudomonas aeruginosa: implication of matrilysis and receptor cleavage.

    PubMed

    Beaufort, Nathalie; Corvazier, Elisabeth; Mlanaoindrou, Saouda; de Bentzmann, Sophie; Pidard, Dominique

    2013-01-01

    , pseudomonal LasB thus appears to induce endothelial anoikis not only via matrilysis, as observed for many pro-apoptotic proteinases, but also via cleavage of some essential cell-to-cell and cell-to-matrix adhesion receptors implicated in the maintenance of the endothelial barrier.

  6. Does Cleavage Work at Work? Men, but Not Women, Falsely Believe Cleavage Sells a Weak Product

    ERIC Educational Resources Information Center

    Glick, Peter; Chrislock, Karyna; Petersik, Korinne; Vijay, Madhuri; Turek, Aleksandra

    2008-01-01

    We examined whether men, but not women, would be distracted by a female sales representative's exposed cleavage, leading to greater perceived efficacy for a weak, but not for a strong product. A community sample of 88 men and 97 women viewed a video of a female pharmaceutical sales representative who (a) had exposed cleavage or dressed modestly…

  7. Selective cleavage of pepsin by molybdenum metallopeptidase

    SciTech Connect

    Yenjai, Sudarat; Malaikaew, Pinpinat; Liwporncharoenvong, Teerayuth; Buranaprapuk, Apinya

    2012-03-02

    Graphical abstract: Molybdenum metallopeptidase: the Mo(VI) cluster with six molybdenum cations has the ability to cleave protein under mild conditions (37 Degree-Sign C, pH 7) without reducing agents. The reaction required only low concentration of ammonium heptamolybdatetetrahydrate ((NH{sub 4}){sub 6}Mo{sub 7}O{sub 24}{center_dot}4H{sub 2}O) (0.125 mM). The reaction undergoes possibly via a hydrolytic mechanism. This is the first demonstration of protein cleavage by a molybdenum cluster. Highlights: Black-Right-Pointing-Pointer This is the first demonstration of protein cleavage by a Mo(VI) cluster with six molybdenum cations. Black-Right-Pointing-Pointer The cleavage reaction undergoes at mild conditions. Black-Right-Pointing-Pointer No need of reducing agents. Black-Right-Pointing-Pointer Only low concentration of Mo(VI) cluster and short time of incubation are needed. -- Abstract: In this study, the cleavage of protein by molybdenum cluster is reported for the first time. The protein target used is porcine pepsin. The data presented in this study show that pepsin is cleaved to at least three fragments with molecular weights of {approx}23, {approx}19 and {approx}16 kDa when the mixture of the protein and ammonium heptamolybdate tetrahydrate ((NH{sub 4}){sub 6}Mo{sub 7}O{sub 24}{center_dot}4H{sub 2}O) was incubated at 37 Degree-Sign C for 24 h. No self cleavage of pepsin occurs at 37 Degree-Sign C, 24 h indicating that the reaction is mediated by the metal ions. N-terminal sequencing of the peptide fragments indicated three cleavage sites of pepsin between Leu 112-Tyr 113, Leu 166-Leu 167 and Leu 178-Asn 179. The cleavage reaction occurs after incubation of the mixture of pepsin and (NH{sub 4}){sub 6}Mo{sub 7}O{sub 24}{center_dot}4H{sub 2}O) only for 2 h. However, the specificity of the cleavage decreases when incubation time is longer than 48 h. The mechanism for cleavage of pepsin is expected to be hydrolytic chemistry of the amide bonds in the protein

  8. Structural basis of cohesin cleavage by separase

    PubMed Central

    Lin, Zhonghui; Luo, Xuelian; Yu, Hongtao

    2016-01-01

    Accurate chromosome segregation requires timely dissolution of chromosome cohesion after chromosomes are properly attached to the mitotic spindle. Separase is absolutely essential for cohesion dissolution in organisms from yeast to man1,2. It cleaves the kleisin subunit of cohesin and opens the cohesin ring to allow chromosome segregation. Cohesin cleavage is spatiotemporally controlled by separase-associated regulatory proteins, including the inhibitory chaperone securin3–6, and by phosphorylation of both the enzyme and substrates7–12. Dysregulation of this process causes chromosome missegregation and aneuploidy, contributing to cancer and birth defects. Despite its essential functions, atomic structures of separase have not been determined. Here, we report crystal structures of the separase protease domain from Chaetomium thermophilum, alone or covalently bound to unphosphorylated and phosphorylated inhibitory peptides derived from a cohesin cleavage site. These structures reveal how separase recognizes cohesin and how cohesin phosphorylation by polo-like kinase 1 (Plk1) enhances cleavage. Consistent with a previous cellular study13, mutating two securin residues in a conserved motif that partially matches the separase cleavage consensus converts securin from a separase inhibitor to a substrate. Our study establishes atomic mechanisms of substrate cleavage by separase and suggests competitive inhibition by securin. PMID:27027290

  9. In vitro activation of the rhesus macaque myeloid alpha-defensin precursor proRMAD-4 by neutrophil serine proteinases.

    PubMed

    Kamdar, Karishma; Maemoto, Atsuo; Qu, Xiaoqing; Young, Steven K; Ouellette, André J

    2008-11-21

    Alpha-defensins are mammalian antimicrobial peptides expressed mainly by cells of myeloid lineage or small intestinal Paneth cells. The peptides are converted from inactive 8.5-kDa precursors to membrane-disruptive forms by post-translational proteolytic events. Because rhesus myeloid pro-alpha-defensin-4 (proRMAD-4((20-94))) lacks bactericidal peptide activity in vitro, we tested whether neutrophil azurophil granule serine proteinases, human neutrophil elastase (NE), cathepsin G (CG), and proteinase-3 (P3) have in vitro convertase activity. Only NE cleaved proRMAD-4((20-94)) at the native RMAD-4 N terminus to produce fully processed, bactericidal RMAD-4((62-94)). The final CG cleavage product was RMAD-4((55-94)), and P3 produced both RMAD-4((55-94)) and RMAD-4(57-94). Nevertheless, NE, CG, and P3 digests of proRMAD4 and purified RMAD-4((62-94)), RMAD-4((55-94)), and RMAD-4(57-94) peptides had equivalent in vitro bactericidal activities. Bactericidal peptide activity assays of proRMAD-4((20-94)) variants containing complete charge-neutralizing D/E to N/Q or D/E to A substitutions showed that (DE/NQ)-proRMAD-4((20-94)) and (DE/A)-proRMAD-4((20-94)) were as active as mature RMAD-4((62-94)). Therefore, proregion Asp and Glu side chains inhibit the RMAD-4 component of full-length proRMAD-4((20-94)), perhaps by a combination of charge-neutralizing and hydrogen-bonding interactions. Although native RMAD-4((62-94)) resists NE, CG, and P3 proteolysis completely, RMAD-4((62-94)) variants with disulfide pairing disruptions or lacking disulfide bonds were degraded extensively, evidence that the disulfide array protects the alpha-defensin moiety from degradation by the myeloid converting enzymes. These in vitro analyses support the conclusion that rhesus macaque myeloid pro-alpha-defensins are converted to active forms by serine proteinases that co-localize in azurophil granules.

  10. Nanomechanical cleavage of molybdenum disulphide atomic layers.

    PubMed

    Tang, Dai-Ming; Kvashnin, Dmitry G; Najmaei, Sina; Bando, Yoshio; Kimoto, Koji; Koskinen, Pekka; Ajayan, Pulickel M; Yakobson, Boris I; Sorokin, Pavel B; Lou, Jun; Golberg, Dmitri

    2014-04-03

    The discovery of two-dimensional materials became possible due to the mechanical cleavage technique. Despite its simplicity, the as-cleaved materials demonstrated surprising macro-continuity, high crystalline quality and extraordinary mechanical and electrical properties that triggered global research interest. Here such cleavage processes and associated mechanical behaviours are investigated by a direct in situ transmission electron microscopy probing technique, using atomically thin molybdenum disulphide layers as a model material. Our technique demonstrates layer number selective cleavage, from a monolayer to double layer and up to 23 atomic layers. In situ observations combined with molecular dynamics simulations reveal unique layer-dependent bending behaviours, from spontaneous rippling (<5 atomic layers) to homogeneous curving (~ 10 layers) and finally to kinking (20 or more layers), depending on the competition of strain energy and interfacial energy.

  11. Nanomechanical cleavage of molybdenum disulphide atomic layers

    NASA Astrophysics Data System (ADS)

    Tang, Dai-Ming; Kvashnin, Dmitry G.; Najmaei, Sina; Bando, Yoshio; Kimoto, Koji; Koskinen, Pekka; Ajayan, Pulickel M.; Yakobson, Boris I.; Sorokin, Pavel B.; Lou, Jun; Golberg, Dmitri

    2014-04-01

    The discovery of two-dimensional materials became possible due to the mechanical cleavage technique. Despite its simplicity, the as-cleaved materials demonstrated surprising macro-continuity, high crystalline quality and extraordinary mechanical and electrical properties that triggered global research interest. Here such cleavage processes and associated mechanical behaviours are investigated by a direct in situ transmission electron microscopy probing technique, using atomically thin molybdenum disulphide layers as a model material. Our technique demonstrates layer number selective cleavage, from a monolayer to double layer and up to 23 atomic layers. In situ observations combined with molecular dynamics simulations reveal unique layer-dependent bending behaviours, from spontaneous rippling (<5 atomic layers) to homogeneous curving (~ 10 layers) and finally to kinking (20 or more layers), depending on the competition of strain energy and interfacial energy.

  12. Cleavage fracture in bainitic and martensitic microstructures

    SciTech Connect

    Zhang, X.Z.; Knott, J.F.

    1999-09-29

    This paper addresses the mechanisms of cleavage fracture in the pressure-vessel steel A533B. Microstructures of single bainite microstructures exhibit a higher propensity for brittle cleavage fracture than do those of auto-tempered martensites. The K{sub 1c} values of mixed microstructures are determined by the statistical distribution of the two phases and the range of the values is bounded by limits set by those for the single-phase microstructures. The results are explained in terms of the RKR model, which involves a local cleavage stress {sigma}*{sub F} and a distance ahead of the macrocrack tip, X, as two critical parameters. It is found that the carbides or carbide colonies act as critical microcrack nuclei, and hence play a key role in determining the fracture toughness, although packet boundaries in bainite may give rise to pop-in arrests in displacement-controlled tests.

  13. Limited caspase cleavage of human BAP31.

    PubMed

    Määttä, J; Hallikas, O; Welti, S; Hildén, P; Schröder, J; Kuismanen, E

    2000-11-10

    Human BAP31 was cleaved at both of its two identical caspase cleavage sites in two previously reported models of apoptosis. We show here that only the most carboxy-terminal site is cleaved during apoptosis induced in HeLa cells by tunicamycin, tumor necrosis factor and cycloheximide, or staurosporine. Similar results were obtained in HL-60 cells using Fas/APO-1 antibodies, or cycloheximide. This limited cleavage, which is inhibited by several caspase inhibitors, removes eight amino acids from human BAP31 including the KKXX coat protein I binding motif. Ectopic expression of the resulting cleavage product induces redistribution of mannosidase II from the Golgi and prevents endoplasmic reticulum to Golgi transport of virus glycoproteins.

  14. Validation of a mutant of the pore-forming toxin sticholysin-I for the construction of proteinase-activated immunotoxins.

    PubMed

    Pentón, David; Pérez-Barzaga, Victor; Díaz, Iscel; Reytor, Mey L; Campos, Javier; Fando, Rafael; Calvo, Loany; Cilli, Eduardo M; Morera, Vivian; Castellanos-Serra, Lila R; Pazos, Fabiola; Lanio, María E; Alvarez, Carlos; Pons, Tirso; Tejuca, Mayra

    2011-06-01

    The use of pore-forming toxins from sea anemones (actinoporins) in the construction of immunotoxins (ITs) against tumour cells is an alternative for cancer therapy. However, the main disadvantage of actinoporin-based ITs obtained so far has been the poor cellular specificity associated with the toxin's ability to bind and exert its activity in almost any cell membrane. Our final goal is the construction of tumour proteinase-activated ITs using a cysteine mutant at the membrane binding region of sticholysin-I (StI), a cytolysin isolated from the sea anemone Stichodactyla helianthus. The mutant and the ligand moiety would be linked by proteinase-sensitive peptides through the StI cysteine residue blocking the toxin binding region and hence the IT non-specific killing activity. To accomplish this objective the first step was to obtain the mutant StI W111C, and to evaluate the impact of mutating tryptophan 111 by cysteine on the toxin pore-forming capacity. After proteolysis of the cleavage sequence, a short peptide would remain attached to the toxin. The next step was to evaluate whether this mutant is able to form pores even with a residual peptide linked to cysteine 111. In this work we demonstrated that (i) StI W111C shows pore-forming capacity in a nanomolar range, although it is 8-fold less active than the wild-type recombinant StI, corroborating the previously reported importance of residue 111 for the binding of StI to membranes, and (ii) the mutant is able to form pores even with a residual seven-residue peptide linked to cysteine 111. In addition, it was demonstrated that binding of a large molecule to cysteine 111 renders an inactive toxin that is no longer able to bind to the membrane. These results validate the mutant StI W111C for its use in the construction of tumour proteinase-activated ITs.

  15. The role of fungal proteinases in pathophysiology of Stachybotrys chartarum.

    PubMed

    Yike, Iwona; Rand, Thomas; Dearborn, Dorr G

    2007-10-01

    The adverse health effects of Stachybotrys chartarum have often been linked to exposure to the trichothecene mycotoxins. Recent studies have shown that in addition to mycotoxins this fungus is capable of producing and secreting in vivo proteins such as hemolysins and proteinases. Spore extracts obtained from a high trichothecene producing isolate JS 58-17 exhibited a significantly lower proteolytic activity compared to the low trichothecene producer, JS 58-06. Growing isolates on rice or potato dextrose agar results in higher proteolytic activity of the spores compared to those grown on drywall. Proteinases in the spore extracts can hydrolyze gelatin and collagen I and IV. Analysis of zymograms shows the presence of several proteins with proteolytic activity in the spores of S. chartarum. Human tracheal epithelial cells exposed to spore extracts produced significantly higher levels of IL-6, IL-8, and TNF-alpha than control cells. This stimulation of cytokine production was completely abolished by Pefabloc, a serine protease inhibitor. Neutrophil numbers and proinflammatory cytokine (IL1-beta and TNF-alpha) concentrations were highly elevated in the lungs of 7 day old rat pups exposed intratracheally to 4 x 10(4) spores/gm body weight compared to control. No significant differences in those inflammatory indices in vivo were noted between the treatments with the high trichothecene producer, isolate JS 58-17 and JS 58-06, which does not produce macrocyclic trichothecenes. Immunohistochemistry revealed reduced collagen IV labeling in spore-induced lung granulomas in rat pups exposed to both isolates. These results suggest that proteinases from S. chartarum spores significantly contribute to lung inflammation and injury.

  16. Purification and characterization of an alpha-macroglobulin proteinase inhibitor from the mollusc Octopus vulgaris.

    PubMed Central

    Thøgersen, I B; Salvesen, G; Brucato, F H; Pizzo, S V; Enghild, J J

    1992-01-01

    The cell-free haemolymph of the mollusc Octopus vulgaris inhibited the proteolytic activity of the thermolysin against the high-molecular-mass substrate hide powder azure. The purified inhibitor was a glycoprotein composed of two identical 180 kDa disulphide-linked subunits. In addition to the inhibition of the metalloproteinase thermolysin, the protein inhibited the serine proteinases human neutrophil elastase, pig pancreatic elastase, bovine chymotrypsin, bovine trypsin and the cysteine proteinase papain. A fraction of the proteinase-inhibitor complex resisted dissociation after denaturation indicating that some of the proteinase molecules became covalently bound. The nucleophile beta-aminopropionitrile decreased the covalent binding of proteinases to the Octopus vulgaris protein, suggesting that this interaction is mediated by an internal thiol ester; the reactivity and the amino acid sequence flanking the reactive residues of the putative thiol ester were consistent with this hypothesis. Bound trypsin remained active against the low-molecular-mass chromatogenic substrate H-D-Pro-Phe-Arg p-nitroanilide and was protected from inhibition by active-site-directed protein inhibitors of trypsin; however, the bound trypsin was readily inhibited by small synthetic inhibitors. This indicates that the inhibition of proteinases is accomplished by steric hindrance. The proteinase-inhibitory activity of this protein is characteristic of inhibition by mammalian alpha-macroglobulins and the presence of a putative thiol ester suggests that the Octopus vulgaris proteinase inhibitor is a homologue of human alpha 2-macroglobulin. Images Fig. 1. Fig. 2. Fig. 3. Fig. 5. PMID:1379044

  17. Autoantibodies against the multicatalytic proteinase in patients with systemic lupus erythematosus

    PubMed Central

    1991-01-01

    Sera from patients with systemic lupus erythematosus contain specific autoantibodies directed against different polypeptide components of the multicatalytic proteinase (also known as proteasome or prosome). These human autoantibodies, in contrast to polyclonal antibodies obtained in rabbits against the purified enzyme, recognize highly conserved epitopes of the multicatalytic proteinase polypeptides from yeast to human. PMID:1703207

  18. Antibody in sera of patients infected with Trichomonas vaginalis is to trichomonad proteinases.

    PubMed Central

    Alderete, J F; Newton, E; Dennis, C; Neale, K A

    1991-01-01

    BACKGROUND--A recent report demonstrated the immunogenic character of the cysteine proteinases of Trichomonas vaginalis. It was of interest, therefore, to examine for the presence of serum anti-proteinase antibody among patients with trichomoniasis. METHODS--An immunoprecipitation assay was used involving protein A-bearing Staphylococcus aureus first coated with the IgG fraction of goat anti-human Ig and then mixed with individual sera of patients to bind human antibody. These antibody-coated bacteria were then added to detergent extracts of T vaginalis. Bound immune complexes on S aureus were washed and solubilised for electrophoretic analysis on acrylamide copolymerised with gelatin for detection of proteinase activity. RESULTS--Sera from patients (50/50), but none from sera of normal, uninfected women, possessed IgG to numerous trichomonad cysteine proteinases. The presence of this serum anti-proteinase antibody disappeared after drug treatment and cure of patients of the T vaginalis infection. CONCLUSIONS--The commonality of the anti-proteinase antibody in the sera of patients with trichomoniasis provided evidence for the expression of the same repertoire of parasite proteinases during infection. These observations have important implications for the in vivo relevance of the proteinases and indicate that strategies to use a specific serum antibody response for diagnosis of this infection may be possible. Images PMID:1916796

  19. Roles for proteinases in the pathogenesis of chronic obstructive pulmonary disease

    PubMed Central

    Owen, Caroline A

    2008-01-01

    Since the early 1960s, a compelling body of evidence has accumulated to show that proteinases play critical roles in airspace enlargement in chronic obstructive pulmonary disease (COPD). However, until recently the causative enzymes and their exact roles in pathologic processes in COPD have not been clear. Recent studies of gene-targeted mice in murine models of COPD have confirmed roles for proteinases not only in airspace enlargement, but also in airway pathologies in COPD. These studies have also shed light on the specific proteinases involved in COPD pathogenesis, and the mechanisms by which these proteinases injure the lung. They have also identified important interactions between different classes of proteinases, and between proteinases and other molecules that amplify lung inflammation and injury. This review will discuss the biology of proteinases and the mechanisms by which they contribute to the pathogenesis of COPD. In addition, I will discuss the potential of proteinase inhibitors and anti-inflammatory drugs as new treatment strategies for COPD patients. PMID:18686734

  20. The 2.5 A X-ray crystal structure of the acid-stable proteinase inhibitor from human mucous secretions analysed in its complex with bovine alpha-chymotrypsin.

    PubMed Central

    Grütter, M G; Fendrich, G; Huber, R; Bode, W

    1988-01-01

    Orthorhombic crystals of the complex formed between bovine alpha-chymotrypsin and a recombinant human mucous proteinase inhibitor (SLPI) were grown. Data to 2.3 A resolution were collected on the area-detector diffractometer FAST. The crystal structure of the complex was solved by Patterson search techniques using chymotrypsin as a search model. A cyclic procedure of modeling and crystallographic refinement enabled the determination of the SLPI structure. The current crystallographic R-value is 0.19. SLPI has a boomerang-like shape with both wings comprising two well separated domains of similar architecture. In each domain the polypeptide chain is arranged like a stretched spiral. Two internal strands form a regular beta-hairpin loop which is accompanied by two external strands linked by the proteinase binding segment. The polypeptide segment of each domain is interconnected by four disulfide bridges with a connectivity pattern hitherto unobserved. The reactive site loop of the second domain has elastase and chymotrypsin binding properties. It contains the scissile peptide bond between Leu72I and Met73I and has a similar conformation to that observed in other serine proteinase protein inhibitors. Eight residues of this loop, two of the adjacent hairpin loop, the C-terminal segment and Trp30I are in direct contact with the cognate enzyme. The binding loop of the first domain (probably with anti-trypsin activity) is disordered due to proteolytic cleavage occurring in the course of crystallization. PMID:3366116

  1. [Structural regularities in activated cleavage sites of thrombin receptors].

    PubMed

    Mikhaĭlik, I V; Verevka, S V

    1999-01-01

    Comparison of thrombin receptors activation splitting sites sequences testifies to their similarity both in activation splitting sites of protein precursors and protein proteinase inhibitors reactive sites. In all these sites corresponded to effectory sites P2'-positions are placed by hydrophobic amino-acids only. The regularity defined conforms with previous thesis about the role of effectory S2'-site in regulation of the processes mediated by serine proteinases.

  2. Expression of virus-encoded proteinases: functional and structural similarities with cellular enzymes.

    PubMed Central

    Dougherty, W G; Semler, B L

    1993-01-01

    Many viruses express their genome, or part of their genome, initially as a polyprotein precursor that undergoes proteolytic processing. Molecular genetic analyses of viral gene expression have revealed that many of these processing events are mediated by virus-encoded proteinases. Biochemical activity studies and structural analyses of these viral enzymes reveal that they have remarkable similarities to cellular proteinases. However, the viral proteinases have evolved unique features that permit them to function in a cellular environment. In this article, the current status of plant and animal virus proteinases is described along with their role in the viral replication cycle. The reactions catalyzed by viral proteinases are not simple enzyme-substrate interactions; rather, the processing steps are highly regulated, are coordinated with other viral processes, and frequently involve the participation of other factors. Images PMID:8302216

  3. Activation of Proteinase 3 Contributes to Nonalcoholic Fatty Liver Disease and Insulin Resistance

    PubMed Central

    Toonen, Erik JM; Mirea, Andreea-Manuela; Tack, Cees J; Stienstra, Rinke; Ballak, Dov B; van Diepen, Janna A; Hijmans, Anneke; Chavakis, Triantafyllos; Dokter, Wim H; Pham, Christine TN; Netea, Mihai G; Dinarello, Charles A; Joosten, Leo AB

    2016-01-01

    Activation of inflammatory pathways is known to accompany development of obesity-induced nonalcoholic fatty liver disease (NAFLD), insulin resistance and type 2 diabetes. In addition to caspase-1, the neutrophil serine proteases proteinase 3, neutrophil elastase and cathepsin G are able to process the inactive proinflammatory mediators interleukin (IL)-1β and IL-18 to their bioactive forms, thereby regulating inflammatory responses. In this study, we investigated whether proteinase 3 is involved in obesity-induced development of insulin resistance and NAFLD. We investigated the development of NAFLD and insulin resistance in mice deficient for neutrophil elastase/proteinase 3 and neutrophil elastase/cathepsin G and in wild-type mice treated with the neutrophil serine proteinase inhibitor human α-1 antitrypsin. Expression profiling of metabolically relevant tissues obtained from insulin-resistant mice showed that expression of proteinase 3 was specifically upregulated in the liver, whereas neutrophil elastase, cathepsin G and caspase-1 were not. Neutrophil elastase/proteinase 3-deficient mice showed strongly reduced levels of lipids in the liver after being fed a high-fat diet. Moreover, these mice were resistant to high–fat–diet-induced weight gain, inflammation and insulin resistance. Injection of proteinase 3 exacerbated insulin resistance in caspase-1–/– mice, indicating that proteinase 3 acts independently of caspase-1. Treatment with α-1 antitrypsin during the last 10 d of a 16-wk high-fat diet reduced hepatic lipid content and decreased fasting glucose levels. We conclude that proteinase 3 is involved in NAFLD and insulin resistance and that inhibition of proteinase 3 may have therapeutic potential. PMID:27261776

  4. Activation of proteinase 3 contributes to Non-alcoholic Fatty Liver Disease (NAFLD) and insulin resistance.

    PubMed

    Toonen, Erik J M; Mirea, Andreea-Manuela; Tack, Cees J; Stienstra, Rinke; Ballak, Dov B; van Diepen, Janna A; Hijmans, Anneke; Chavakis, Triantafyllos; Dokter, Wim H; Pham, Christine T N; Netea, Mihai G; Dinarello, Charles A; Joosten, Leo A B

    2016-05-24

    Activation of inflammatory pathways is known to accompany development of obesity-induced non-alcoholic fatty liver disease (NAFLD), insulin resistance and type 2 diabetes. In addition to caspase-1, the neutrophil serine proteases proteinase 3, neutrophil elastase and cathepsin G are able to process the inactive pro-inflammatory mediators IL-1β and IL-18 to their bioactive forms, thereby regulating inflammatory responses. In the present study, we investigated whether proteinase 3 is involved in obesity-induced development of insulin resistance and NAFLD. We investigated the development of NAFLD and insulin resistance in mice deficient for neutrophil elastase/proteinase 3 and neutrophil elastase/cathepsin G and in wild-type mice treated with the neutrophil serine proteinase inhibitor human alpha-1 antitrypsin. Expression profiling of metabolically relevant tissues obtained from insulin resistant mice showed that expression of proteinase 3 was specifically upregulated in the liver, whereas neutrophil elastase, cathepsin G and caspase-1 were not. Neutrophil elastase/proteinase 3 deficient mice showed strongly reduced levels of lipids in the liver after fed a high fat diet. Moreover, these mice were resistant to high fat diet-induced weight gain, inflammation and insulin resistance. Injection of proteinase 3 exacerbated insulin resistance in caspase-1(-/-) mice, indicating that proteinase 3 acts independently of caspase-1. Treatment with alpha-1 antitrypsin during the last 10 days of a 16 week high fat diet reduced hepatic lipid content and decreased fasting glucose levels. We conclude that proteinase 3 is involved in NAFLD and insulin resistance and that inhibition of proteinase 3 may have therapeutic potential.

  5. Diisopropyl fluorophosphate labeling of sperm-associated proteinases

    SciTech Connect

    Odem, R.R.; Willand, J.L.; Polakoski, K.L. )

    1990-02-01

    Proteinase inhibitors have been shown to be capable of preventing various aspects of fertilization. Diisopropyl fluorophosphate (DFP) is an irreversible inhibitor of trypsin-like enzymes that is commercially available in a radiolabeled form. The experiments described herein were designed to determine if DFP would prevent sperm function in live, motile sperm and to identify the sperm proteins bound with DFP. DFP at 5 mM concentrations had no observable effect on sperm motility, but inhibited the penetration of zona-free hamster ova by human sperm (5.5%) compared to controls (33.5%). Acid extracts of motile sperm that had been incubated with radiolabeled DFP and collected by the swim-up procedure demonstrated the presence of radiolabeled DFP, and the autoradiography of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels of these extracts localized the uptake of radiolabeled DFP to proteins in the molecular weight region of the proacrosin-acrosin system. Acid-extracted proteinases from semen samples incubated with DFP demonstrated a concentration-dependent inhibition of both esterolytic hydrolysis of benzoyl-arginine ethyl ester on spectrophotometric analysis and proteolytic activity on gelatin SDS-PAGE zymography. DFP-labeled proteins were precipitated by highly specific antibodies to proacrosin. These results demonstrated that DFP is capable of inhibiting sperm function, and that it associates with the proacrosin-acrosin system in live motile sperm.

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

    PubMed

    Tati, Ramesh; Kristoffersson, Ann-Charlotte; Manea Hedström, Minola; Mörgelin, Matthias; Wieslander, Jörgen; van Kooten, Cees; Karpman, Diana

    2017-02-01

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

  7. Modeling the 3D structure of wheat subtilisin/chymotrypsin inhibitor (WSCI). Probing the reactive site with two susceptible proteinases by time-course analysis and molecular dynamics simulations.

    PubMed

    Facchiano, Angelo M; Costantini, Susan; Di Maro, Antimo; Panichi, Daniela; Chambery, Angela; Parente, Augusto; Di Gennaro, Simone; Poerio, Elia

    2006-07-01

    Comparative modeling and time-course hydrolysis experiments have been applied to investigate two enzyme-inhibitor complexes formed between the wheat subtilisin-chymotrypsin inhibitor (WSCI) and two susceptible proteinases. WSCI represents the first case of a wheat protein inhibitor active against animal chymotrypsins and bacterial subtilisins. The model was created using as template structure that of the CI-2A inhibitor from barley (PDB code: 2CI2), which shares 87% sequence identity with WSCI. Under these conditions of high similarity, the comparative modeling approach can be successfully applied. We predicted the WSCI 3D model and used it to investigate enzyme-inhibitor complex systems. Experimental observations indicated that chymotrypsin, but not subtilisin, in addition to cleavage at the primary reactive site Met48-Glu49, is able to hydrolyze a second peptide bond between Phe58 and Val59. Here, we report on cleavage of the peptide bond at the inhibitor's reactive site (Met48-Glu49) determined using time-course hydrolysis experiments; the same event was investigated for both subtilisin/WSCI and chymotrypsin/WSCI complexes using molecular dynamics simulations. The molecular details of the initial inhibitor-enzyme interactions, as well as of the changes observed during the simulations, allow us to speculate on the different fates of the two WSCI-proteinase complexes.

  8. Enhancing the specificity of the enterokinase cleavage reaction to promote efficient cleavage of a fusion tag.

    PubMed

    Shahravan, S Hesam; Qu, Xuanlu; Chan, I-San; Shin, Jumi A

    2008-06-01

    In our work with designed minimalist proteins based on the bZIP motif, we have found our His-tagged proteins to be prone to inclusion body formation and aggregation; we suspect this problem is largely due to the His tag, known to promote aggregation. Using AhR6-C/EBP, a hybrid of the AhR basic region and C/EBP leucine zipper, as representative of our bZIP-like protein family, we attempted removal of the His tag with enterokinase (EK) but obtained the desired cleavage product in very small yield. EK is known for proteolysis at noncanonical sites, and most cleavage occurred at unintended sites. We manipulated experimental conditions to improve specificity of proteolysis and analyzed the cleavage products; no effect was observed after changing pH, temperature, or the amount of EK. We then suspected the accessibility of the EK site was impeded due to protein aggregation. We found that the easily implemented strategy of addition of urea (1-4 M) greatly improved EK cleavage specificity at the canonical site and reduced adventitious cleavage. We believe that this enhancement in specificity is due to a more "open" protein structure, in which the now accessible canonical target can compete effectively with adventitious cleavage sites of related sequence.

  9. A role of secreted proteinase of Candida albicans for the invasion of chick chorio-allantoic membrane.

    PubMed

    Kobayashi, I; Kondoh, Y; Shimizu, K; Tanaka, K

    1989-01-01

    The invasion of Candida albicans strains into the chorio-allantoic membrane (CAM) of a developing chick was studied by light and electron microscopy. A proteinase-producing strain, NUM961, invaded into intact CAM, but proteinase-deficient strain NUM678 cells remained on the surface of the CAM with no evidence of damage to the host cells. However, NUM678 cells invaded into the ectoderm-damaged CAM, or proteinase-treated one. Electron microscopy revealed that treatment with purified Candida proteinase disorganized the ectoderm tissue by disrupting the intercellular junctions. These results suggest that Candida proteinase damages the CAM surface, which enables the invasion of the growing hyphae.

  10. Characterization of a chimeric foot-and-mouth disease virus bearing a bovine rhinitis B virus leader proteinase.

    PubMed

    Uddowla, Sabena; Pacheco, Juan M; Larson, Christopher; Bishop, Elizabeth; Rodriguez, Luis L; Rai, Devendra K; Arzt, Jonathan; Rieder, Elizabeth

    2013-12-01

    Bovine rhinitis B virus (BRBV) shares many motifs and sequence similarities with foot-and-mouth disease virus (FMDV). This study examined if the BRBV leader proteinase (L(pro) ) could functionally replace that of FMDV. A mutant A24LBRV3DYR FMDV engineered with the BRBV L(pro) and an antigenic marker in the 3D polymerase exhibited growth properties and eIF4G cleavage similar to parental A24WT virus. The A24LBRV3DYR type I interferon activity in infected bovine cells resembled that of A24LL virus that lacks L(pro), but this effect was less pronounced for A24LBRV3DYR infected porcine cells. In vivo studies showed that the A24LBRV3DYR virus was attenuated in cattle, and exhibited low virulence in pigs exposed by direct contact. The mutant virus induced protective immunity in cattle against challenge with parental A24WT. These results provide evidence that L(pro) of different Aphthoviruses are not fully functionally interchangeable and have roles that may depend on the nature of the infected host.

  11. Unexpected Trypsin Cleavage at Ubiquitinated Lysines

    PubMed Central

    2015-01-01

    Unexpected tryptic cleavage has been characterized at modified K48 residues in polyubiquitins. In particular, the tryptic products of all seven of the lysine-linked dimers of ubiquitin and of three trimers—linear Ub–48Ub–48Ub, linear Ub–63Ub–63Ub, and the branched trimer [Ub]2–6,48Ub—have been analyzed. In addition to the peptide products expected under commonly used tryptic conditions, we observe that peptides are formed with an unexpected ε-glycinylglycinyl-Lys carboxyl terminus when the site of linkage is Lys48. Trypsin from three different commercial sources exhibited this aberration. Initial cleavage at R74 is proposed in a distal ubiquitin to produce a glycinylglycinyl-lysine residue which is bound by trypsin. PMID:26182167

  12. Limited proteolysis by macrophage elastase inactivities human. cap alpha. /sub 1/-proteinase inhibitor

    SciTech Connect

    Banda, M.J.; Clark, E.J.; Werb, Z.

    1980-12-01

    Ever since the initial description of ..cap alpha../sub 1/-proteinase inhibitor (..cap alpha../sub 1/PI), the role of this plasma glycoprotein and its allelic polymorphism in disease and in healthy physiology has been the subject of much investigation, ..cap alpha../sub 1/PI inactivates a number of serine proteinases, including granulocyte elastase, and thus affords protection from the connective tissue degradation mediated by this class of proteinases. Because an imbalance in the ratio between ..cap alpha../sub 1/PI and proteinase may contribute to the development of destructive lung diseases, proteinases have been implicated in the pathogenesis of pulmonary emphysema. Both macrophages and polymorphonuclear leukocytes have been implicated in disruption of the ..cap alpha../sub 1/PI-proteinase balance. In this report, a new mechanism for alteration of the ..cap alpha../sub 1/PI-proteinase balance is demonstrated. It was found that the purified form of macrophage elastase catalytically degrades and inactivates ..cap alpha../sub 1/PI so that it no longer inhibits the elastinolytic activity of granulocyte elastase.

  13. [High molecular weight chitosan and sodium alginate effect on secretory acid proteinase of Candida albicans].

    PubMed

    Calamari, Silvia; Bojanich, Alejandra; Barembaum, Silvina; Azcurra, Ana; Virga, Carolina; Dorronsoro, Susana

    2004-12-01

    The effect of high molecular weight chitosan (HMWCh) and sodium alginate (NaAL) on acid proteinase secretion of Candida albicans (one of culture collection and five isolates) was evaluated. The secretion of acid proteinase was induced in the presence and the absence of these polymers in different concentrations and their enzymatic activity was determined. HMWCh and NaAL significantly diminished the enzymatic activity (>76% for the collection strains and > 89% for the isolates, p < 0.05). HMWCh did not modify protein concentrations, but NaAL did. It can be concluded that both polymers can inhibit the proteinase activity of Candida albicans.

  14. Structural and functional properties of kunitz proteinase inhibitors from leguminosae: a mini review.

    PubMed

    Oliva, Maria Luiza Vilela; Ferreira, Rodrigo da Silva; Ferreira, Joana Gasperazzo; de Paula, Cláudia Alessandra Andrade; Salas, Carlos E; Sampaio, Misako Uemura

    2011-08-01

    Seed proteins that inhibit proteinases are classified in families based on amino acid sequence similarity, nature of reactive site and mechanism of action, and are used as tools for investigating proteinases in physiological and pathological events. More recently, the plant Kunitz family of inhibitors with two disulphide bridges was enlarged with members containing variable number of cysteine residues, ranging from no cysteine at all to more than four residues. The characteristic of these proteins, as well the interactions with their target proteinases, are briefly discussed.

  15. Triacontanol negatively modulates the jasmonic acid-stimulated proteinase inhibitors in tomato (Lycopersicon esculentum).

    PubMed

    Ramanarayan, Krishnamurthy; Swamy, Gangadharamurthy Sivakumar

    2004-04-01

    Triacontanol (TRIA), a long chain aliphatic alcohol (C30H61OH) reverses the effect of jasmonic acid (JA) in inducing proteinase inhibitors (PIs) in tomato leaves. Porcine pancreas trypsin and Spodoptera litura gut proteinases were inhibited in the presence of leaf proteins treated with JA, and TRIA partially reverses this effect. Spodoptera litura larvae fed with tomato leaves treated with JA were reduced in body weight and TRIA is able to partially reverse this JA-induced effect. These results reflect the partial reversal effect of TRIA in down regulating the JA-induced production of proteinase inhibitors.

  16. Homology models of main proteinase from coronavirus associated with SARS

    NASA Astrophysics Data System (ADS)

    Liu, Hsuan-Liang; Lin, Jin-Chung; Ho, Yih; Chen, Chin-Wen

    2005-01-01

    In this study, two homology models of the main proteinase (M pro) from the novel coronavirus associated with severe acute respiratory syndrome (SARS-CoV) were constructed. These models reveal three distinct functional domains, in which an intervening loop connecting domains II and III as well as a catalytic cleft containing the substrate binding subsites S1 and S2 between domains I and II are observed. S2 exhibits structural variations more significantly than S1 during the 200 ps molecular dynamics simulations because it is located at the open mouth of the catalytic cleft and the amino acid residues lining up this subsite are least conserved. In addition, the higher structural variation of S2 makes it flexible enough to accommodate a bulky hydrophobic residue from the substrate.

  17. Novel distribution of the secretory leucocyte proteinase inhibitor in kidney.

    PubMed Central

    Ohlsson, S; Ljungkrantz, I; Ohlsson, K; Segelmark, M; Wieslander, J

    2001-01-01

    The secretory leucocyte proteinase inhibitor (SLPI) is a low molecular weight, tissue-specific inhibitor of, for example, elastase and cathepsin G, which also have antimicrobial capacity. SLPI has been localised to the respiratory, gastrointestinal and genital tracts, but so far not to the kidney. The presence of SLPI in renal tubuli cells was demonstrated using immunohistochemistry and, by means of in situ hybridisation on human renal biopsies, we were able to demonstrate SLPI production. In various inflammatory conditions in the kidneys, the protease-antiprotease balance is disturbed. For this reason, as well as the possible role in the defence against ascending urinary tract infections, it is interesting to establish a source of SLPI in renal tubuli cells. PMID:11817677

  18. Alpha and omega of carotenoid cleavage.

    PubMed

    Lakshman, M R

    2004-01-01

    In early 1900s, based on indirect evidence, Steenbock and Morton independently predicted that beta-carotene could be the biological precursor of vitamin A, although this notion was contested by others. In the 1930s, Thomas Moore showed the in vivo formation of vitamin A from beta-carotene. But it was not until Jim Olson and DeWitt Goodman independently showed in 1965 the formation of retinal, the aldehyde form of vitamin A from beta-carotene in cell-free extracts of liver and intestine, that this vital pathway of beta-carotene was recognized. Despite compelling evidence in several experimental systems for the central cleavage of beta-carotene to retinal by many investigators, there were some careful independent studies by Glover et al., Ganguly et al., Hansen and Meret and Krinsky et al. showing the eccentric cleavage of beta-carotene resulting in the formation of apocarotenoids both in vivo and in vitro. In an attempt to resolve this controversial issue, we revisited this problem in 1989 and showed beyond doubt the formation of retinal as the sole enzymatic product of a cytosolic enzyme from rabbit and rat intestinal mucosa by mass spectrometry and tracer analysis of the crystallized product. This was confirmed in 1996 by Nagao using the pig intestinal extract. Yeum et al. confirmed in 2000 that retinal is the sole product of beta-carotene cleavage in the presence of alpha-tocopherol, and that the observed formation of apocarotenoids occurs only in the absence of an antioxidant like alpha-tocopherol. In the same year, Barua and Olson also concluded from their in vivo studies in rats that central cleavage is by far the major pathway for the formation of vitamin A from beta-carotene. Beta, beta-carotene 15,15'-dioxygenase (EC 1.13.11.21) is the key enzyme that cleaves beta-carotene into two molecules of retinal. It is a cytosolic enzyme primarily localized in the duodenal mucosa although it has been found in liver. It is a 66 kDa sulfhydryl protein, requires

  19. Altered Expression of Brain Proteinase-Activated Receptor-2, Trypsin-2 and Serpin Proteinase Inhibitors in Parkinson's Disease.

    PubMed

    Hurley, Michael J; Durrenberger, Pascal F; Gentleman, Steve M; Walls, Andrew F; Dexter, David T

    2015-09-01

    Neuroinflammation is thought to contribute to cell death in neurodegenerative disorders, but the factors involved in the inflammatory process are not completely understood. Proteinase-activated receptor-2 (PAR2) expression in brain is increased in Alzheimer's disease and multiple sclerosis, but the status of PAR2 in Parkinson's disease is unknown. This study examined expression of PAR2 and endogenous proteinase activators (trypsin-2, mast cell tryptase) and proteinase inhibitors (serpin-A5, serpin-A13) in areas vulnerable and resistant to neurodegeneration in Parkinson's disease at different Braak α-synuclein stages of the disease in post-mortem brain. In normal aged brain, expression of PAR-2, trypsin-2, and serpin-A5 and serpin-A13 was found in neurons and microglia, and alterations in the amount of immunoreactivity for these proteins were found in some brain regions. Namely, there was a decrease in neurons positive for serpin-A5 in the dorsal motor nucleus, and serpin-A13 expression was reduced in the locus coeruleus and primary motor cortex, while expression of PAR2, trypsin-2 and both serpins was reduced in neurons within the substantia nigra. There was an increased number of microglia that expressed serpin-A5 in the dorsal motor nucleus of vagus and elevated numbers of microglia that expressed serpin-A13 in the substantia nigra of late Parkinson's disease cases. The number of microglia that expressed trypsin-2 increased in primary motor cortex of incidental Lewy body disease cases. Analysis of Parkinson's disease cases alone indicated that serpin-A5 and serpin-A13, and trypsin-2 expression in midbrain and cerebral cortex was different in cases with a high incidence of L-DOPA-induced dyskinesia and psychosis compared to those with low levels of these treatment-induced side effects. This study showed that there was altered expression in brain of PAR2 and some proteins that can control its function in Parkinson's disease. Given the role of PAR2 in

  20. Effect of a Bowman-Birk proteinase inhibitor from Phaseolus coccineus on Hypothenemus hampei gut proteinases in vitro.

    PubMed

    de Azevedo Pereira, Railene; Valencia-Jiménez, Arnubio; Magalhães, Cláudio Picanço; Prates, Maura Vianna; Melo, Jorge Alex Taquita; de Lima, Liziane Maria; de Sales, Maurício Pereira; Tempel Nakasu, Erich Yukio; da Silva, Maria Cristina Mattar; Grossi-de-Sá, Maria Fátima

    2007-12-26

    The coffee berry borer, Hypothenemus hampei (Ferrari), is an important devastating coffee pest worldwide. Both trypsin and chymotrypsin enzyme activities from H. hampei larval midgut can be inactivated by proteinaceous enzyme-inhibitors. A serine proteinase inhibitor belonging to the Bowman-Birk class was purified from a wild accession of Phaseolus coccineus L. seeds. The inhibitor (PcBBI1) is a cysteine-rich protein that is heat-stable at alkaline pH. MALDI-TOF/MS analysis showed that PcBBI1 occurs in seeds as a monomer (8689 Da) or dimer (17,378 Da). Using in vitro inhibition assays, it was found that PcBBI1 has a high inhibitory activity against H. hampei trypsin-like enzymes, bovine pancreatic chymotrypsin, and trypsin. According to this, PcBBI1 could be a promising tool to make genetically modified coffee with resistance to coffee berry borer.

  1. Circular dichroism of stem bromelain: a third spectral class within the family of cysteine proteinases.

    PubMed Central

    Arroyo-Reyna, A; Hernandez-Arana, A; Arreguin-Espinosa, R

    1994-01-01

    Two forms of stem bromelain (EC 3.4.22.4) were isolated from commercial, crude and chromatographically purified preparations of the enzyme by means of gel-filtration and cation-exchange liquid chromatography. These forms possess nearly identical secondary and tertiary structures, as judged from their circular dichroism (c.d.) spectra. The spectral characteristics of stem bromelain suggest that this enzyme belongs to the alpha + beta protein class, as other cysteine proteinases do. In agreement with these results, quantitative estimation of secondary structures yielded amounts similar to those for papain and proteinase omega. However, the bromelain c.d. curve is clearly distinguishable from those reported for papain and proteinase omega, on one hand, and that of chymopapain, on the other. Thus, it is apparent that there are at least three types of c.d. spectra associated with the family of cysteine proteinases. PMID:8198520

  2. [Properties of Bacillus pumilus subtilisin like proteinase secreted from recombinant strain on different growth stages].

    PubMed

    Balaban, N P; Danilova, Iu V; Shamsutdinov, T R; Mardanova, A M; Cheremin, A M; Rudenskaia, G N; Sharipova, M R

    2013-01-01

    Bacillus pumilus 3-19 glutamylendopeptidase has been isolated from culture liquid of Bacillus subtilis recombinant strain on different growth stages: growth retardation (early enzyme) and stationary phase (late enzyme). The effect of purified proteinase of different growth stages on insulin beta-chain, protein and oligopeptide substrates has been studied. Comparative study of physicochemical properties of early and late proteinases was carried out. Two protein fractions were different in catalytic characteristics and demonstrated different sensitivity to the presence of metal cations.

  3. Trypanosoma cruzi: insights into naphthoquinone effects on growth and proteinase activity.

    PubMed

    Bourguignon, Saulo C; Cavalcanti, Danielle F B; de Souza, Alessandra M T; Castro, Helena C; Rodrigues, Carlos R; Albuquerque, Magaly G; Santos, Dilvani O; da Silva, Gabriel Gomes; da Silva, Fernando C; Ferreira, Vitor F; de Pinho, Rosa T; Alves, Carlos R

    2011-01-01

    In this study we compared the effects of naphthoquinones (α-lapachone, β-lapachone, nor-β-lapachone and Epoxy-α-lap) on growth of Trypanosoma cruzi epimastigotes forms, and on viability of VERO cells. In addition we also experimentally analyzed the most active compounds inhibitory profile against T. cruzi serine- and cysteine-proteinases activity and theoretically evaluated them against cruzain, the major T. cruzi cysteine proteinase by using a molecular docking approach. Our results confirmed β-lapachone and Epoxy-α-lap with a high trypanocidal activity in contrast to α-lapachone and nor-β-lapachone whereas Epoxy-α-lap presented the safest toxicity profile against VERO cells. Interestingly the evaluation of the active compounds effects against T. cruzi cysteine- and serine-proteinases activities revealed different targets for these molecules. β-Lapachone is able to inhibit the cysteine-proteinase activity of T. cruzi proteic whole extract and of cruzain, similar to E-64, a classical cysteine-proteinase inhibitor. Differently, Epoxy-α-lap inhibited the T. cruzi serine-proteinase activity, similar to PMSF, a classical serine-proteinase inhibitor. In agreement to these biological profiles in the enzymatic assays, our theoretical analysis showed that E-64 and β-lapachone interact with the cruzain specific S2 pocket and active site whereas Epoxy-α-lap showed no important interactions. Overall, our results infer that β-lapachone and Epoxy-α-lap compounds may inhibit T. cruzi epimastigotes growth by affecting T. cruzi different proteinases. Thus the present data shows the potential of these compounds as prototype of protease inhibitors on drug design studies for developing new antichagasic compounds.

  4. Effect of carbon and nitrogen sources on neutral proteinase production by Pseudomonas aeruginosa.

    PubMed

    Nigam, J N; Pillai, K R; Baruah, J N

    1981-01-01

    A strain of Pseudomonas aeruginosa from soil produced large quantities of extracellular neutral proteinase and could utilize several organic substances as carbon and nitrogen sources for enzyme production. The growth media required the presence of a high amount of phosphate when glucose was the carbon source. The intermediates of citric-acid cycle acids supported the proteinase production more than any other carbon sources. However, complex nitrogenous substances supported enzyme production more efficiently. Higher concentration of casamino acids suppressed the protinase synthesis.

  5. [Activity dynamics of proteinases and glycosidases of fish chymus with exposure in fresh and brackish water].

    PubMed

    Kuz'mina, V V; Shekhovtsova, N V; Bolobonina, V E

    2010-01-01

    Activity of proteinases of the content of intestines (chymus) of the benthos-eater Carassius carassius fed different diets during prolonged exposure to water is studied. In the process of exposure of the chymus to water, the activity of proteinases decreases. Activity of glycosidases may increase, maximally during the first three days of exposure. This phenomenon suggests the important role of enzymes of the enteral microflora in processes of destruction of proteinaceous and carbohydrate components of the suspension and especially of organic detritus.

  6. Bitter gourd proteinase inhibitors: potential growth inhibitors of Helicoverpa armigera and Spodoptera litura.

    PubMed

    Telang, Manasi; Srinivasan, Ajay; Patankar, Aparna; Harsulkar, Abhay; Joshi, Vijay; Damle, Archana; Deshpande, Vasanti; Sainani, Mohini; Ranjekar, Prabhakar; Gupta, Gorakh; Birah, Ajanta; Rani, Seema; Kachole, Manavendra; Giri, Ashok; Gupta, Vidya

    2003-07-01

    Proteinase inhibitors (PIs) from the seeds of bitter gourd (Momordica charantia L.) were identified as strong inhibitors of Helicoverpa armigera gut proteinases (HGP). Biochemical investigations showed that bitter gourd PIs (BGPIs) inhibited more than 80% HGP activity. Electrophoretic analysis revealed the presence of two major proteins (BGPI-1 and-2) and two minor proteins (BGPI-3 and-4) having inhibitory activity against both trypsin and HGP. The major isoforms BGPI-1 and BGPI-2 have molecular mass of 3.5 and 3.0 kDa, respectively. BGPIs inhibited HGP activity of larvae fed on different host plants, on artificial diet with or without added PIs and proteinases excreted in fecal matter. Degradation of BGPI-1 by HGP showed direct correlation with accumulation of BGPI-2-like peptide, which remained stable and active against high concentrations of HGP up to 3 h. Chemical inhibitors of serine proteinases offered partial protection to BGPI-1 from degradation by HGP, suggesting that trypsin and chymotrypsin like proteinases are involved in degradation of BGPI-1. In larval feeding studies, BGPIs were found to retard growth and development of two lepidopteran pests namely Helicoverpa armigera and Spodoptera litura. This is the first report showing that BGPIs mediated inhibition of insect gut proteinases directly affects fertility and fecundity of both H. armigera and S. litura. The results advocate use of BGPIs to introduce insect resistance in otherwise susceptible plants.

  7. A serine proteinase of an archaebacterium, Halobacterium mediterranei. A homologue of eubacterial subtilisins.

    PubMed Central

    Stepanov, V M; Rudenskaya, G N; Revina, L P; Gryaznova, Y B; Lysogorskaya, E N; Filippova IYu; Ivanova, I I

    1992-01-01

    A homogeneous serine proteinase secreted by the extreme halophilic bacterium Halobacterium mediterranei 1538 was isolated by affinity chromatography on bacitracin-Sepharose with a yield of 48% (260-fold purification). The enzyme reveals an optimum for pyroglutamyl-Ala-Ala-Leu p-nitroanilide hydrolysis at pH 8.0-8.5 (Km 0.14 mM; k(cat). 36.9 s-1). Its activity increases linearly with NaCl concentration over the range 2-5 M. The substrate specificity of the enzyme is comparable with that of secretory subtilisins, the extent of protein degradation approaching that attained with proteinase K. The enzyme has a molecular mass of 41 kDa and a pI of 7.5. The N-terminal sequence of H. mediterranei serine proteinase reveals a 50% identity with that of Thermoactinomyces vulgaris serine proteinases, indicating that the enzyme belongs to the subtilisin family. Hence the serine proteinase secreted by the halophilic bacterium should be considered as a functional analogue, and a structural homologue, of eubacterial serine proteinases (subtilisins). Images Fig. 3. PMID:1637313

  8. Involement of an acrosinlike proteinase in the sulfhydryl-induced degradation of rabbit sperm nuclear protamine

    PubMed Central

    Zirkin, BR; Chang, TSK; Heaps, J

    1980-01-01

    Previous studies demonstrated that proteolytic activity is associated with isolated rabbit sperm nuclei and is responsible for the degradation of nuclear protamine that occurs during thiol-induced in vitro decondensation of the nuclei (Zirkin and Chang, 1977; Chang and Zirkin, 1978). In this study, we present the results of experiments designed to characterize this proteolytic activity. Basic protein isolated from rabbit sperm nuclei incubated with 5 mM dithiothreitol (DTT) and 1 percent Triton X-100 for increasing periods of time exhibited progressively faster migrating bands on acid-urea polyacrylamide gels, reflection the progressive degradation of protamine. Ultimately, a specific and characteristic peptide banding pattern resulted. When sperm nuclei were treated with the esterase inhibitor nitrophenyl-p-guanidino benzoate (NPGB) to inhibit the nuclear-associated proteolytic activity and then incubated with one of several exogenous proteinases in addition to DTT and Triton X-100, characteristic peptide banding patterns were seen for each exogenous proteinase employed. For trypsin, chymotrypsin, pronase, and papain, the peptide banding patterns differed from one another and from the pattern characteristic of protamine degradation by the nuclear-associated proteinase. By contrast, when rabbit acrosin served as the exogenous proteinase, the peptide banding pattern seen was identical to the pattern characteristic of the nuclear-associated proteinase. These results demonstrate directly that the proteinase associated with rabbit sperm nuclei and involved in sperm nuclear decondensation in vitro is acrosinlike. PMID:6988441

  9. General up regulation of Spodoptera frugiperda trypsins and chymotrypsins allows its adaptation to soybean proteinase inhibitor.

    PubMed

    Brioschi, Daniela; Nadalini, Larissa D; Bengtson, Mario H; Sogayar, Mari Cleide; Moura, Daniel S; Silva-Filho, Marcio C

    2007-12-01

    The existence of a diverse serine proteinase gene family in lepidopteran insects suggests they play a significant role in the insect adaptation to plant proteinase inhibitors. These proteinases have been shown to be involved in the process of proteolytic digestion in insect larvae. We carried out a selective transcriptome study of midguts from Spodoptera frugiperda larvae fed on a diet supplemented with soybean proteinase inhibitor (SPI). Using subtracted cDNA libraries made of gut-expressed transcripts, a total of 2100 partial sequences were obtained, of those 38% were related to digestive process. Two large and diverse groups of chymotrypsins and trypsins were obtained, and some of these proteinase-encoding genes were further characterized by quantitative RT-PCR. The transcription analyses revealed two groups: one group of genes constitutively expressed in the control larvae that is up regulated by introducing SPI to the diet, and a second group that is absent in the control but is induced by the SPI-rich diet. This observation suggests that adaptation of S. frugiperda to SPI involves de novo synthesis and also up regulation of existing enzymes. Proteases from intestines of larvae reared on a diet with SPI showed insensitivity to the inhibitor. The proteases were also insensitive to a broad-spectrum potato proteinase inhibitor preparation. We propose that adaptation of S. frugiperda to SPI follows a "shotgun" approach, based on a general up regulation of a large set of endoproteinases.

  10. Corticosteroid-binding globulin cleavage is paradoxically reduced in alpha-1 antitrypsin deficiency: Implications for cortisol homeostasis.

    PubMed

    Nenke, Marni A; Holmes, Mark; Rankin, Wayne; Lewis, John G; Torpy, David J

    2016-01-15

    High-affinity corticosteroid-binding globulin (haCBG) is cleaved by neutrophil elastase (NE) resulting in permanent transition to the low cortisol-binding affinity form (laCBG), thereby increasing cortisol availability at inflammatory sites. Alpha-1 antitrypsin (AAT) is the major inhibitor of NE. AAT deficiency (AATD) predisposes patients to early-onset emphysema due to increased proteolytic destruction from the inherent proteinase-antiproteinase imbalance. We hypothesized that AATD may result in increased CBG cleavage in vivo. We collected demographic data and blood samples from 10 patients with AATD and 28 healthy controls measuring total CBG and haCBG levels by parallel in-house ELISAs, as well as AAT, total and free cortisol levels. haCBG was higher (median [range]); 329 [210-551] vs. 250 [175-365] nmol/L; P<0.005, and laCBG lower; 174 [68-229] vs. 220 [119-348] nmol/L; P=0.016 in the AATD group, compared with controls. The ratio of haCBG:total CBG was also higher in AATD; 72 [53-83] vs. 54 [41-72] %; P=0.0001). There was a negative correlation between haCBG:total CBG and AAT levels (P<0.05, R=-0.64). Paradoxically, proteolytic cleavage of CBG was reduced in AATD, despite the recognized increase in NE activity. This implies that NE activity is not the mechanism for systemic CBG cleavage in basal, low inflammatory conditions. Relatively low levels of laCBG may have implications for cortisol action in AATD.

  11. Purification and characterization of a subtilisin-like proteinases secreted in the stationary growth phase of Bacillus amyloliquefaciens H2.

    PubMed

    Balaban, N P; Malikova, L A; Mardanova, A M; Rudenskaya, G N; Sharipova, M R

    2007-04-01

    Proteinases secreted during the early and late stationary phases have been isolated from the culture liquid of Bacillus amyloliquefaciens H2 using CM-cellulose ion-exchange chromatography with subsequent FPLC on a Mono S column. Considering the character of hydrolysis of specific chromogenic substrates and the type of inhibition, these enzymes were identified as subtilisin-like proteinases. The molecular weight of both proteinases is 29 kD. The proteolytic activity of the proteinases secreted during the early and late stationary phases towards the synthetic substrate Z-Ala-Ala-Leu-pNA was maximal at pH 8.5 and 9.0, respectively. The maximal activity of both proteinases was observed at 37 degrees C, and the proteins were stable within the pH range of 7.2-9.5. The subtilisin-like proteinases from B. amyloliquefaciens were shown to catalyze synthesis of peptide bonds.

  12. Additive Promotion of Viral Internal Ribosome Entry Site-Mediated Translation by Far Upstream Element-Binding Protein 1 and an Enterovirus 71-Induced Cleavage Product

    PubMed Central

    Hung, Chuan-Tien; Kung, Yu-An; Li, Mei-Ling; Lee, Kuo-Ming; Liu, Shih-Tung; Shih, Shin-Ru

    2016-01-01

    The 5' untranslated region (5' UTR) of the enterovirus 71 (EV71) RNA genome contains an internal ribosome entry site (IRES) that is indispensable for viral protein translation. Due to the limited coding capacity of their RNA genomes, EV71 and other picornaviruses typically recruit host factors, known as IRES trans-acting factors (ITAFs), to mediate IRES-dependent translation. Here, we show that EV71 viral proteinase 2A is capable of cleaving far upstream element-binding protein 1 (FBP1), a positive ITAF that directly binds to the EV71 5' UTR linker region to promote viral IRES-driven translation. The cleavage occurs at the Gly-371 residue of FBP1 during the EV71 infection process, and this generates a functional cleavage product, FBP11-371. Interestingly, the cleavage product acts to promote viral IRES activity. Footprinting analysis and gel mobility shift assay results showed that FBP11-371 similarly binds to the EV71 5' UTR linker region, but at a different site from full-length FBP1; moreover, FBP1 and FBP11-371 were found to act additively to promote IRES-mediated translation and virus yield. Our findings expand the current understanding of virus-host interactions with regard to viral recruitment and modulation of ITAFs, and provide new insights into translational control during viral infection. PMID:27780225

  13. A single proteolytic cleavage within the lower hinge of trastuzumab reduces immune effector function and in vivo efficacy

    PubMed Central

    2012-01-01

    Introduction Recent studies reported that human IgG antibodies are susceptible to specific proteolytic cleavage in their lower hinge region, and the hinge cleavage results in a loss of Fc-mediated effector functions. Trastuzumab is a humanized IgG1 therapeutic monoclonal antibody for the treatment of HER2-overexpressing breast cancers, and its mechanisms of action consist of inhibition of HER2 signaling and Fc-mediated antibody-dependent cellular cytotoxicity (ADCC). The objective of this study is to investigate the potential effect of proteinase hinge cleavage on the efficacy of trastuzumab using both a breast cancer cell culture method and an in vivo mouse xenograft tumor model. Methods Trastuzumab antibody was incubated with a panel of human matrix metalloproteinases, and proteolytic cleavage in the lower hinge region was detected using both western blotting and mass spectrometry. Single hinge cleaved trastuzumab (scIgG-T) was purified and evaluated for its ability to mediate ADCC and inhibition of breast cancer cell proliferation in vitro as well as anti-tumor efficacy in the mouse xenograft tumor model. Infiltrated immune cells were detected in tumor tissues by immunohistochemistry. Results scIgG-T retains HER2 antigen binding activity and inhibits HER2-mediated downstream signaling and cell proliferation in vitro when compared with the intact trastuzumab. However, scIgG-T lost Fc-mediated ADCC activity in vitro, and had significantly reduced anti-tumor efficacy in a mouse xenograft tumor model. Immunohistochemistry showed reduced immune cell infiltration in tumor tissues treated with scIgG-T when compared with those treated with the intact trastuzumab, which is consistent with the decreased ADCC mediated by scIgG-T in vitro. Conclusion Trastuzumab can be cleaved by matrix metalloproteinases within the lower hinge. scIgG-T exhibited a significantly reduced anti-tumor efficacy in vivo due to the weakened immune effector function such as ADCC. The results

  14. [On the Features of Embryonic Cleavage in Diverse Fish Species].

    PubMed

    Desnitskiy, A G

    2015-01-01

    Literature on the earliest steps of fish embryogenesis (including a number of "non-model" species) has been considered. The main attention has been paid to the loss of cleavage division synchrony and the first latitudinal cleavage furrow. In teleostean embryos, the features of their meroblastic cleavage are not rigidly associated with egg size. The midblastula transition (in a form clearly enough) occurs in some chondrostean and teleostean fishes, but it has not been detected in the representatives of sarcopterygian and chondrichthyan fishes.

  15. Early cleavage in Phoronis muelleri (Phoronida) displays spiral features.

    PubMed

    Pennerstorfer, Markus; Scholtz, Gerhard

    2012-01-01

    The view that early cleavage in Phoronida follows a radial pattern is widely accepted. However, data supporting this characterization are ambiguous. Studies have been repeatedly reporting variation between individual embryos, and the occurrence of embryos exhibiting oblique divisions or nonradial cell arrangements. Such embryos were often considered to represent variation within radial cleavage, or artificial appearances. Cleavage in Phoronis muelleri was previously characterized as "derived radial," but also oblique spindles and cell elongations, and shifted cell arrangements were observed. We studied the early cleavage in P. muelleri applying 4D microscopy, fluorescent staining, and confocal laser scanning microscopy. To deal with the problem of variation we provide statistical evaluations of our data. These show that oblique divisions do not represent variational abnormalities. In fact, they reveal that most cells divide obliquely from the third cleavage onwards. What is more, in almost all cells the axis of the third cleavage is inclined dextrally. The fourth cleavage is even stronger sinistrally pronounced. Subsequently, the pattern of alternating cleavage orientation is largely restricted to animal and vegetal blastomeres. As a result of the obliqueness of divisions, four cells encircle the poles in most embryos. Cross furrows are occasionally present. We found no indications for radial cleavage in P. muelleri. In contrast, the observed cleavage displays several characters consistent with the pattern of spiral cleavage. A close relation of phoronid and spiralian cleavage is also suggested by molecular phylogenies, allying both groups in the Lophotrochozoa. We suggest our findings to represent morphological support for this lophotrochozoan/spiralian affinity of Phoronida.

  16. Proteinase 3, Wegener's autoantigen: from gene to antigen.

    PubMed

    van der Geld, Y M; Limburg, P C; Kallenberg, C G

    2001-02-01

    Proteinase 3 (PR3) is one of four serine protease homologues in the azurophilic granules of neutrophils and granules of monocytes. It is of importance that anti-neutrophil cytoplasmic antibodies (ANCA) in patients with Wegener's granulomatosis (WG) are mainly directed against PR3 only. Furthermore, PR3 is overexpressed in a variety of acute and chronic myeloid leukemia cells. Cytotoxic T lymphocytes specific for a PR3-derived peptide have been shown to specifically lyse leukemia cells that overexpress PR3. This review will focus on PR3 and the characteristics of PR3 that might implicate this particular antigen in the pathogenesis of WG and as target for immunotherapy in myeloid leukemias. We will discuss the genetic localization and gene regulation of PR3, the processing, storage, and expression of the PR3 protein, and the physiological functions of PR3, and compare this with the three other neutrophil-derived serine proteases: human leukocyte elastase, cathepsin G, and azurocidin. Three main differences are described between PR3 and the other serine proteases. This makes PR3 a very intriguing protein with a large array of physiological functions, some of which may play a role in ANCA-associated vasculitidis and myeloid leukemia.

  17. Functional role of aspartic proteinase cathepsin D in insect metamorphosis

    PubMed Central

    Gui, Zhong Zheng; Lee, Kwang Sik; Kim, Bo Yeon; Choi, Yong Soo; Wei, Ya Dong; Choo, Young Moo; Kang, Pil Don; Yoon, Hyung Joo; Kim, Iksoo; Je, Yeon Ho; Seo, Sook Jae; Lee, Sang Mong; Guo, Xijie; Sohn, Hung Dae; Jin, Byung Rae

    2006-01-01

    Background Metamorphosis is a complex, highly conserved and strictly regulated development process that involves the programmed cell death of obsolete larval organs. Here we show a novel functional role for the aspartic proteinase cathepsin D during insect metamorphosis. Results Cathepsin D of the silkworm Bombyx mori (BmCatD) was ecdysone-induced, differentially and spatially expressed in the larval fat body of the final instar and in the larval gut of pupal stage, and its expression led to programmed cell death. Furthermore, BmCatD was highly induced in the fat body of baculovirus-infected B. mori larvae, suggesting that this gene is involved in the induction of metamorphosis of host insects infected with baculovirus. RNA interference (RNAi)-mediated BmCatD knock-down inhibited programmed cell death of the larval fat body, resulting in the arrest of larval-pupal transformation. BmCatD RNAi also inhibited the programmed cell death of larval gut during pupal stage. Conclusion Based on these results, we concluded that BmCatD is critically involved in the programmed cell death of the larval fat body and larval gut in silkworm metamorphosis. PMID:17062167

  18. Conformational Plasticity of the 2A Proteinase from Enterovirus 71

    PubMed Central

    Cai, Qixu; Yameen, Muhammad; Liu, Weihua; Gao, Zhenting; Li, Yaozong; Peng, Xuanjia; Cai, Yaxian; Wu, Caiming; Zheng, Qian

    2013-01-01

    The 2A proteinase (2Apro) is an enterovirally encoded cysteine protease that plays essential roles in both the processing of viral precursor polyprotein and the hijacking of host cell translation and other processes in the virus life cycle. Crystallographic studies of 2Apro from enterovirus 71 (EV71) and its interaction with the substrate are reported here. EV71 2Apro was comprised of an N-terminal domain of a four-stranded antiparallel β sheet and a C-terminal domain of a six-stranded antiparallel β barrel with a tightly bound zinc atom. Unlike in other 2Apro structures, there is an open cleft across the surface of the protein in an open conformation. As demonstrated by the crystallographic studies and modeling of the complex structure, the open cleft could be fitted with the substrate. On comparison 2Apro of EV71 to those of the human rhinovirus 2 and coxsackievirus B4, the open conformation could be closed with a hinge motion in the bII2 and cII β strands. This was supported by molecular dynamic simulation. The structural variation among different 2Apro structures indicates a conformational flexibility in the substrate-binding cleft. The open structure provides an accessible framework for the design and development of therapeutics against the viral target. PMID:23616646

  19. Role of saliva proteinase 3 in dental caries

    PubMed Central

    Yang, Teng-Yu; Zhou, Wen-Jie; Du, Yue; Wu, Song-Tao; Yuan, Wen-Wen; Yu, Yu; Su, Lin; Luo, Yang; Zhang, Jie-Hua; Lu, Wan-Lu; Wang, Xiao-Qian; Chen, Jiao; Feng, Yun; Zhou, Xue-Dong; Zhang, Ping

    2015-01-01

    Salivary analysis can be used to assess the severity of caries. Of the known salivary proteins, a paucity of information exists concerning the role of proteinase 3 (PR3), a serine protease of the chymotrypsin family, in dental caries. Whole, unstimulated saliva was collected from children with varying degrees of active caries and tested using a Human Protease Array Kit and an enzyme-linked immunosorbent assay. A significantly decreased concentration of salivary PR3 was noted with increasing severity of dental caries (P<0.01); a positive correlation (r=0.87; P<0.01; Pearson's correlation analysis) was also observed between salivary pH and PR3 concentration. In an antibacterial test, a PR3 concentration of 250 ng·mL−1 or higher significantly inhibited Streptococcus mutans UA159 growth after 12 h of incubation (P<0.05). These studies indicate that PR3 is a salivary factor associated with the severity of dental caries, as suggested by the negative relationship between salivary PR3 concentration and the severity of caries as well as the susceptibility of S. mutans to PR3. PMID:26756046

  20. Proteinase 3-ANCA Vasculitis versus Myeloperoxidase-ANCA Vasculitis

    PubMed Central

    Hilhorst, Marc; van Paassen, Pieter

    2015-01-01

    In patients with GN or vasculitis, ANCAs are directed against proteinase 3 (PR3) or myeloperoxidase (MPO). The differences between PR3-ANCA-associated vasculitis (AAV) and MPO-AAV described in the past have been supplemented during the last decade. In this review, we discuss the differences between these two small-vessel vasculitides, focusing especially on possible etiologic and pathophysiologic differences. PR3-AAV is more common in northern parts of the world, whereas MPO-AAV is more common in southern regions of Europe, Asia, and the Pacific, with the exception of New Zealand and Australia. A genetic contribution has been extensively studied, and there is a high prevalence of the HLA-DPB1*04:01 allele in patients with PR3-AAV as opposed to patients with MPO-AAV and/or healthy controls. Histologically, MPO-AAV and PR3-AAV are similar but show qualitative differences when analyzed carefully. Clinically, both serotypes are difficult to distinguish, but quantitative differences are present. More organs are affected in PR3-AAV, whereas renal limited vasculitis occurs more often in patients with MPO-AAV. For future clinical trials, we advocate classifying patients by ANCA serotype as opposed to the traditional disease type classification. PMID:25956510

  1. Substrate and inhibitor studies with human gastric aspartic proteinases.

    PubMed Central

    Baxter, A; Campbell, C J; Grinham, C J; Keane, R M; Lawton, B C; Pendlebury, J E

    1990-01-01

    The separation of pepsin isoenzymes 1, 2, 3 and 5 (gastricsin) in human gastric juice was effected by chromatography on Mono Q ion-exchanger, and slow-moving proteinase was purified to homogeneity by using a modified procedure incorporating a novel affinity-chromatography step. The pH-activity profiles of these enzymes with mucus glycoprotein and basement-membrane substrates were determined; the profiles for pepsin 2 were noticeably different, and, in general, the pH optima for the hydrolysis of basement membrane were more acidic. Pepsin 1 expressed larger specificity constants (kcat./Km) than pepsin 3 with a series of synthetic peptide substrates, reflecting greater binding (smaller Km) by pepsin 1. Inhibitor studies at pH 1.7 and 4.5 with a series of P2-substituted lactoyl-pepstatins implied that valine at position P2 was optimal for inhibiting pepsins 1, 2 and 3 but detrimental for pepsin 5, whereas lysine at position P2 was tolerated well by pepsin 5 but not by pepsins 1, 2 and 3. The potency of lactoyl-pepstatin with lysine at position P2 did not increase as a function of pH. P2-substituted lactoyl-pepstatins failed to show any inhibitory selectivity among pepsins 1, 2 and 3. PMID:2111133

  2. Interference of Wegener's granulomatosis autoantibodies with neutrophil Proteinase 3 activity.

    PubMed Central

    van de Wiel, B A; Dolman, K M; van der Meer-Gerritsen, C H; Hack, C E; von dem Borne, A E; Goldschmeding, R

    1992-01-01

    Classic anti-neutrophil cytoplasmic autoantibodies (C-ANCA) are disease-specific markers of Wegener's granulomatosis (WG). The possible pathogenetic role of these autoantibodies, which are directed against Proteinase 3 (PR3), is not yet clear. We studied the effect of C-ANCA on PR3 proteolytic activity and on the complexation of PR3 with alpha 1-antitrypsin (alpha 1AT). C-ANCA IgG from eight patients with active WG significantly inhibited PR3 proteolytic activity, particularly towards elastin (median 84.2% inhibition). C-ANCA IgG significantly inhibited the complexation of PR3 with alpha 1AT (median 58.8% inhibition). Moreover, addition of purified PR3 to C-ANCA-positive sera from WG patients yielded less complexes with alpha 1AT (median 44.8%) compared with sera containing perinuclear anti-neutrophil cytoplasmic autoantibodies (P-ANCA) or ANCA-negative sera. These findings indicate the existence of a hitherto unknown property of C-ANCA, which may be of importance in the pathogenesis of WG. PMID:1458677

  3. Alkaline proteinase inhibitor of Pseudomonas aeruginosa: a mutational and molecular dynamics study of the role of N-terminal residues in the inhibition of Pseudomonas alkaline proteinase.

    PubMed

    Feltzer, Rhona E; Trent, John O; Gray, Robert D

    2003-07-11

    Alkaline proteinase inhibitor of Pseudomonas aeruginosa is a 11.5-kDa, high affinity inhibitor of the serralysin class of zinc-dependent proteinases secreted by several Gram-negative bacteria. X-ray crystallography of the proteinase-inhibitor complex reveals that five N-terminal inhibitor residues occupy the extended substrate binding site of the enzyme and that the catalytic zinc is chelated by the alpha-amino and carbonyl groups of the N-terminal residue of the inhibitor. In this study, we assessed the effect of alteration of inhibitor residues 2-5 on its affinity for Pseudomonas alkaline proteinase (APR) as derived from the ratio of the dissociation and associate rate constants for formation of the enzyme-inhibitor complex. The largest effect was observed at position Ser-2, which occupies the S1' pocket of the enzyme and donates a hydrogen bond to the carboxyl group of the catalytic Glu-177 of the proteinase. Substitution of Asp, Arg, or Trp at this position increased the dissociation constant KD by 35-, 180-, and 13-fold, respectively. Mutation at positions 3-5 of the trunk also resulted in a reduction in enzyme-inhibitor affinity, with the exception of an I4W mutant, which exhibited a 3-fold increase in affinity. Molecular dynamics simulation of the complex formation between the catalytic domain of APR and the S2D mutant showed that the carboxyl of Asp-2 interacts with the catalytic zinc, thereby partially neutralizing the negative charge that otherwise would clash with the carboxyl group of Glu-177 of APR. Simulation of the interaction between the alkaline proteinase and the I4W mutant revealed a major shift in the loop comprised of residues 189-200 of the enzyme that allowed formation of a stacking interaction between the aromatic rings of Ile-4 of the inhibitor and Tyr-158 of the proteinase. This new interaction could account for the observed increase in enzyme-inhibitor affinity.

  4. Brittle to ductile transition in cleavage fracture

    SciTech Connect

    Argon, A.S.; Berg, Q.

    1992-09-30

    The problem of interpretation of fracture transition from brittle to ductile or vice versa is the subject of study. An instrumented tapered double cantilever beam (TDCB) has been developed as a definitive tool in the study of the intrinsic mechanism in single crystalline samples. In this experiment, the crack velocity is directly proportional to actuator velocity. In experiments performed on TDCB shaped Si single crystals, oriented for cleavage on either [l brace]111[r brace] or [l brace]110[r brace] planes, a number of troubling features of jerky carck extension were encountered. Evidence suggests that nucleation of dislocation loops from crack tip is easier than moving these dislocations away from crack tip. 14 refs, 1 fig.

  5. OH cleavage from tyrosine: debunking a myth

    PubMed Central

    Bury, Charles S.; Carmichael, Ian; Garman, Elspeth F

    2017-01-01

    During macromolecular X-ray crystallography experiments, protein crystals held at 100 K have been widely reported to exhibit reproducible bond scission events at doses on the order of several MGy. With the objective to mitigate the impact of radiation damage events on valid structure determination, it is essential to correctly understand the radiation chemistry mechanisms at play. OH-cleavage from tyrosine residues is regularly cited as amongst the most available damage pathways in protein crystals at 100 K, despite a lack of widespread reports of this phenomenon in protein crystal radiation damage studies. Furthermore, no clear mechanism for phenolic C—O bond cleavage in tyrosine has been reported, with the tyrosyl radical known to be relatively robust and long-lived in both aqueous solutions and the solid state. Here, the initial findings of Tyr –OH group damage in a myrosinase protein crystal have been reviewed. Consistent with that study, at increasing doses, clear electron density loss was detectable local to Tyr –OH groups. A systematic investigation performed on a range of protein crystal damage series deposited in the Protein Data Bank has established that Tyr –OH electron density loss is not generally a dominant damage pathway in protein crystals at 100 K. Full Tyr aromatic ring displacement is here proposed to account for instances of observable Tyr –OH electron density loss, with the original myrosinase data shown to be consistent with such a damage model. Systematic analysis of the effects of other environmental factors, including solvent accessibility and proximity to di­sulfide bonds or hydrogen bond interactions, is also presented. Residues in known active sites showed enhanced sensitivity to radiation-induced disordering, as has previously been reported. PMID:28009542

  6. Kazal-type serine proteinase inhibitors in the midgut of Phlebotomus papatasi

    PubMed Central

    Sigle, Leah Theresa; Ramalho-Ortigão, Marcelo

    2013-01-01

    Sandflies (Diptera: Psychodidae) are important disease vectors of parasites of the genus Leishmania, as well as bacteria and viruses. Following studies of the midgut transcriptome of Phlebotomus papatasi, the principal vector of Leishmania major, two non-classical Kazal-type serine proteinase inhibitors were identified (PpKzl1 and PpKzl2). Analyses of expression profiles indicated that PpKzl1 and PpKzl2 transcripts are both regulated by blood-feeding in the midgut of P. papatasi and are also expressed in males, larva and pupa. We expressed a recombinant PpKzl2 in a mammalian expression system (CHO-S free style cells) that was applied to in vitro studies to assess serine proteinase inhibition. Recombinant PpKzl2 inhibited α-chymotrypsin to 9.4% residual activity and also inhibited α-thrombin and trypsin to 33.5% and 63.9% residual activity, suggesting that native PpKzl2 is an active serine proteinase inhibitor and likely involved in regulating digestive enzymes in the midgut. Early stages of Leishmania are susceptible to killing by digestive proteinases in the sandfly midgut. Thus, characterising serine proteinase inhibitors may provide new targets and strategies to prevent transmission of Leishmania. PMID:24037187

  7. Taraxalisin -- a serine proteinase from dandelion Taraxacum officinale Webb s.l.

    PubMed

    Rudenskaya, G N; Bogacheva, A M; Preusser, A; Kuznetsova, A V; Dunaevsky YaE; Golovkin, B N; Stepanov, V M

    1998-10-23

    Latex of dandelion roots contains a serine proteinase that hydrolyzes a chromogenic peptide substrate Glp-Ala-Ala-Leu-pNA optimally at pH 8.0. Maximal activity of the proteinase in the roots is attained in April, at the beginning of plant development after the winter period. The protease was isolated by ammonium sulfate precipitation of the root extract followed by affinity chromatography on a Sepharose-Ala-Ala-Leu-mrp and gel filtration on Superose 6R performed in FPLC regime. Pure serine proteinase named taraxalisin was inactivated by specific inhibitors of serine proteinases, diisopropylfluorophosphate (DFP) and phenylmethylsulfonylfluoride (PMSF). Its molecular mass is 67 kDa and pI 4.5. pH stability range is 6-9 in the presence of 2 mM Ca2+, temperature optimum is at 40 degrees C; Km=0.37+/-0.06 mM. The substrate specificity of taraxalisin towards synthetic peptides and insulin B-chain is comparable with that of two other subtilisin-like serine proteinases, cucumisin and macluralisin. The taraxalisin N-terminal sequence traced for 15 residues revealed 40% coinciding residues when aligned with that of subtilisin Carlsberg.

  8. [Progresses in the structure and function of Kazal-type proteinase inhibitors].

    PubMed

    Zheng, Qing-Liang; Sheng, Qing; Zhang, Yao-Zhou

    2006-09-01

    Proteinase inhibitors are widely distributed in many living organisms and play crucial roles in many biological processes, particularly in regulating the proteinase activity spatially and temporally. However, The Kazal family of serine protease inhibitors is one of the most important and extensively studied protease inhibitor families. This type of protease inhibitor normally consists of one or several domains. Every domain has a highly conserved sequence structure and molecular conformation. It is found that contact residues are hyper variable, which are responsible for the interaction of inhibitors and proteinases. Most of them are in the solvent exposed loop. But P1 residue is the key active site of the interaction between inhibitor and enzyme. The types of the amino acid at P1 site likely play an important role in causing different inhibitory activity. The substitutions at the contact residues cause significant effects on the association constant. By using the Laskowski algorithm, the Ki values of a Kazal domain against six serine proteinases can be predicted from the domain' s sequence alone. At present there are many Kazal proteinase inhibitors found in the organisms, which show important biological functions. This article gives a comprehensive review of the newer developments in the characters and the interaction of the Kazal-type inhibitors.

  9. [Biosynthesis of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant Bacillus subtilis strain].

    PubMed

    Kirillova, Iu M; Mikhaĭlova, E O; Balaban, N P; Mardanova, A M; Kaiumov, A R; Rudenskaia, G N; Kostrov, S V; Sharipova, M R

    2006-01-01

    The effect of certain nutrients on the growth and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant strain Bacillus subtilis AJ73(pCS9) was studied. Glucose was found to inhibit the synthesis of proteinase in the early (28 h of growth) but not in the late stationary phase (48 h of growth). The inhibitory effect of the other mono- and disaccharides studied was less pronounced. Casamino acids added to the medium at concentrations of 0.1-1% as an additional carbon and nitrogen source stimulated enzyme biosynthesis. Individual amino acids (cysteine, asparagine, glutamine, tryptophan, histidine, and glutamate) also stimulated enzyme biosynthesis in the early stationary phase by 25-30%, whereas other amino acids (valine, leucine, alanine, and aspartate) were ineffective or even slightly inhibitory to enzyme production. The stimulatory effect of the first group of amino acids on the synthesis of proteinase in the late stationary phase was negligible. In contrast, the bivalent ions Ca2+, Mg2+, and Mn2+ stimulated biosynthesis of proteinase in the late stationary phase (by 20-60%) and not in the early stationary phase. The data indicate that there are differences in the biosyntheses of proteinase by the recombinant B. subtilis strain during the early and late periods of the stationary phases.

  10. Purification and characterization of serine proteinase 2 from Bacillus intermedius 3-19.

    PubMed

    Balaban, N P; Mardanova, A M; Sharipova, M R; Gabdrakhmanova, L A; Sokolova, E A; Rudenskaya, G N; Leshchinskaya, I B

    2004-04-01

    A proteinase secreted in the late stationary phase was isolated from the culture fluid of Bacillus intermedius 3-19 by ion-exchange chromatography on CM-cellulose followed by FPLC on a Mono S column. The enzyme was completely inhibited by the serine proteinase inhibitors diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride. The maximum proteolytic activity against the synthetic chromogenic substrate Z-Ala-Ala-Leu-pNA was observed at pH 9.0. The molecular weight of the enzyme is 28 kD and its isoelectric point is 9.2. We have also determined pH- and thermostability and Km and kcat of this proteinase. The enzyme has been classified as a thiol-dependent serine proteinase. N-Terminal amino acid sequence (10 residues) and amino acid composition of the protein were also determined. By the mode of hydrolysis of peptide bonds in the oxidized B-chain of insulin, this enzyme is similar to the thiol-dependent serine proteinase 1 from B. intermedius 3-19 secreted during vegetative growth.

  11. Pacifastin, a novel 155-kDa heterodimeric proteinase inhibitor containing a unique transferrin chain

    PubMed Central

    Liang, Zicai; Sottrup-Jensen, Lars; Aspán, Anna; Hall, Martin; Söderhäll, Kenneth

    1997-01-01

    A 155-kDa proteinase inhibitor, pacifastin, from plasma of the freshwater crayfish, Pacifastacus leniusculus, was found to be composed of two covalently linked subunits. The two subunits are encoded by two different mRNAs, which were cloned and sequenced. The heavy chain of pacifastin (105 kDa) is related to transferrins, containing three transferrin lobes, two of which seem to be active for iron binding. The light chain of pacifastin (44 kDa) is the inhibitory subunit, and has nine cysteine-rich inhibitory domains that are homologous to each other and to low molecular weight proteinase inhibitors isolated from the grasshopper, Locusta migratoria. The nine light chain domains and the Locusta inhibitors share a characteristic cysteine array (Cys-Xaa9–12-Cys-Xaa2-Cys-Xaa-Cys-Xaa6–8-Cys-Xaa4-Cys) distinct from any described proteinase inhibitor family, suggesting that they constitute a new family of proteinase inhibitors. Pacifastin is the first known protein that has combined properties of a transferrin-like molecule and a proteinase inhibitor. PMID:9192625

  12. Leishmania (Viannia) braziliensis: influence of successive in vitro cultivation on the expression of promastigote proteinases.

    PubMed

    Rebello, Karina Mastropasqua; Britto, Constança; Pereira, Bernardo Acácio Santini; Pita-Pereira, Daniela de; Moraes, Milton Ozório; Ferreira, Anna Beatriz Robottom; Cysne-Finkelstein, Léa; Otto, Thomas Dan; Côrtes, Luzia Monteiro de Castro; da-Silva, Gabriel Gomes; Alves, Carlos Roberto

    2010-12-01

    Cysteine proteinases are an important virulence factor in Leishmania parasites. In this study we analyzed the cysteine proteinase expression of infective Leishmania (Viannia) braziliensis promastigotes, examining the expression induced by successive in vitro passages in culture. We observed that this parasite presents a decrease in its virulence over BALB/c macrophages, after successive passages in culture, but still they present proteinase activity, being capable of hydrolyzing the substrate pGlu-Phe-Leu-p Nitroanilide at pH 7.0. This proteinase activity also decreases in the course of the successive passages. Additionally, the decrease in the amount of CPB proteins following successive passages of promastigotes was verified by immunoblotting assays, using an anti-CPB antiserum. Real-time PCR assays were performed to assess the relative cpb expression when compared to a housekeeping gene in promastigote cDNA preparations from the first, fourth and seventh passages. Interestingly, the data indicate a relative increase in cpb gene transcripts as the promastigotes were maintained under in vitro culture: 2.2 times higher for fourth and 2.7 times higher for seventh passages when compared to the first passage. Thus, the information gathered here shows that the expression of cysteine proteinases is modified during in vitro cultivation of L. (V.) braziliensis promastigotes.

  13. HIV proteinase inhibitors target the Ddi1-like protein of Leishmania parasites

    PubMed Central

    White, Rhian E.; Powell, David J.; Berry, Colin

    2011-01-01

    HIV proteinase inhibitors reduce the levels of Leishmania parasites in vivo and in vitro, but their biochemical target is unknown. We have identified an ortholog of the yeast Ddi1 protein as the only member of the aspartic proteinase family in Leishmania parasites, and in this study we investigate this protein as a potential target for the drugs. To date, no enzyme assay has been developed for the Ddi1 proteins, but Saccharomyces cerevisiae lacking the DDI1 gene secrete high levels of protein into the medium. We developed an assay in which these knockout yeast were functionally complemented to low secretion by introduction of genes encoding Ddi1 orthologs from Leishmania major or humans. Plasmid alone controls gave no complementation. Treatment of the Ddi1 transformants with HIV proteinase inhibitors showed differential effects dependent on the origin of the Ddi1. Dose responses allowed calculation of IC50 values; e.g., for nelfinavir, of 3.4 μM (human Ddi1) and 0.44 μM (Leishmania Ddi1). IC50 values with Leishmania constructs mirror the potency of inhibitors against parasites. Our results show that Ddi1 proteins are targets of HIV proteinase inhibitors and indicates the Leishmania Ddi1 as the likely target for these drugs and a potential target for antiparasitic therapy.—White, R. E., Powell, D. J., Berry, C. HIV proteinase inhibitors target the Ddi1-Like protein of Leishmania parasites. PMID:21266539

  14. Differential antibiosis against Helicoverpa armigera exerted by distinct inhibitory repeat domains of Capsicum annuum proteinase inhibitors.

    PubMed

    Joshi, Rakesh S; Gupta, Vidya S; Giri, Ashok P

    2014-05-01

    Plant defensive serine proteinase inhibitors (PIs) are known to have negative impact on digestive physiology of herbivore insects and thus have a crucial role in plant protection. Here, we have assessed the efficacy and specificity of three previously characterized inhibitory repeat domain (IRD) variants from Capsicum annuum PIs viz., IRD-7, -9 and -12 against gut proteinases from Helicoverpa armigera. Comparative study of in silico binding energy revealed that IRD-9 possesses higher affinity towards H. armigera serine proteinases as compared to IRD-7 and -12. H. armigera fed on artificial diet containing 5 TIU/g of recombinant IRD proteins exhibited differential effects on larval growth, survival rate and other nutritional parameters. Major digestive gut trypsin and chymotrypsin genes were down regulated in the IRD fed larvae, while few of them were up-regulated, this indicate alterations in insect digestive physiology. The results corroborated with proteinase activity assays and zymography. These findings suggest that the sequence variations among PIs reflect in their efficacy against proteinases in vitro and in vivo, which also could be used for developing tailor-made multi-domain inhibitor gene(s).

  15. Neutrophil elastase and proteinase 3 trafficking routes in myelomonocytic cells

    SciTech Connect

    Kaellquist, Linda; Rosen, Hanna; Nordenfelt, Pontus; Calafat, Jero; Janssen, Hans; Persson, Ann-Maj; Hansson, Markus; Olsson, Inge

    2010-11-15

    Neutrophil elastase (NE) and proteinase 3 (PR3) differ in intracellular localization, which may reflect different trafficking mechanisms of the precursor forms when synthesized at immature stages of neutrophils. To shed further light on these mechanisms, we compared the trafficking of precursor NE (proNE) and precursor PR3 (proPR3). Like proNE [1], proPR3 interacted with CD63 upon heterologous co-expression in COS cells but endogenous interaction was not detected although cell surface proNE/proPR3/CD63 were co-endocytosed in myelomonocytic cells. Cell surface proNE/proPR3 turned over more rapidly than cell surface CD63 consistent with processing/degradation of the pro-proteases but recycling of CD63. Colocalization of proNE/proPR3/CD63 with clathrin and Rab 7 suggested trafficking through coated vesicles and late endosomes. Partial caveolar trafficking of proNE/CD63 but not proPR3 was suggested by colocalization with caveolin-1. Blocking the C-terminus of proNE/proPR3 by creating a fusion with FK506 binding protein inhibited endosomal re-uptake of proNE but not proPR3 indicating 'pro{sub C}'-peptide-dependent structural/conformational requirements for proNE but not for proPR3 endocytosis. The NE aminoacid residue Y199 of a proposed NE sorting motif that interacts with AP-3 [2] was not required for proNE processing, sorting or endocytosis in rat basophilic leukemia (RBL) cells expressing heterologous Y199-deleted proNE; this suggests operation of another AP-3-link for proNE targeting. Our results show intracellular multi-step trafficking to be different between proNE and proPR3 consistent with their differential subcellular NE/PR3 localization in neutrophils.

  16. Pathway-selective antagonism of proteinase activated receptor 2

    PubMed Central

    Suen, J Y; Cotterell, A; Lohman, R J; Lim, J; Han, A; Yau, M K; Liu, L; Cooper, M A; Vesey, D A; Fairlie, D P

    2014-01-01

    Background and Purpose Proteinase activated receptor 2 (PAR2) is a GPCR associated with inflammation, metabolism and disease. Clues to understanding how to block PAR2 signalling associated with disease without inhibiting PAR2 activation in normal physiology could be provided by studies of biased signalling. Experimental Approach PAR2 ligand GB88 was profiled for PAR2 agonist and antagonist properties by several functional assays associated with intracellular G-protein-coupled signalling in vitro in three cell types and with PAR2-induced rat paw oedema in vivo. Key Results In HT29 cells, GB88 was a PAR2 antagonist in terms of Ca2+ mobilization and PKC phosphorylation, but a PAR2 agonist in attenuating forskolin-induced cAMP accumulation, increasing ERK1/2 phosphorylation, RhoA activation, myosin phosphatase phosphorylation and actin filament rearrangement. In CHO-hPAR2 cells, GB88 inhibited Ca2+ release, but activated Gi/o and increased ERK1/2 phosphorylation. In human kidney tubule cells, GB88 inhibited cytokine secretion (IL6, IL8, GM-CSF, TNF-α) mediated by PAR2. A rat paw oedema induced by PAR2 agonists was also inhibited by orally administered GB88 and compared with effects of locally administered inhibitors of G-protein coupled pathways. Conclusions and Implications GB88 is a biased antagonist of PAR2 that selectively inhibits PAR2/Gq/11/Ca2+/PKC signalling, leading to anti-inflammatory activity in vivo, while being an agonist in activating three other PAR2-activated pathways (cAMP, ERK, Rho) in human cells. These findings highlight opportunities to design drugs to block specific PAR2-linked signalling pathways in disease, without blocking beneficial PAR2 signalling in normal physiology, and to dissect PAR2-associated mechanisms of disease in vivo. PMID:24821440

  17. Site specificity of DSP-PP cleavage by BMP1.

    PubMed

    Yang, Robert T; Lim, Glendale L; Yee, Colin T; Fuller, Robert S; Ritchie, Helena H

    2014-08-01

    Bone morphogenic protein 1 (BMP1), a metalloproteinase, is known to cleave a wide variety of extracellular matrix proteins, suggesting that a consensus substrate cleavage amino acid sequence might exist. However, while such a consensus sequence has been proposed based on P4 to P4' (i.e. the four amino acids flanking either side of the BMP1 cleavage site; P4P3P2P1|P1'P2'P3'P4') sequence homologies between two BMP1 substrates, dentin matrix protein 1 and dentin sialoprotein phosphophoryn (DSP-PP) (i.e. xMQx|DDP), no direct testing has so far been attempted. Using an Sf9 cell expression system, we have been able to produce large amounts of uncleaved DSP-PP. Point mutations introduced into this recombinant DSP-PP were then tested for their effects on DSP-PP cleavage by either Sf9 endogenous tolloid-related protein 1 (TLR-1) or by its human homolog, BMP1. Here, we have measured DSP-PP cleavage efficiencies after modifications based on P4-P4' sequence comparisons with dentin matrix protein 1, as well as for prolysyl oxidase and chordin, two other BMP1 substrates. Our results demonstrate that any mutations within or outside of the DSP-PP P4 to P4' cleavage site can block, impair or accelerate DSP-PP cleavage, and suggest that its BMP1 cleavage site is highly conserved in order to regulate its cleavage efficiency, possibly with additional assistance from its conserved exosites. Thus, BMP1 cleavage cannot be based on a consensus substrate cleavage site.

  18. Thrombin-induced regulation of CD95(Fas) expression in the N9 microglial cell line: evidence for involvement of proteinase-activated receptor(1) and extracellular signal-regulated kinase 1/2.

    PubMed

    Weinstein, Jonathan R; Zhang, Matthew; Kutlubaev, Mansur; Lee, Richard; Bishop, Caroline; Andersen, Henrik; Hanisch, Uwe-Karsten; Möller, Thomas

    2009-03-01

    Microglia are the immune cells of the CNS. Brain injury triggers phenotypic changes in microglia including regulation of surface antigens. The serine proteinase alpha-thrombin can induce profound changes in neural cell physiology via cleavage of proteinase-activated receptors (PARs). We recently demonstrated that pharmaceutical-grade recombinant human alpha-thrombin (rh-thr) induces a restricted set of proteolysis-dependent changes in microglia. CD95(Fas) is a cell-death receptor that is up-regulated in microglia by inflammatory stimuli. Here we characterized the effect of rh-thr on CD95(Fas) expression in the N9 microglial cell line. Dose-response and time course studies demonstrated maximal effects at 100 U/ml and 24 h, respectively. Regulation of expression was seen at both the surface protein and steady-state mRNA levels. The rh-thr-induced effects were mimicked by PAR(1) agonist peptides and blocked by pharmacologic inhibitors selective for extracellular signal-regulated kinase 1/2 (ERK 1/2). Rh-thr also induced a rapid and sustained phosphorylation of ERK 1/2. Thrombin-induced regulation of CD95(Fas) could modulate the neuroinflammatory response in a variety of neurological disorders.

  19. Several properties of the partially purified proteinase inhibitor in eggplant exocarp.

    PubMed

    Kanamori, M; Ibuki, F; Yamada, M; Tashiro, M; Miyoshi, M

    1975-01-01

    A proteinase inhibitor was isolated and partially purified from the exocarp of eggplant, Solanum melongena L., by means of acetate buffer extraction, heat treatment, salting-out and column chromatography on DEAE-cellulose. This preparation showed inhibitory activities on various proteinases; trypsin [EC 3.4.4.4] and Pronase were strongly inhibited while alpha-chymotrypsin [EC 3.4.4.5] and Nagarse were weakly inhibited. The inhibitor was a protein substance, and, therefore, it was gradually inactivated by the long-time incubation with Pronase. The inhibition mode was non-competitive on trypsin and competitive on Pronase on the basis of Lineweaver-Burk plots. The investigations on the inhibition behavior in the co-existence of two kinds of proteinases suggested that the inhibitor was not of multi-headed type.

  20. Purification and Characterization of an Extracellular Proteinase from Brevibacterium linens ATCC 9174

    PubMed Central

    Rattray, F. P.; Bockelmann, W.; Fox, P. F.

    1995-01-01

    An extracellular serine proteinase from Brevibacterium linens ATCC 9174 was purified to homogeneity. pH and temperature optima were 8.5 and 50(deg)C, respectively. The results for the molecular mass of the proteinase were 56 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 126 kDa by gel filtration, indicating that the native enzyme exists as a dimer. Mg(sup2+) and Ca(sup2+) activated the proteinase, as did NaCl; however, Hg(sup2+), Fe(sup2+), and Zn(sup2+) caused strong inhibition. The sequence of the first 20 N-terminal amino acids was NH(inf2)-Ala-Lys-Asn-Asp-Ala-Val-Gly-Gly-Met-Gly-Tyr-Leu-Ser-Met-Ile-Pro-Se r-Gln-Pro-Gly. PMID:16535130

  1. Characteristics of a Proteinase of a Trichosporon Species Isolated from Dungeness Crab Meat

    PubMed Central

    Groninger, Herman S.; Eklund, M. W.

    1966-01-01

    The proteinase of a Trichosporon species was partially purified by dialysis, ammonium sulfate fractionation, and Sephadex G-100 gel filtration. A 170-fold purification of the enzyme with a 1.4% recovery of the activity was achieved. The proteinase was separated into a major component and possibly two minor components by starch gel electrophoresis. The pH optimum of the enzyme was 5.8 to 6.2. It was active against casein, hemoglobin, and crab protein substrates, but inactive against bovine serum albumin, lysozyme, and benzoylarginine ethyl ester. It was slightly activated by 10 mm cysteine, 0.1 mm ethylenediaminetetraacetic acid, and 0.1 mm Co++. There was slight inhibition by 10 mm Co++ and 0.1 mm phenylmethylsulfonylfluoride, and total inhibition by 1 mmp-chloromercuribenzoate. The proteinase was completely inactivated by heating at 60 C for 10 min. PMID:5914489

  2. Purification and partial characterization of proteinase inhibitors of equine seminal plasma.

    PubMed

    Vasconcelos, André Belico; Santos, Alexandre Martins Costa; Oliveira, Jamil Silvano; Lagares, Monique de Albuquerque; Santoro, Marcelo Matos

    2009-07-01

    The aims of the study were: 1/ to isolate and identify equine seminal plasma proteinase inhibitors, 2/ to evaluate their inhibitory potential, and 3/ to test a correlation between protein concentration in seminal plasma supernatant (obtained after precipitation with 36% ammonium sulfate) and stallion sexual maturity. Seminal plasma proteins obtained from six stallions were chromatographed in a Superose 12 (FPLC system) column followed by C(18) HPLC reverse-phase. Inhibition of trypsin amidase activity was evaluated in the collected fractions. Active proteins with a molecular mass of 6.3-7.0 KDa were identified using mass spectrometry. The older stallions showed a reduction in total seminal plasma protein concentration, but had similar concentrations of proteinase inhibitors (0.28+/-0.10 mg/ml) in seminal plasma supernatant. Different proteinase inhibitor isoforms were found in semen of all stallions which suggests that the isoforms may be used as biomarkers of individual animals.

  3. Biological roles of cysteine proteinases in the pathogenesis of Trichomonas vaginalis.

    PubMed

    Hernández, Hilda M; Marcet, Ricardo; Sarracent, Jorge

    2014-01-01

    Human trichomonosis, infection with Trichomonas vaginalis, is the most common non-viral sexually transmitted disease in the world. The host-parasite interaction and pathophysiological processes of trichomonosis remain incompletely understood. This review focuses on the advancements reached in the area of the pathogenesis of T. vaginalis, especially in the role of the cysteine proteinases. It highlights various approaches made in this field and lists a group of trichomonad cysteine proteinases involved in diverse processes such as invasion of the mucous layer, cytoadherence, cytotoxicity, cytoskeleton disruption of red blood cells, hemolysis, and evasion of the host immune response. A better understanding of the biological roles of cysteine proteinases in the pathogenesis of this parasite could be used in the identification of new chemotherapeutic targets. An additional advantage could be the development of a vaccine in order to reduce transmission of T. vaginalis.

  4. Toll-like receptors recognize distinct proteinase-resistant glycoconjugates in Campylobacter jejuni and Escherichia coli.

    PubMed

    Phongsisay, Vongsavanh; Hara, Hiromitsu; Fujimoto, Shuji

    2015-03-01

    Campylobacter jejuni causes gastroenteritis and autoimmune neuropathy Guillain-Barré syndrome. The mechanism by which C. jejuni infection results in such the hyperimmunity is not completely understood. Host immunity plays an important role in the disease pathogenesis; however, little is known how immune system recognizes this human pathogen. In this study, we report that Toll-like receptors recognize distinct proteinase K-resistant glycoconjugates in C. jejuni and Escherichia coli. Lipopolysaccharide is solely proteinase-resistant glycoconjugate in E. coli. In contrast, C. jejuni possesses at least five different components that are resistant to proteinase digestion and are capable of inducing NF-κB activation through TLR2 and TLR4. Possession of multiple activators of Toll-like receptors may be the unique strategy of C. jejuni to trigger hyperimmunity.

  5. Purification and Characterization of an Extracellular Proteinase from Brevibacterium linens ATCC 9174.

    PubMed

    Rattray, F P; Bockelmann, W; Fox, P F

    1995-09-01

    An extracellular serine proteinase from Brevibacterium linens ATCC 9174 was purified to homogeneity. pH and temperature optima were 8.5 and 50(deg)C, respectively. The results for the molecular mass of the proteinase were 56 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 126 kDa by gel filtration, indicating that the native enzyme exists as a dimer. Mg(sup2+) and Ca(sup2+) activated the proteinase, as did NaCl; however, Hg(sup2+), Fe(sup2+), and Zn(sup2+) caused strong inhibition. The sequence of the first 20 N-terminal amino acids was NH(inf2)-Ala-Lys-Asn-Asp-Ala-Val-Gly-Gly-Met-Gly-Tyr-Leu-Ser-Met-Ile-Pro-Se r-Gln-Pro-Gly.

  6. Consequences of manganese replacement of copper for prion protein function and proteinase resistance

    PubMed Central

    Brown, David R.; Hafiz, Farida; Glasssmith, Leslie L.; Wong, Boon-Seng; Jones, Ian M.; Clive, Christine; Haswell, Stephen J.

    2000-01-01

    The prion protein (PrP) binds copper and has antioxidant activity enhancing the survival of neurones in culture. The ability of the PrP to bind other cations was tested and it was found that only manganese could substitute for copper. Although initially manganese-loaded PrP exhibited similar structure and activity to copper-loaded PrP, after aging, manganese-loaded PrP became proteinase resistant and lost function. It was also found that manganese could be incorporated into PrP expressed by astrocytes and that this PrP was partially proteinase resistant. These results show that it is possible to generate proteinase-resistant PrP from cells and suggest a possible mechanism for the formation of the scrapie isoform of the PrP as generated in sporadic prion disease. PMID:10716918

  7. Molecular investigation on the interaction of spermine with proteinase K by multispectroscopic techniques and molecular simulation studies.

    PubMed

    Hosseini-Koupaei, Mansoore; Shareghi, Behzad; Saboury, Ali Akbar; Davar, Fateme

    2017-01-01

    The alteration in structure, function and stability of proteinase K in the presence of spermine was investigated using spectroscopic methods and simulation techniques. The stability and enzyme activity of proteinase K-spermine complex were significantly enhanced as compared to that of the pure enzyme. The increase in the value of Vmax and the catalytic efficiency of Proteinase K in presence of spermine confirmed that the polyamine could bring the enzyme hyperactivation. UV-vis spectroscopy, intrinsic fluorescence and circular dichroism methods demonstrated that the binding of spermine changed the microenvironment and structure of proteinase K. The fluorescence studies, showing that spermine quenched the intensity of proteinase K with static mechanism. Thermodynamic parameters analysis suggested that hydrogen bond and van der Waals forces play a key role in complex stability which is in agreement with modeling studies. The CD spectra represented the secondary structure alteration of proteinase K with an increase in α-helicity and a decrease in β-sheet of proteinase K upon spermine conjugation. The molecular simulation results proposed that spermine could interact with proteinase K spontaneously at single binding site, which is in agreement with spectroscopic results. This agreement between experimental and theoretical results may be a worth method for protein-ligand complex studies.

  8. The characterization of SaPIN2b, a plant trichome-localized proteinase inhibitor from Solanum americanum.

    PubMed

    Luo, Ming; Ding, Ling-Wen; Ge, Zhi-Juan; Wang, Zhen-Yu; Hu, Bo-Lun; Yang, Xiao-Bei; Sun, Qiao-Yang; Xu, Zeng-Fu

    2012-11-16

    Proteinase inhibitors play an important role in plant resistance of insects and pathogens. In this study, we characterized the serine proteinase inhibitor SaPIN2b, which is constitutively expressed in Solanum americanum trichomes and contains two conserved motifs of the proteinase inhibitor II (PIN2) family. The recombinant SaPIN2b (rSaPIN2b), which was expressed in Escherichia coli, was demonstrated to be a potent proteinase inhibitor against a panel of serine proteinases, including subtilisin A, chymotrypsin and trypsin. Moreover, rSaPIN2b also effectively inhibited the proteinase activities of midgut trypsin-like proteinases that were extracted from the devastating pest Helicoverpa armigera. Furthermore, the overexpression of SaPIN2b in transgenic tobacco plants resulted in enhanced resistance against H. armigera. Taken together, our results demonstrated that SaPIN2b is a potent serine proteinase inhibitor that may act as a protective protein in plant defense against insect attacks.

  9. Regulation of alpha 1 proteinase inhibitor function by rabbit alveolar macrophages. Evidence for proteolytic rather than oxidative inactivation.

    PubMed Central

    Banda, M J; Clark, E J; Werb, Z

    1985-01-01

    Rabbit alveolar macrophages were cultured in an environment conducive to the secretion of both reactive oxygen and proteinases, so that the relative importance of proteolytic and oxidative inactivation of alpha 1-proteinase inhibitor by alveolar macrophages could be evaluated. The inactivation of alpha 1-proteinase inhibitor was proportional to its proteolysis, and there was no detectable inactivation in the absence of proteolysis. Although the live macrophages were capable of secreting reactive oxygen, they did not inactivate alpha 1-proteinase inhibitor by oxidation. The inactivation of alpha 1-proteinase inhibitor by proteolysis was proportional to the secretion of elastinolytic activity by the alveolar macrophages. The inability of the alveolar macrophages to oxidize alpha 1-proteinase inhibitor was attributed to the methionine in the macrophages, in secreted proteins, and in the culture medium competing for oxidants. The data suggest that proteolytic inactivation of alpha 1-proteinase inhibitor may be important in vivo and that the methionine concentration in vivo may protect alpha 1-proteinase inhibitor from significant oxidative inactivation. Images PMID:2989330

  10. Immobilization of a proteinase from the extremely thermophilic organism Thermus Rt41A.

    PubMed

    Wilson, S A; Peek, K; Daniel, R M

    1994-02-05

    An extracellular proteinase from Thermus strain Rt41A was immobilized to controlled pore glass (CPG) beads. The properties of the free and CPG-immobilized enzymes were compared using both a large (azocasein) and a small (peptidase) substrate. The specific activity of the immobilized proteinase was 5284 azoU/mg with azocasein and 144 sucU/mg for SucAAPFpNA. The percentage recovery of enzyme activity was unaffected by pore size when it was immobilized at a fixed level of activity/g of beads, whereas it increased with increasing pore size when added at a fixed level/m(2) of support. Saturation of the CPG beads was observed at 540 azoU/m(2) of 105-nm beads. Lower levels (50 azoU/m(2) of 50-nm beads) were used in characterization experiments. The pH optimum of the immobilized Rt41A proteinase was 8.0 for azocasein and 9.5 for SucAAPFpNA, compared with the free proteinase which was 10.5 for both substrates. The immobilized enzyme retained 65% of its maximum activity against azocasein at pH 12, whereas the free proteinase retained less than 10% under the same conditions. Stability at 80 degrees C increased on immobilization at all pH values between 5 and 11, the greatest increase in half-life being approximately 12-fold at pH 7.0. Temperature-activity profiles for both the free and immobilized enzymes were similar for both substrates. The stability of the immobilized proteinase, however, was higher than that of the free enzyme in the absence and presence of CaCl(2). Overall, the results show that low levels of calcium (10 muM) protect against thermal denaturation, but that high calcium or immobilization are required to protect against autolysis. (c) 1994 John Wiley & Sons, Inc.

  11. Serine proteinases of mast cell and leukocyte granules. A league of their own.

    PubMed

    Caughey, G H

    1994-12-01

    Serine proteinases are hydrolases that use serine's side chain hydroxyl group to attack and cleave internal peptide bonds in peptides and proteins. They reside in all mammalian tissues, including the lung and airway. As a group, they vary tremendously in form and target specificity and have a vast repertoire of functions, many of which are critical for life. A subset of these proteinases is expressed primarily in the cytosolic granules of leukocytes from bone marrow, including mast cells. Examples are elastase-related proteinases and cathepsin G of monocytes and neutrophils, the many "granzymes" of cytotoxic T lymphocytes and natural killer (NK) cells, and the tryptases and chymases of mast cells. The pace of discovery and characterization of these granule-associated serine proteinases, fueled by technical advances in molecular biology, has accelerated rapidly in the past few years. Progress has been made in assigning possible functions to individual proteinases. However, the burgeoning numbers of these enzymes; their cell, tissue and species-dependent differences in expression; and their variety of action in vitro (despite, in many cases, shared modes of activation and recent divergence in protein evolution) have vexed and challenged those of us who are anxious to establish their roles in mammalian biology. Certainly, much remains to be discovered and clarified. The purpose of this overview is to capture the state of the art in this field, stressing the similarities as well as the differences among individual granule-associated proteinases and focusing particularly on those enzymes likely to be important in the human lung and airways.

  12. Analysis of human immunoglobulin-degrading cysteine proteinases of Trichomonas vaginalis.

    PubMed Central

    Provenzano, D; Alderete, J F

    1995-01-01

    Trichomonas vaginalis is a protozoan parasite that causes a widely distributed sexually transmitted disease (STD). Since immunoglobulin G (IgG) antibodies to specific trichomonad immunogens are found in serum and vaginal washes (VWs) from patients with trichomoniasis, a potential mechanism of immune evasion by this parasite might be the ability of T. vaginalis proteinases to degrade human immunoglobulins (Igs). Incubation of human IgG with lysates of T. vaginalis organisms resulted in time- and concentration-dependent degradation of the heavy chain. Secretory IgA was degraded similarly. Inhibitors of cysteine proteinases, when added to trichomonal lysates, abolished IgG and IgA degradation, while EDTA, a metalloproteinase inhibitor, did not. Substrate-gel electrophoresis with human IgG, IgM, or IgA copolymerized with acrylamide revealed several distinct cysteine proteinases in both lysates and culture supernatants from logarithmically growing parasites that degraded all classes of human antibodies. Trichomonal lysates and supernatants of numerous isolates tested all had Ig-degrading activity. Finally, proteolytic activity against IgG was detected in most (26 of 33; 78%) VWs from patients with trichomoniasis. In contrast, 18 of 28 (65%) VWs from women without trichomoniasis or from patients infected with other STDs had no detectable proteinases when tested in an identical manner. The other 10 of these 28 VWs (35%) had smaller amounts of detectable Ig-degrading proteinases. These differences in Ig-degrading proteinase activity between patients with and without trichomoniasis, regardless of coinfecting STDs, were statistically significant (P = 0.001). These results illustrate that T. vaginalis is capable of degrading human Igs. PMID:7642267

  13. Cloning and rational mutagenesis of kexstatin I, a potent proteinaceous inhibitor of Kex2 proteinase.

    PubMed Central

    Oda, K; Oyama, H; Ito, S; Fukiharu, M; Miyagawa, Y; Takahashi, S; Hirose, M; Kikuchi, N; Nakayama, T; Shibano, Y

    2001-01-01

    Kexstatin I is a potent proteinaceous inhibitor of Kex2 proteinase (EC 3.4.21.61). In the present study we show the molecular cloning, primary structure determination and expression of the gene encoding kexstatin I. We also demonstrate its enhanced activity and specificity for Kex2 proteinase inhibition by rational mutagenesis. The cloned kexstatin I gene encoded a protein of 145 amino acid residues, including the 35-residue signal sequence for secretion. The amino acid sequence showed 52% identity with those of the Streptomyces subtilisin inhibitors (SSIs). Thus kexstatin I is the first SSI-family member that can inhibit Kex2 proteinase. The reactive site of the inhibitor was determined to be -Thr(69)-Lys(70) downward arrowGlu(71)-, where downward arrow indicates the reactive site. Because Kex2 proteinase generally shows the highest affinity for substrates with basic amino acid residues at the P(1) and P(2) sites, conversion of the Thr(69)-Lys(70) segment of the inhibitor into dibasic motifs was expected to result in enhanced inhibitory activities. Thus we constructed kexstatin I mutants, in which the Thr(69)-Lys(70) sequence was replaced by the Thr(69)-Arg(70), Lys(69)-Lys(70) and Lys(69)-Arg(70) sequences using PCR-based mutagenesis, and analysed them kinetically. Among these mutants, the Lys(69)-Arg(70) mutant was the most potent inhibitor. The K(i) for Kex2 proteinase was 3.2x10(-10) M, which was 140-fold lower than that of the inhibitor with the Thr(69)-Lys(70) sequence. Although kexstatin I could also inhibit subtilisin, the enhancement of inhibitory activity upon such mutations was specific for Kex2 proteinase inhibition. PMID:11284720

  14. 3-Keto-5-aminohexanoate Cleavage Enzyme

    PubMed Central

    Bellinzoni, Marco; Bastard, Karine; Perret, Alain; Zaparucha, Anne; Perchat, Nadia; Vergne, Carine; Wagner, Tristan; de Melo-Minardi, Raquel C.; Artiguenave, François; Cohen, Georges N.; Weissenbach, Jean; Salanoubat, Marcel; Alzari, Pedro M.

    2011-01-01

    The exponential increase in genome sequencing output has led to the accumulation of thousands of predicted genes lacking a proper functional annotation. Among this mass of hypothetical proteins, enzymes catalyzing new reactions or using novel ways to catalyze already known reactions might still wait to be identified. Here, we provide a structural and biochemical characterization of the 3-keto-5-aminohexanoate cleavage enzyme (Kce), an enzymatic activity long known as being involved in the anaerobic fermentation of lysine but whose catalytic mechanism has remained elusive so far. Although the enzyme shows the ubiquitous triose phosphate isomerase (TIM) barrel fold and a Zn2+ cation reminiscent of metal-dependent class II aldolases, our results based on a combination of x-ray snapshots and molecular modeling point to an unprecedented mechanism that proceeds through deprotonation of the 3-keto-5-aminohexanoate substrate, nucleophilic addition onto an incoming acetyl-CoA, intramolecular transfer of the CoA moiety, and final retro-Claisen reaction leading to acetoacetate and 3-aminobutyryl-CoA. This model also accounts for earlier observations showing the origin of carbon atoms in the products, as well as the absence of detection of any covalent acyl-enzyme intermediate. Kce is the first representative of a large family of prokaryotic hypothetical proteins, currently annotated as the “domain of unknown function” DUF849. PMID:21632536

  15. Lesion Recognition and Cleavage by Endonuclease V

    PubMed Central

    Lin, Jun; Gao, Honghai; Schallhorn, Kathryn A.; Harris, Rebecca M.; Cao, Weiguo; Ke, Pu Chun

    2008-01-01

    Endonuclease V (endo V) recognizes and cleaves deoxyinosine in deaminated DNA. These enzymatic activities are precursors of DNA repair and are fueled by metal ions such as Ca2+ and Mg2+, with the former being associated with protein binding and the latter with DNA cleavage. Using the technique of fluorescence resonance energy transfer (FRET) we determined the single-molecule kinetics of endo V in a catalytic cycle using a substrate of deoxyinosine-containing single-stranded DNA (ssDNA). The ssDNA was labeled with TAMRA, a fluorescence donor, while the endo V was labeled with Cy5, a fluorescence acceptor. The time lapses of FRET, resulting from the sequential association, recognition, and dissociation of the deoxyinosine by the endo V, were determined at 5.9 s, 14.5 s, and 9.1 s, respectively, in the presence of Mg2+. In contrast, the process of deoxyinosine recognition appeared little affected by the metal type. The prolonged association and dissociation events in the presence of the Ca2+-Mg2+ combination, as compared to that of Mg2+ alone, support the hypothesis that endo V has two metal binding sites to regulate its enzymatic activities. PMID:17521169

  16. Measurement of the cleavage energy of graphite

    PubMed Central

    Wang, Wen; Dai, Shuyang; Li, Xide; Yang, Jiarui; Srolovitz, David J.; Zheng, Quanshui

    2015-01-01

    The basal plane cleavage energy (CE) of graphite is a key material parameter for understanding many of the unusual properties of graphite, graphene and carbon nanotubes. Nonetheless, a wide range of values for the CE has been reported and no consensus has yet emerged. Here we report the first direct, accurate experimental measurement of the CE of graphite using a novel method based on the self-retraction phenomenon in graphite. The measured value, 0.37±0.01 J m−2 for the incommensurate state of bicrystal graphite, is nearly invariant with respect to temperature (22 °C≤T≤198 °C) and bicrystal twist angle, and insensitive to impurities from the atmosphere. The CE for the ideal ABAB graphite stacking, 0.39±0.02 J m−2, is calculated based on a combination of the measured CE and a theoretical calculation. These experimental measurements are also ideal for use in evaluating the efficacy of competing theoretical approaches. PMID:26314373

  17. Peptidase specificity from the substrate cleavage collection in the MEROPS database and a tool to measure cleavage site conservation

    PubMed Central

    Rawlings, Neil D.

    2016-01-01

    One peptidase can usually be distinguished from another biochemically by its action on proteins, peptides and synthetic substrates. Since 1996, the MEROPS database (http://merops.sanger.ac.uk) has accumulated a collection of cleavages in substrates that now amounts to 66,615 cleavages. The total number of peptidases for which at least one cleavage is known is 1700 out of a total of 2457 different peptidases. This paper describes how the cleavages are obtained from the scientific literature, how they are annotated and how cleavages in peptides and proteins are cross-referenced to entries in the UniProt protein sequence database. The specificity profiles of 556 peptidases are shown for which ten or more substrate cleavages are known. However, it has been proposed that at least 40 cleavages in disparate proteins are required for specificity analysis to be meaningful, and only 163 peptidases (6.6%) fulfil this criterion. Also described are the various displays shown on the website to aid with the understanding of peptidase specificity, which are derived from the substrate cleavage collection. These displays include a logo, distribution matrix, and tables to summarize which amino acids or groups of amino acids are acceptable (or not acceptable) in each substrate binding pocket. For each protein substrate, there is a display to show how it is processed and degraded. Also described are tools on the website to help with the assessment of the physiological relevance of cleavages in a substrate. These tools rely on the hypothesis that a cleavage site that is conserved in orthologues is likely to be physiologically relevant, and alignments of substrate protein sequences are made utilizing the UniRef50 database, in which in each entry sequences are 50% or more identical. Conservation in this case means substitutions are permitted only if the amino acid is known to occupy the same substrate binding pocket from at least one other substrate cleaved by the same peptidase. PMID

  18. Changes in midgut endopeptidase activity of Spodoptera frugiperda (Lepidoptera: Noctuidae) are responsible for adaptation to soybean proteinase inhibitors.

    PubMed

    Paulillo, L C; Lopes, A R; Cristofoletti, P T; Parra, J R; Terra, W R; Silva-Filho, M C

    2000-06-01

    The development of transgenic maize plants expressing soybean proteinase inhibitors could reduce the economic damage of one of the major maize pests in Brazil, the fall armyworm, Spodoptera frugiperda (J.E. Smith, 1797). We examined the influence of soybean proteinase inhibitors on digestive enzyme properties and development of S. frugiperda larvae. The inhibition of trypsin and chymotrypsin activities in vitro by soybean proteinase inhibitors suggested that either Kunitz (SBTI) or Bowman-Birk (SBBI) would have a potential antimetabolic effect when ingested by insect larvae. However, chronic ingestion of semipurified soybean inhibitors did not result in a significant reduction of growth and development of fall armyworm. Therefore, digestive serine proteinase activities (trypsin and chymotrypsin) of fall armyworm larvae were characterized. The results suggest that S. frugiperda was able to physiologically adapt to dietary proteinase inhibitors by altering the complement of proteolytic enzymes in the insect midguts.

  19. Understanding and targeting a novel plant viral proteinase/substrate interaction. Final report, July 1, 1989--June 30, 1995

    SciTech Connect

    Dougherty, W.

    1995-10-01

    The past 3 years of funding have focused our efforts on trying to understand the molecular basis of a unique substrate interaction displayed by a viral proteinase. We have made good progress and during this funding period we have made four contributions to the scientific literature and have developed the application of the proteinase in the expression and purification of recombinant fusion proteins. A comprehensive review of virus-encoded proteinases, written during the funding period, emphazing the tremendous similarity of viral proteinases with their cellular counterparts and at the same time detail the unique characteristics which permit them to function in a cellular environment. The focus of the research effort was the tobacco etch virus (TEV) 27kDa NIa proteinase.

  20. A practical total synthesis of the microbial alkaline proteinase inhibitor (MAPI).

    PubMed

    Haebich, Dieter; Hillisch, Alexander; El Sheikh, Sherif

    2009-12-01

    Diverse serine and cysteine proteases as well as alkaline proteinases and elastases play a crucial role in numerous biological processes. Natural peptide aldehydes such as the "microbial alkaline proteinase inhibitor" (MAPI, 1) are valuable tools to characterize novel enzymes and to study their function in nature. Within a drug discovery program we wanted to design and explore non-natural MAPI congeners with novel biological profiles. To that end we devised a simple, practical, and scalable synthesis of MAPI 1 from readily available amino acid building blocks. The modular nature of our approach allows convenient structural modification of the MAPI backbone.

  1. Effects of E-64, a cysteine proteinase inhibitor, on cowpea weevil growth, development, and fecundity

    SciTech Connect

    Murdock, L.L.; Shade, R.E.; Pomeroy, M.A.

    1988-06-01

    E-64, a specific inhibitor of cysteine proteinases, was incorporated into artificial seeds at low levels (0.01-0.25% by weight). It prolonged developmental time and increased mortality of the larval cowpea weevil, Callosobruchus maculatus (F.), in direct proportion to its concentration in the artificial seeds. The fecundity of females emerging from the artificial seeds was significantly decreased by E-64 concentrations of 0.06% and higher. These observations are compatible with the hypothesis that the midgut cysteine proteinase in C. maculatus is essential for normal growth and development.

  2. Quantification of DNA cleavage specificity in Hi-C experiments.

    PubMed

    Meluzzi, Dario; Arya, Gaurav

    2016-01-08

    Hi-C experiments produce large numbers of DNA sequence read pairs that are typically analyzed to deduce genomewide interactions between arbitrary loci. A key step in these experiments is the cleavage of cross-linked chromatin with a restriction endonuclease. Although this cleavage should happen specifically at the enzyme's recognition sequence, an unknown proportion of cleavage events may involve other sequences, owing to the enzyme's star activity or to random DNA breakage. A quantitative estimation of these non-specific cleavages may enable simulating realistic Hi-C read pairs for validation of downstream analyses, monitoring the reproducibility of experimental conditions and investigating biophysical properties that correlate with DNA cleavage patterns. Here we describe a computational method for analyzing Hi-C read pairs to estimate the fractions of cleavages at different possible targets. The method relies on expressing an observed local target distribution downstream of aligned reads as a linear combination of known conditional local target distributions. We validated this method using Hi-C read pairs obtained by computer simulation. Application of the method to experimental Hi-C datasets from murine cells revealed interesting similarities and differences in patterns of cleavage across the various experiments considered.

  3. The VHSE-based prediction of proteasomal cleavage sites.

    PubMed

    Xie, Jiangan; Xu, Zhiling; Zhou, Shangbo; Pan, Xianchao; Cai, Shaoxi; Yang, Li; Mei, Hu

    2013-01-01

    Prediction of proteasomal cleavage sites has been a focus of computational biology. Up to date, the predictive methods are mostly based on nonlinear classifiers and variables with little physicochemical meanings. In this paper, the physicochemical properties of 14 residues both upstream and downstream of a cleavage site are characterized by VHSE (principal component score vector of hydrophobic, steric, and electronic properties) descriptors. Then, the resulting VHSE descriptors are employed to construct prediction models by support vector machine (SVM). For both in vivo and in vitro datasets, the performance of VHSE-based method is comparatively better than that of the well-known PAProC, MAPPP, and NetChop methods. The results reveal that the hydrophobic property of 10 residues both upstream and downstream of the cleavage site is a dominant factor affecting in vivo and in vitro cleavage specificities, followed by residue's electronic and steric properties. Furthermore, the difference in hydrophobic potential between residues flanking the cleavage site is proposed to favor substrate cleavages. Overall, the interpretable VHSE-based method provides a preferable way to predict proteasomal cleavage sites.

  4. Use of Cleavage as an Aid in the Optical Determination of Minerals.

    ERIC Educational Resources Information Center

    Ehlers, Ernest G.

    1980-01-01

    Described is the use of cleavage as an aid to microscopic determination of unknown minerals by immersion methods. Cleavages are examined in relation to fragment shapes, types of extinction, and cleavage-optical relationships. (Author/DS)

  5. A comparative study of the role of the major proteinases of germinated common bean (Phaseolus vulgaris L.) and soybean (Glycine max (L.) Merrill) seeds in the degradation of their storage proteins.

    PubMed

    Zakharov, A; Carchilan, M; Stepurina, T; Rotari, V; Wilson, K; Vaintraub, I

    2004-10-01

    Two types of cysteine proteases, low-specificity enzymes from the papain family and Asn-specific from the legumain family are generally considered to be the major endopeptidases responsible for the degradation of seed storage proteins during early seedling growth. The action of the corresponding enzymes (CPPh1 and LLP, respectively) from common bean (Phaseolus vulgaris L.) on phaseolin (the common bean storage protein), and on the homologous soybean (Glycine max (L.) Merrill) storage protein, beta-conglycinin, was studied. Under the action of LLP, proteolysis of phaseolin was limited to cleavage of its interdomain linker. No cleavage of the interdomain linker occurred in beta-conglycinin with LLP. LLP action was restricted to splitting off the disordered N-terminal extensions of alpha and alpha' subunits. No extensive hydrolysis (degradation to short TCA-soluble peptides) of either protein occurred under the action of LLP. CPPh1 cleaved the phaseolin subunits into roughly half-sized fragments at the onset of proteolysis. The cleavage was accompanied by a small (8-10%) decrease of protein. No decrease of protein occurred with further incubation. Thus the two most active proteinases detected in common bean seedlings individually were incapable of the extensive degradation of phaseolin. Extensive hydrolysis of phaseolin was only achieved by the consecutive action of LLP and CPPh1. Similar cleavages occurred during the action of CPPh1 on beta-conglycinin. However, by contrast with phaseolin, CPPh1 by itself accomplished the extensive hydrolysis of beta-conglycinin. The differences in the course of proteolysis of the proteins studied were determined by their structural peculiarities.

  6. Identification of the aspartic proteinases from human erythrocyte membranes and gastric mucosa (slow-moving proteinase) as catalytically equivalent to cathepsin E.

    PubMed Central

    Jupp, R A; Richards, A D; Kay, J; Dunn, B M; Wyckoff, J B; Samloff, I M; Yamamoto, K

    1988-01-01

    Three aspartic proteinases with similar Mr values (approx. 80,000) but from distinct sources (human gastric mucosa, human erythrocyte membranes and rat spleen) were shown to have immunological cross-reactivity and comparable mobilities when subjected to polyacrylamide-gel electrophoresis under non-denaturing conditions. Kinetic parameters (kcat, Km and Ki) were determined for the interactions of the three enzymes with two synthetic chromogenic substrates and five inhibitors (naturally occurring and synthetic). On this basis it would appear that all of the enzymes should be considered equivalent to cathepsin E. pH-activity measurements indicated that the aspartic proteinase that originated from the erythrocyte membranes retained activity at a higher pH value than either of its readily soluble counterparts. Images Fig. 1. Fig. 2. PMID:3058118

  7. Specific oxidative cleavage of carotenoids by VP14 of maize

    SciTech Connect

    Schwartz, S.H.; Zeevaart, J.A.D.; Gage, D.A.; Tan, Bao Cai

    1997-06-20

    The plant growth regulator abscisic acid (ABA) is formed by the oxidative cleavage of an epoxy-carotenoid. The synthesis of other apocarotenoids, such as vitamin A in animals, may occur by a similar mechanism. In ABA biosynthesis, oxidative cleavage is the first committed reaction and is believed to be the key regulatory step. A new ABA-deficient mutant of maize has been identified and the corresponding gene, Vp14, has been cloned. The recombinant VP14 protein catalyzes the cleavage of 9-cis-epoxy-carotenoids to form C{sub 25} apo-aldehydes and xanthoxin, a precursor of ABA in higher plants.

  8. Detection of nucleic acid sequences by invader-directed cleavage

    DOEpatents

    Brow, Mary Ann D.; Hall, Jeff Steven Grotelueschen; Lyamichev, Victor; Olive, David Michael; Prudent, James Robert

    1999-01-01

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The 5' nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based by charge.

  9. High-molecular-mass multicatalytic proteinase complexes produced by the nitrogen-fixing actinomycete Frankia strain BR.

    PubMed Central

    Benoist, P; Müller, A; Diem, H G; Schwencke, J

    1992-01-01

    A major-high-molecular mass proteinase and seven latent minor proteinases were found in cell extracts and in concentrates of culture medium from Frankia sp. strain BR after nondenaturing electrophoresis in mixed gelatin-polyacrylamide gels. All of these complexes showed multicatalytic properties. Their molecular masses and their sedimentation coefficients varied from 1,300 kDa (28S) to 270 kDa (12S). The electroeluted 1,300-kDa proteinase complex dissociated into 11 low-molecular-mass proteinases (40 to 19 kDa) after sodium dodecyl sulfate activation at 30 degrees C and electrophoresis under denaturing conditions. All of these electroeluted proteinases hydrolyzed N-carbobenzoxy-Pro-Ala-Gly-Pro-4-methoxy-beta- naphthylamide, D-Val-Leu-Arg-4-methoxy-beta-naphthylamide, and Boc-Val-Pro-Arg-4-methyl-7-coumarylamide, whereas Suc-Leu-Leu-Val-Tyr-4-methyl-7-coumarylamide was cleaved only by the six lower-molecular-mass proteinases (27.5 to 19 kDa). Examination by electron microscopy of uranyl acetate-stained, electroeluted 1,300- and 650-kDa intracellular and extracellular proteinase complexes showed ring-shaped and cylindrical particles (10 to 11 nm in diameter, 15 to 16 nm long) similar to those of eukaryotic prosomes and proteasomes. Polyclonal antibodies raised against rat skeletal muscle proteasomes cross-reacted with all of the high-molecular-mass proteinase complexes and, after denaturation of the electroeluted 1,300-kDa band, with polypeptides of 35 to 38, 65, and 90 kDa. Electrophoresis of the activated cell extracts under denaturing conditions revealed 11 to 17 gelatinases from 40 to 19 kDa, including the 11 proteinases of the 1,300-kDa proteinase complex. The inhibition pattern of these proteinases is complex. Thiol-reactive compounds and 1-10-phenanthroline strongly inhibited all of the proteinases, but inhibitors against serine-type proteinases were also effective for most of them. Images PMID:1537794

  10. Isolation and characterization of a subtilisin-like proteinase of Bacillus intermedius secreted by the Bacillus subtilis recombinant strain AJ73 at different growth stages.

    PubMed

    Mikhailova, E O; Mardanova, A M; Balaban, N P; Rudenskaya, G N; Sharipova, M R

    2007-02-01

    Two subtilisin-like serine proteinases of Bacillus intermedius secreted by the Bacillus subtilis recombinant strain AJ73 (pCS9) on the 28th and 48th h of culture growth (early and late proteinase, respectively) have been isolated by ion-exchange chromatography on CM-cellulose and by FPLC. Molecular weights of both proteinases were determined. The N-terminal sequences of the recombinant protein and mature proteinases of the original strain were compared. Kinetic parameters and substrate specificities of the early and late proteinase were analyzed. Physicochemical properties of the enzymes were studied.

  11. Detection of nucleic acids by multiple sequential invasive cleavages

    SciTech Connect

    Hall, Jeff G; Lyamichev, Victor I; Mast, Andrea L; Brow, Mary Ann D

    2012-10-16

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of human cytomegalovirus nucleic acid in a sample.

  12. Detection of nucleic acids by multiple sequential invasive cleavages 02

    DOEpatents

    Hall, Jeff G.; Lyamichev, Victor I.; Mast, Andrea L.; Brow, Mary Ann D.

    2002-01-01

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of human cytomegalovirus nucleic acid in a sample.

  13. Detection of nucleic acids by multiple sequential invasive cleavages

    DOEpatents

    Hall, Jeff G.; Lyamichev, Victor I.; Mast, Andrea L.; Brow, Mary Ann D.

    1999-01-01

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of human cytomegalovirus nucleic acid in a sample.

  14. Surface located procollagen N-propeptides on dermatosparactic collagen fibrils are not cleaved by procollagen N-proteinase and do not inhibit binding of decorin to the fibril surface.

    PubMed

    Watson, R B; Holmes, D F; Graham, H K; Nusgens, B V; Kadler, K E

    1998-04-24

    Dermatosparaxis is a recessive disorder of animals (including man) which is caused by mutations in the gene for the enzyme procollagen N-proteinase and is characterised by extreme skin fragility. Partial loss of enzyme activity results in accumulation of pNcollagen (collagen with N-propeptides) and abnormal collagen fibrils in the fragile skin. How the N-propeptides persist in the tissue and how abnormal fibril morphology results in fragile skin is poorly understood. Using biochemical and quantitative mass mapping electron microscopy we showed that the collagen fibrils in the skin of a dermatosparactic calf contained 57% type I pNcollagen and 43% type I collagen and the fibrils were irregularly arranged in bundles and hieroglyphic in cross-section. Image analysis of the fibril cross-sections suggested that the deviation from circularity of dermatosparactic fibrils was caused by N-propeptides of pNcollagen being located at the fibril surface. Comparison of experimental and theoretical axial mass distributions of the fibrils showed that the N-propeptides were located to the overlap zone of the fibril D-period (where D=67 nm, the characteristic axial periodicity of collagen fibrils). Treatment of the dermatosparactic fibrils with N-proteinase did not remove the N-propeptides from the fibrils, although the N-propeptides were efficiently removed by trypsin and chymotrypsin. However, the N-propeptides were efficiently cleaved by the N-proteinase when the pNcollagen molecules were extracted from the fibrils. These results are consistent with close packing of N-propeptides at the fibril surface which prevented cleavage by the N-proteinase. Long-range axial mass determination along the fibril length showed gross non-uniformity with multiple mass bulges. Of note is the skin fragility in dermatosparaxis, and also the appearance of mass bulges along the fibril long axis symptomatic of the fragile skin of mice which lack decorin. Western blot analysis showed that the

  15. Mechanisms for ribotoxin-induced ribosomal RNA cleavage

    SciTech Connect

    He, Kaiyu; Zhou, Hui-Ren; Pestka, James J.

    2012-11-15

    The Type B trichothecene deoxynivalenol (DON), a ribotoxic mycotoxin known to contaminate cereal-based foods, induces ribosomal RNA (rRNA) cleavage in the macrophage via p38-directed activation of caspases. Here we employed the RAW 264.7 murine macrophage model to test the hypothesis that this rRNA cleavage pathway is similarly induced by other ribotoxins. Capillary electrophoresis confirmed that the antibiotic anisomycin (≥ 25 ng/ml), the macrocylic trichothecene satratoxin G (SG) (≥ 10 ng/ml) and ribosome-inactivating protein ricin (≥ 300 ng/ml) induced 18s and 28s rRNA fragmentation patterns identical to that observed for DON. Also, as found for DON, inhibition of p38, double-stranded RNA-activated kinase (PKR) and hematopoietic cell kinase (Hck) suppressed MAPK anisomycin-induced rRNA cleavage, while, in contrast, their inhibition did not affect SG- and ricin-induced rRNA fragmentation. The p53 inhibitor pifithrin-μ and pan caspase inhibitor Z-VAD-FMK suppressed rRNA cleavage induced by anisomycin, SG and ricin, indicating that these ribotoxins shared with DON a conserved downstream pathway. Activation of caspases 8, 9 and 3 concurrently with apoptosis further suggested that rRNA cleavage occurred in parallel with both extrinsic and intrinsic pathways of programmed cell death. When specific inhibitors of cathepsins L and B (lysosomal cysteine cathepsins active at cytosolic neutral pH) were tested, only the former impaired anisomycin-, SG-, ricin- and DON-induced rRNA cleavage. Taken together, the data suggest that (1) all four ribotoxins induced p53-dependent rRNA cleavage via activation of cathepsin L and caspase 3, and (2) activation of p53 by DON and anisomycin involved p38 whereas SG and ricin activated p53 by an alternative mechanism. Highlights: ► Deoxynivalenol (DON) anisomycin, satratoxin G (SG) and ricin are ribotoxins. ► Ribotoxins induce 18s and 28s rRNA cleavage in the RAW 264.7 macrophage model. ► Ribotoxins induce rRNA cleavage via

  16. Prevalence, susceptibility profile and proteinase production of yeasts causing vulvovaginitis in Turkish women.

    PubMed

    Ozcan, Sema Keceli; Budak, Fatma; Yucesoy, Gulseren; Susever, Serdar; Willke, Ayse

    2006-02-01

    In this study the prevalence of vulvovaginal candidiasis (VVC), antifungal susceptibility and proteinase production of isolated Candida species were investigated. Vaginal swabs were collected from symptomatic women with vulvovaginitis attending the Obstetrics and Gynecology Clinic of Kocaeli University, Turkey. The relation between risk factors, such as pregnancy, diabetes mellitus, antibiotic and corticosteroid use, history of sexually transmitted diseases and contraceptive methods, was recorded. Candida spp. were identified by conventional methods, then evaluated for proteinase secretion in a medium containing casein. Antifungal susceptibility was determined according to the NCCLS microdilution method. The prevalence of women with vulvovaginitis was 35.7% (170/6080) and 16% (28/170) of them were diagnosed as VVC. Candida albicans was the dominant species: 21 (75%), followed by 4 C. glabrata (14%), 2 C. tropicalis (7%), and one C. krusei (3.5%). All isolates were susceptible to fluconazole, itraconazole and amphotericin B, except one C. krusei, one C. glabrata and one C. albicans that were resistant to fluconazole. Proteinase production was determined in 19 (90.5%) C. albicans and in all C. tropicalis isolates. Proteinase activity was not associated with antifungal resistance. No association was found between risk factors and VVC.

  17. Detergents modify proteinase K resistance of PrPSc in different transmissible spongiform encephalopathies (TSEs)

    PubMed Central

    Breyer, Johanna; Wemheuer, Wiebke M.; Wrede, Arne; Graham, Catherine; Benestad, Sylvie L.; Brenig, Bertram; Richt, Jürgen A.; Schulz-Schaeffer, Walter J.

    2012-01-01

    Prion diseases are diagnosed by the detection of their proteinase K-resistant prion protein fragment (PrPSc). Various biochemical protocols use different detergents for the tissue preparation. We found that the resistance of PrPSc against proteinase K may vary strongly with the detergent used. In our study, we investigated the influence of the most commonly used detergents on eight different TSE agents derived from different species and distinct prion disease forms. For a high throughput we used a membrane adsorbtion assay to detect small amounts of prion aggregates, as well as Western blotting. Tissue lysates were prepared using DOC, SLS, SDS or Triton X-100 in different concentrations and these were digested with various amounts of proteinase K. Detergents are able to enhance or diminish the detectability of PrPSc after proteinase K digestion. Depending on the kind of detergent, its concentration - but also on the host species that developed the TSE and the disease form or prion type - the detectability of PrPSc can be very different. The results obtained here may be helpful during the development or improvement of a PrPSc detection method and they point towards a detergent effect that can be additionally used for decontamination purposes. A plausible explanation for the detergent effects described in this article could be an interaction with the lipids associated with PrPSc that may stabilize the aggregates. PMID:22226540

  18. Subunit structure of karatasin, the proteinase isolated from Bromelia plumieri (karatas).

    PubMed

    Montes, C; Amador, M; Cuevas, D; Cordoba, F

    1990-01-01

    Close to 15% of the karatasin proteinase activity in the fruit juice of Bromelia plumieri (karatas) is present outside dialysis Visking tubing in 7 days in 0.2 M acetate buffer (pH) 3.5 or 6.5) containing phenyl mercuric acetate. The small proteinase(s), distinct from the 85% activity in juice due to nondialysable karatasin with a reported Mr of 24,868, separates across Spectrapore (13 kDa) membranes but not across Spectrapore with 3.5 kDa average pore diameter. The dialyzed proteinase is named karatasin-D (K-D). Purified non-Dialysable karatasin can be dissociated to what seems to be K-D by incubation in a buffer solution, containing SDS and 2-mercaptoethanol with phenyl mercuric acetate, in dialysis experiments for 8 days at room temperature using Spectrapore 13 kDa tubing. Thus, native karatasin in B. plumieri fruit juice seem to be the result of association of 2 small molecular mass K-D subunits, linked together by disulfide bonds and electrostatic forces, in equilibrium with small amounts of free K-D molecules. The amino acid composition and partial sequence of karatasin up to the 14th position from the amino terminus have discrete analogies with papain and with stem bromelain.

  19. Proteinase K and the structure of PrPse: the good, the bad, and the ugly

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infectious proteins (prions) are, ironically, defined by their resistance to proteolytic digestion. A defining characteristic of the transmissible isoform of the prion protein (PrPSc) is its partial resistance to proteinase K (PK) digestion. Diagnosis of prion disease typically relies upon immunod...

  20. Insect and wound induced GUS gene expression from a Beta vulgaris proteinase inhibitor gene promoter

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Inducible gene promoters that are specifically activated by pathogen invasion or insect pest attack are needed for effective expression of resistance genes to control plant diseases. In the present study, a promoter from a serine proteinase inhibitor gene (BvSTI) shown to be up-regulated in resist...

  1. Detergents modify proteinase K resistance of PrP Sc in different transmissible spongiform encephalopathies (TSEs).

    PubMed

    Breyer, Johanna; Wemheuer, Wiebke M; Wrede, Arne; Graham, Catherine; Benestad, Sylvie L; Brenig, Bertram; Richt, Jürgen A; Schulz-Schaeffer, Walter J

    2012-05-25

    Prion diseases are diagnosed by the detection of their proteinase K-resistant prion protein fragment (PrP(Sc)). Various biochemical protocols use different detergents for the tissue preparation. We found that the resistance of PrP(Sc) against proteinase K may vary strongly with the detergent used. In our study, we investigated the influence of the most commonly used detergents on eight different TSE agents derived from different species and distinct prion disease forms. For a high throughput we used a membrane adsorption assay to detect small amounts of prion aggregates, as well as Western blotting. Tissue lysates were prepared using DOC, SLS, SDS or Triton X-100 in different concentrations and these were digested with various amounts of proteinase K. Detergents are able to enhance or diminish the detectability of PrP(Sc) after proteinase K digestion. Depending on the kind of detergent, its concentration - but also on the host species that developed the TSE and the disease form or prion type - the detectability of PrP(Sc) can be very different. The results obtained here may be helpful during the development or improvement of a PrP(Sc) detection method and they point towards a detergent effect that can be additionally used for decontamination purposes. A plausible explanation for the detergent effects described in this article could be an interaction with the lipids associated with PrP(Sc) that may stabilize the aggregates.

  2. A new subtilisin-like proteinase from roots of the dandelion Taraxacum officinale Webb S. L.

    PubMed

    Bogacheva, A M; Rudenskaya, G N; Preusser, A; Tchikileva, I O; Dunaevsky, Y E; Golovkin, B N; Stepanov, V M

    1999-09-01

    A serine proteinase from roots of Taraxacum officinale Webb S. L. was isolated by affinity chromatography and gel-filtration on Superose 6R using FPLC. The enzyme is a 67-kD glycoprotein containing 54% carbohydrate which we have named taraxalisin. The substrate specificity of taraxalisin toward synthetic peptides and oxidized insulin B-chain is comparable with that of cucumisin from Cucumis melo and the subtilisin-like serine proteinase macluralisin from Maclura pomifera. The proteinase is inactivated by DFP and PMSF. Taraxalisin exhibits maximal activity at pH 8.0. The pH range for stability of the enzyme is narrow--6.0-9.0. The temperature optimum for the subtilisin-like activity is 40 degrees C. The N-terminal sequence of taraxalisin has 40% of its residues identical to those of subtilisin Carlsberg. Thus, the serine proteinase from dandelion roots is a member of the subtilisin family, which is evidently widespread in the plant kingdom.

  3. Proteinase from germinating bean cotyledons. Evidence for involvement of a thiol group in catalysis.

    PubMed

    Csoma, C; Polgár, L

    1984-09-15

    To degrade storage proteins germinating seeds synthesize proteinases de novo that can be inhibited by thiol-blocking reagents [Baumgartner & Chrispeels (1977) Eur. J. Biochem. 77, 223-233]. We have elaborated a procedure for isolation of such a proteinase from the cotyledons of Phaseolus vulgaris. The purification procedure involved fractionation of the cotyledon homogenate with acetone and with (NH4)2SO4 and successive chromatographies on DEAE-cellulose, activated thiol-Sepharose Sepharose and Sephacryl S-200. The purified enzyme has an Mr of 23,400, proved to be highly specific for the asparagine side chain and blocking of its thiol group resulted in loss of the catalytic activity. The chemical properties of the thiol group of the bean enzyme were investigated by acylation with t-butyloxycarbonyl-L-asparagine p-nitro-phenyl ester and by alkylations with iodoacetamide and iodoacetate. Deviations from normal pH-rate profile were observed, which indicated that the thiol group is not a simple functional group, but constitutes a part of an interactive system at the active site. The pKa value for acylation and the magnitude of the rate constant for alkylation with iodoacetate revealed that the bean proteinase possesses some properties not shared by papain and the other cysteine proteinases studied to date.

  4. Purification and characterization of a milk-clotting aspartic proteinase from globe artichoke (Cynara scolymus L.).

    PubMed

    Llorente, Berta E; Brutti, Cristina B; Caffini, Néstor O

    2004-12-29

    The study of proteinase expression in crude extracts from different organs of the globe artichoke (Cynara scolymus L.) disclosed that enzymes with proteolytic and milk-clotting activity are mainly located in mature flowers. Maximum proteolytic activity was recorded at pH 5.0, and inhibition studies showed that only pepstatin, specific for aspartic proteinases, presented a significant inhibitory effect. Such properties, in addition to easy enzyme inactivation by moderate heating, make this crude protease extract potentially useful for cheese production. Adsorption with activated carbon, together with anion exchange and affinity chromatography, led to the isolation of a heterodimeric milk-clotting proteinase consisting of 30- and 15-kDa subunits. MALDI-TOF MS of the 15-kDa chain determined a 15.358-Da mass, and the terminal amino sequence presented 96% homology with the smaller cardosin A subunit. The amino terminal sequence of the 30-kDa chain proved to be identical to the larger cardosin A subunit. Electrophoresis evidenced proteinase self-processing that was confirmed by immunoblots presenting 62-, 30-, and 15-kDa bands.

  5. LEKTI domain 15 is a functional Kazal-type proteinase inhibitor.

    PubMed

    Vitzithum, Klaus; Lauber, Thomas; Kreutzmann, Peter; Schulz, Axel; Sommerhoff, Christian P; Rösch, Paul; Marx, Ute C

    2008-01-01

    The multidomain proteinase inhibitor LEKTI (lympho-epithelial Kazal-type related inhibitor) consists of 15 potential serine proteinase inhibitory domains. In various diseases such as the severe skin disorder Netherton syndrome as well as atopy, defects in the gene encoding LEKTI have been identified that generate premature termination codons of translation, suggesting a specific role of the COOH-terminal part of LEKTI in healthy individuals. We overexpressed and purified a sequence comprising the 15th domain of LEKTI for further characterisation. Here, we present a high yield expression system for recombinant production and efficient purification of LEKTI domain 15 as a highly soluble protein with a uniform disulfide pattern that is identical to that of other known Kazal-type inhibitors. Also, the expected P1P1' site was confirmed. LEKTI domain 15 is a well-structured protein as verified by circular dichroism (CD) spectroscopy and a tight-binding and stable inhibitor of the serine proteinase trypsin. These findings confirm the designation of domain 15 as a proteinase inhibitor of the Kazal family.

  6. Insect resistance to sugar beet pests mediated by a Beta vulgaris proteinase inhibitor transgene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We transformed sugar beet (Beta vulgaris) hairy roots and Nicotiana benthamiana plants with a Beta vulgaris root gene (BvSTI) that codes for a serine proteinase inhibitor. BvSTI is a root gene cloned from the F1016 breeding line that has moderate levels of resistance to the sugar beet root maggot ...

  7. Applicability of Yeast Extracellular Proteinases in Brewing: Physiological and Biochemical Aspects

    PubMed Central

    Bilinski, Carl A.; Russell, Inge; Stewart, Graham G.

    1987-01-01

    A general screening survey for expression of extracellular acid proteinase production was performed on over 100 cultures belonging to the genus Saccharomyces. Although two strains of Saccharomyces cerevisiae showed positive extracellular proteinase phenotypes in plate tests, it was not possible to demonstrate proteolytic activities in cell-free culture supernatants in assays performed at beer pH values. Of several yeasts from other genera examined, Saccharomycopsis fibuligera and Torulopsis magnoliae produced extracellular proteinases with desirable properties. Proteolytic activities were detected in assays performed at beer pH values and at lower temperature. Brewer's wort served as a highly inducing medium for extracellular proteinase production, with T. magnoliae yielding enzyme of highest specific activity. In fact, commencement of enzyme production was detected shortly after the onset of exponential growth in brewer's wort. Inclusion of crude enzyme preparations in brewer's wort inoculated simultaneously with brewer's yeast reduced final ethanol yields slightly and was found to be effective in reducing chill haze formation in bottled beer. PMID:16347298

  8. Nutritional Requirements and Nitrogen-Dependent Regulation of Proteinase Activity of Lactobacillus helveticus CRL 1062

    PubMed Central

    Hebert, Elvira M.; Raya, Raul R.; De Giori, Graciela S.

    2000-01-01

    The nutritional requirements of Lactobacillus helveticus CRL 1062 were determined with a simplified chemically defined medium (SCDM) and compared with those of L. helveticus CRL 974 (ATCC 15009). Both strains were found to be prototrophic for alanine, glycine, asparagine, glutamine, and cysteine. In addition, CRL 1062 also showed prototrophy for lysine and serine. The microorganisms also required riboflavin, calcium pantothenate, pyridoxal, nicotinic acid, and uracil for growth in liquid SCDM. The growth rate and the synthesis of their cell membrane-bound serine proteinases, but not of their intracellular leucyl-aminopeptidases, were influenced by the peptide content of the medium. The highest proteinase levels were found during cell growth in basal SCDM, while the synthesis of this enzyme was inhibited in SCDM supplemented with Casitone, Casamino Acids, or β-casein. Low-molecular-mass peptides (<3,000 Da), extracted from Casitone, and the dipeptide leucylproline (final concentration, 5 mM) play important roles in the medium-dependent regulation of proteinase activity. The addition of the dipeptide leucylproline (5 mM) to SCDM reduced proteinase activity by 25%. PMID:11097908

  9. Microbial cleavage of organic C-S bonds

    DOEpatents

    Kilbane, II, John J.

    1994-01-01

    A microbial process for selective cleavage of organic C--S bonds which may be used for reducing the sulfur content of sulfur-containing organic carbonaceous materials, Microorganisms of Rhodococcus rhodochrous and Bacillus sphaericus have been found which have the ability of selective cleavage of organic C--S bonds. Particularly preferred microorganisms are Rhodococcus rhodochrous strain ATCC 53968 and Bacillus sphaericus strain ATCC 53969 and their derivatives.

  10. Microbial cleavage of organic C-S bonds

    DOEpatents

    Kilbane, J.J. II.

    1994-10-25

    A microbial process is described for selective cleavage of organic C-S bonds which may be used for reducing the sulfur content of sulfur-containing organic carbonaceous materials. Microorganisms of Rhodococcus rhodochrous and Bacillus sphaericus have been found which have the ability of selective cleavage of organic C-S bonds. Particularly preferred microorganisms are Rhodococcus rhodochrous strain ATCC 53968 and Bacillus sphaericus strain ATCC 53969 and their derivatives.

  11. Cleavage of a specific bond in troponin C by thrombin.

    PubMed

    Leavis, P C; Rosenfeld, S; Lu, R C

    1978-08-21

    Limited proteolysis of rabbit skeletal troponin C with bovine thrombin yielded two fragments, TH1 (Mr = 11000) containing Ca2+ binding regions I--III and TH2 (Mr = 6000) containing region IV. Determination of the partial sequences of the fragments established the site of cleavage at Arg120-Ala121. Secondary cleavage by thrombin at other arginyl or lysyl residues in troponin C was ruled out by the sequence data and by the amino acid compositions of the two fragments.

  12. A statistical model for cleavage fracture of low alloy steel

    SciTech Connect

    Chen, J.H.; Wang, G.Z.; Wang, H.J.

    1996-10-01

    A new statistical model for cleavage fracture of the low alloy steel is proposed. This model is based on a recently suggested physical model and takes account of the effect of the preceding loading processes. This statistical model satisfactorily describes the failure probability distribution of 42 precracked specimens fractured at various loads at a test temperature of {minus}100 C. The micromechanisms of cleavage fracture of low alloy steel are also further discussed.

  13. Proteinase inhibitors in severe inflammatory processes (septic shock and experimental endotoxaemia): biochemical, pathophysiological and therapeutic aspects.

    PubMed

    Fritz, H

    1979-01-01

    Plasma levels of antithrombin III, alpha 2-macroglobulin and inter-alpha-trypsin inhibitor, as well as those of various clotting, complement and other plasma factors, were significantly decreased in 18 patients suffering from hyperdynamic septic shock. A similar statistically significant reduction of the concentrations of several plasma factors (prothrombin and antithrombin III, plasminogen and alpha 2-plasmin inhibitor, complement factor C3 and clotting factor XIII) was observed in experimental endotoxaemia. In this model the reduction in the plasma levels of these factors was considerably diminished by the intravenous injection of a granulocytic elastase--cathepsin G inhibitor of lower molecular weight from soybeans. The results of both studies indicate that consumption of plasma factors in the course of Gram-negative sepsis proceeds not only via the classical routes (by activation of the clotting, fibrinolytic and complement cascades by system-specific proteinases such as thrombokinase or the plasminogen activator) but also to an appreciable degree of unspecific degradation of plasma factors by neutral proteinases such as elastase and cathepsin G. The endotoxin-induced release of both sorts of proteinases, the system-specific ones and the unspecific lysosomal proteinases from leucocytes and other cells, is likely to be mainly responsible for the consumption of antithrombin III and alpha-2-macroglobulin via complex formation (followed by elimination of the complexes) and the increased turnover of the inter-alpha-trypsin inhibitor as observed in the clinical study. The therapeutic use of an exogenous elastase--cathepsin G inhibitor in the experimental model was stimulated by the observation that human mucous secretions contain and acid-stable inhibitor of the neutral granulocytic proteinases, called HUSI-I or antileucoproteinase. This inhibitor protects mucous membranes and soluble proteins against proteolytic attack by leucocytic proteinases released in the

  14. Enzymatic response of the eucalypt defoliator Thyrinteina arnobia (Stoll) (Lepidoptera: Geometridae) to a bis-benzamidine proteinase Inhibitor. i.

    PubMed

    Marinho-Prado, Jeanne Scardini; Lourenção, A L; Guedes, R N C; Pallini, A; Oliveira, J A; Oliveira, M G A

    2012-10-01

    Ingestion of proteinase inhibitors leads to hyperproduction of digestive proteinases, limiting the bioavailability of essential amino acids for protein synthesis, which affects insect growth and development. However, the effects of proteinase inhibitors on digestive enzymes can lead to an adaptive response by the insect. In here, we assessed the biochemical response of midgut proteinases from the eucalypt defoliator Thyrinteina arnobia (Stoll) to different concentrations of berenil, a bis-benzamidine proteinase inhibitor, on eucalyptus. Eucalyptus leaves were immersed in berenil solutions at different concentrations and fed to larvae of T. arnobia. Mortality was assessed daily. The proteolytic activity in the midgut of T. arnobia was assessed after feeding on plants sprayed with aqueous solutions of berenil, fed to fifth instars of T. arnobia for 48 h before midgut removal for enzymatic assays. Larvae of T. arnobia were able to overcome the effects of the lowest berenil concentrations by increasing their trypsin-like activity, but not as berenil concentration increased, despite the fact that the highest berenil concentration resulted in overproduction of trypsin-like proteinases. Berenil also prevented the increase of the cysteine proteinases activity in response to trypsin inhibition.

  15. Distinct OGT-Binding Sites Promote HCF-1 Cleavage

    PubMed Central

    Bhuiyan, Tanja; Waridel, Patrice; Kapuria, Vaibhav; Zoete, Vincent; Herr, Winship

    2015-01-01

    Human HCF-1 (also referred to as HCFC-1) is a transcriptional co-regulator that undergoes a complex maturation process involving extensive O-GlcNAcylation and site-specific proteolysis. HCF-1 proteolysis results in two active, noncovalently associated HCF-1N and HCF-1C subunits that regulate distinct phases of the cell-division cycle. HCF-1 O-GlcNAcylation and site-specific proteolysis are both catalyzed by O-GlcNAc transferase (OGT), which thus displays an unusual dual enzymatic activity. OGT cleaves HCF-1 at six highly conserved 26 amino acid repeat sequences called HCF-1PRO repeats. Here we characterize the substrate requirements for OGT cleavage of HCF-1. We show that the HCF-1PRO-repeat cleavage signal possesses particular OGT-binding properties. The glutamate residue at the cleavage site that is intimately involved in the cleavage reaction specifically inhibits association with OGT and its bound cofactor UDP-GlcNAc. Further, we identify a novel OGT-binding sequence nearby the first HCF-1PRO-repeat cleavage signal that enhances cleavage. These results demonstrate that distinct OGT-binding sites in HCF-1 promote proteolysis, and provide novel insights into the mechanism of this unusual protease activity. PMID:26305326

  16. Granzyme B mediates both direct and indirect cleavage of extracellular matrix in skin after chronic low-dose ultraviolet light irradiation

    PubMed Central

    Parkinson, Leigh G; Toro, Ana; Zhao, Hongyan; Brown, Keddie; Tebbutt, Scott J; Granville, David J

    2015-01-01

    Extracellular matrix (ECM) degradation is a hallmark of many chronic inflammatory diseases that can lead to a loss of function, aging, and disease progression. Ultraviolet light (UV) irradiation from the sun is widely considered as the major cause of visible human skin aging, causing increased inflammation and enhanced ECM degradation. Granzyme B (GzmB), a serine protease that is expressed by a variety of cells, accumulates in the extracellular milieu during chronic inflammation and cleaves a number of ECM proteins. We hypothesized that GzmB contributes to ECM degradation in the skin after UV irradiation through both direct cleavage of ECM proteins and indirectly through the induction of other proteinases. Wild-type and GzmB-knockout mice were repeatedly exposed to minimal erythemal doses of solar-simulated UV irradiation for 20 weeks. GzmB expression was significantly increased in wild-type treated skin compared to nonirradiated controls, colocalizing to keratinocytes and to an increased mast cell population. GzmB deficiency significantly protected against the formation of wrinkles and the loss of dermal collagen density, which was related to the cleavage of decorin, an abundant proteoglycan involved in collagen fibrillogenesis and integrity. GzmB also cleaved fibronectin, and GzmB-mediated fibronectin fragments increased the expression of collagen-degrading matrix metalloproteinase-1 (MMP-1) in fibroblasts. Collectively, these findings indicate a significant role for GzmB in ECM degradation that may have implications in many age-related chronic inflammatory diseases. PMID:25495009

  17. Pest protection conferred by a Beta vulgaris serine proteinase inhibitor gene.

    PubMed

    Smigocki, Ann C; Ivic-Haymes, Snezana; Li, Haiyan; Savić, Jelena

    2013-01-01

    Proteinase inhibitors provide a means of engineering plant resistance to insect pests. A Beta vulgaris serine proteinase inhibitor gene (BvSTI) was fused to the constitutive CaMV35S promoter for over-expression in Nicotiana benthamiana plants to study its effect on lepidopteran insect pests. Independently derived BvSTI transgenic tobacco T2 homozygous progeny were shown to have relatively high BvSTI gene transcript levels. BvSTI-specific polyclonal antibodies cross-reacted with the expected 30 kDA recombinant BvSTI protein on Western blots. In gel trypsin inhibitor activity assays revealed a major clear zone that corresponded to the BvSTI proteinase inhibitor that was not detected in the untransformed control plants. BvSTI-transgenic plants were bioassayed for resistance to five lepidopteran insect pests. Spodoptera frugiperda, S. exigua and Manduca sexta larvae fed BvSTI leaves had significant reductions in larval weights as compared to larvae fed on untransformed leaves. In contrast, larval weights increased relative to the controls when Heliothis virescens and Agrotis ipsilon larvae were fed on BvSTI leaves. As the larvae entered the pupal stage, pupal sizes reflected the overall larval weights. Some developmental abnormalities of the pupae and emerging moths were noted. These findings suggest that the sugar beet BvSTI gene may prove useful for effective control of several different lepidopteran insect pests in genetically modified tobacco and other plants. The sugar beet serine proteinase inhibitor may be more effective for insect control because sugar beet is cropped in restricted geographical areas thus limiting the exposure of the insects to sugar beet proteinase inhibitors and build up of non-sensitive midgut proteases.

  18. Activities of amylase, proteinase, and lipase enzymes from Lactococcus chungangensis and its application in dairy products.

    PubMed

    Konkit, Maytiya; Kim, Wonyong

    2016-07-01

    Several enzymes are involved in the process of converting milk to lactic acid and coagulated milk to curd and, therefore, are important in dairy fermented products. Amylase, proteinase, and lipase are enzymes that play an important role in degrading milk into monomeric molecules such as oligosaccharides, amino acids, and fatty acids, which are the main molecules responsible for flavors in cheese. In the current study, we determined the amylase, proteinase, and lipase activities of Lactococcus chungangensis CAU 28(T), a bacterial strain of nondairy origin, and compared them with those of the reference strain, Lactococcus lactis ssp. lactis KCTC 3769(T), which is commonly used in the dairy industry. Lactococcus chungangensis CAU 28(T) and L. lactis ssp. lactis KCTC 3769(T) were both found to have amylase, proteinase, and lipase activities in broth culture, cream cheese, and yogurt. Notably, the proteinase and lipase activities of L. chungangensis CAU 28(T) were higher than those of L. lactis ssp. lactis KCTC 3769(T), with proteinase activity of 10.50 U/mL in tryptic soy broth and 8.64 U/mL in cream cheese, and lipase activity of 100 U/mL of tryptic soy broth, and 100 U/mL of cream cheese. In contrast, the amylase activity was low, with 5.28 U/mL in tryptic soy broth and 8.86 U/mL in cream cheese. These enzyme activities in L. chungangensis CAU 28(T) suggest that this strain has potential to be used for manufacturing dairy fermented products, even though the strain is of nondairy origin.

  19. A chymotrypsin-like proteinase from the midgut of Tenebrio molitor larvae.

    PubMed

    Elpidina, E N; Tsybina, T A; Dunaevsky, Y E; Belozersky, M A; Zhuzhikov, D P; Oppert, B

    2005-08-01

    A chymotrypsin-like proteinase was isolated from the posterior midgut of larvae of the yellow mealworm, Tenebrio molitor, by ion-exchange and gel filtration chromatography. The enzyme, TmC1, was purified to homogeneity as determined by SDS-PAGE and postelectrophoretic activity detection. TmC1 had a molecular mass of 23.0 kDa, pI of 8.4, a pH optimum of 9.5, and the optimal temperature for activity was 51 degrees C. The proteinase displayed high stability at temperatures below 43 degrees C and in the pH range 6.5-11.2, which is inclusive of the pH of the posterior and middle midgut. The enzyme hydrolyzed long chymotrypsin peptide substrates SucAAPFpNA, SucAAPLpNA and GlpAALpNA and did not hydrolyze short chymotrypsin substrates. Kinetic parameters of the enzymatic reaction demonstrated that the best substrate was SucAAPFpNA, with k(cat app) 36.5 s(-1) and K(m) 1.59 mM. However, the enzyme had a lower K(m) for SucAAPLpNA, 0.5 mM. Phenylmethylsulfonyl fluoride (PMSF) was an effective inhibitor of TmC1, and the proteinase was not inhibited by either tosyl-l-phenylalanine chloromethyl ketone (TPCK) or N(alpha)-tosyl-l-lysine chloromethyl ketone (TLCK). However, the activity of TmC1 was reduced with sulfhydryl reagents. Several plant and insect proteinaceous proteinase inhibitors were active against the purified enzyme, the most effective being Kunitz soybean trypsin inhibitor (STI). The N-terminal sequence of the enzyme was IISGSAASKGQFPWQ, which was up to 67% similar to other insect chymotrypsin-like proteinases and 47% similar to mammalian chymotrypsin A. The amino acid composition of TmC1 differed significantly from previously isolated T. molitor enzymes.

  20. Proteinases in Naegleria Fowleri (strain NF3), a pathogenic amoeba: a preliminary study.

    PubMed

    Mat Amin, Nakisah

    2004-12-01

    Naegleria fowleri is a free-living amoeba, known as a causative agent for a fatal disease of the central nervous system (CNS) in man such as Primary amoebic meningoencephalitis (PAM). Factors contributing to its pathogenicity and its distribution in the environment have been investigated by previous researchers. In case of its pathogenicity, several enzymes such as phospolipase A and sphingomyelinase, have been proposed to probably act as aggressors in promoting PAM but no study so far have been conducted to investigate the presence of proteinase enzyme in this amoeba although a 56kDa cystein proteinase enzyme has been identified in Entamoeba histolytica as an important contributing factor in the amoeba's virulence. In this preliminary study, a pathogenic amoeba, Naegleria fowleri (strain NF3) was examined for the presence of proteinases. Samples of enzymes in this amoeba were analysed by electrophoresis using SDS-PAGE-gelatin gels. The results showed that this amoeba possesses at least two high molecular weight proteinases on gelatin gels; their apparent molecular weights are approximately 128 kDa and approximately 170 kDa. Band of approximately 128 kDa enzyme is membrane-associated and its activity is higher at alkaline pH compared with lower pH; at lower pH, its activity is greatly stimulated by DTT. The approximately 170 kDa band enzyme appears to be inactivated at pH 8.0, at lower ph its activity is higher and DTT-dependance. The activity of this enzyme is partially inhibited by inhibitor E-64 but markedly inhibited to antipain suggesting it belongs to the cysteine proteinase group.

  1. Induction of a heparin-stimulated serine proteinase in sex accessory gland tumors of the Lobund-Wistar rat.

    PubMed

    Wilson, Michael J; Lind, Jeremy; Sinha, Akhouri A

    2015-08-01

    Induction of new proteinase activities that may process growth factors, modify cell surface receptors, cleave extracellular matrix proteins, etc. is considered fundamental in carcinogenesis. The purpose of this study was to characterize a novel proteinase activity induced in sex accessory gland cancers (about 70% in seminal vesicles) of adult male Lobund-Wistar rats by a single injection of N-nitroso-N-methylurea (NMU; 25mg/kg) followed by implanted testosterone propionate (45mg in silastic tubing every 2months) treatment for 10-14months. A 28kDa proteinase activity was detected in tumor extracts using SDS-gelatin gel zymography with incubations done without CaCl2. Its activity was stimulated 15 fold by heparin (optimal activity 1.5-3.0μg/lane) added to the tissue extract-SDS sample buffer prior to electrophoresis. No 28kDa heparin-stimulated proteinase (H-SP) was found in the dorsal, lateral and anterior (coagulating gland) prostate lobes or seminal vesicles of untreated adult rats, but there was a 26-30kDa Ca(2+)-independent proteinase activity in the ventral prostate that showed limited heparin stimulation. The 28kDa H-SP was completely inhibited by 1.0mM 4-(2-aminoethyl)benzenesulfonylfluoride (AESBF) indicating that it was a serine-type proteinase. Other types of proteinase inhibitors were without effect, including serine proteinase inhibitors benzamidine, tranexamic acid and ε-aminocaproic acid. Proteinase activities of about 28kDa were found with casein, fibrinogen or carboxymethylated transferrin as substrate, however, these activities were not stimulated by heparin. Similar levels of activities of the 28kDa H-SP were found in primary tumors and their metastases, but little/no activity was detected in serum, even from rats with large tumor volume and metastases. These data demonstrate overexpression of a heparin-stimulated 28kDa serine proteinase in the primary tumors of sex accessory gland cancers and their metastases. This proteinase either does not

  2. PCR cloning and expression analysis of cDNAs encoding cysteine proteinases from germinating seeds of Vicia sativa L.

    PubMed

    Becker, C; Fischer, J; Nong, V H; Münitz, K

    1994-11-01

    cDNA clones encoding cysteine proteinases from cotyledons of germinated seeds of Vicia sativa L. have been obtained by means of PCR. Degenerate oligonucleotide primers were designed according to conserved amino acid regions of known cysteine proteinases. The deduced amino acid sequences of the cDNA clones encoding VSCYSPR1 and VSCYSPR2 display strong homology to cysteine proteinases of the so called papain superfamily. Northern analyses revealed developmentally regulated expression of both the mRNAs in germinating seeds. The transcripts were shown to be products of two distinct single genes, each exhibiting structural polymorphisms as exposed in few nucleotide substitutions.

  3. Assessing Activity and Inhibition of Middle East Respiratory Syndrome Coronavirus Papain-Like and 3C-Like Proteases Using Luciferase-Based Biosensors

    PubMed Central

    Kilianski, Andy; Mielech, Anna M.; Deng, Xufang

    2013-01-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) is associated with an outbreak of more than 90 cases of severe pneumonia with high mortality (greater than 50%). To date, there are no antiviral drugs or specific therapies to treat MERS-CoV. To rapidly identify potential inhibitors of MERS-CoV replication, we expressed the papain-like protease (PLpro) and the 3-chymotrypsin-like protease (3CLpro) from MERS-CoV and developed luciferase-based biosensors to monitor protease activity in cells. We show that the expressed MERS-CoV PLpro recognizes and processes the canonical CoV-PLpro cleavage site RLKGG in the biosensor. However, existing CoV PLpro inhibitors were unable to block MERS-CoV PLpro activity, likely due to the divergence of the amino acid sequence in the drug binding site. To investigate MERS-CoV 3CLpro activity, we expressed the protease in context with flanking nonstructural protein 4 (nsp4) and the amino-terminal portion of nsp6 and detected processing of the luciferase-based biosensors containing the canonical 3CLpro cleavage site VRLQS. Importantly, we found that a small-molecule inhibitor that blocks replication of severe acute respiratory syndrome (SARS) CoV and murine CoV also inhibits the activity of MERS-CoV 3CLpro. Overall, the protease expression and biosensor assays developed here allow for rapid evaluation of viral protease activity and the identification of protease inhibitors. These biosensor assays can now be used to screen for MERS-CoV-specific or broad-spectrum coronavirus PLpro and 3CLpro inhibitors. PMID:23986593

  4. Cleavage of the Bloom’s syndrome gene product during apoptosis by caspase-3 results in an impaired interaction with topoisomerase IIIα

    PubMed Central

    Freire, Raimundo; d’Adda di Fagagna, Fabrizio; Wu, Leonard; Pedrazzi, Graziella; Stagljar, Igor; Hickson, Ian D.; Jackson, Stephen P.

    2001-01-01

    In higher eukaryotes, the integration of signals triggered in response to certain types of stress can result in programmed cell death. Central to these events is the sequential activation of a cascade of proteinases known as caspases. The final activated effector caspases of this cascade digest a number of cellular proteins, in some cases increasing their enzymatic activity, in others destroying their function. Of the proteins shown to be targets for caspase-mediated proteolysis, a surprisingly large proportion are proteins involved in the signalling or repair of DNA damage. Here we investigate whether BLM, the product of the gene mutated in Bloom’s syndrome, a human autosomal disease characterised by cancer predisposition and sunlight sensitivity, is cleaved during apoptosis. BLM interacts with topoisomerase IIIα and has been proposed to play an important role in maintaining genomic integrity through its roles in DNA repair and replication. We show that BLM is cleaved during apoptosis by caspase-3 and reveal that the main cleavage site is located at the junction between the N-terminal and central helicase domains of BLM. Proteolytic cleavage by caspase-3 produces a 120 kDa fragment, which contains the intact helicase domain and three smaller fragments, the relative amounts of which depend on time of incubation with caspase-3. The 120 kDa fragment retains the helicase activity of the intact BLM protein. However, its interaction with topoisomerase IIIα is severely impaired. Since the BLM–topoisomerase interaction is believed to be necessary for many of the replication and recombination functions of BLM, we suggest that caspase-3 cleavage of BLM could alter the localisation and/or function of BLM and that these changes may be important in the process of apoptosis. PMID:11470874

  5. [Expression of Chinese sturgeon cystatin in yeast Pichia pastoris and its proteinase inhibitory activity analysis].

    PubMed

    Ma, Dong-Mei; Bai, Jun-Jie; Jian, Qing; Lao, Hai-Hua; Ye, Xing; Luo, Jian-Ren

    2003-09-01

    Cystatin, which widely distributed in both tissues and body fluids of animal and plant, was a superfamily of cysteine proteinase inhibitors. It could form activity-inhibitor complexes with cysteine proteinases to inhibit the hydrolytic activity of proteinases. Cystatin played important roles not only in the inhibition of the proteolytic degradation of fish muscle, but also in biological defense systems against invaders. To explore the functions of fish cystatin and the potential values in fish disease prevention and cure, as well as seafood processing, the recombinant yeast strains which could express Chinese sturgeon cystatin were constructed. First, the cystatin cDNA of Chinese sturgeon, which had been PCR modified, was subcloned into yeast integrated vector pPICZaA. After extracted and purified, the recombinant plasmids were linearized by Sac I. The yeast Pichia pastoris GS115 strain was transformed by use of the Lithium Chloride transformation method, and the recombinant cystatin yeast strains got. After 0.5% methanol induction, SDS-PAGE analysis of the culture supernatant indicated that the yield of recombinant cystatin was about 215mg x L(-1) with the percentage about 73.6%. The recombinant cystatin was purified through Q-Sepharose anion-exchange chromatography, and the purity reached about 94.2%. The inhibitory activity of recombinant cystatin was measured by inhibiting the proteinase activity of papain. The results showed that about 1 microg recombinant cystatin could inhibit the activity of 15 microg papain. Heat stability assay results showed that there was a decrease in inhibitory activity of cystatin with the increasing of temperature. When solution of recombinant cystatin was kept at 70 degrees C for 5min, the inhibitory activity reduced fast. While the recombinant cystatin was heated to 90 degrees C for 5min, the inhibitory activity of recombinant cystatin was undetected. The inhibitory activity for recombinant Chinese sturgeon cystatin was higher

  6. Crystal structure of viral serpin crmA provides insights into its mechanism of cysteine proteinase inhibition.

    PubMed Central

    Simonovic, M.; Gettins PGW; Volz, K.

    2000-01-01

    CrmA is an unusual viral serpin that inhibits both cysteine and serine proteinases involved in the regulation of host inflammatory and apoptosis processes. It differs from other members of the serpin superfamily by having a reactive center loop that is one residue shorter, and by its apparent inability to form SDS-stable covalent complexes with cysteine proteinases. To obtain insight into the inhibitory mechanism of crmA, we determined the crystal structure of reactive center loop-cleaved crmA to 2.9 A resolution. The structure, which is the first of a viral serpin, suggests that crmA can inhibit cysteine proteinases by a mechanism analogous to that used by other serpins against serine proteinases. However, one striking difference from other serpins, which may be significant for in vivo function, is an additional highly charged antiparallel strand for b sheet A, whose sequence and length are unique to crmA. PMID:10975564

  7. Ring cleavage of sulfur heterocycles: how does it happen?

    PubMed

    Bressler, D C; Norman, J A; Fedorak, P M

    Sulfur heterocycles are common constituents of petroleum and liquids derived from coal, and they are found in some secondary metabolites of microorganisms and plants. They exist primarily as saturated rings and thiophenes. There are two major objectives driving investigations of the microbial metabolism of organosulfur compounds. One is the quest to develop a process for biodesulfurization of fossil fuels, and the other is to understand the fates of organosulfur compounds in petroleum- or creosote-contaminated environments which is important in assessing bioremediation processes. For these processes to be successful, cleavage of different types of sulfur heterocyclic rings is paramount. This paper reviews the evidence for microbial ring cleavage of a variety of organosulfur compounds and discusses the few well-studied cases which have shown that the C-S bond is most susceptible to breakage leading to disruption of the ring. In most cases, the introduction of one or more oxygen atom(s) onto the adjacent C atom and/or onto the S atom weakens the C-S bond, facilitating its cleavage. Although much is known about the thiophene ring cleavage in dibenzothiophene, there is still a great deal to be learned about the cleavage of other sulfur heterocycles.

  8. Cleavage of Signal Regulatory Protein α (SIRPα) Enhances Inflammatory Signaling.

    PubMed

    Londino, James D; Gulick, Dexter; Isenberg, Jeffrey S; Mallampalli, Rama K

    2015-12-25

    Signal regulatory protein α (SIRPα) is a membrane glycoprotein immunoreceptor abundant in cells of monocyte lineage. SIRPα ligation by a broadly expressed transmembrane protein, CD47, results in phosphorylation of the cytoplasmic immunoreceptor tyrosine-based inhibitory motifs, resulting in the inhibition of NF-κB signaling in macrophages. Here we observed that proteolysis of SIRPα during inflammation is regulated by a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), resulting in the generation of a membrane-associated cleavage fragment in both THP-1 monocytes and human lung epithelia. We mapped a charge-dependent putative cleavage site near the membrane-proximal domain necessary for ADAM10-mediated cleavage. In addition, a secondary proteolytic cleavage within the membrane-associated SIRPα fragment by γ-secretase was identified. Ectopic expression of a SIRPα mutant plasmid encoding a proteolytically resistant form in HeLa cells inhibited activation of the NF-κB pathway and suppressed STAT1 phosphorylation in response to TNFα to a greater extent than expression of wild-type SIRPα. Conversely, overexpression of plasmids encoding the proteolytically cleaved SIRPα fragments in cells resulted in enhanced STAT-1 and NF-κB pathway activation. Thus, the data suggest that combinatorial actions of ADAM10 and γ-secretase on SIRPα cleavage promote inflammatory signaling.

  9. Cleavage Entropy as Quantitative Measure of Protease Specificity

    PubMed Central

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

    2013-01-01

    A purely information theory-guided approach to quantitatively characterize protease specificity is established. We calculate an entropy value for each protease subpocket based on sequences of cleaved substrates extracted from the MEROPS database. We compare our results with known subpocket specificity profiles for individual proteases and protease groups (e.g. serine proteases, metallo proteases) and reflect them quantitatively. Summation of subpocket-wise cleavage entropy contributions yields a measure for overall protease substrate specificity. This total cleavage entropy allows ranking of different proteases with respect to their specificity, separating unspecific digestive enzymes showing high total cleavage entropy from specific proteases involved in signaling cascades. The development of a quantitative cleavage entropy score allows an unbiased comparison of subpocket-wise and overall protease specificity. Thus, it enables assessment of relative importance of physicochemical and structural descriptors in protease recognition. We present an exemplary application of cleavage entropy in tracing substrate specificity in protease evolution. This highlights the wide range of substrate promiscuity within homologue proteases and hence the heavy impact of a limited number of mutations on individual substrate specificity. PMID:23637583

  10. Binding and cleavage of nucleic acids by the "hairpin" ribozyme.

    PubMed

    Chowrira, B M; Burke, J M

    1991-09-03

    The "hairpin" ribozyme derived from the minus strand of tobacco ringspot virus satellite RNA [(-)sTRSV] efficiently catalyzes sequence-specific RNA hydrolysis in trans (Feldstein et al., 1989; Hampel & Triz, 1989; Haseloff & Gerlach, 1989). The ribozyme does not cleave DNA. An RNA substrate analogue containing a single deoxyribonucleotide residue 5' to the cleavage site (A-1) binds to the ribozyme efficiently but cannot be cleaved. A DNA substrate analogue with a ribonucleotide at A-1 is cleaved; thus A-1 provides the only 2'-OH required for cleavage. These results support cleavage via a transphosphorylation mechanism initiated by attack of the 2'-OH of A-1 on the scissile phosphodiester. The ribozyme discriminates between DNA and RNA in both binding and cleavage. Results indicate that the 2'-OH of A-1 functions in complex stabilization as well as cleavage. The ribozyme efficiently cleaves a phosphorothioate diester linkage, suggesting that the pro-Rp oxygen at the scissile phosphodiester does not coordinate Mg2+.

  11. Cleavage of Signal Regulatory Protein α (SIRPα) Enhances Inflammatory Signaling*

    PubMed Central

    Londino, James D.; Gulick, Dexter; Isenberg, Jeffrey S.; Mallampalli, Rama K.

    2015-01-01

    Signal regulatory protein α (SIRPα) is a membrane glycoprotein immunoreceptor abundant in cells of monocyte lineage. SIRPα ligation by a broadly expressed transmembrane protein, CD47, results in phosphorylation of the cytoplasmic immunoreceptor tyrosine-based inhibitory motifs, resulting in the inhibition of NF-κB signaling in macrophages. Here we observed that proteolysis of SIRPα during inflammation is regulated by a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), resulting in the generation of a membrane-associated cleavage fragment in both THP-1 monocytes and human lung epithelia. We mapped a charge-dependent putative cleavage site near the membrane-proximal domain necessary for ADAM10-mediated cleavage. In addition, a secondary proteolytic cleavage within the membrane-associated SIRPα fragment by γ-secretase was identified. Ectopic expression of a SIRPα mutant plasmid encoding a proteolytically resistant form in HeLa cells inhibited activation of the NF-κB pathway and suppressed STAT1 phosphorylation in response to TNFα to a greater extent than expression of wild-type SIRPα. Conversely, overexpression of plasmids encoding the proteolytically cleaved SIRPα fragments in cells resulted in enhanced STAT-1 and NF-κB pathway activation. Thus, the data suggest that combinatorial actions of ADAM10 and γ-secretase on SIRPα cleavage promote inflammatory signaling. PMID:26534964

  12. Cleavage entropy as quantitative measure of protease specificity.

    PubMed

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

    2013-04-01

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

  13. Increased expression of Candida albicans secretory proteinase, a putative virulence factor, in isolates from human immunodeficiency virus-positive patients.

    PubMed Central

    Ollert, M W; Wende, C; Görlich, M; McMullan-Vogel, C G; Borg-von Zepelin, M; Vogel, C W; Korting, H C

    1995-01-01

    The increased prevalence and the severity of oropharyngeal candidiasis in human immunodeficiency virus (HIV)-positive patients are attributed exclusively to the virus-induced immune deficiency of the host. The present study was aimed at answering the question of whether Candida albicans secretory proteinase, a putative virulence factor of the opportunistic C. albicans yeast, has any potential influence on the clinical manifestation of oropharyngeal candidiasis in HIV-positive patients. We measured the secretory proteinase activities of clinical C. albicans isolates from the oropharynges of either HIV-positive individuals (n = 100) or a control group (n = 122). The mean secretory proteinase activity of C. albicans isolates from the HIV-positive group (4,255 +/- 2,372 U/liter) was significantly higher compared with that of isolates from the control group (2,324 +/- 1,487 U/liter) (P < 0.05). The higher level of secretory proteinase activity in the culture supernatants of individual C. albicans isolates correlated with the increased level of proteinase expression on the cell surface, as revealed by cytofluorometry, and with higher levels of secretion of the immunodetectable protein, as shown by Western blotting (immunoblotting). Proteinase activity within the population of C. albicans isolates from HIV-positive individuals was independent of the patient's clinical disease stage and the CD4+/CD8+ cell numbers. Furthermore, no correlation of the proteinase activities with the C. albicans serotype was found, although C. albicans serotype B was significantly more frequent in the HIV-positive group (40%) compared with that in the control group (12%). However, a positive correlation of proteinase activity to antifungal susceptibility was evident.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8567880

  14. Specificity of an extracellular proteinase from Conidiobolus coronatus and its inhibition by an inhibitor from insect hemolymph.

    PubMed

    Bania, Jacek; Samborski, Jaroslaw; Bogus, Mieczyslawa; Polanowski, Antoni

    2006-08-01

    The relatively little-investigated entomopathogen Conidiobolus coronatus secretes several proteinases into culture broth. Using a combination of ion-exchange and size-exclusion chromatography, we purified to homogeneity a serine proteinase of Mr 30,000-32,000, as ascertained by SDS-PAGE. The purified enzyme showed subtilisin-like activity. It very effectively hydrolyzed N-Suc-Ala(2)-Pro-Phe-pNa with a Km-1.36 x 10(-4) M and Kcat-24 s(-1), and N-Suc-Ala(2)-Pro-Leu-pNa with Km-6.65 x 10(-4) M and Kcat-11 s(-1). The specificity index k(cat)/K(m) for the tested substrates was calculated to be 176,340 s(-1) M(-1) and 17,030 s(-1) M(-1), respectively. Using oxidized insulin B chain as a substrate, the purified proteinase exhibited specificity to aromatic and hydrophobic amino-acid residues, such as Phe, Leu, and Gly at the P1 position, splitting primarily the peptide bonds: Phe(1)-Val(2), Leu(15)-Tyr(16), and Gly(23)-Phe(24). The proteinase appeared to be sensitive to the specific synthetic inhibitors of the serine proteinases DFP (diisopropyl flourophosphate) and PMSF (phenyl-methylsulfonyl fluoride) as well as to some naturally occurring protein inhibitors of chymotrypsin. It is worth noting that the enzyme exhibited the highest sensitivity to inhibition by AMCI-1 (with an association constant of 3 x 10(10) M(-1)), an inhibitor of cathepsin G/chymotrypsin from the larval hemolymph of Apis mellifera, reinforcing the possibility of involvement of inhibitors from hemolymph in insect innate immunity. The substrate specificity and proteinase inhibitor effects indicate that the purified proteinase from the fermentation broth of Conidiobolus coronatus is a subtilisin-like serine proteinase.

  15. Experimental verification of cleavage characteristic stress vs grain size

    SciTech Connect

    Lei, W. . Dept. of Mechanical Engineering); Li, D.; Yao, M. . School of Materials Science and Engineering)

    1994-07-01

    Instead of the accepted cleavage fracture stress [sigma][sub f] proposed by Knott et al, a new parameter S[sub co], named as ''cleavage characteristic stress,'' has been recently recommended to characterize the microscopic resistance to cleavage fracture. To give a definition, S[sub co] is the fracture stress at the brittle/ductile transition temperature of steels in plain tension, below which the yield strength approximately equals the true fracture stress combined with an abrupt curtailment of ductility. By considering a single-grain microcrack arrested at a boundary, Huang and Yao set up an expression of S[sub co] as a function of grain size. The present work was arranged to provide an experimental verification of S[sub co] vs grain size.

  16. A trypsin-like proteinase in the midgut of Ectomyelois ceratoniae Zeller (Lepidoptera: Pyralidae): purification, characterization, and host plant inhibitors.

    PubMed

    Ranjbar, Mina; Zibaee, Arash; Sendi, Jalal Jalali

    2014-01-01

    A trypsin-like proteinase was purified and characterized in the midgut of Ectomyelois ceratoniae. A purification process that used Sepharyl G-100 and DEAE-cellulose fast flow chromatographies revealed a proteinase with specific activity of 66.7 μmol/min/mg protein, recovery of 27.04 and purification fold of 23.35. Molecular weight of the purified protein was found to be 35.8 kDa. Optimal pH and temperature were obtained 9 and 20°C for the purified trypsin proteinase, respectively. The purified enzyme was significantly inhibited by PMSF, TLCK, and SBTI as specific inhibitors of trypsins in which TLCK showed the highest inhibitory effect. Trypsin proteinase inhibitors were extracted from four varieties of pomegranate including Brait, Torsh-Sabz, May-Khosh, and Shirin by ion exchange chromatography. It was found that fractions 17-20 of Brait; fractions 18 and 21-26 of Torsh-Sabz; fractions 1-7, 11-17, and 19-21 of May-Khosh and fraction 8 for Shirin showed presence of trypsin inhibitor in these host. Comparison of their inhibitory effects on the purified trypsin proteinase of E. ceratoniae demonstrated that fractions from May-khosh variety had the highest effect on the enzyme among other extracted fractions. Characterization of serine proteinases of insects mainly trypsins is one of the promising methods to decrease population and damages via extracting their inhibitors and providing resistant varieties.

  17. New insight into the cleavage reaction of Nostoc sp. strain PCC 7120 carotenoid cleavage dioxygenase in natural and nonnatural carotenoids.

    PubMed

    Heo, Jinsol; Kim, Se Hyeuk; Lee, Pyung Cheon

    2013-06-01

    Carotenoid cleavage dioxygenases (CCDs) are enzymes that catalyze the oxidative cleavage of carotenoids at a specific double bond to generate apocarotenoids. In this study, we investigated the activity and substrate preferences of NSC3, a CCD of Nostoc sp. strain PCC 7120, in vivo and in vitro using natural and nonnatural carotenoid structures. NSC3 cleaved β-apo-8'-carotenal at 3 positions, C-13 C-14, C-15 C-15', and C-13' C-14', revealing a unique cleavage pattern. NSC3 cleaves the natural structure of carotenoids 4,4'-diaponeurosporene, 4,4'-diaponeurosporen-4'-al, 4,4'-diaponeurosporen-4'-oic acid, 4,4'-diapotorulene, and 4,4'-diapotorulen-4'-al to generate novel cleavage products (apo-14'-diaponeurosporenal, apo-13'-diaponeurosporenal, apo-10'-diaponeurosporenal, apo-14'-diapotorulenal, and apo-10'-diapotorulenal, respectively). The study of carotenoids with natural or nonnatural structures produced by using synthetic modules could provide information valuable for understanding the cleavage reactions or substrate preferences of other CCDs in vivo and in vitro.

  18. New Insight into the Cleavage Reaction of Nostoc sp. Strain PCC 7120 Carotenoid Cleavage Dioxygenase in Natural and Nonnatural Carotenoids

    PubMed Central

    Heo, Jinsol; Kim, Se Hyeuk

    2013-01-01

    Carotenoid cleavage dioxygenases (CCDs) are enzymes that catalyze the oxidative cleavage of carotenoids at a specific double bond to generate apocarotenoids. In this study, we investigated the activity and substrate preferences of NSC3, a CCD of Nostoc sp. strain PCC 7120, in vivo and in vitro using natural and nonnatural carotenoid structures. NSC3 cleaved β-apo-8′-carotenal at 3 positions, C-13C-14, C-15C-15′, and C-13′C-14′, revealing a unique cleavage pattern. NSC3 cleaves the natural structure of carotenoids 4,4′-diaponeurosporene, 4,4′-diaponeurosporen-4′-al, 4,4′-diaponeurosporen-4′-oic acid, 4,4′-diapotorulene, and 4,4′-diapotorulen-4′-al to generate novel cleavage products (apo-14′-diaponeurosporenal, apo-13′-diaponeurosporenal, apo-10′-diaponeurosporenal, apo-14′-diapotorulenal, and apo-10′-diapotorulenal, respectively). The study of carotenoids with natural or nonnatural structures produced by using synthetic modules could provide information valuable for understanding the cleavage reactions or substrate preferences of other CCDs in vivo and in vitro. PMID:23524669

  19. Molecular cloning of Kazal-type proteinase inhibitor of the shrimp Fenneropenaeus chinensis.

    PubMed

    Kong, Hee Jeong; Cho, Hyun Kook; Park, Eun-Mi; Hong, Gyeong-Eun; Kim, Young-Ok; Nam, Bo-Hye; Kim, Woo-Jin; Lee, Sang-Jun; Han, Hyon Sob; Jang, In-Kwon; Lee, Chang Hoon; Cheong, Jaehun; Choi, Tae-Jin

    2009-01-01

    Proteinase inhibitors play important roles in host defence systems involving blood coagulation and pathogen digestion. We isolated and characterized a cDNA clone for a Kazal-type proteinase inhibitor (KPI) from a hemocyte cDNA library of the oriental white shrimp Fenneropenaeus chinensis. The KPI gene consists of three exons and two introns. KPI cDNA contains an open reading frame of 396 bp, a polyadenylation signal sequence AATAAA, and a poly (A) tail. KPI cDNA encodes a polypeptide of 131 amino acids with a putative signal peptide of 21 amino acids. The deduced amino acid sequence of KPI contains two homologous Kazal domains, each with six conserved cysteine residues. The mRNA of KPI is expressed in the hemocytes of healthy shrimp, and the higher expression of KPI transcript is observed in shrimp infected with the white spot syndrome virus (WSSV), suggesting a potential role for KPI in host defence mechanisms.

  20. Analysis of the autoproteolytic activity of the recombinant helper component proteinase from zucchini yellow mosaic virus.

    PubMed

    Boonrod, Kajohn; Füllgrabe, Marc W; Krczal, Gabi; Wassenegger, Michael

    2011-10-01

    The multifunctional helper component proteinase (HC-Pro) of potyviruses contains an autoproteolytic function that, together with the protein 1 (P1) and NIa proteinase, processes the polyprotein into mature proteins. In this study, we analysed the autoproteolytic active domain of zucchini yellow mosaic virus (ZYMV) HC-Pro. Several Escherichia coli-expressed MBP:HC-Pro:GFP mutants containing deletions or point mutations at either the N- or C-terminus of the HC-Pro protein were examined. Our results showed that amino acids essential for the proteolytic activity of ZYMV HC-Pro are distinct from those of the tobacco etch virus HC-Pro, although the amino acid sequences in the proteolytic active domain are conserved among potyviruses.

  1. Purification and Characterization of a Keratinolytic Serine Proteinase from Streptomyces albidoflavus

    PubMed Central

    Bressollier, Philippe; Letourneau, François; Urdaci, Maria; Verneuil, Bernard

    1999-01-01

    Streptomyces strain K1-02, which was identified as a strain of Streptomyces albidoflavus, secreted at least six extracellular proteases when it was cultured on feather meal-based medium. The major keratinolytic serine proteinase was purified to homogeneity by a two-step procedure. This enzyme had a molecular weight of 18,000 and was optimally active at pH values ranging from 6 to 9.5 and at temperatures ranging from 40 to 70°C. Its sensitivity to protease inhibitors, its specificity on synthetic substrates, and its remarkably high level of NH2-terminal sequence homology with Streptomyces griseus protease B (SGPB) showed that the new enzyme, designated SAKase, was homologous to SGPB. We tested the activity of SAKase with soluble and fibrous substrates (elastin, keratin, and type I collagen) and found that it was very specific for keratinous substrates compared to SGPB and proteinase K. PMID:10347045

  2. Purification and characterization of a keratinolytic serine proteinase from Streptomyces albidoflavus.

    PubMed

    Bressollier, P; Letourneau, F; Urdaci, M; Verneuil, B

    1999-06-01

    Streptomyces strain K1-02, which was identified as a strain of Streptomyces albidoflavus, secreted at least six extracellular proteases when it was cultured on feather meal-based medium. The major keratinolytic serine proteinase was purified to homogeneity by a two-step procedure. This enzyme had a molecular weight of 18,000 and was optimally active at pH values ranging from 6 to 9.5 and at temperatures ranging from 40 to 70 degrees C. Its sensitivity to protease inhibitors, its specificity on synthetic substrates, and its remarkably high level of NH2-terminal sequence homology with Streptomyces griseus protease B (SGPB) showed that the new enzyme, designated SAKase, was homologous to SGPB. We tested the activity of SAKase with soluble and fibrous substrates (elastin, keratin, and type I collagen) and found that it was very specific for keratinous substrates compared to SGPB and proteinase K.

  3. A Kunitz proteinase inhibitor from corms of Xanthosoma blandum with bactericidal activity.

    PubMed

    Lima, Thaís B; Silva, Osmar N; Migliolo, Ludovico; Souza-Filho, Carlos R; Gonçalves, Eduardo G; Vasconcelos, Ilka M; Oliveira, José T A; Amaral, André C; Franco, Octávio L

    2011-05-27

    Bacterial infections directly affect the world's population, and this situation has been aggravated by indiscriminate use of antimicrobial agents, which can generate resistant microorganisms. In this report, an initial screening of proteins with antibacterial activity from corms of 15 species of the Xanthosoma genus was conducted. Since Xanthosoma blandum corms showed enhanced activity toward bacteria, a novel protein with bactericidal activity was isolated from this particular species. Edman degradation was used for protein N-termini determination; the primary structure showed similarities with Kunitz inhibitors, and this protein was named Xb-KTI. This protein was further challenged against serine proteinases from different sources, showing clear inhibitory activities. Otherwise, no hemolytic activity was observed for Xb-KTI. The results demonstrate the biotechnological potential of Xb-KTI, the first proteinase inhibitor with antimicrobial activity described in the Xanthosoma genus.

  4. On the modeling of snake venom serine proteinase interactions with benzamidine-based thrombin inhibitors

    PubMed Central

    Henriques, Elsa S.; Fonseca, Nelson; Ramos, Maria João

    2004-01-01

    Pit viper venoms contain a number of serine proteinases that exhibit one or more thrombin-like activities on fibrinogen and platelets, this being the case for the kinin-releasing and fibrinogen-clotting KN-BJ from the venom of Bothrops jararaca. A three-dimensional structural model of the KN-BJ2 serine proteinase was built by homology modeling using the snake venom plasminogen activator TSV-PA as a major template and porcine kallikrein as additional structural support. A set of intrinsic buried waters was included in the model and its behavior under dynamic conditions was molecular dynamics simulated, revealing a most interesting similarity pattern to kallikrein. The benzamidine-based thrombin inhibitors α-NAPAP, 3-TAPAP, and 4-TAPAP were docked into the refined model, allowing for a more insightful functional characterization of the enzyme and a better understanding of the reported comparatively low affinity of KN-BJ2 toward those inhibitors. PMID:15322279

  5. Inactivation of alpha 1-proteinase inhibitor by Cu(II) and hydrogen peroxide.

    PubMed

    Kwon, N S; Chan, P C; Kesner, L

    1990-03-01

    When alpha 1-proteinase inhibitor was treated with 1-5 microM CuSO4 in the presence of H2O2 (250-1000 microM), its elastase inhibitory capacity was markedly decreased. Several other metal ions tested had either very little or no effect. The Cu(II)-catalyzed decreased in the inhibition of elastase activity can also be demonstrated in dialyzed plasma. These results are consistent with the hypothesis that in several pathological conditions in which extracellular copper levels are elevated, Cu(II)-catalyzed peroxidation of alpha 1-proteinase inhibitor may occur at sites of inflammation where H2O2 is secreted as a major product by activated phagocytes.

  6. The Role of Cysteine Proteinases and their Inhibitors in the Host-Pathogen Cross Talk

    PubMed Central

    Kopitar-Jerala, Nataša

    2012-01-01

    Proteinases and their inhibitors play essential functional roles in basic biological processes in both hosts and pathogens. Endo/lysosomal cathepsins participate in immune response in pathogen recognition and elimination. They are essential for both antigen processing and presentation (host adaptive immune response) and activation of endosomal Toll like receptors (innate immune response). Pathogens can produce proteases and also natural inhibitors to subvert the host immune response. Several pathogens are sensed through the intracellular pathogen recognition receptors, but only some of them use the host proteolytic system to escape into the cytosol. In this review, I provide an update on the most recent developments regarding the role of proteinases and their inhibitors in the initiation and regulation of immune responses. PMID:23305363

  7. Degradation of immunoglobulins, protease inhibitors, and interleukin-1 by a secretory proteinase of Acanthamoeba castellanii

    PubMed Central

    Na, Byoung-Kuk; Cho, Jong-Hwa; Song, Chul-Yong; Kim, Tong-Soo

    2002-01-01

    The effect of a secretory proteinase from the pathogenic amoebae Acanthamoeba castellanii on host's defense-oriented or regulatory proteins such as immunoglobulins, interleukin-1, and protease inhibitors was investigated. The enzyme was found to degrade secretory immunoglobulin A (sIgA), IgG, and IgM. It also degraded interleukin-1α (IL-1α) and IL-1β. Its activity was not inhibited by endogenous protease inhibitors, such as α2-macroglobulin, α1-trypsin inhibitor, and α2-antiplasmin. Furthermore, the enzyme rapidly degraded those endogenous protease inhibitors as well. The degradation of host's defense-oriented or regulatory proteins by the Acanthamoeba proteinase suggested that the enzyme might be an important virulence factor in the pathogenesis of Acanthamoeba infection. PMID:12073735

  8. Kinetic analysis of a general model of activation of aspartic proteinase zymogens.

    PubMed

    Varón, R; García-Moreno, M; Valera-Ruipérez, D; García-Molina, F; García-Cánovas, F; Ladrón-de Guevara, R G; Masiá-Pérez, J; Havsteen, B H

    2006-10-07

    Starting from a simple general reaction mechanism of activation of aspartic proteinase zymogens involving an uni- and a bimolecular simultaneous route, the time course equation of the concentration of the zymogen and of the activated enzyme have been derived. From these equations, an analysis quantifying the relative contribution to the global process of the two routes has been carried out for the first time. This analysis suggests a way to predict the time course of the relative contribution as well as the effect of the initial zymogen and activating enzyme concentrations, on the relative weight. An experimental design and kinetic data analysis is suggested to estimate the kinetic parameters involved in the reaction mechanism proposed. Finally, we apply some of our results to experimental data obtained by other authors in experimental studies of the activation of some aspartic proteinase zymogens.

  9. Identification of monomeric alpha-macroglobulin proteinase inhibitors in birds, reptiles, amphibians and mammals, and purification and characterization of a monomeric alpha-macroglobulin proteinase inhibitor from the American bullfrog Rana catesbeiana.

    PubMed Central

    Rubenstein, D S; Thøgersen, I B; Pizzo, S V; Enghild, J J

    1993-01-01

    The alpha-macroglobulins are classified as broad-spectrum inhibitors because of their ability to entrap proteinases of different specificities and catalytic class. Tetrameric and dimeric alpha-macroglobulins have been identified in a wide variety of organisms including those as primitive as the mollusc Octopus vulgaris; however, monomeric alpha-macroglobulin proteinase inhibitors have been previously identified only in rodents. The monomeric alpha-macroglobulin proteinase inhibitors are believed to be analogous to the evolutionary precursor of the multimeric members of this family exemplified by the tetrameric human alpha 2-macroglobulin. Until now, monomeric alpha-macroglobulin proteinase inhibitors have only been identified in rodents and have therefore been considered an evolutionary anomaly. However, in this report we have utilized several sensitive assays to screen various plasmas and sera for the presence of monomeric alpha-macroglobulins, and our results suggest that monomeric alpha-macroglobulin proteinase inhibitors are present in organisms belonging to the avian, reptilian, amphibian and mammalian classes of the chordate phylum. This indicates that these proteins are more widespread than previously recognized and that their presence in rodents is not an anomaly. To demonstrate further that the identified proteins were indeed monomeric alpha-macroglobulin proteinase inhibitors, we purified the monomeric alpha-macroglobulin from the American bullfrog Rana catesbeiana. We conclude that this protein is a monomer of 180 kDa on the basis of its behaviour on (i) pore-limit gel electrophoresis, (ii) non-reducing and reducing SDS/PAGE and (iii) gel-filtration chromatography. In addition, we demonstrate that this protein is an alpha-macroglobulin proteinase inhibitor by virtue of (i) its ability to inhibit proteinases of different catalytic class, (ii) the presence of a putative internal beta-cysteinyl-gamma-glutamyl thioester and (iii) an inhibitory mechanism

  10. SARS CoV main proteinase: The monomer-dimer equilibrium dissociation constant.

    PubMed

    Graziano, Vito; McGrath, William J; Yang, Lin; Mangel, Walter F

    2006-12-12

    The SARS coronavirus main proteinase (SARS CoV main proteinase) is required for the replication of the severe acute respiratory syndrome coronavirus (SARS CoV), the virus that causes SARS. One function of the enzyme is to process viral polyproteins. The active form of the SARS CoV main proteinase is a homodimer. In the literature, estimates of the monomer-dimer equilibrium dissociation constant, KD, have varied more than 65,0000-fold, from <1 nM to more than 200 microM. Because of these discrepancies and because compounds that interfere with activation of the enzyme by dimerization may be potential antiviral agents, we investigated the monomer-dimer equilibrium by three different techniques: small-angle X-ray scattering, chemical cross-linking, and enzyme kinetics. Analysis of small-angle X-ray scattering data from a series of measurements at different SARS CoV main proteinase concentrations yielded KD values of 5.8 +/- 0.8 microM (obtained from the entire scattering curve), 6.5 +/- 2.2 microM (obtained from the radii of gyration), and 6.8 +/- 1.5 microM (obtained from the forward scattering). The KD from chemical cross-linking was 12.7 +/- 1.1 microM, and from enzyme kinetics, it was 5.2 +/- 0.4 microM. While each of these three techniques can present different, potential limitations, they all yielded similar KD values.

  11. A heat-stable serine proteinase from the extreme thermophilic archaebacterium Sulfolobus solfataricus.

    PubMed

    Burlini, N; Magnani, P; Villa, A; Macchi, F; Tortora, P; Guerritore, A

    1992-08-21

    A proteinase was purified to electrophoretic homogeneity from crude extracts of the thermoacidophilic archaebacterium Sulfolobus solfataricus. Molecular mass values assessed by SDS-PAGE and gel filtration were 54 and 118 kDa, respectively, which points to a dimeric structure of the molecule. An isoelectric point of 5.6 was also determined. The enzyme behaved as a chymotrypsin-like serine proteinase, as shown by the inhibitory effects exerted by phenylmethanesulfonyl fluoride, 3,4-dichloroisocoumarin, tosylphenylalaninechloromethyl ketone and chymostatin. Consistently with the inhibition pattern, the enzyme cleaved chromogenic substrates at the carboxyl side of aromatic or bulky aliphatic amino acids; however, it effectively attacked only a small number of such substrates, thus, displaying a specificity much narrower than and clearly different from that of chymotrypsin. This was confirmed by its inability to digest a set of natural substrate proteins, as well as insulin chains A and B; only after alkylation casein was degraded to some extent. Proteinase activity was significantly stimulated by Mn2+ which acted as a mixed-type nonessential activator. The enzyme also displayed a broad pH optimum in the range 6.5-8.0. Furthermore, it was completely stable up to 90 degrees C; above this temperature it underwent first-order thermal inactivation with half-lives ranging from 342 min (92 degrees C) to 7 min (101 degrees C). At 50 degrees C it could withstand 6 M urea and, to some extent, different organic solvents; however, at 95 degrees C it was extensively inactivated by all of these compounds. None of the chemical physical properties of the enzyme, including amino-acid analysis, provided evidence of a possible relation to other well-known microbial serine proteinases.

  12. Crystal quality and inhibitor binding by aspartic proteinases; preparation of high quality crystals of mouse renin

    NASA Astrophysics Data System (ADS)

    Badasso, M.; Sibanda, B. L.; Cooper, J. B.; Dealwis, C. G.; Wood, S. P.

    1992-08-01

    Renin from mouse submandibular glands has been highly purified and co-crystallized with a synthetic nonapeptide fragment of rat angiotensionogen in which the scissile Leu-Leu bond has been modified as a hydroxyethylene mimic of the transition state. The strong diffraction from these crystals compared to the native form is discussed in relation to the behaviour of other members of the aspartic proteinase family in crystallisation.

  13. Crystal structure of 2A proteinase from hand, foot and mouth disease virus.

    PubMed

    Mu, Zhixia; Wang, Bei; Zhang, Xiaoyu; Gao, Xiaopan; Qin, Bo; Zhao, Zhendong; Cui, Sheng

    2013-11-15

    EV71 is responsible for several epidemics worldwide; however, the effective antiviral drug is unavailable to date. The 2A proteinase (2A(pro)) of EV71 presents a promising drug target due to its multiple roles in virus replication, inhibition of host protein synthesis and evasion of innate immunity. We determined the crystal structure of EV71 2A(pro) at 1.85Å resolution, revealing that the proteinase maintains a chymotrypsin-like fold. The active site is composed of the catalytic triads C110A, H21 and D39 with the geometry similar to that in other picornaviral 2A(pro), 3C(pro) and serine proteinases. The cI-to-eI2 loop at the N-terminal domain of EV71 2A(pro) adopts a highly stable conformation and contributes to the hydrophilic surface property, which are strikingly different in HRV2 2A(pro) but are similar in CVB4 2A(pro). We identified a hydrophobic motif "LLWL" followed by an acidic motif "DEE" at the C-terminus of EV71 2A(pro). The "LLWL" motif is folded into the β-turn structure that is essential for the positioning of the acidic motif. Our structural and mutagenesis study demonstrated that both the negative charging and the correct positioning of the C-terminus are essential for EV71 replication. Deletion of the "LLWL" motif abrogated the proteolytic activity, indicating that the motif is critical for maintaining the active proteinase conformation. Our findings provide the structural and functional insights into EV71 2A(pro) and establish a framework for structure-based inhibitor design.

  14. Novel Aggregation Properties of Candida albicans Secreted Aspartyl Proteinase Sap6 Mediate Virulence in Oral Candidiasis.

    PubMed

    Kumar, Rohitashw; Saraswat, Darpan; Tati, Swetha; Edgerton, Mira

    2015-07-01

    Candida albicans, a commensal fungus of the oral microbiome, causes oral candidiasis in humans with localized or systemic immune deficiencies. Secreted aspartic proteinases (Saps) are a family of 10 related proteases and are virulence factors due to their proteolytic activity, as well as their roles in adherence and colonization of host tissues. We found that mice infected sublingually with C. albicans cells overexpressing Sap6 (SAP6 OE and a Δsap8 strain) had thicker fungal plaques and more severe oral infection, while infection with the Δsap6 strain was attenuated. These hypervirulent strains had highly aggregative colony structure in vitro and higher secreted proteinase activity; however, the levels of proteinase activity of C. albicans Saps did not uniformly match their abilities to damage cultured oral epithelial cells (SCC-15 cells). Hyphal induction in cells overexpressing Sap6 (SAP6 OE and Δsap8 cells) resulted in formation of large cell-cell aggregates. These aggregates could be produced in germinated wild-type cells by addition of native or heat-inactivated Sap6. Sap6 bound only to germinated cells and increased C. albicans adhesion to oral epithelial cells. The adhesion properties of Sap6 were lost upon deletion of its integrin-binding motif (RGD) and could be inhibited by addition of RGD peptide or anti-integrin antibodies. Thus, Sap6 (but not Sap5) has an alternative novel function in cell-cell aggregation, independent of its proteinase activity, to promote infection and virulence in oral candidiasis.

  15. SARS CoV Main Proteinase: The Monomer-Dimer Equilibrium Dissociation Constant

    SciTech Connect

    Graziano,V.; McGrath, W.; Yang, L.; Mangel, W.

    2006-01-01

    The SARS coronavirus main proteinase (SARS CoV main proteinase) is required for the replication of the severe acute respiratory syndrome coronavirus (SARS CoV), the virus that causes SARS. One function of the enzyme is to process viral polyproteins. The active form of the SARS CoV main proteinase is a homodimer. In the literature, estimates of the monomer-dimer equilibrium dissociation constant, K{sub D}, have varied more than 650000-fold, from <1 nM to more than 200 {mu}M. Because of these discrepancies and because compounds that interfere with activation of the enzyme by dimerization may be potential antiviral agents, we investigated the monomer-dimer equilibrium by three different techniques: small-angle X-ray scattering, chemical cross-linking, and enzyme kinetics. Analysis of small-angle X-ray scattering data from a series of measurements at different SARS CoV main proteinase concentrations yielded K{sub D} values of 5.8 {+-} 0.8 {mu}M (obtained from the entire scattering curve), 6.5 {+-} 2.2 {mu}M (obtained from the radii of gyration), and 6.8 {+-} 1.5 {mu}M (obtained from the forward scattering). The K{sub D} from chemical cross-linking was 12.7 {+-} 1.1 {mu}M, and from enzyme kinetics, it was 5.2 {+-} 0.4 {mu}M. While each of these three techniques can present different, potential limitations, they all yielded similar K{sub D} values.

  16. Effect of acute ozone exposure on the proteinase-antiproteinase balance in the rat lung

    SciTech Connect

    Pickrell, J.A.; Gregory, R.E.; Cole, D.J.; Hahn, F.F.; Henderson, R.F.

    1987-04-01

    Lung disease may result from a persisting proteinase excess or a depletion of antiproteinase in pulmonary parenchyma. We investigated the in vivo effect of a 48-hr exposure to ozone at 0.5, 1.0, or 1.5 ppm on proteinase and antiproteinase activity of rat lungs. Elastase inhibitory capacities of serum, lung tissue, and airway washings were measured as indicators of antielastase activity. Trypsin inhibitory capacity was measured using an esterolytic procedure. Proteinase was measured as radioactive release from a /sup 14/C-globin substrate. The 48-hr exposures to O/sub 3/ at levels up to 1 ppm produced concentration-dependent decreases of 35-80% of antiproteinase activities in serum and in lung tissue. However, exposure to 1.5 ppm O/sub 3/ resulted in no decrease in antiproteinase activities. Acid proteinase activities (pH 4.2) were increased 65-120% by exposure to 1 or 1.5 ppm O/sub 3/, which correlated with inflammatory cells noted histologically. At 1.5 ppm O/sub 3/, pulmonary edema and hemorrhage were noted in histologic sections. These changes led to a flooding of the alveoli with up to 40 times normal protein levels and a greater than fivefold increase in airway antiproteinase. These data suggest that serum and soluble lung tissue antiproteinase activity decreased upon exposure to low levels of ozone. However, if O/sub 3/ exposure is high enough to produce pulmonary hemorrhage, antiproteinase may increase following serum exudation. These changes may be important in the development of ozone-induced lung diseases, especially emphysema.

  17. Novel Aggregation Properties of Candida albicans Secreted Aspartyl Proteinase Sap6 Mediate Virulence in Oral Candidiasis

    PubMed Central

    Kumar, Rohitashw; Saraswat, Darpan; Tati, Swetha

    2015-01-01

    Candida albicans, a commensal fungus of the oral microbiome, causes oral candidiasis in humans with localized or systemic immune deficiencies. Secreted aspartic proteinases (Saps) are a family of 10 related proteases and are virulence factors due to their proteolytic activity, as well as their roles in adherence and colonization of host tissues. We found that mice infected sublingually with C. albicans cells overexpressing Sap6 (SAP6 OE and a Δsap8 strain) had thicker fungal plaques and more severe oral infection, while infection with the Δsap6 strain was attenuated. These hypervirulent strains had highly aggregative colony structure in vitro and higher secreted proteinase activity; however, the levels of proteinase activity of C. albicans Saps did not uniformly match their abilities to damage cultured oral epithelial cells (SCC-15 cells). Hyphal induction in cells overexpressing Sap6 (SAP6 OE and Δsap8 cells) resulted in formation of large cell-cell aggregates. These aggregates could be produced in germinated wild-type cells by addition of native or heat-inactivated Sap6. Sap6 bound only to germinated cells and increased C. albicans adhesion to oral epithelial cells. The adhesion properties of Sap6 were lost upon deletion of its integrin-binding motif (RGD) and could be inhibited by addition of RGD peptide or anti-integrin antibodies. Thus, Sap6 (but not Sap5) has an alternative novel function in cell-cell aggregation, independent of its proteinase activity, to promote infection and virulence in oral candidiasis. PMID:25870228

  18. Isolation and characterization of two forms of an acidic bromelain stem proteinase.

    PubMed

    Harrach, T; Eckert, K; Maurer, H R; Machleidt, I; Machleidt, W; Nuck, R

    1998-05-01

    Two forms of an acidic bromelain proteinase isolated from crude bromelain, an extract from pineapple stem, were found by a two-step FPLC purification procedure. The basic main components were removed by cation exchange chromatography and the breakthrough fraction was further resolved by anion exchange chromatography into 15 protein fractions, only two of which, called SBA/a and SBA/b, were proteolytically active. These components were characterized by electrospray mass spectroscopy (ESMS), isoelectric focusing, N-terminal amino acid sequence analysis, monosaccharide analysis, and enzymatic parameters. The molecular masses of SBA/a and SBA/b were determined by ESMS to be 23,550 and 23,560, respectively. The isoelectric points (pI) of the two bands of SBA/a were 4.8 and 4.9; SBA/b focused as a single band at pI = 4.8. Partial N-terminal amino acid sequences (11 residues) were identical to SBA/a and SBA/b and identical with those of stem bromelain, the basic main proteinase of the pineapple stem, and fruit bromelain, the acidic main proteinase of the pineapple fruit. Both components are highly glycosylated; hydrolysis of SBA/a yielded about twofold more monosaccharide per protein than SBA/b. The comparison of the catalytic properties of SBA/a with those of SBA/b revealed no relevant differences in the hydrolysis of three peptidyl-NH-Mec substrates and in the inhibition profiles using chicken cystatin and E-64, indicating that these components can be considered as two forms of a single enzyme. Both forms are scarcely inhibited by chicken cystatin and slowly inactivated by E-64, hence are nontypical cysteine proteinases of the papain superfamily.

  19. Digestion of human immunoglobulin G by the major cysteine proteinase (cruzipain) from Trypanosoma cruzi.

    PubMed

    Bontempi, E; Cazzulo, J J

    1990-08-01

    The major cysteine proteinase (cruzipain) from Trypanosoma cruzi was able to digest human IgG, as shown by polyacrylamide gel electrophoresis in the presence of SDS, and by gel filtration on a Superose 12 column, in a FPLC system. The Fab fragment of IgG was only slightly degraded, but Fc was extensively hydrolyzed to small peptides. The results suggest that cruzipain might be involved in the defense mechanisms of the parasite against the immune response of the host.

  20. Abnormal early cleavage events predict early embryo demise: sperm oxidative stress and early abnormal cleavage.

    PubMed

    Burruel, Victoria; Klooster, Katie; Barker, Christopher M; Pera, Renee Reijo; Meyers, Stuart

    2014-10-13

    Human embryos resulting from abnormal early cleavage can result in aneuploidy and failure to develop normally to the blastocyst stage. The nature of paternal influence on early embryo development has not been directly demonstrated although many studies have suggested effects from spermatozoal chromatin packaging, DNA damage, centriolar and mitotic spindle integrity, and plasma membrane integrity. The goal of this study was to determine whether early developmental events were affected by oxidative damage to the fertilizing sperm. Survival analysis was used to compare patterns of blastocyst formation based on P2 duration. Kaplan-Meier survival curves demonstrate that relatively few embryos with short (<1 hr) P2 times reached blastocysts, and the two curves diverged beginning on day 4, with nearly all of the embryos with longer P2 times reaching blastocysts by day 6 (p < .01). We determined that duration of the 2nd to 3rd mitoses were sensitive periods in the presence of spermatozoal oxidative stress. Embryos that displayed either too long or too short cytokineses demonstrated an increased failure to reach blastocyst stage and therefore survive for further development. Although paternal-derived gene expression occurs later in development, this study suggests a specific role in early mitosis that is highly influenced by paternal factors.

  1. Comparison of self-processing of foot-and-mouth disease virus leader proteinase and porcine reproductive and respiratory syndrome virus leader proteinase nsp1α.

    PubMed

    Steinberger, Jutta; Kontaxis, Georg; Rancan, Chiara; Skern, Tim

    2013-09-01

    The foot-and-mouth disease virus leader proteinase (Lb(pro)) cleaves itself off the nascent viral polyprotein. NMR studies on the monomeric variant Lb(pro) L200F provide structural evidence for intramolecular self-processing. (15)N-HSQC measurements of Lb(pro) L200F showed specifically shifted backbone signals in the active and substrate binding sites compared to the monomeric variant sLb(pro), lacking six C-terminal residues. This indicates transient intramolecular interactions between the C-terminal extension (CTE) of one molecule and its own active site. Contrastingly, the porcine reproductive and respiratory syndrome virus (PRRSV) leader proteinase nsp1α, with a papain-like fold like Lb(pro), stably binds its own CTE. Parts of the β-sheet domains but none of the α-helical domains of Lb(pro) and nsp1α superimpose; consequently, the α-helical domain of nsp1α is oriented differently relative to its β-sheet domain. This provides a large interaction surface for the CTE with the globular domain, stabilising the intramolecular complex. Consequently, self-processing inactivates nsp1α but not Lb(pro).

  2. Proteinase K enhanced immunoreactivity of the prion protein-specific monoclonal antibody 2A11.

    PubMed

    Brun, Alejandro; Castilla, Joaquín; Ramírez, Miguel A; Prager, Kai; Parra, Beatriz; Salguero, Francisco J; Shiveral, Diane; Sánchez, Carmen; Sánchez-Vizcaíno, José M; Douglas, Alastair; Torres, Juan M

    2004-01-01

    Here, we report the development and further characterisation of a novel PrP-specific monoclonal antibody: 2A11. By Western blot analysis, 2A11 reacts with PrPC from a variety of species including cow, sheep, pig, hamster, rabbit, cat, dog, deer and mouse but fails to react with human, chicken and turtle PrP. Reactivity to PrPC in Western blot was found to be dependent on the redox state of the protein since binding of mAb 2A11 to its epitope was more effective in reducing conditions. 2A11 binding site was mapped within a region comprised by residues 171-179 (six octarepeats bovine PrP notation; 163-171 for the ovine PrP notation). Interestingly, in immunohistochemistry (IHC) analysis, immunoreactivity was greatly enhanced after proteinase K (PK) sample treatment, while little or no reaction was observed in non-PK-treated BSE samples and samples from healthy animals. Quantitative differences in reactivity to BSE prions after PK treatment were also observed, to a lesser extent, by Western blot analysis. Since definitive diagnosis of prion diseases rely on IHC assays of proteinase K-treated samples, the use of mAb 2A11 might contribute to reduce the occurrence of false positive detection due to incomplete proteinase K digestion.

  3. Three low molecular weight cysteine proteinase inhibitors of human seminal fluid: purification and enzyme kinetic properties.

    PubMed

    Yadav, Vikash Kumar; Chhikara, Nirmal; Gill, Kamaldeep; Dey, Sharmistha; Singh, Sarman; Yadav, Savita

    2013-08-01

    The cystatins form a superfamily of structurally related proteins with highly conserved structural folds. They are all potent, reversible, competitive inhibitors of cysteine proteinases (CPs). Proteins from this group present differences in proteinase inhibition despite their high level of structural similarities. In this study, three cysteine proteinase inhibitors (CPIs) of low molecular weight were isolated from human seminal fluid (HSF) by affinity chromatography on carboxymethyl (CM)-papain-Sepharose column, purified using various chromatographic procedures and checked for purity on sodium-dodecyl PAGE (SDS-PAGE). Matrix-assisted laser desorption-ionization-time-of flight-mass spectrometry (MALDI-TOF-MS) identified these proteins as cystatin 9, cystatin SN, and SAP-1 (an N-terminal truncated form of cystatin S). All three CPIs suppressed the activity of papain potentially and showed remarkable heat stability. Interestingly SAP-1 also inhibits the activity of trypsin, chymotrypsin, pepsin, and PSA (prostate specific antigen) and acts as a cross-class protease inhibitor in in vitro studies. Using Surface Plasmon Resonance, we have also observed that SAP-1 shows a significant binding with all these proteases. These studies suggest that SAP-1 is a cross-class inhibitor that may regulate activity of various classes of proteases within the reproductive systems. To our knowledge, this is the first report about purification of CPIs from HSF; the identification of such proteins could provide better insights into the physiological processes and offer intimation for further research.

  4. Neutrophil Elastase, Proteinase 3, and Cathepsin G as Therapeutic Targets in Human Diseases

    PubMed Central

    Horwitz, Marshall S.; Jenne, Dieter E.; Gauthier, Francis

    2010-01-01

    Polymorphonuclear neutrophils are the first cells recruited to inflammatory sites and form the earliest line of defense against invading microorganisms. Neutrophil elastase, proteinase 3, and cathepsin G are three hematopoietic serine proteases stored in large quantities in neutrophil cytoplasmic azurophilic granules. They act in combination with reactive oxygen species to help degrade engulfed microorganisms inside phagolysosomes. These proteases are also externalized in an active form during neutrophil activation at inflammatory sites, thus contributing to the regulation of inflammatory and immune responses. As multifunctional proteases, they also play a regulatory role in noninfectious inflammatory diseases. Mutations in the ELA2/ELANE gene, encoding neutrophil elastase, are the cause of human congenital neutropenia. Neutrophil membrane-bound proteinase 3 serves as an autoantigen in Wegener granulomatosis, a systemic autoimmune vasculitis. All three proteases are affected by mutations of the gene (CTSC) encoding dipeptidyl peptidase I, a protease required for activation of their proform before storage in cytoplasmic granules. Mutations of CTSC cause Papillon-Lefèvre syndrome. Because of their roles in host defense and disease, elastase, proteinase 3, and cathepsin G are of interest as potential therapeutic targets. In this review, we describe the physicochemical functions of these proteases, toward a goal of better delineating their role in human diseases and identifying new therapeutic strategies based on the modulation of their bioavailability and activity. We also describe how nonhuman primate experimental models could assist with testing the efficacy of proposed therapeutic strategies. PMID:21079042

  5. Fibronectin-Degrading Activity of Trypanosoma cruzi Cysteine Proteinase Plays a Role in Host Cell Invasion

    PubMed Central

    Maeda, Fernando Yukio; Cortez, Cristian; Izidoro, Mario Augusto; Juliano, Luiz

    2014-01-01

    Trypanosoma cruzi, the agent of Chagas disease, binds to diverse extracellular matrix proteins. Such an ability prevails in the parasite forms that circulate in the bloodstream and contributes to host cell invasion. Whether this also applies to the insect-stage metacyclic trypomastigotes, the developmental forms that initiate infection in the mammalian host, is not clear. Using T. cruzi CL strain metacyclic forms, we investigated whether fibronectin bound to the parasites and affected target cell invasion. Fibronectin present in cell culture medium bound to metacyclic forms and was digested by cruzipain, the major T. cruzi cysteine proteinase. G strain, with negligible cruzipain activity, displayed a minimal fibronectin-degrading effect. Binding to fibronectin was mediated by gp82, the metacyclic stage-specific surface molecule implicated in parasite internalization. When exogenous fibronectin was present at concentrations higher than cruzipain can properly digest, or fibronectin expression was stimulated by treatment of epithelial HeLa cells with transforming growth factor beta, the parasite invasion was reduced. Treatment of HeLa cells with purified recombinant cruzipain increased parasite internalization, whereas the treatment of parasites with cysteine proteinase inhibitor had the opposite effect. Metacyclic trypomastigote entry into HeLa cells was not affected by anti-β1 integrin antibody but was inhibited by anti-fibronectin antibody. Overall, our results have indicated that the cysteine proteinase of T. cruzi metacyclic forms, through its fibronectin-degrading activity, is implicated in host cell invasion. PMID:25267835

  6. Structurally unique recombinant Kazal-type proteinase inhibitor retains activity when terminally extended and glycosylated.

    PubMed

    Kludkiewicz, Barbara; Kodrík, Dalibor; Grzelak, Krystyna; Nirmala, Xavier; Sehnal, Frantisek

    2005-10-01

    Recombinant derivatives of the Kazal-type serine proteinase inhibitor GmSPI2 (36 amino acid residues), which is a component of insect silk, were prepared in the expression vector Pichia pastoris. The rhSPI2 had a C-terminal hexahistidine tag attached to the GmSPI2 sequence, rtSPI2 was extended with GluAlaAla at the N-terminus, and rfSPI2 included this N-terminal extension and a C-terminal tail of 22 residues (myc epitope and hexahistidine). A portion of the secreted rfSI2 was O-glycosylated with a trimannosyl or hexamannosyl. The native inhibitor was active slightly on trypsin and highly on subtilisin and proteinase K. The extended C-terminus in rhSPI2 and rfSPI2 enhanced activity on the two latter enzymes and rendered rfSPI2 active on elastase and pronase, but abolished the inhibition of trypsin. The glycosylation of rfSPI2 reduced its inhibitory activity to a level comparable with the native inhibitor. The rtSPI2 with tripeptide extension at the N-terminus and no C-terminal modification was clearly less active than the native inhibitor. None of the tested compounds inhibited alpha-chymotrypsin and the non-serine proteinases.

  7. Characterization of the mature cell surface proteinase of Lactobacillus delbrueckii subsp. lactis CRL 581.

    PubMed

    Villegas, Josefina M; Brown, Lucía; Savoy de Giori, Graciela; Hebert, Elvira M

    2015-05-01

    The cell envelope-associated proteinase (CEP) of Lactobacillus delbrueckii subsp. lactis CRL 581 (PrtL) has an essential role in bacterial growth, contributes to the flavor and texture development of fermented products, and can release bioactive health-beneficial peptides during milk fermentation. The genome of L. delbrueckii subsp. lactis CRL 581 possesses only one gene that encodes PrtL, which consists of 1924 amino acids and is a multidomain protein anchored to the cell via its W domain. PrtL was extracted from the cell under high ionic strength conditions using NaCl, suggesting an electrostatic interaction between the proteinase and the cell envelope. The released PrtL was purified and biochemically characterized; its activity was maximal at temperatures between 37 and 40 °C and at pH between 7 and 8. Under optimal conditions, PrtL exhibited higher affinity for succinyl-alanyl-alanyl-prolyl-phenylalanine-p-nitroanilide than for succinyl-alanyl-glutamyl-prolyl-phenylalanine-p-nitroanilide, while methoxy-succinyl-arginyl-prolyl-tyrosyl-p-nitroanilide was not degraded. A similar α- and β-casein degradation pattern was observed with the purified and the cell envelope-bound proteinase. Finally, on the basis of its specificity towards caseins and the unique combination of amino acids at residues thought to be involved in substrate specificity, PrtL can be classified as a representative of a new group of CEP.

  8. Activation of intracellular serine proteinase in Bacillus subtilis cells during sporulation.

    PubMed Central

    Burnett, T J; Shankweiler, G W; Hageman, J H

    1986-01-01

    Cells of Bacillus subtilis 168 (trpC2) growing and sporulating in a single chemically defined medium carried out intracellular protein degradation and increased their levels of intracellular serine protease-1 in a manner very similar to what had previously been reported for cells sporulating in nutrient broth. The results were interpreted to mean that these processes are intrinsic to sporulation rather than medium dependent. To determine the cause of these increases in specific activity of proteinases, we purified the protease, prepared rabbit immunoglobulins directed against it, and monitored changes in protease antigen levels by performing rocket immunoelectrophoresis. In cells sporulating in nutrient broth, the protease antigen levels increased about 7-fold, whereas the specific activity increased about 150-fold, for an activation of about 20-fold. In cells sporulating in the single chemically defined sporulation medium, the protease antigen increased about 10-fold, whereas the specific activity increased at least 400-fold, for an activation of about 40-fold. These results were interpreted to mean that a posttranslational event activated the protease in vivo; a previously described endogenous proteinase inhibitor was confirmed to be present in the strain used. Chloramphenicol added to the cultures inhibited both the increases in antigen levels and in the specific activity of the proteinase. PMID:3079745

  9. Proteolytic activity and fatal gram-negative sepsis in burned mice: effect of exogenous proteinase inhibition.

    PubMed Central

    Neely, A N; Miller, R G; Holder, I A

    1994-01-01

    Circulating proteolytic activity (PA) increases following burn or surgical trauma. Challenging traumatized mice with the yeast Candida albicans further increases PA. Once a PA threshold has been passed, mortality increases as PA increases. The purposes of this study were to determine (i) if gram-negative bacterial challenge affects circulating PA and mortality as Candida challenge does and (ii) if proteinase inhibitor treatment with aprotinin, antithrombin III, and alpha 1-proteinase inhibitor decreases circulating PA and increases the survival of burned mice infected with a bacterium. For all bacteria tested (Proteus mirabilis, Pseudomonas aeruginosa, and Klebsiella pneumoniae), burn plus challenge significantly elevated PA and mortality above levels in mice that were only burned or only challenged. Quantitative culture counts indicated that the mice died of sepsis. Proteinase inhibitor treatment of mice burned and challenged with K. pneumoniae significantly decreased circulating PA, decreased the hepatic microbial load, and increased survival. Hence, in traumatized mice challenged with either C. albicans or gram-negative bacteria, a relationship exists between proteolytic load and subsequent septic death. Parallels between these animal studies and human studies are discussed. PMID:8188336

  10. [Conditions of the biosynthesis of an extracellular subtilisin-like proteinase by Bacillus pumilus KMM 62].

    PubMed

    Malikova, L A; Mardanova, A M; Sokolova, O V; Balaban, N P; Rudenskaia, G N; Sharipova, M R

    2007-01-01

    The influence of the cultivation conditions on Bacillus pumilus KMM 62 growth and effectiveness of the production of a subtilisin-like serine proteinase were investigated. Enzyme accumulation in the culture fluid reached the maximum value after 32 and 46-48 h of growth; it depends on the composition of the nutrient medium. The ratio of the concentrations of two main components of the medium, peptone and inorganic phosphate, which was optimal for enzyme biosynthesis was determined by multifactor experiments. Ammonium salts, when introduced as an additional nitrogen source, had different effects on the proteinase biosynthesis at different growth stages: they suppress enzyme production at the early stationary growth phase and stimulate the biosynthesis of the enzyme after 46-48 h of growth. Complex organic substrates (albumin, casein, hemoglobin, and gelatin) have a repressive effect on the biosynthesis of the enzyme. The effect of amino acids on culture growth and enzyme biosynthesis during the early and late stationary growth phase is different. Hydrophilic amino acids, glutamine, and glutamic acid exhibit the most pronounced repressive action on biosynthesis. The activity of different regulatory mechanisms for the synthesis of this proteinase is assumed at the early and late stationary stages of growth.

  11. Limited proteolysis and sequence analysis of the 2-oxo acid dehydrogenase complexes from Escherichia coli. Cleavage sites and domains in the dihydrolipoamide acyltransferase components.

    PubMed Central

    Packman, L C; Perham, R N

    1987-01-01

    The structures of the dihydrolipoamide acyltransferase (E2) components of the 2-oxo acid dehydrogenase complexes from Escherichia coli were investigated by limited proteolysis. Trypsin and Staphylococcus aureus V8 proteinase were used to excise the three lipoyl domains from the E2p component of the pyruvate dehydrogenase complex and the single lipoyl domain from the E2o component of the 2-oxoglutarate dehydrogenase complex. The principal sites of action of these enzymes on each E2 chain were determined by sequence analysis of the isolated lipoyl fragments and of the truncated E2p and E2o chains. Each of the numerous cleavage sites (12 in E2p, six in E2o) fell within similar segments of the E2 chains, namely stretches of polypeptide rich in alanine, proline and/or charged amino acids. These regions are clearly accessible to proteinases of Mr 24,000-28,000 and, on the basis of n.m.r. spectroscopy, some of them have previously been implicated in facilitating domain movements by virtue of their conformational flexibility. The limited proteolysis data suggest that E2p and E2o possess closer architectural similarities than would be predicted from inspection of their amino acid sequences. As a result of this work, an error was detected in the sequence of E2o inferred from the previously published sequence of the encoding gene, sucB. The relevant peptides from E2o were purified and sequenced by direct means; an amended sequence is presented. Images Fig. 1. Fig. 2. PMID:3297046

  12. Peptide substrate specificities and protein cleavage sites of human endometase/matrilysin-2/matrix metalloproteinase-26.

    PubMed

    Park, Hyun I; Turk, Benjamin E; Gerkema, Ferry E; Cantley, Lewis C; Sang, Qing-Xiang Amy

    2002-09-20

    Human endometase/matrilysin-2/matrix metalloproteinase-26 (MMP-26) is a novel epithelial and cancer-specific metalloproteinase. Peptide libraries were used to profile the substrate specificity of MMP-26 from the P4-P4' sites. The optimal cleavage motifs for MMP-26 were Lys-Pro-Ile/Leu-Ser(P1)-Leu/Met(P1')-Ile/Thr-Ser/Ala-Ser. The strongest preference was observed at the P1' and P2 sites where hydrophobic residues were favored. Proline was preferred at P3, and Serine was preferred at P1. The overall specificity was similar to that of other MMPs with the exception that more flexibility was observed at P1, P2', and P3'. Accordingly, synthetic inhibitors of gelatinases and collagenases inhibited MMP-26 with similar efficacy. A pair of stereoisomers had only a 40-fold difference in K(i)(app) values against MMP-26 compared with a 250-fold difference against neutrophil collagenase, indicating that MMP-26 is less stereoselective for its inhibitors. MMP-26 autodigested itself during the folding process. Two of the major autolytic sites were Leu(49)-Thr(50) and Ala(75)-Leu(76), which still left the cysteine switch sequence (PHC(82)GVPD) intact. This suggests that Cys(82) may not play a role in the latency of the zymogen. Interestingly, inhibitor titration studies revealed that only approximately 5% of the total MMP-26 molecules was catalytically active, indicating that the thiol groups of Cys(82) in the active molecules may be dissociated or removed from the active site zinc ions. MMP-26 cleaved Phe(352)-Leu(353) and Pro(357)-Met(358) in the reactive loop of alpha(1)-proteinase inhibitor and His(140)-Val(141) in insulin-like growth factor-binding protein-1, probably rendering these substrates inactive. Among the fluorescent peptide substrates analyzed, Mca-Pro-Leu-Ala-Nva-Dpa-Ala-Arg-NH(2) displayed the highest specificity constant (30,000/molar second) with MMP-26. This report proposes a working model for the future studies of pro-MMP-26 activation, the design of inhibitors

  13. Proteinase A, a storage-globulin-degrading endopeptidase of vetch (Vicia sativa L.) seeds, is not involved in early steps of storage-protein mobilization.

    PubMed

    Becker, C; Senyuk, V I; Shutov, A D; Nong, V H; Fischer, J; Horstmann, C; Müntz, K

    1997-09-01

    Proteinase A is a papain-like cysteine endopeptidase of vetch (Vicia sativa L.) which was assumed to initiate storage-globulin breakdown just after the onset of seed germination. This enzyme was purified from cotyledons of vetch seedlings. On gelatin-containg SDS gels, active proteinase A migrated with an apparent molecular mass of 21 kDa, whereas after heat denaturation its molecular size on SDS/PAGE was 29 kDa. Although proteinase A is capable of hydrolyzing storage globulins in vitro it could not be localized in the protein-body fraction of cotyledons from germinating seeds. cDNA clones encoding proteinase A precursor have been obtained by PCR. The precursor is composed of an N-terminal signal sequence followed by a propeptide, the region encoding mature proteinase A, and a C-terminal KDEL sequence. Mature proteinase A with a derived molecular mass of 25,244 Da does not have the KDEL sequence. The derived amino acid sequence of the proteinase A precursor is 78.2% identical to sulfhydryl-endopeptidase (SH-EP), a cysteine endopeptidase from germinating Vigna mungo seedlings. Northern blot analysis indicated that proteinase A mRNA appears de novo in cotyledons of 1-day-germinated vetch seeds, where its amount increases up to day 6. No proteinase A mRNA was detected in other vetch organs, not even in the embryo axis, which contains stored globulins. By means of antibodies raised against the purified and against recombinantly produced proteinase A, the 29-kDa bands of mature proteinase A were detected in cotyledon extracts of 6-day-germinated seeds when globulin degradation has already far proceeded. The reported data do not agree with the proposed triggering role of proteinase A in storage-globulin breakdown during germination.

  14. Evolution of development in the sea star genus Patiriella: clade-specific alterations in cleavage.

    PubMed

    Cerra, Anna; Byrne, Maria

    2004-01-01

    Examination of early development in five species of the Patiriella sea star species complex indicates that the ancestral-type radial holoblastic cleavage (Type I) is characteristic of P. regularis and P. exigua, whereas cleavage in species from the calcar clade followed multiple alternatives (Types II-IV) from holoblastic to meroblastic. Considering that invariant radial cleavage is thought to play a role in embryonic axis formation in echinoderms, we documented the details of blastomere formation in Patiriella sp. and followed development of the embryos. In Type II cleavage, the first and second cleavage planes appeared simultaneously at one pole of the embryo, dividing it directly into four equally sized blastomeres. In Type III cleavage, the first and second cleavage planes appeared simultaneously, followed promptly by the third cleavage plane, dividing the embryo directly into eight equally sized blastomeres. In Type IV cleavage, numerous furrows appeared simultaneously at one end of the embryo, dividing it into 32-40 equally sized blastomeres. Confocal sections revealed that embryos with cleavage Types II-IV were initially syncytial. The timing of karyokinesis in embryos with Types II and III cleavage was similar to that seen in clutch mates with Type I cleavage. Karyokinesis in embryos with Type IV cleavage, however, differed in timing compared with Type I clutch mates. Alteration in cleavage was not associated with polarized distribution of maternally provided nutrients. For each cleavage type, development was normal to the competent larval stage. Although variable blastomere configuration in the calcar clade may be linked to possession of a lecithotrophic development, other Patiriella species with this mode of development have typical cleavage. The presence of variable cleavage in all calcar clade species indicates that phylogenetic history has played a role in the distribution of this embryonic trait in Patiriella. The plasticity in early cleavage in these

  15. Modeling Radial Holoblastic Cleavage: A Laboratory Activity for Developmental Biology.

    ERIC Educational Resources Information Center

    Ellis, Linda K.

    2000-01-01

    Introduces a laboratory activity designed for an undergraduate developmental biology course. Uses Play-Doh (plastic modeling clay) to build a multicellular embryo in order to provide a 3-D demonstration of cleavage. Includes notes for the instructor and student directions. (YDS)

  16. Cleavage sites within the poliovirus capsid protein precursors

    SciTech Connect

    Larsen, G.R.; Anderson, C.W.; Dorner, A.J.; Semler, B.L.; Wimmer, E.

    1982-01-01

    Partial amino-terminal sequence analysis was performed on radiolabeled poliovirus capsid proteins VP1, VP2, and VP3. A computer-assisted comparison of the amino acid sequences obtained with that predicted by the nucleotide sequence of the poliovirus genome allows assignment of the amino terminus of each capsid protein to a unique position within the virus polyprotein. Sequence analysis of trypsin-digested VP4, which has a blocked amino terminus, demonstrates that VP4 is encoded at or very near to the amino terminus of the polyprotein. The gene order of the capsid proteins is VP4-VP2-VP3-VP1. Cleavage of VP0 to VP4 and VP2 is shown to occur between asparagine and serine, whereas the cleavages that separate VP2/VP3 and VP3/VP1 occur between glutamine and glycine residues. This finding supports the hypothesis that the cleavage of VP0, which occurs during virion morphogenesis, is distinct from the cleavages that separate functional regions of the polyprotein.

  17. A review of statistical methods for prediction of proteolytic cleavage.

    PubMed

    duVerle, David A; Mamitsuka, Hiroshi

    2012-05-01

    A fundamental component of systems biology, proteolytic cleavage is involved in nearly all aspects of cellular activities: from gene regulation to cell lifecycle regulation. Current sequencing technologies have made it possible to compile large amount of cleavage data and brought greater understanding of the underlying protein interactions. However, the practical impossibility to exhaustively retrieve substrate sequences through experimentation alone has long highlighted the need for efficient computational prediction methods. Such methods must be able to quickly mark substrate candidates and putative cleavage sites for further analysis. Available methods and expected reliability depend heavily on the type and complexity of proteolytic action, as well as the availability of well-labelled experimental data sets: factors varying greatly across enzyme families. For this review, we chose to give a quick overview of the general issues and challenges in cleavage prediction methods followed by a more in-depth presentation of major techniques and implementations, with a focus on two particular families of cysteine proteases: caspases and calpains. Through their respective differences in proteolytic specificity (high for caspases, broader for calpains) and data availability (much lower for calpains), we aimed to illustrate the strengths and limitations of techniques ranging from position-based matrices and decision trees to more flexible machine-learning methods such as hidden Markov models and Support Vector Machines. In addition to a technical overview for each family of algorithms, we tried to provide elements of evaluation and performance comparison across methods.

  18. Mitochondria Localize to the Cleavage Furrow in Mammalian Cytokinesis

    PubMed Central

    Lawrence, Elizabeth J.; Mandato, Craig A.

    2013-01-01

    Mitochondria are dynamic organelles with multiple cellular functions, including ATP production, calcium buffering, and lipid biosynthesis. Several studies have shown that mitochondrial positioning is regulated by the cytoskeleton during cell division in several eukaryotic systems. However, the distribution of mitochondria during mammalian cytokinesis and whether the distribution is regulated by the cytoskeleton has not been examined. Using live spinning disk confocal microscopy and quantitative analysis of mitochondrial fluorescence intensity, we demonstrate that mitochondria are recruited to the cleavage furrow during cytokinesis in HeLa cells. After anaphase onset, the mitochondria are recruited towards the site of cleavage furrow formation, where they remain enriched as the furrow ingresses and until cytokinesis completion. Furthermore, we show that recruitment of mitochondria to the furrow occurs in multiple mammalian cells lines as well as in monopolar, bipolar, and multipolar divisions, suggesting that the mechanism of recruitment is conserved and robust. Using inhibitors of cytoskeleton dynamics, we show that the microtubule cytoskeleton, but not actin, is required to transport mitochondria to the cleavage furrow. Thus, mitochondria are specifically recruited to the cleavage furrow in a microtubule-dependent manner during mammalian cytokinesis. Two possible reasons for this could be to localize mitochondrial function to the furrow to facilitate cytokinesis and / or ensure accurate mitochondrial inheritance. PMID:23991162

  19. Stimulation of cleavage of membrane proteins by calmodulin inhibitors.

    PubMed Central

    Díaz-Rodríguez, E; Esparís-Ogando, A; Montero, J C; Yuste, L; Pandiella, A

    2000-01-01

    The ectodomain of several membrane-bound proteins can be shed by proteolytic cleavage. The activity of the proteases involved in shedding is highly regulated by several intracellular second messenger pathways, such as protein kinase C (PKC) and intracellular Ca(2+). Recently, the shedding of the adhesion molecule L-selectin has been shown to be regulated by the interaction of calmodulin (CaM) with the cytosolic tail of L-selectin. Prevention of CaM-L-selectin interaction by CaM inhibitors or mutation of a CaM binding site in L-selectin induced L-selectin ectodomain shedding. Whether this action of CaM inhibitors also affects other membrane-bound proteins is not known. In the present paper we show that CaM inhibitors also stimulate the cleavage of several other transmembrane proteins, such as the membrane-bound growth factor precursors pro-transforming growth factor-alpha and pro-neuregulin-alpha2c, the receptor tyrosine kinase, TrkA, and the beta-amyloid precursor protein. Cleavage induced by CaM inhibitors was a rapid event, and resulted from the activation of a mechanism that was independent of PKC or intracellular Ca(2+) increases, but was highly sensitive to hydroxamic acid-based metalloprotease inhibitors. Mutational analysis of the intracellular domain of the TrkA receptor indicated that CaM inhibitors may stimulate membrane-protein ectodomain cleavage by mechanisms independent of CaM-substrate interaction. PMID:10677354

  20. Mitochondria localize to the cleavage furrow in mammalian cytokinesis.

    PubMed

    Lawrence, Elizabeth J; Mandato, Craig A

    2013-01-01

    Mitochondria are dynamic organelles with multiple cellular functions, including ATP production, calcium buffering, and lipid biosynthesis. Several studies have shown that mitochondrial positioning is regulated by the cytoskeleton during cell division in several eukaryotic systems. However, the distribution of mitochondria during mammalian cytokinesis and whether the distribution is regulated by the cytoskeleton has not been examined. Using live spinning disk confocal microscopy and quantitative analysis of mitochondrial fluorescence intensity, we demonstrate that mitochondria are recruited to the cleavage furrow during cytokinesis in HeLa cells. After anaphase onset, the mitochondria are recruited towards the site of cleavage furrow formation, where they remain enriched as the furrow ingresses and until cytokinesis completion. Furthermore, we show that recruitment of mitochondria to the furrow occurs in multiple mammalian cells lines as well as in monopolar, bipolar, and multipolar divisions, suggesting that the mechanism of recruitment is conserved and robust. Using inhibitors of cytoskeleton dynamics, we show that the microtubule cytoskeleton, but not actin, is required to transport mitochondria to the cleavage furrow. Thus, mitochondria are specifically recruited to the cleavage furrow in a microtubule-dependent manner during mammalian cytokinesis. Two possible reasons for this could be to localize mitochondrial function to the furrow to facilitate cytokinesis and / or ensure accurate mitochondrial inheritance.

  1. Site-selective chemical cleavage of peptide bonds.

    PubMed

    Elashal, Hader E; Raj, Monika

    2016-05-07

    Site-selective cleavage of extremely unreactive peptide bonds is a very important chemical modification that provides invaluable information regarding protein sequence, and it acts as a modulator of protein structure and function for therapeutic applications. For controlled and selective cleavage, a daunting task, chemical reagents must selectively recognize or bind to one or more amino acid residues in the peptide chain and selectively cleave a peptide bond. Building on this principle, we have developed an approach that utilizes a chemical reagent to selectively modify the serine residue in a peptide chain and leads to the cleavage of a peptide backbone at the N-terminus of the serine residue. After cleavage, modified residues can be converted back to the original fragments. This method exhibits broad substrate scope and selectively cleaves various bioactive peptides with post-translational modifications (e.g. N-acetylation and -methylation) and mutations (d- and β-amino acids), which are a known cause of age related diseases.

  2. Gleevec shifts APP processing from a β-cleavage to a nonamyloidogenic cleavage

    PubMed Central

    Netzer, William J.; Bettayeb, Karima; Sinha, Subhash C.; Flajolet, Marc; Bustos, Victor

    2017-01-01

    Neurotoxic amyloid-β peptides (Aβ) are major drivers of Alzheimer’s disease (AD) and are formed by sequential cleavage of the amyloid precursor protein (APP) by β-secretase (BACE) and γ-secretase. Our previous study showed that the anticancer drug Gleevec lowers Aβ levels through indirect inhibition of γ-secretase activity. Here we report that Gleevec also achieves its Aβ-lowering effects through an additional cellular mechanism. It renders APP less susceptible to proteolysis by BACE without inhibiting BACE enzymatic activity or the processing of other BACE substrates. This effect closely mimics the phenotype of APP A673T, a recently discovered mutation that protects carriers against AD and age-related cognitive decline. In addition, Gleevec induces formation of a specific set of APP C-terminal fragments, also observed in cells expressing the APP protective mutation and in cells exposed to a conventional BACE inhibitor. These Gleevec phenotypes require an intracellular acidic pH and are independent of tyrosine kinase inhibition, given that a related compound lacking tyrosine kinase inhibitory activity, DV2-103, exerts similar effects on APP metabolism. In addition, DV2-103 accumulates at high concentrations in the rodent brain, where it rapidly lowers Aβ levels. This study suggests that long-term treatment with drugs that indirectly modulate BACE processing of APP but spare other BACE substrates and achieve therapeutic concentrations in the brain might be effective in preventing or delaying the onset of AD and could be safer than nonselective BACE inhibitor drugs. PMID:28115709

  3. Kazal-type proteinase inhibitor from disk abalone (Haliotis discus discus): molecular characterization and transcriptional response upon immune stimulation.

    PubMed

    Wickramaarachchi, W D Niroshana; De Zoysa, Mahanama; Whang, Ilson; Wan, Qiang; Lee, Jehee

    2013-09-01

    Proteinases and proteinase inhibitors are involved in several biological and physiological processes in all multicellular organisms. Proteinase inhibitors play a key role in regulating the activity of the respective proteinases. Among serine proteinase inhibitors, kazal-type proteinase inhibitors (KPIs) are widely found in mammals, avians, and a variety of invertebrates. In this study, we describe the identification of a kazal-type serine proteinase inhibitor (Ab-KPI) from the disk abalone, Haliotis discus discus, which is presumably involved in innate immunity. The full-length cDNA of Ab-KPI includes 600 bp nucleotides with an open reading frame (ORF) encoding a polypeptide of 143 amino acids. The deduced amino acid sequence of Ab-KPI contains a putative 17-amino acid signal peptide and two tandem kazal domains with high similarity to other kazal-type SPIs. Each kazal domain consists of reactive site (P1) residue containing a leucine (L), and a threonine (T) located in the second amino acid position after the second conserved cysteine of each domain. Temporal expression of Ab-KPI was assessed by real time quantitative PCR in hemocytes and mantle tissue following bacterial and viral hemorrhagic septicemia virus (VHSV) challenge, and tissue injury. At 6 h post-bacterial and -VHSV challenge, Ab-KPI expression in hemocytes was increased 14-fold and 4-fold, respectively, compared to control samples. The highest up-regulations upon tissue injury were shown at 9 h and 12 h in hemocytes and mantle, respectively. The transcriptional modulation of Ab-KPI following bacterial and viral challenges and tissue injury indicates that it might be involved in immune defense as well as wound healing process in abalone.

  4. Phospholipase and Aspartyl Proteinase Activities of Candida Species Causing Vulvovaginal Candidiasis in Patients with Type 2 Diabetes Mellitus.

    PubMed

    Bassyouni, Rasha H; Wegdan, Ahmed Ashraf; Abdelmoneim, Abdelsamie; Said, Wessam; AboElnaga, Fatma

    2015-10-01

    Few research had investigated the secretion of phospholipase and aspartyl proteinase from Candida spp. causing infection in females with type 2 diabetes mellitus. This research aimed to investigate the prevalence of vulvovaginal candidiasis (VVC) in diabetic versus non-diabetic women and compare the ability of identified Candida isolates to secrete phospholipases and aspartyl proteinases with characterization of their genetic profile. The study included 80 females with type 2 diabetes mellitus and 100 non-diabetic females within the child-bearing period. Candida strains were isolated and identified by conventional microbiological methods and by API Candida. The isolates were screened for their extracellular phospholipase and proteinase activities by culturing them on egg yolk and bovine serum albumin media, respectively. Detection of aspartyl proteinase genes (SAP1 to SAP8) and phospholipase genes (PLB1, PLB2) were performed by multiplex polymerase chain reaction. Our results indicated that vaginal candidiasis was significantly higher among the diabetic group versus nondiabetic group (50% versus 20%, respectively) (p = 0.004). C. albicans was the most prevalent species followed by C. glabrata in both groups. No significant association between diabetes mellitus and phospholipase activities was detected (p = 0.262), whereas high significant proteinase activities exhibited by Candida isolated from diabetic females were found (82.5%) (p = 0.000). Non-significant associations between any of the tested proteinase or phospholipase genes and diabetes mellitus were detected (p > 0.05). In conclusion, it is noticed that the incidence of C. glabrata causing VVC is increased. The higher prevalence of vaginal candidiasis among diabetics could be related to the increased aspartyl proteinase production in this group of patients.

  5. Purification and characterization of a 39,000-Da serine proteinase from the hemolymph of a solitary ascidian, Halocynthia roretzi.

    PubMed

    Shishikura, F; Abe, T; Ohtake, S; Tanaka, K

    1997-09-01

    A new endogenous serine proteinase from the cell-free hemolymph of a solitary ascidian, Halocythia roretzi, was purified by a combination of ammonium sulfate fractionation, hydrophobic interaction chromatography on TSKgel Toyopearl HW 65 F, ion exchange chromatography on TSKgel DEAE-Toyopearl 650 M, affinity chromatography on Arginine-Sepharose 4B, gel filtration on TSKgel Toyopearl HW 65F and hydroxyapatite chromatography on Bio-Gel HT. The serine proteinase is a single polypeptide chain whose molecular weight and isoelectric point are 39 kDa and about 7.6 pI, respectively. The most susceptible substrate was Boc-Leu-Gly-Arg-4-methyl-coumaryl-7-amide (MCA), and activity was optimal at pH 8. The enzyme was relatively stable at high temperatures; about 50% activity was retained even at 60 degrees C for 30 min in 50 mM Tris-HCl, pH 8.0, containing 0.5 M NaCl, and 0.05% Brij-35. The enzyme was characterized by the inhibitory effects of synthetic or natural inhibitors, substrate specificity toward 26 peptidyl-MCAs, proteinase activity toward natural proteins and complex formation with a serine proteinase inhibitor (58 kDa) previously found in H. roretzi hemolymph, indicating that the enzyme was a member of serine proteinases and strongly inhibited by the 58 kDa serine proteinase inhibitor as well as human antithrombin III. We also demonstrated the clotting enzyme activity of the purified serine proteinase toward bovine fibrinogen and Limulus coagulogen, a fibrinogen-like clottable protein of horseshoe crabs.

  6. Site-specific laser modification (cleavage) of oligodeoxynucleotides.

    PubMed

    Benimetskaya, L Z; Bulychev, N V; Kozionov, A L; Koshkin, A A; Lebedev, A V; Novozhilov SYu; Stockman, M I

    1989-06-01

    Sequence-specific photomodification of oligodeoxynucleotide pAGAGTATTGACTTA ("a target") has been carried out with the aid of complementary fluorescent probes. Such a probe consisted of oligodeoxynucleotide pAATACTCT and a chromophore group attached to its 5' end. Three different derivatives of ethidium bromide were used as a chromophore. The photomodification was induced by nitrogen laser radiation (337 nm, 15 MW/cm2). The irradiation induces the following photodamages: target cleavage at the specific binding site with a cutting off of the 8-mer from its 5' end (yield up to 12%), formation of specific covalent adduct target-probe with a yield of 20-70%, and piperidine-sensitive target modifications with a 7-27% yield (for different chromophores). The total yield of specific photodamages of all kinds is 50-80%. The target cleavage and generation of piperidine-sensitive modifications are optically nonlinear processes. Piperidine treatment of the irradiated samples led to specific cleavage of the target with the yield up to 40%. All kinds of observed modifications are not influenced by high concentrations of free radical scavengers: 1.3M tBuOH and 10 mM cystamine. The pattern of cleavage indicates that the most probable position of the chromophore is between T8 and G9 of the target, i.e., the chromophore stacks on top of the last A.T base pair of the duplex. The aggregate of evidence is in agreement with the mechanism of nonlinear photomodification (the cleavage and generation of piperidine-sensitive modifications) based on the transfer of two-photon excitation energy from the chromophore to the target.

  7. von Willebrand factor storage requires intact prosequence cleavage site.

    PubMed

    Journet, A M; Saffaripour, S; Cramer, E M; Tenza, D; Wagner, D D

    1993-02-01

    Large multimers of the adhesive glycoprotein von Willebrand factor (vWf) are stored in endothelial cells in rod-shaped granules called Weibel-Palade bodies, while small multimers are secreted constitutively. Expression of pro-vWf in other cells with a regulated pathway of secretion, results in formation of vWf-containing storage granules that have a morphology similar to Weibel-Palade bodies. vWf expressed without its prosequence is not stored. To evaluate the importance of prosequence cleavage in vWf storage, the Arg at position -1, known to be necessary for cleavage, was mutated to Gly. Transfection of this cleavage mutant into two cell lines with a regulated pathway of secretion (RIN 5F and AtT-20 cells) led to the formation of large multimers. However, treatment of the cell lysates by the enzyme endoglycosidase H (Endo-H) did not reveal significant amounts of intracellular Endo-H-resistant vWf, which indicates the absence of a pool of stored processed vWf. In addition, no Weibel-Palade body-like structure was detected in these cells by immunofluorescence labeling with anti-vWf antiserum. Electron microscopy and immunocytochemistry of RIN 5F cells expressing the pro-vWf mutant confirmed the absence of Weibel-Palade body-like structures. In addition, anti-vWf-linked gold particles were found in the ER, occasionally in rounded granules and particularly in lysosomal structures which were abundant. We conclude that the formation of large aggregates is not sufficient to induce efficient vWf storage, and that the lack of cleavage of the prosequence may direct the mutant pro-vWf molecule to a degradative pathway. Therefore, the prosequence cleavage is a requirement for vWf storage.

  8. Crack tip blunting and cleavage under dynamic conditions

    NASA Astrophysics Data System (ADS)

    Rajan, V. P.; Curtin, W. A.

    2016-05-01

    In structural materials with both brittle and ductile phases, cracks often initiate within the brittle phase and propagate dynamically towards the ductile phase. The macroscale, quasistatic toughness of the material thus depends on the outcome of this microscale, dynamic process. Indeed, dynamics has been hypothesized to suppress dislocation emission, which may explain the occurrence of brittle transgranular fracture in mild steels at low temperatures (Lin et al., 1987). Here, crack tip blunting and cleavage under dynamic conditions are explored using continuum mechanics and molecular dynamics simulations. The focus is on two questions: (1) whether dynamics can affect the energy barriers for dislocation emission and cleavage, and (2) what happens in the dynamic "overloaded" situation, in which both processes are energetically possible. In either case, dynamics may shift the balance between brittle cleavage and ductile blunting, thereby affecting the intrinsic ductility of the material. To explore these effects in simulation, a novel interatomic potential is used for which the intrinsic ductility is tunable, and a novel simulation technique is employed, termed as a "dynamic cleavage test", in which cracks can be run dynamically at a prescribed energy release rate into a material. Both theory and simulation reveal, however, that the intrinsic ductility of a material is unaffected by dynamics. The energy barrier to dislocation emission appears to be identical in quasi-static and dynamic conditions, and, in the overloaded situation, ductile crack tip behavior ultimately prevails since a single emission event can blunt and arrest the crack, preventing further cleavage. Thus, dynamics cannot embrittle a ductile material, and the origin of brittle failure in certain alloys (e.g., mild steels) appears unrelated to dynamic effects at the crack tip.

  9. Primary structure of the A chain of human complement-classical-pathway enzyme C1r. N-terminal sequences and alignment of autolytic fragments and CNBr-cleavage peptides.

    PubMed Central

    Gagnon, J; Arlaud, G J

    1985-01-01

    Activated human complement-classical-pathway enzyme C1r has previously been shown to undergo autolytic cleavages occurring in the A chain [Arlaud, Villiers, Chesne & Colomb (1980) Biochim. Biophys. Acta 616, 116-129]. Chemical analysis of the autolytic products confirms that the A chain undergoes two major cleavages, generating three fragments, which have now been isolated and characterized. The N-terminal alpha fragment (approx. 210 residues long) has a blocked N-terminus, as does the whole A chain, whereas N-terminal sequences of fragments beta and gamma (approx. 66 and 176 residues long respectively) do not, and their N-terminal sequences were determined. Fragments alpha, beta and gamma, which are not interconnected by disulphide bridges, are located in this order within C1r A chain. Fragment gamma is disulphide-linked to the B chain of C1r, which is C-terminal in the single polypeptide chain of precursor C1r. CNBr cleavage of C1r A chain yields seven major peptides, CN1b, CN4a, CN2a, CN1a, CN3, CN4b and CN2b, which were positioned in that order, on the basis of N-terminal sequences of the methionine-containing peptides generated from tryptic cleavage of the succinylated (3-carboxypropionylated) C1r A chain. About 60% of the sequence of C1r A chain (440-460 residues long) was determined, including the complete sequence of the C-terminal 95 residues. This region shows homology with the corresponding parts of plasminogen and chymotrypsinogen and, more surprisingly, with the alpha 1 chain of human haptoglobin 1-1, a serine proteinase homologue. PMID:2983658

  10. Iron-mediated cleavage of C-C bonds in vicinal tricarbonyl compounds in water.

    PubMed

    Mecinović, Jasmin; Hamed, Refaat B; Schofield, Christopher J

    2009-01-01

    Three of a kind: Vicinal tricarbonyl compounds undergo C-C cleavage mediated by ferric ions (see scheme). The observed cleavage of ninhydrin and dehydroascorbic acid has relevance for amino acid detection and the metabolism of vitamin C.

  11. [Growth conditions and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant Bacillus subtilis strain].

    PubMed

    Kirillova, Iu M; Mikhaĭlova, E O; Balaban, N P; Mardanova, A M; Rudenskaia, G N; Kostrov, S V; Sharipova, M R

    2006-01-01

    The effect of the components of the nutrient medium on growth and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant strain Bacillus subtilis AJ73(pCS9) was studied. The production of proteinase was found to be dependent on the composition of the nutrient medium and showed two peaks, at the 28th and 48th h of growth. The concentrations of the main components of the nutrient medium (peptone and inorganic phosphate) optimal for the biosynthesis of subtilisin-like serine proteinase at the 28th and 48th h of growth were determined in factorial experiments. Complex organic substances, casein at concentrations of 0.5-1%, gelatin at concentrations of 0.5-1%, and yeast extract at a concentration of 0.5%, stimulated the production of subtilisin-like serine proteinase by the recombinant strain. The study of the sporulation dynamics in this strain showed that the proteinase peaks at the 28th and 48th h of growth correspond, respectively, to the initial stage of sporulation and to the terminal stages of endospore formation (V-VII stages of sporulation).

  12. Protein digestion in cereal aphids (Sitobion avenae) as a target for plant defence by endogenous proteinase inhibitors.

    PubMed

    Pyati, Prashant; Bandani, Ali R; Fitches, Elaine; Gatehouse, John A

    2011-07-01

    Gut extracts from cereal aphids (Sitobion avenae) showed significant levels of proteolytic activity, which was inhibited by reagents specific for cysteine proteases and chymotrypsin-like proteases. Gut tissue contained cDNAs encoding cathepsin B-like cysteine proteinases, similar to those identified in the closely related pea aphid (Acyrthosiphon pisum). Analysis of honeydew (liquid excreta) from cereal aphids fed on diet containing ovalbumin showed that digestion of ingested proteins occurred in vivo. Protein could partially substitute for free amino acids in diet, although it could not support complete development. Recombinant wheat proteinase inhibitors (PIs) fed in diet were antimetabolic to cereal aphids, even when normal levels of free amino acids were present. PIs inhibited proteolysis by aphid gut extracts in vitro, and digestion of protein fed to aphids in vivo. Wheat subtilisin/chymotrypsin inhibitor, which was found to inhibit serine and cysteine proteinases, was more effective in both inhibitory and antimetabolic activity than wheat cystatin, which inhibited cysteine proteases only. Digestion of ingested protein is unlikely to contribute significantly to nutritional requirements when aphids are feeding on phloem, and the antimetabolic activity of dietary proteinase inhibitors is suggested to result from effects on proteinases involved in degradation of endogenous proteins.

  13. Preoviposition activation of cathepsin-like proteinases in degenerating ovarian follicles of the mosquito Culex pipiens pallens.

    PubMed

    Uchida, K; Ohmori, D; Ueno, T; Nishizuka, M; Eshita, Y; Fukunaga, A; Kominami, E

    2001-09-01

    Within developing ovaries of many insects, some developing follicles or oocytes usually degenerate (follicular atresia or oosorption), while the others may continue to grow to maturity, thus maintaining the balance between the number of eggs and reproductive circumstances such as available nutrients. To help clarify the phenomenon of follicular atresia during ovarian development, we examined cysteine proteinases stored in mosquito Culex pipiens pallens ovaries. First, analysis using synthesized substrates showed that cathepsin B- and L-like proteinases gradually accumulated in the developing ovaries after a blood meal, which required more than 10 min of preincubation under acidic conditions to reach their maximum activities. However, homogenates of degenerating follicles 3 days after feeding showed proteolytic activities without acid treatment, suggesting that the proteinases had already been activated, while the extract of normally developing follicles collected from the same ovaries required more than 10 min of acid preincubation to reach the optimum activities, suggesting that the enzymes remained as inactive forms. Chemical and immunohistochemical analyses showed that more proteinases are located in the cytoplasm, rather than being associated with yolk granules. Ovarian proteinases, which are believed to become activated at the onset of embryogenesis, should also be activated during oogenesis, presumably to enhance oosorption.

  14. Comparison of self-processing of foot-and-mouth disease virus leader proteinase and porcine reproductive and respiratory syndrome virus leader proteinase nsp1α

    SciTech Connect

    Steinberger, Jutta; Rancan, Chiara; Skern, Tim

    2013-09-01

    The foot-and-mouth disease virus leader proteinase (Lb{sup pro}) cleaves itself off the nascent viral polyprotein. NMR studies on the monomeric variant Lb{sup pro} L200F provide structural evidence for intramolecular self-processing. {sup 15}N-HSQC measurements of Lb{sup pro} L200F showed specifically shifted backbone signals in the active and substrate binding sites compared to the monomeric variant sLb{sup pro}, lacking six C-terminal residues. This indicates transient intramolecular interactions between the C-terminal extension (CTE) of one molecule and its own active site. Contrastingly, the porcine reproductive and respiratory syndrome virus (PRRSV) leader proteinase nsp1α, with a papain-like fold like Lb{sup pro}, stably binds its own CTE. Parts of the β-sheet domains but none of the α-helical domains of Lb{sup pro} and nsp1α superimpose; consequently, the α-helical domain of nsp1α is oriented differently relative to its β-sheet domain. This provides a large interaction surface for the CTE with the globular domain, stabilising the intramolecular complex. Consequently, self-processing inactivates nsp1α but not Lb{sup pro}. - Highlights: • We examine self-processing of the leader protease of foot-and-mouth disease virus. • NMR analysis strongly supports intramolecular self-processing. • Self-processing is a dynamic process with no stable complex. • Structural comparison with nsp1α of PRRSV which forms stable intramolecular complex. • Subdomain orientation explains differences in stability of intramolecular complexes.

  15. Liquid Chromatography-Tandem Mass Spectrometry to Define Sortase Cleavage Products.

    PubMed

    Duong, Andrew; Koteva, Kalinka; Sexton, Danielle L; Elliot, Marie A

    2016-01-01

    Sortase enzymes have specific endopeptidase activity, cleaving within a defined pentapeptide sequence at the C-terminal end of their protein substrates. Here, we describe how monitoring sortase cleavage activity can be achieved using peptide substrates. Peptide cleavage can be readily analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS), which allows for the precise definition of cleavage sites. This technique could be used to analyze the peptidase activity of any enzyme, and identify sites of cleavage within any peptide.

  16. Cleavage patterns and the topology of the metazoan tree of life

    PubMed Central

    Valentine, James W.

    1997-01-01

    Several major alliances of metazoan phyla have been identified by small subunit rRNA sequence comparisons. It is possible to arrange the phyla to produce a parsimonious distribution of cleavage types, requiring only one change from a radial ancestral condition to spiral cleavage and one other to “idiosyncratic” cleavage; this arrangement is consistent with most of the recent molecular phylogenies. The cleavage shifts are correlated with changes in many of the features that once were used to distinguish Protostomia and Deuterostomia. It is hypothesized that changes in cleavage direction are causally associated with changes in blastomere fates and thus that cleavage type correlates with such features as the identity of mesoderm founder cells, which in turn can constrain the mode of origination of the eucelom. Cleavage changes may also affect the timing of cell fate specification. In a tree that emphasizes cleavage parsimony, radial cleavage, regulative development, and enterocely are ancestral within the Bilateria, and spiral or idiosyncratic cleavages, mosaic development, and schizocely are associated with a change in cleavage direction. Deuterostomy is presumably ancestral and is correlated with radial cleavage for this reason, rather than mechanistically. PMID:9223303

  17. Stille coupling via C–N bond cleavage

    PubMed Central

    Wang, Dong-Yu; Kawahata, Masatoshi; Yang, Ze-Kun; Miyamoto, Kazunori; Komagawa, Shinsuke; Yamaguchi, Kentaro; Wang, Chao; Uchiyama, Masanobu

    2016-01-01

    Cross-coupling is a fundamental reaction in the synthesis of functional molecules, and has been widely applied, for example, to phenols, anilines, alcohols, amines and their derivatives. Here we report the Ni-catalysed Stille cross-coupling reaction of quaternary ammonium salts via C–N bond cleavage. Aryl/alkyl-trimethylammonium salts [Ar/R–NMe3]+ react smoothly with arylstannanes in 1:1 molar ratio in the presence of a catalytic amount of commercially available Ni(cod)2 and imidazole ligand together with 3.0 equivalents of CsF, affording the corresponding biaryl with broad functional group compatibility. The reaction pathway, including C–N bond cleavage step, is proposed based on the experimental and computational findings, as well as isolation and single-crystal X-ray diffraction analysis of Ni-containing intermediates. This reaction should be widely applicable for transformation of amines/quaternary ammonium salts into multi-aromatics. PMID:27686744

  18. Stille coupling via C-N bond cleavage

    NASA Astrophysics Data System (ADS)

    Wang, Dong-Yu; Kawahata, Masatoshi; Yang, Ze-Kun; Miyamoto, Kazunori; Komagawa, Shinsuke; Yamaguchi, Kentaro; Wang, Chao; Uchiyama, Masanobu

    2016-09-01

    Cross-coupling is a fundamental reaction in the synthesis of functional molecules, and has been widely applied, for example, to phenols, anilines, alcohols, amines and their derivatives. Here we report the Ni-catalysed Stille cross-coupling reaction of quaternary ammonium salts via C-N bond cleavage. Aryl/alkyl-trimethylammonium salts [Ar/R-NMe3]+ react smoothly with arylstannanes in 1:1 molar ratio in the presence of a catalytic amount of commercially available Ni(cod)2 and imidazole ligand together with 3.0 equivalents of CsF, affording the corresponding biaryl with broad functional group compatibility. The reaction pathway, including C-N bond cleavage step, is proposed based on the experimental and computational findings, as well as isolation and single-crystal X-ray diffraction analysis of Ni-containing intermediates. This reaction should be widely applicable for transformation of amines/quaternary ammonium salts into multi-aromatics.

  19. Enhancing PDT drug delivery by enzymatic cleavage of porphyrin phosphates

    NASA Astrophysics Data System (ADS)

    Xu, Bing; Liang, Gaolin; Wang, Ling; Yang, Zhimou; Chan, Kalok; Chang, Chi K.

    2007-02-01

    A new anionic porphyrin-phosphate conjugate has been made as the substrate of phosphatase to evaluate its cellular-uptake and potential targeting on cancer cells, taking advantage of the over-expression of phosphatases associated with the development of cancers. The phosphate groups increase the hydrophilicity of porphyrin dityrosine phosphate and facilitate its formulation in aqueous solvent. Upon hydrolysis by phosphatase after cellular uptaking, the more hydrophobic porphyrin-dityrosine promises to give better cellular retention. Indeed, the phosphate conjugate displayed a much better PDT effect than that of the parent porphyrin at the same concentration (10 μM) and light dosage on HeLa cells, indicating the enzyme-cleavage reaction occurred in HeLa cells plays a role. Photosenzitizers utilizing enzyme-cleavage might be a promising approach for photodynamic therapy.

  20. Sequence specificity of DNA cleavage by Micrococcus luteus. gamma. endonuclease

    SciTech Connect

    Hentosh, P.; Henner, W.D.; Reynolds, R.J.

    1985-04-01

    DNA fragments of defined sequence have been used to determine the sites of cleavage by ..gamma..-endonuclease activity in extracts prepared from Micrococcus luteus. End-labeled DNA restriction fragments of pBR322 DNA that had been irradiated under nitrogen in the presence of potassium iodide or t-butanol were treated with M. luteus ..gamma.. endonuclease and analyzed on irradiated DNA preferentially at the positions of cytosines and thymines. DNA cleavage occurred immediately to the 3' side of pyrimidines in irradiated DNA and resulted in fragments that terminate in a 5'-phosphoryl group. These studies indicate that both altered cytosines and thymines may be important DNA lesions requiring repair after exposure to ..gamma.. radiation.

  1. Hydrolytic cleavage of DNA by quercetin zinc(II) complex.

    PubMed

    Jun, Tan; Bochu, Wang; Liancai, Zhu

    2007-03-01

    Quercetin zinc(II) complex was investigated focusing on its hydrolytic activity toward DNA. The complex successfully promotes the cleavage of plasmid DNA, producing single and double DNA strand breaks. The amount of conversion of supercoiled form (SC) of plasmid to the nicked circular form (NC) depends on the concentration of the complex as well as the duration of incubation of the complex with DNA. The rate of conversion of SC to NC is 1.68x10(-4) s(-1) at pH 7.2 in the presence of 100 microM of the complex. The hydrolytic cleavage of DNA by the complex is supported by the evidence from free radical quenching, thiobarbituric acid-reactive substances (TBARS) assay, and T4 ligase ligation.

  2. Hydrolytic cleavage of DNA by quercetin manganese(II) complexes.

    PubMed

    Jun, Tan; Bochu, Wang; Liancai, Zhu

    2007-04-01

    Quercetin manganese(II) complexes were investigated focusing on its DNA hydrolytic activity. The complexes successfully promote the cleavage of plasmid DNA, producing single and double DNA strand breaks. The amount of conversion of supercoiled form (SC) of plasmid DNA to the nicked circular form (NC) depends on the concentration of the complex as well as the duration of incubation of the complexes with DNA. The maximum rate of conversion of the supercoiled form to the nicked circular form at pH 7.2 in the presence of 100 microM of the complexes is found to be 1.32 x 10(-4) s(-1). The hydrolytic cleavage of DNA by the complexes was supported by the evidence from free radical quenching, thiobarbituric acid-reactive substances (TBARS) assay and T4 ligase ligation.

  3. Enzymes active in the areas undergoing cartilage resorption during the development of the secondary ossification center in the tibiae of rats ages 0-21 days: I. Two groups of proteinases cleave the core protein of aggrecan.

    PubMed

    Lee, E R; Lamplugh, L; Davoli, M A; Beauchemin, A; Chan, K; Mort, J S; Leblond, C P

    2001-09-01

    The formation of a secondary ossification center in the cartilaginous epiphysis of long bones requires the excavation of canals and marrow space and, therefore, the resorption of cartilage. On the assumption that its resorption requires the lysis of the major cartilage component aggrecan, it was noted that the core protein may be cleaved in vitro by proteinases from two subfamilies: matrix metalloproteinases (MMPs) and aggrecanases. Such cleavage results in aggrecan being replaced by a fragment of itself referred to as a "G1-fragment." To find out if this cleavage occurs in the developing epiphysis of the rat tibia, the approach has been to localize the G1 fragments. For this purpose two neoepitope antisera were applied, one capable of recognizing the MMP-generated G1-fragment that bears the C-terminus ...FVDIPEN341 and the other capable of recognizing the aggrecanase-generated G1-fragment that carries the C-terminus ...NITEGE373. With the aid of these antisera, we report here that aggrecan cleavage is localized to newly developed sites of erosion. Thus, at 6 days of age, canals allowing the entry of capillaries are dug out from the surface of the epiphysis in a radial direction (stage I), whereas immunostaining indicative of aggrecan cleavage by MMPs appears at the blind end of each canal. The next day, the canal blind ends fuse to create a marrow space in the epiphysis (stage II), whereas immunostaining produced by MMPs occurs along the walls of this space. By 9 days, clusters of hypertrophic chondrocytes are scattered along the marrow space wall to initiate the formation of the secondary ossification center (stage III), where the resorption sites are unreactive to either antiserum. From the 9th to the 21st day, the center keeps on enlarging and, as the distal wall of the marrow space recedes, it is intensely immunostained with both antisera indicating that both MMPs and aggrecanases are involved in this resorption. We conclude, that both enzyme subfamilies

  4. Effect of irreversibility on the thermodynamic characterization of the thermal denaturation of Aspergillus saitoi acid proteinase.

    PubMed Central

    Tello-Solis, S R; Hernandez-Arana, A

    1995-01-01

    The thermal denaturation of the acid proteinase from Aspergillus saitoi was studied by CD and differential scanning calorimetry (DSC). This process seemed to be completely irreversible, as protein samples that were heated to temperatures at which the transition had been completed and then cooled at 25 degrees C did not show any reversal of the change in the CD signal. Similar results were obtained with DSC. Nevertheless, we were able to detect the presence of reversibly unfolded species in experiments in which the enzyme solution was heated to a temperature within the transition region, followed by rapid cooling at 25 degrees C. Accordingly, the denaturation of behaviour of the acid proteinase seems to be consistent with the existence of one (or more) reversible unfolding transition followed by an irreversible step. The van't Hoff enthalpy, delta HvH, which corresponds to the reversible transition was calculated from extrapolation to infinite heating rate as 310 kJ.mol-1. This parameter was also determined from direct estimation of the equilibrium constant at several temperatures (delta HvH = 176 kJ.mol-1). Comparison of the average delta HvH with the calorimetric enthalpy (delta Hcal. = 770 kJ.mol-1) gave a value of 3.2 for the delta Hcal./delta HvH ratio, indicating that the molecular structure of the enzyme is probably formed by three or four cooperative regions, a number similar to that of the acid proteinase, pepsin. It should be noted that a completely different conclusion would be obtained from a straightforward analysis of the calorimetric curves, disregarding the effect of irreversibility on the denaturation process. PMID:7487958

  5. Proteinase and Growth Factor Alterations Revealed by Gene Microarray Analysis of Human Diabetic Corneas

    PubMed Central

    Saghizadeh, Mehrnoosh; Kramerov, Andrei A.; Tajbakhsh, Jian; Aoki, Annette M.; Wang, Charles; Chai, Ning-Ning; Ljubimova, Julia Y.; Sasaki, Takako; Sosne, Gabriel; Carlson, Marc R. J.; Nelson, Stanley F.

    2005-01-01

    PURPOSE. To identify proteinases and growth factors abnormally expressed in human corneas of donors with diabetic retinopathy (DR), additional to previously described matrix metalloproteinase (MMP)-10 and -3 and insulin-like growth factor (IGF)-I. METHODS. RNA was isolated from 35 normal, diabetic, and DR autopsy human corneas ex vivo or after organ culture. Amplified cRNA was analyzed using 22,000-gene microarrays (Agi-lent Technologies, Palo Alto, CA). Gene expression in each diabetic corneal cRNA was assessed against pooled cRNA from 7 to 9 normal corneas. Select differentially expressed genes were validated by quantitative real-time RT-PCR (QPCR) and immunohistochemistry. Organ cultures were treated with a cathepsin inhibitor, cystatin C, or MMP-10. RESULTS. More than 100 genes were upregulated and 2200 were downregulated in DR corneas. Expression of cathepsin F and hepatocyte growth factor (HGF) genes was increased in ex vivo and organ-cultured DR corneas compared with normal corneas. HGF receptor c-met, fibroblast growth factor (FGF)-3, its receptor FGFR3, tissue inhibitor of metalloproteinase (TIMP)-4, laminin α4 chain, and thymosin β4 genes were down-regulated. The data were corroborated by QPCR and immuno-histochemistry analyses; main changes of these components occurred in corneal epithelium. In organ-cultured DR corneas, cystatin C increased laminin-10 and integrin α3β1, whereas in normal corneas MMP-10 decreased laminin-10 and integrin α3β1 expression. CONCLUSIONS. Elevated cathepsin F and the ability of its inhibitor to produce a more normal phenotype in diabetic corneas suggest increased proteolysis in these corneas. Proteinase changes may result from abnormalities of growth factors, such as HGF and FGF-3, in DR corneas. Specific modulation of proteinases and growth factors could reduce diabetic corneal epitheliopathy. PMID:16186340

  6. Effects of pH on the association between the inhibitor cystatin and the proteinase chymopapain.

    PubMed

    Reyes-Espinosa, Francisco; Arroyo-Reyna, Alfonso; Garcia-Gutierrez, Ponciano; Serratos, Iris N; Zubillaga, Rafael A

    2014-01-01

    Cysteine proteinases are involved in many aspects of physiological regulation. In humans, some cathepsins have shown another function in addition to their role as lysosomal proteases in intracellular protein degradation; they have been implicated in the pathogenesis of several heart and blood vessel diseases and in cancer development. In this work, we present a fluorometric and computational study of the binding of one representative plant cysteine proteinase, chymopapain, to one of the most studied inhibitors of these proteinases: chicken cystatin. The binding equilibrium constant, Kb, was determined in the pH range between 3.5 and 10.0, revealing a maximum in the affinity at pH 9.0. We constructed an atomic model for the chymopapain-cystatin dimer by docking the individual 3D protein structures; subsequently, the model was refined using a 100 ns NPT molecular dynamics simulation in explicit water. Upon scrutiny of this model, we identified 14 ionizing residues at the interface of the complex using a cutoff distance of 5.0 Å. Using the pKa values predicted with PROPKA and a modified proton-linkage model, we performed a regression analysis on our data to obtain the composite pKavalues for three isoacidic residues. We also calculated the electrostatic component of the binding energy (ΔGb,elec) at different pH values using an implicit solvent model and APBS software. The pH profile of this calculated energy compares well with the experimentally obtained binding energy, ΔGb. We propose that the residues that form an interchain ionic pair, Lys139A from chymopapain and Glu19B from cystatin, as well as Tyr61A and Tyr67A from chymopapain are the main residues responsible for the observed pH dependence in the chymopapain- cystatin affinity.

  7. Nepenthesin, a unique member of a novel subfamily of aspartic proteinases: enzymatic and structural characteristics.

    PubMed

    Takahashi, Kenji; Athauda, Senarath B P; Matsumoto, Koji; Rajapakshe, Sanath; Kuribayashi, Masayuki; Kojima, Masaki; Kubomura-Yoshida, Nobuko; Iwamatsu, Akihiro; Shibata, Chiaki; Inoue, Hideshi

    2005-12-01

    Carnivorous plants are known to secrete acid proteinases to digest prey, mainly insects, for nitrogen uptake. In our recent study, we have purified, for the first time, to homogeneity two acid proteinases, nepenthesin I (Nep I) and nepenthesin II (Nep II) from the pitcher fluid of Nepenthes distillatoria and investigated their enzymatic and structural characteristics. Both enzymes were optimally active at pH approx. 2.6 toward acid-denatured hemoglobin; the specificity of Nep I toward oxidized insulin B chain appears to be similar, but slightly wider than those of other aspartic proteinases (APs). At or below 50 degrees C both enzymes were remarkably stable; especially Nep I was extremely stable over a wide range of pH from 3 to 10 for over 30 days. This suggests an evolutionary adaptation of the enzymes to their specific habitat. We have also cloned the cDNAs and deduced the complete amino acid sequences of the precursors of Nep I and Nep II from the pitcher tissue of Nepenthes gracilis. Although the corresponding mature enzymes are homologous with ordinary pepsin-type APs, both enzymes had a high content of cysteine residues (12 residues per molecule), which are assumed to form six unique disulfide bonds as suggested by computer modeling and are thought to contribute toward the remarkable stability of Neps. Moreover, the amino acid sequence identity of Neps with ordinary APs, including plant vacuolar APs, are remarkably low (approx. 20%), and phylogenetic comparison shows that Neps are distantly related to them to form a novel subfamily of APs with a high content of cysteine residues and a characteristic insertion, named 'the Nep-type AP (NAP)-specific insertion', including a large number of novel, orthologous plant APs emerging in the gene/protein databases.

  8. Enzymic and structural characterization of nepenthesin, a unique member of a novel subfamily of aspartic proteinases.

    PubMed

    Athauda, Senarath B P; Matsumoto, Koji; Rajapakshe, Sanath; Kuribayashi, Masayuki; Kojima, Masaki; Kubomura-Yoshida, Nobuko; Iwamatsu, Akihiro; Shibata, Chiaki; Inoue, Hideshi; Takahashi, Kenji

    2004-07-01

    Carnivorous plants are known to secrete acid proteinases to digest prey, mainly insects, for nitrogen uptake. In the present study, we have purified, for the first time, to homogeneity two acid proteinases (nepenthesins I and II) from the pitcher fluid of Nepenthes distillatoria (a pitcher-plant known locally as badura) and investigated their enzymic and structural characteristics. Both enzymes were optimally active at pH approx. 2.6 towards acid-denatured haemoglobin; the specificity of nepenthesin I towards oxidized insulin B chain appears to be similar, but slightly wider than those of other APs (aspartic proteinases). Among the enzymic properties, however, the most notable is their unusual stability: both enzymes were remarkably stable at or below 50 degrees C, especially nepenthesin I was extremely stable over a wide range of pH from 3 to 10 for over 30 days. This suggests an evolutionary adaptation of the enzymes to their specific habitat. We have also cloned the cDNAs and deduced the complete amino acid sequences of the precursors of nepenthesins I and II (437 and 438 residues respectively) from the pitcher tissue of N. gracilis. Although the corresponding mature enzymes (each 359 residues) are homologous with ordinary pepsin-type APs, both enzymes had a high content of cysteine residues (12 residues/molecule), which are assumed to form six unique disulphide bonds as suggested by computer modelling and are supposed to contribute towards the remarkable stability of nepenthesins. Moreover, the amino acid sequence identity of nepenthesins with ordinary APs, including plant vacuolar APs, is remarkably low (approx. 20%), and phylogenetic comparison shows that nepenthesins are distantly related to them to form a novel subfamily of APs with a high content of cysteine residues and a characteristic insertion, named 'the nepenthesin-type AP-specific insertion', that includes a large number of novel, orthologous plant APs emerging in the gene/protein databases.

  9. Proteinase and phospholipase activities and development at different temperatures of yeasts isolated from bovine milk.

    PubMed

    Melville, Priscilla A; Benites, Nilson R; Ruz-Peres, Monica; Yokoya, Eugenio

    2011-11-01

    The presence of yeasts in milk may cause physical and chemical changes limiting the durability and compromising the quality of the product. Moreover, milk and dairy products contaminated by yeasts may be a potential means of transmission of these microorganisms to man and animals causing several kinds of infections. This study aimed to determine whether different species of yeasts isolated from bovine raw milk had the ability to develop at 37°C and/or under refrigeration temperature. Proteinase and phospholipase activities resulting from these yeasts were also monitored at different temperatures. Five genera of yeasts (Aureobasidium sp., Candida spp., Geotrichum spp., Trichosporon spp. and Rhodotorula spp.) isolated from bovine raw milk samples were evaluated. All strains showed one or a combination of characteristics: growth at 37°C (99·09% of the strains), psychrotrophic behaviour (50·9%), proteinase production (16·81% of the strains at 37°C and 4·09% under refrigeration) and phospholipase production (36·36% of the isolates at 37°C and 10·9% under refrigeration), and all these factors may compromise the quality of the product. Proteinase production was similar for strains incubated at 37°C (16·81% of the isolates) and room temperature (17·27%) but there was less amount of phospholipase-producing strains at room temperature (15·45% of the isolates were positive) when compared with incubation at 37°C (36·36%). Enzymes production at 37°C by yeasts isolated from milk confirmed their pathogenic potential. The refrigeration temperature was found to be most efficient to inhibit enzymes production and consequently ensure better quality of milk. The viability of yeasts and the activity of their enzymes at different temperatures are worrying because this can compromise the quality of dairy products at all stages of production and/or storage, and represent a risk to the consumer.

  10. Influence of immunoprotection on genetic variability of cysteine proteinases from Haemonchus contortus adult worms.

    PubMed

    Martín, S; Molina, J M; Hernández, Y I; Ferrer, O; Muñoz, Ma C; López, A; Ortega, L; Ruiz, A

    2015-11-01

    The limitations associated with the use of anthelmintic drugs in the control of gastrotintestinal nematodosis, such as the emergence of anthelmintic resistance, have stimulated the study of the immunological control of many parasites. In the case of Haemonchus contortus, several vaccination trials using native and recombinant antigens have been conducted. A group of antigens with demonstrated immunoprotective value are cathepsin B - like proteolytic enzymes of the cysteine proteinase type. These enzymes, which have been observed in both excretory-secretory products and somatic extracts of H. contortus, may vary among different geographic isolates and on strains isolated from different hosts, or even from the same host, as has been demonstrated in some comparative studies of genetic variability. In the present study, we evaluated the genetic variability of the worms that fully developed their endogenous cycle in immunised sheep and goat in order to identify the alleles of most immunoprotective value. To address these objectives, groups of sheep and goats were immunised with PBS soluble fractions enriched for cysteine proteinases from adult worms of H. contortus from either a strain of H. contortus isolated from goats of Gran Canaria Island (SP) or a strain isolated from sheep of North America (NA). The results confirmed the immunoprophylactic value of this type of enzyme against haemonchosis in both sheep and goats in association with increased levels of specific IgG. The genetic analysis demonstrated that the immunisation had a genetic selection on proteinase-encoding genes. In all the immunised animals, allelic frequencies were statistically different from those observed in non-immunised control animals in the four analysed genes. The reduction in the allelic frequencies suggests that parasites expressing these proteases are selectively targeted by the vaccine, and hence they should be considered in any subunit vaccine approach to control haemonchosis in small

  11. Exposure to tobacco-derived materials induces overproduction of secreted proteinases in mast cells

    SciTech Connect

    Small-Howard, Andrea; Turner, Helen . E-mail: hturner@queens.org

    2005-04-15

    Mast cells reside at interfaces with the environment, including the mucosa of the respiratory and gastrointestinal tracts. This localization exposes mast cells to inhaled, or ingested, environmental challenges. In the airways of smokers, resident immune cells will be in contact with the condensed components of cigarette smoke. Mast cells are of particular interest due to their ability to promote airway remodeling and mucus hypersecretion. Clinical data show increased levels of mast cell-secreted tryptase and increased numbers of degranulated mast cells in the lavage and bronchial tissue of smokers. Since mast cell-secreted proteinases (MCPTs), including tryptases, contribute to pathological airway remodeling, we investigated the relationship between mast cell proteinases and smoke exposure. We exposed a mast cell line to cigarette smoke condensate (CSC). We show that CSC exposure increases MCPT levels in mast cells using an assay for tryptase-type MCPT activity. We hypothesized that this increase in MCPT activity reflects a CSC-induced increase in the cytosolic pool of proteinase molecules, via stimulation of MCPT transcription. Transcript array data suggested that mRNA changes in response to CSC were limited in number and peaked after 3 h of CSC exposure. However, we noted marked transcriptional regulation of several MCPT genes. CSC-induced changes in the mRNA levels for MCPTs were confirmed using quantitative RT-PCR. Taken together, our data suggest that chronic exposure to cigarette smoke up-regulates MCPT levels in mast cells at both the protein and the mRNA level. We suggest that the pathological airway remodeling that has been described in clinical studies of smoke inhalation may be attributable to MCPT overproduction in vivo.

  12. Mechanism and specificity of RNA cleavage by chemical ribonucleases.

    PubMed

    Beloglazova, N; Vlassov, A; Konevetc, D; Sil'nikov, V; Zenkova, M; Giege, R; Vlassov, V

    1999-01-01

    Cleaving of model RNA substrates by chemical ribonucleases constructed by conjugation of 1,4 diazabicyclo[2,2,2]octane with histamine and histidine was investigated. Similarly to RNase A, the chemical RNases produce fragments with 5' hydroxy-group and 3'-cyclophosphate. The cleavage occurs as the catalytic reaction: more than 150 phosphodiester bonds in RNA can be cleaved by one molecule of RNase mimic.

  13. How the glycosyltransferase OGT catalyzes amide bond cleavage

    PubMed Central

    Janetzko, John; Trauger, Sunia A.; Lazarus, Michael B.; Walker, Suzanne

    2016-01-01

    The essential human enzyme O-GlcNAc transferase (OGT), known for modulating the functions of nuclear and cytoplasmic proteins through Ser/Thr glycosylation, was unexpectedly implicated in the proteolytic maturation of the cell cycle regulator host cell factor-1 (HCF-1). Here we show that HCF-1 cleavage occurs via glycosylation of a glutamate side chain followed by on-enzyme formation of an internal pyroglutamate, which undergoes spontaneous backbone hydrolysis. PMID:27618188

  14. Cleavage Mapping the Topology of Protein Folding Intermediates

    DTIC Science & Technology

    2007-11-02

    investigate the changes that occur in two of these mutants. V66L has a greatly lowered m value while that of A90S is substantially increased (5...stability of the folded state of nuclease. The cleavage technique will be used to investigate the changes that occur in two of these mutants. V66L...Connecticut, 06520 3Instituto de Qufmica y Fisicoquimica Biolögicas, Facultad de Farmacia y Bioqufmica (UBA-CONICET), Buenos Aires, Argentina 4

  15. Mycothiol synthesis by an anomerization reaction through endocyclic cleavage

    PubMed Central

    2016-01-01

    Summary Mycothiol is found in Gram-positive bacteria, where it helps in maintaining a reducing intracellular environment and it plays an important role in protecting the cell from toxic chemicals. The inhibition of the mycothiol biosynthesis is considered as a treatment for tuberculosis. Mycothiol contains an α-aminoglycoside, which is difficult to prepare stereoselectively by a conventional glycosylation reaction. In this study, mycothiol was synthesized by an anomerization reaction from an easily prepared β-aminoglycoside through endocyclic cleavage. PMID:26977192

  16. Glutamic Acid Selective Chemical Cleavage of Peptide Bonds.

    PubMed

    Nalbone, Joseph M; Lahankar, Neelam; Buissereth, Lyssa; Raj, Monika

    2016-03-04

    Site-specific hydrolysis of peptide bonds at glutamic acid under neutral aqueous conditions is reported. The method relies on the activation of the backbone amide chain at glutamic acid by the formation of a pyroglutamyl (pGlu) imide moiety. This activation increases the susceptibility of a peptide bond toward hydrolysis. The method is highly specific and demonstrates broad substrate scope including cleavage of various bioactive peptides with unnatural amino acid residues, which are unsuitable substrates for enzymatic hydrolysis.

  17. Positioning the cleavage furrow: All you need is Rho

    PubMed Central

    Liu, Zairan

    2016-01-01

    RhoA controls cleavage furrow formation during cell division, but whether RhoA suffices to orchestrate spatiotemporal dynamics of furrow formation is unknown. In this issue, Wagner and Glotzer (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201603025) show that RhoA activity can induce furrow formation in all cell cortex positions and cell cycle phases. PMID:27325786

  18. Infected Aortic Aneurysm Mimicking Anti-proteinase 3-Antineutrophil Cytoplasmic Antibody-associated Vasculitis

    PubMed Central

    Hachiya, Kenta; Wakami, Kazuaki; Yoshida, Atsuhiro; Suda, Hisao; Ohte, Nobuyuki

    2016-01-01

    We herein report an unusual case of an infected descending aortic pseudoaneurysm with luminal pathognomonic oscillating vegetation with serological findings and clinical features mimicking anti-proteinase 3-antineutrophil cytoplasmic antibody-associated vasculitis. The positive blood cultures and imaging findings, including a pseudoaneurysm and vegetations in the aorta, suggested the presence of an infected aortic aneurysm. The patient was successfully treated with antibiotics and endovascular aortic repair. A precise diagnosis is crucial in order to avoid inappropriate therapy such as immunosuppressive treatment, which could result in life-threatening consequences in a patient with an infected aortic aneurysm. PMID:27904110

  19. Two distinct phases of apoptosis in mammary gland involution: proteinase-independent and -dependent pathways

    SciTech Connect

    Lund, Leif R; Romer, John; Thomasset, Nicole; Solberg, Helene; Pyke, Charles; Bissell, Mina J; Dano, Keld; Werb, Zena

    1996-01-01

    Postlactational involution of the mammary gland is characterized by two distinct physiological events: apoptosis of the secretory, epithelial cells undergoing programmed cell death, and proteolytic degradation of the mammary gland basement membrane. We examined the spatial and temporal patterns of apoptotic cells in relation to those of proteinases during involution of the BALB/c mouse mammary gland. Apoptosis was almost absent during lactation but became evident at day 2 of involution, when {beta}-casein gene expression was still high. Apoptotic cells were then seen at least up to day 8 of involution, when {beta}-casein gene expression was being extinguished. Expression of sulfated glycoprotein-2 (SGP-2), interleukin-1{beta} converting enzyme (ICE) and tissue inhibitor of metalloproteinases-1 was upregulated at day 2, when apoptotic cells were seen initially. Expression of the matrix metalloproteinases gelatinase A and stromelysin-1 and the serine proteinase urokinase-type plasminogen activator, which was low during lactation, was strongly upregulated in parallel starting at day 4 after weaning, coinciding with start of the collapse of the lobulo-alveolar structures and the intensive tissue remodeling in involution. The major sites of mRNA synthesis for these proteinases were fibroblast-like cells in the periductal stroma and stromal cells surrounding the collapsed alveoli, suggesting that the degradative phase of involution is due to a specialized mesenchymal-epithelial interaction. To elucidate the functional role of these proteinases during involution, at the onset of weaning we treated mice systemically with the glucocorticoid hydrocortisone, which is known to inhibit mammary gland involution. Although the initial wave of apoptotic cells appeared in the lumina of the gland, the dramatic regression and tissue remodeling usually evident by day 5 was substantially inhibited by systemic treatment with hydrocortisone. mRNA and protein for gelatinase A, stromelysin

  20. Investigation of Serine-Proteinase-Catalyzed Peptide Splicing in Analogues of Sunflower Trypsin Inhibitor 1 (SFTI-1).

    PubMed

    Karna, Natalia; Łęgowska, Anna; Malicki, Stanisław; Dębowski, Dawid; Golik, Przemysław; Gitlin, Agata; Grudnik, Przemysław; Wladyka, Benedykt; Brzozowski, Krzysztof; Dubin, Grzegorz; Rolka, Krzysztof

    2015-09-21

    Serine-proteinase-catalyzed peptide splicing was demonstrated in analogues of the trypsin inhibitor SFTI-1: both single peptides and two-peptide chains (C- and N-terminal peptide chains linked by a disulfide bridge). In the second series, peptide splicing with catalytic amount of proteinase was observed only when formation of acyl-enzyme intermediate was preceded by hydrolysis of the substrate Lys-Ser peptide bond. Here we demonstrate that with an equimolar amount of the proteinase, splicing occurs in all the two-peptide-chain analogues. This conclusion was supported by high resolution crystal structures of selected analogues in complex with trypsin. We showed that the process followed a direct transpeptidation mechanism. Thus, the acyl-enzyme intermediate was formed and was immediately used for a new peptide bond formation; products associated with the hydrolysis of the acyl-enzyme were not observed. The peptide splicing was sequence- not structure-specific.

  1. [Activity of cytoplasmic proteinases from rat liver in Heren's carcinoma during tumor growth and treatment with medicinal herbs].

    PubMed

    Marchenko, M M; Kopyl'chuk, H P; Hrygor'ieva, O V

    2000-01-01

    The dynamics of the acid and neutral proteinases general enzymes activity change in the hepatocytes postnuclear fraction in the rats suffering from the Heren's carcinoma was investigated. It was determined that in the tumor development of the enzyme activity level of both the acid and neutral proteinases increased 2,6-fold. The natural preparation of the herbs (Calendula officinalis L., Echinacea purpurea L., Scorzonera humilis L., Aconitum moldavicum Hacq.) normalizes both the activity of the investigated enzymes and coefficients of the liver weights of the sick animals. The chemical medicinal preparation 5,6-benzcumarine-5-uracil normalizes the activity of the neutral cytoplasmatic proteinases and reduces the level of the proteolytic activity of the acid enzymes in comparison with the control group of the animals as well as increases of the liver weight coefficients.

  2. Purification, crystallization and preliminary crystallographic studies of a Kunitz-type proteinase inhibitor from tamarind (Tamarindus indica) seeds

    PubMed Central

    Patil, Dipak N.; Preeti; Chaudhry, Anshul; Sharma, Ashwani K.; Tomar, ­Shailly; Kumar, Pravindra

    2009-01-01

    A Kunitz-type proteinase inhibitor has been purified from tamarind (Tamarindus indica) seeds. SDS–PAGE analysis of a purified sample showed a homogeneous band corresponding to a molecular weight of 21 kDa. The protein was identified as a Kunitz-type proteinase inhibitor based on N-terminal amino-acid sequence analysis. It was crystallized by the vapour-diffusion method using PEG 6000. The crystals belonged to the orthorhombic space group C2221, with unit-cell parameters a = 37.2, b = 77.1, c = 129.1 Å. Diffraction data were collected to a resolution of 2.7 Å. Preliminary crystallographic analysis indicated the presence of one proteinase inhibitor molecule in the asymmetric unit, with a solvent content of 44%. PMID:19574654

  3. Cloning eleven midgut trypsin cDNAs and evaluating the interaction of proteinase inhibitors with Cry1Ac against the tobacco budworm Heliothis virescens (F.) (Lepidoptera: Noctuidae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Midgut trypsins are associated with Bt protoxin activation and toxin degradation. Proteinase inhibitors have potential insecticidal toxicity against a wide range of insect species. Proactive action to examine trypsin gene profiles and proteinase inhibitors for interaction with Bt toxin is necessary ...

  4. Potential Use of Proteinase Inhibitors, Avidin, and Other Bio-reagents for Synergizing Bt Performance and Delaying Resistance Development to Bt

    Technology Transfer Automated Retrieval System (TEKTRAN)

    After being ingested by target insects, the insecticidal proteins from Bacillus thuringiensis (Bt) need to go through a proteolytic process by insect midgut proteinases to become activated. At the same time, Bt can be hydrolyzed and degraded by midgut proteinases to become non-toxic to target insect...

  5. Elasto-viscoplastic phase field modelling of anisotropic cleavage fracture

    NASA Astrophysics Data System (ADS)

    Shanthraj, P.; Svendsen, B.; Sharma, L.; Roters, F.; Raabe, D.

    2017-02-01

    A finite-strain anisotropic phase field method is developed to model the localisation of damage on a defined family of crystallographic planes, characteristic of cleavage fracture in metals. The approach is based on the introduction of an undamaged configuration, and the inelastic deformation gradient mapping this configuration to a damaged configuration is microstructurally represented by the opening of a set of cleavage planes in the three fracture modes. Crack opening is modelled as a dissipative process, and its evolution is thermodynamically derived. To couple this approach with a physically-based phase field method for brittle fracture, a scalar measure of the overall local damage is introduced, whose evolution is determined by the crack opening rates, and weakly coupled with the non-local phase field energy representing the crack opening resistance in the classical sense of Griffith. A finite-element implementation of the proposed model is employed to simulate the crack propagation path in a laminate and a polycrystalline microstructure. As shown in this work, it is able to predict the localisation of damage on the set of pre-defined cleavage planes, as well as the kinking and branching of the crack resulting from the crystallographic misorientation across the laminate boundary and the grain boundaries respectively.

  6. Cleavage and activation of human factor IX by serine proteases

    SciTech Connect

    Enfield, D.L.; Thompson, A.R.

    1984-10-01

    Human factor IX circulates as a single-chain glycoprotein. Upon activation in vitro, it is cleaved into disulfide-linked light and heavy chains and an activation peptide. After reduction of activated /sup 125/I-factor IX, the heavy and light chains are readily identified by gel electrophoresis. A direct, immunoradiometric assay for factor IXa was developed to assess activation of factor IX for proteases that cleaved it. The assay utilized radiolabeled antithrombin III with heparin to identify the active site and antibodies to distinguish factor IX. After cleavage of factor IX by factor XIa, factor VIIa-tissue thromboplastin complex, or the factor X-activating enzyme from Russell's viper venom, antithrombin III bound readily to factor IXa. Cleavage of /sup 125/I-factor IX by trypsin, chymotrypsin, and granulocyte elastase in the presence of calcium yielded major polypeptide fragments of the sizes of the factor XIa-generated light and heavy chains. When the immunoradiometric assay was used to assess trypsin-cleaved factor IX, the product bound antithrombin III, but not maximally. After digesting with insolubilized trypsin, clotting activity confirmed activation. In evaluating activation of factor IX, physical evidence of activation cleavages does not necessarily correlate with generation of an active site.

  7. DNA photoreacts by nucleobase ring cleavage to form labile isocyanates.

    PubMed

    Buschhaus, Laura; Rolf, Josefin; Kleinermanns, Karl

    2013-11-14

    Differential infrared absorption spectroscopy was used to study the formation of isocyanates and further photo-products in the oligonucleotides dG10, dC10 and dT10 and in their mononucleosides by ultraviolet light at 266 nm. We find that α-cleavage takes place in oligonucleotides and mononucleosides both in films and in solution. The very intense and spectrally isolated isocyanate (N=C=O) asymmetric stretch vibration at 2277 cm(-1) is used as a spectroscopic marker for detection of the photo-product. The band disappears upon reaction with small amounts of water vapour as expected for isocyanates. Quantum yields for isocyanate formation by nucleobase ring cleavage in the α-position to the carbonyl group are ∼5 × 10(-5) in the mononucleosides and up to 5 × 10(-4) in the oligonucleotides. In the mixed oligonucleotides dG10/dC10 and dA10/dT10 the quantum yield of α-cleavage drops by a factor of 10 compared to the single oligonucleotides. Implications for DNA repair and photo-induced DNA-protein cross-linking via isocyanate reaction with NH2 groups of amino acids are discussed.

  8. Failure of cell cleavage induces senescence in tetraploid primary cells.

    PubMed

    Panopoulos, Andreas; Pacios-Bras, Cristina; Choi, Justin; Yenjerla, Mythili; Sussman, Mark A; Fotedar, Rati; Margolis, Robert L

    2014-10-15

    Tetraploidy can arise from various mitotic or cleavage defects in mammalian cells, and inheritance of multiple centrosomes induces aneuploidy when tetraploid cells continue to cycle. Arrest of the tetraploid cell cycle is therefore potentially a critical cellular control. We report here that primary rat embryo fibroblasts (REF52) and human foreskin fibroblasts become senescent in tetraploid G1 after drug- or small interfering RNA (siRNA)-induced failure of cell cleavage. In contrast, T-antigen-transformed REF52 and p53+/+ HCT116 tumor cells rapidly become aneuploid by continuing to cycle after cleavage failure. Tetraploid primary cells quickly become quiescent, as determined by loss of the Ki-67 proliferation marker and of the fluorescent ubiquitination-based cell cycle indicator/late cell cycle marker geminin. Arrest is not due to DNA damage, as the γ-H2AX DNA damage marker remains at control levels after tetraploidy induction. Arrested tetraploid cells finally become senescent, as determined by SA-β-galactosidase activity. Tetraploid arrest is dependent on p16INK4a expression, as siRNA suppression of p16INK4a bypasses tetraploid arrest, permitting primary cells to become aneuploid. We conclude that tetraploid primary cells can become senescent without DNA damage and that induction of senescence is critical to tetraploidy arrest.

  9. Telomere lengths in human oocytes, cleavage stage embryos and blastocysts

    PubMed Central

    Turner, S.; Wong, H.P.; Rai, J.; Hartshorne, G.M.

    2010-01-01

    Telomeres are repeated sequences that protect the ends of chromosomes and harbour DNA repair proteins. Telomeres shorten during each cell division in the absence of telomerase. When telomere length becomes critically short, cell senescence occurs. Telomere length therefore reflects both cellular ageing and capacity for division. We have measured telomere length in human germinal vesicle (GV) oocytes and preimplantation embryos, by quantitative fluorescence in situ hybridization (Q-FISH), providing baseline data towards our hypothesis that telomere length is a marker of embryo quality. The numbers of fluorescent foci suggest that extensive clustering of telomeres occurs in mature GV stage oocytes, and in preimplantation embryos. When calculating average telomere length by assuming that each signal presents one telomere, the calculated telomere length decreased from the oocyte to the cleavage stages, and increased between the cleavage stages and the blastocyst (11.12 versus 8.43 versus 12.22 kb, respectively, P < 0.001). Other methods of calculation, based upon expected maximum and minimum numbers of telomeres, confirm that telomere length in blastocysts is significantly longer than cleavage stages. Individual blastomeres within an embryo showed substantial variation in calculated average telomere length. This study implies that telomere length changes according to the stage of preimplantation embryo development. PMID:20573647

  10. Specific cleavage of DJ-1 under an oxidative condition.

    PubMed

    Ooe, Hiromasa; Maita, Chinatsu; Maita, Hiroshi; Iguchi-Ariga, Sanae M M; Ariga, Hiroyoshi

    2006-10-09

    DJ-1 was initially identified by us as a novel oncogene and has recently been found to be a causative gene for familial Parkinson's disease (PD) PARK7. DJ-1 plays roles in transcriptional regulation and in oxidative stress function, and its oxidative state at cysteine residues determines activities of DJ-1. In this study, we found that recombinant DJ-1 expressed in and purified from E. coli was specifically cleaved between glycine and proline at amino acid numbers 157 and 158, respectively, by treatment of DJ-1 with H2O2. A substitution mutant of DJ-1 from cysteine to serine at amino acid number 106, a major oxidation site of DJ-1, was found not to be cleaved under an oxidative condition, suggesting oxidation-dependent cleavage of DJ-1. Cleavage of DJ-1 was also observed in human SH-SY5Y cells that had been treated with H2O2. These results suggest that oxidative stress-induced cleavage of DJ-1 regulates functions of DJ-1.

  11. DNAzyme-Controlled Cleavage of Dimer and Trimer Origami Tiles.

    PubMed

    Wu, Na; Willner, Itamar

    2016-04-13

    Dimers of origami tiles are bridged by the Pb(2+)-dependent DNAzyme sequence and its substrate or by the histidine-dependent DNAzyme sequence and its substrate to yield the dimers T1-T2 and T3-T4, respectively. The dimers are cleaved to monomer tiles in the presence of Pb(2+)-ions or histidine as triggers. Similarly, trimers of origami tiles are constructed by bridging the tiles with the Pb(2+)-ion-dependent DNAzyme sequence and the histidine-dependent DNAzyme sequence and their substrates yielding the trimer T1-T5-T4. In the presence of Pb(2+)-ions and/or histidine as triggers, the programmed cleavage of trimer proceeds. Using Pb(2+) or histidine as trigger cleaves the trimer to yield T5-T4 and T1 or the dimer T1-T5 and T4, respectively. In the presence of Pb(2+)-ions and histidine as triggers, the cleavage products are the monomer tiles T1, T5, and T4. The different cleavage products are identified by labeling the tiles with 0, 1, or 2 streptavidin labels and AFM imaging.

  12. Mechanism of metabolic cleavage of a furan ring

    SciTech Connect

    Kobayashi, T.; Sugihara, J.; Harigaya, S.

    1987-11-01

    We studied the mechanism of metabolic cleavage of a furan ring, using a new hypolipidemic agent, ethyl 2-(4-chlorophenyl)-5-(2-furyl)oxazole-4-acetate (TA-1801), as a model compound. A TA-1801 analogue labeled with deuterium at the 5-position of its furan ring was administered orally to rats. The analysis of urinary metabolites by GC/MS revealed that the deuterium of the furan was retained in the ring-opened metabolite (M3). Metabolic cleavage of furan has been generally considered to proceed by hydroxylation of the 5-position followed by tautomerism and hydrolysis of the resulting 5-hydroxyfuran derivative. However, if the cleavage proceeded by this pathway, the deuterium of the 5-position would be eliminated during hydroxylation. Therefore, we propose that the ring was cleaved directly to form an unsaturated aldehyde, considering the mechanism of oxidation by cytochrome P-450. Although this intermediate was not detected in the biological specimens, a synthetic unsaturated aldehyde was transformed to the actual urinary metabolites M2 and M3 (major ring-opened metabolites) in the isolated rat liver.

  13. Small Molecule-Mediated Cleavage of RNA in Living Cells

    PubMed Central

    Guan, Lirui

    2013-01-01

    Antisense oligonucleotides and small interfering RNAs (siRNAs) control gene expression by triggering the degradation of a mRNA via recruitment of RNase H or the RNA-induced silencing complex (RISC), respectively.[1] These approaches are hampered, however, by the poor cellular permeability of oligonucleotides. A small molecule approach to cleave RNA targets could obviate uptake issues. Several compounds can induce RNA cleavage in vitro,[2] however, to the best of our knowledge no small molecules have been previously described to cleave RNA in living cells. Herein, we describe the development of a potentially general approach to design small molecules that specifically cleave an RNA in a living cell, affecting biological function. Specifically, a designed, modularly assembled small molecule that binds the RNA that causes myotonic dystrophy type 1 (DM1)[3] was appended with a moiety that generates hydroxyl radicals upon irradiation. Cleavage of the transcript improves DM1-associated defects in cell culture, and compounds are non-toxic at an efficacious dose as determined by a MTT viability assay. This approach may allow for the site-specific cleavage and inactivation of other cellular RNAs.[4] Compounds that bind to and cleave RNA have the potential to serve as chemical genetics probes of function or lead therapeutics with spatial and temporal control. PMID:23280953

  14. Demethylation and cleavage of dimethylsulfoniopropionate in marine intertidal sediments

    USGS Publications Warehouse

    Visscher, P.T.; Kiene, R.P.; Taylor, B.F.

    1994-01-01

    Demethylation and cleavage of dimethylsulfoniopropionate (DMSP) was measured in three different types of,intertidal marine sediments: a cyanobacterial mat, a diatom-covered tidal flat and a carbonate sediment. Consumption rates of added DMSP were highest in cyanobacterial mat slurries (59 ?? mol DMSP l-1 slurry h-1) and lower in slurries from a diatom mat and a carbonate tidal sediment (24 and 9 ??mol DMSP l-1 h-1, respectively). Dimethyl sulfide (DMS) and 3-mercaptopropionate (MPA) were produced simultaneously during DMSP consumption, indicating that cleavage and demethylation occurred at the same time. Viable counts of DMSP-utilizing bacteria revealed a population of 2 x 107 cells cm-3 sediment (90% of these cleaved DMSP to DMS, 10% demethylated DMSP to MPA) in the cyanobacterial mat, 7 x 105 cells cm-3 in the diatom mat (23% cleavers, 77% demethylators), and 9 x 104 cells cm-3 (20% cleavers and 80% demethylators) in the carbonate sediment. In slurries of the diatom mat, the rate of MPA production from added 3-methiolpropionate (MMPA) was 50% of the rate of MPA formation from DMSP. The presence of a large population of demethylating bacteria and the production of MPA from DMSP suggest that the demethylation pathway, in addition to cleavage, contributes significantly to DMSP consumption in coastal sediments.

  15. Precocious (pre-anaphase) cleavage furrows in Mesostoma spermatocytes.

    PubMed

    Forer, Arthur; Pickett-Heaps, Jeremy

    2010-08-01

    It generally is assumed that cleavage furrows start ingression at anaphase, but this is not always true. Cleavage furrows are initiated during prometaphase in spermatocytes of the flatworm Mesostoma, becoming detectable soon after the spindles achieve bipolarity. The furrows deepen during prometaphase, but ingression soon arrests. After anaphase the pre-existing furrow recommences its ingression and rapidly cleaves the cell. Such "precocious" furrowing also commonly occurs in diatoms and other algae. The position of the "precocious" cleavage furrow changes when there are changes in the distribution of chromosomes. Each of the 4 unipolarly-oriented univalent chromosomes moves to a pole at the start of prometaphase but later in prometaphase may move to the opposite pole. The furrow position adjusts during prometaphase according to the numbers of univalents at the two poles: when there are two univalent chromosomes at each pole the furrow is symmetrical at the spindle equator, but when there are unequal numbers at the poles the furrow shifts 2-3 microm toward the half-spindle with fewer univalents. Nocodazole causes spindle microtubules to disappear. After addition of nocodazole, bivalents become detached from one pole and move toward the other, which causes the furrow to shift 2-3 microm toward the pole with fewer chromosomes. Furrow positioning thus is sensitive to the positioning of chromosomes in the spindle and furrow positions change in the absence of spindle microtubules.

  16. Extracellular cleavage of E-cadherin promotes epithelial cell extrusion.

    PubMed

    Grieve, Adam G; Rabouille, Catherine

    2014-08-01

    Epithelial cell extrusion and subsequent apoptosis is a key mechanism to prevent the accumulation of excess cells. By contrast, when driven by oncogene expression, apical cell extrusion is followed by proliferation and represents an initial step of tumorigenesis. E-cadherin (E-cad), the main component of adherens junctions, has been shown to be essential for epithelial cell extrusion, but its mechanistic contribution remains unclear. Here, we provide clear evidence that cell extrusion can be driven by the cleavage of E-cad, both in a wild-type and an oncogenic environment. We first show that CDC42 activation in a single epithelial cell results in its efficient matrix metalloproteinase (MMP)-sensitive extrusion through MEK signalling activation and this is supported by E-cad cleavage. Second, using an engineered cleavable form of E-cad, we demonstrate that, by itself, truncation of extracellular E-cad at the plasma membrane promotes apical extrusion. We propose that extracellular cleavage of E-cad generates a rapid change in cell-cell adhesion that is sufficient to drive apical cell extrusion. Whereas in normal epithelia, extrusion is followed by apoptosis, when combined with active oncogenic signalling, it is coupled to cell proliferation.

  17. Asynchronous reformation of individual kallikrein-related secretory proteinases in rat submandibular glands following degranulation by cyclocytidine.

    PubMed

    Proctor, G B; Shori, D K; Chan, K M; Garrett, J R

    1993-10-01

    Time scales for the reformation of the secretory granules in granular tubules and their constituent proteinases were assessed after inducing a massive degranulation by intraperitoneal injection of cyclocytidine in conscious animals. The minimum working dose of cyclocytidine to produce the maximum degranulation and depletion of proteinase activity, at 3 h after injection, was 75 mg/kg. Histologically, although most granular tubule cells then appeared to be extensively degranulated, isolated individual cells showing little or no degranulation always persisted. Acinar cells also showed some depletion of secretory material. At 15 h after injecting cyclocytidine the formation of new granules had begun in the granular tubule cells, but it was not extensive or uniform in adjacent cells; however, the acinar cells already appeared to be regranulated. The pattern of granule reformation in granular tubule cells progressed gradually, so that 7-10 days after cyclocytidine-induced degranulation the cells were mostly packed with granules and showed similar appearances to those of normal resting control glands. Individual proteinases in extracts of the glands were assayed specifically using fluorogenic oligopeptide amidase substrates, with and without appropriate inhibitors. This revealed a 95% reduction in total proteinase activity 3 h after cyclocytidine (75 mg/kg). In the same extracts, acinar peroxidase was reduced by 28%. Peroxidase levels recovered to control values within 15 h after cyclocytidine but recovery of proteinases progressed more gradually and did not occur uniformly for the different constituent proteinases. Tissue kallikrein (rK1) showed the most rapid recovery and had reached levels approaching normal within 3 days.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. The proteolytic system of Lactobacillus sanfrancisco CB1: purification and characterization of a proteinase, a dipeptidase, and an aminopeptidase.

    PubMed Central

    Gobbetti, M; Smacchi, E; Corsetti, A

    1996-01-01

    A cell envelope 57-kDa proteinase, a cytoplasmic 65-kDa dipeptidase, and a 75-kDa aminopeptidase were purified from Lactobacillus sanfrancisco CB1 sourdough lactic acid bacterium by sequential fast protein liquid chromatography steps. All of the enzymes are monomers. The proteinase was most active at pH 7.0 and 40 degrees C, while aminopeptidase and dipeptidase had optima at pH 7.5 and 30 to 35 degrees C. Relatively high activities were observed at the pH and temperature of the sourdough fermentation. The proteinase is a serine enzyme. Urea-polyacrylamide gel electrophoresis of digest of alpha s1- and beta-caseins showed differences in the pattern of peptides released by the purified proteinase and those produced by crude preparations of the cell envelope proteinases of Lactobacillus delbrueckii subsp. bulgaricus B397 and Lactococcus lactis subsp. lactis SK11. Reversed-phase fast protein liquid chromatography of gliadin digests showed a more-complex peptide pattern produced by the proteinase of Lactobacillus sanfrancisco CB1. The dipeptidase is a metalloenzyme with high affinity for dipeptides containing hydrophobic amino acids but had no activity on tripeptides or larger peptides. The aminopeptidase was also inhibited by metal-chelating agents, and showed a broad N-terminal hydrolytic activity including di- and tripeptides. Km values of 0.70 and 0.44 mM were determined for the dipeptidase on Leu-Leu and the aminopeptidase on Leu-p-nitroanilide, respectively. PMID:8795211

  19. Deformation-dependent enzyme mechanokinetic cleavage of type I collagen.

    PubMed

    Wyatt, Karla E-K; Bourne, Jonathan W; Torzilli, Peter A

    2009-05-01

    Collagen is a key structural protein in the extracellular matrix of many tissues. It provides biological tissues with tensile mechanical strength and is enzymatically cleaved by a class of matrix metalloproteinases known as collagenases. Collagen enzymatic kinetics has been well characterized in solubilized, gel, and reconstituted forms. However, limited information exists on enzyme degradation of structurally intact collagen fibers and, more importantly, on the effect of mechanical deformation on collagen cleavage. We studied the degradation of native rat tail tendon fibers by collagenase after the fibers were mechanically elongated to strains of epsilon=1-10%. After the fibers were elongated and the stress was allowed to relax, the fiber was immersed in Clostridium histolyticum collagenase and the decrease in stress (sigma) was monitored as a means of calculating the rate of enzyme cleavage of the fiber. An enzyme mechanokinetic (EMK) relaxation function T(E)(epsilon) in s(-1) was calculated from the linear stress-time response during fiber cleavage, where T(E)(epsilon) corresponds to the zero order Michaelis-Menten enzyme-substrate kinetic response. The EMK relaxation function T(E)(epsilon) was found to decrease with applied strain at a rate of approximately 9% per percent strain, with complete inhibition of collagen cleavage predicted to occur at a strain of approximately 11%. However, comparison of the EMK response (T(E) versus epsilon) to collagen's stress-strain response (sigma versus epsilon) suggested the possibility of three different EMK responses: (1) constant T(E)(epsilon) within the toe region (epsilon<3%), (2) a rapid decrease ( approximately 50%) in the transition of the toe-to-heel region (epsilon congruent with3%) followed by (3) a constant value throughout the heel (epsilon=3-5%) and linear (epsilon=5-10%) regions. This observation suggests that the mechanism for the strain-dependent inhibition of enzyme cleavage of the collagen triple helix may

  20. Use of recombinant Entamoeba histolytica cysteine proteinase 1 to identify a potent inhibitor of amebic invasion in a human colonic model.

    PubMed

    Meléndez-López, Samuel G; Herdman, Scott; Hirata, Ken; Choi, Min-Ho; Choe, Youngchool; Craik, Charles; Caffrey, Conor R; Hansell, Elisabeth; Chávez-Munguía, Bibiana; Chen, Yen Ting; Roush, William R; McKerrow, James; Eckmann, Lars; Guo, Jianhua; Stanley, Samuel L; Reed, Sharon L

    2007-07-01

    Cysteine proteinases are key virulence factors of the protozoan parasite Entamoeba histolytica. We have shown that cysteine proteinases play a central role in tissue invasion and disruption of host defenses by digesting components of the extracellular matrix, immunoglobulins, complement, and cytokines. Analysis of the E. histolytica genome project has revealed more than 40 genes encoding cysteine proteinases. We have focused on E. histolytica cysteine proteinase 1 (EhCP1) because it is one of two cysteine proteinases unique to invasive E. histolytica and is highly expressed and released. Recombinant EhCP1 was expressed in Escherichia coli and refolded to an active enzyme with a pH optimum of 6.0. We used positional-scanning synthetic tetrapeptide combinatorial libraries to map the specificity of the P1 to P4 subsites of the active site cleft. Arginine was strongly preferred at P2, an unusual specificity among clan CA proteinases. A new vinyl sulfone inhibitor, WRR483, was synthesized based on this specificity to target EhCP1. Recombinant EhCP1 cleaved key components of the host immune system, C3, immunoglobulin G, and pro-interleukin-18, in a time- and dose-dependent manner. EhCP1 localized to large cytoplasmic vesicles, distinct from the sites of other proteinases. To gain insight into the role of secreted cysteine proteinases in amebic invasion, we tested the effect of the vinyl sulfone cysteine proteinase inhibitors K11777 and WRR483 on invasion of human colonic xenografts. The resultant dramatic inhibition of invasion by both inhibitors in this human colonic model of amebiasis strongly suggests a significant role of secreted amebic proteinases, such as EhCP1, in the pathogenesis of amebiasis.

  1. The house dust mite allergen Der p 1, unlike Der p 3, stimulates the expression of interleukin-8 in human airway epithelial cells via a proteinase-activated receptor-2-independent mechanism.

    PubMed

    Adam, Emmanuelle; Hansen, Kristina K; Astudillo Fernandez, Olaya; Astudillo, Olaya Fernandez; Coulon, Ludivine; Bex, Françoise; Duhant, Xavier; Jaumotte, Erika; Hollenberg, Morley D; Jacquet, Alain

    2006-03-17

    We investigated and compared the mechanisms by which two dust mite proteolytic allergens, Der p 1 and Der p 3, and a peptide agonist of proteinase-activated receptor 2 (PAR(2)AP) trigger interleukin (IL)-8 release from human pulmonary epithelial cells (A549). Although all three stimuli tested induced the up-regulation of IL-8 (mRNA and protein), the Der p 1-mediated signaling events did not exactly match those induced by PAR(2)AP and Der p 3. First, Der p 1 was less effective in stimulating IL-8 gene transcriptional activity than PAR(2)AP and Der p 3. Second, Der p 1-mediated IL-8 expression was mainly dependent on NF-kappaB, whereas Der p 3 and PAR(2)AP regulated IL-8 expression through the activation of both NF-kappaB and AP-1. Third, although all three MAP kinases, ERK1/2, p38, and JNK, were activated, Der p 1 induced IL-8 release exclusively via the ERK1/2 signaling pathway, whereas PAR(2)AP and Der p 3 also involved the other kinases. Fourth, in HeLa cells, Der p 1 was able to up-regulate IL-8 secretion independent of PAR(2) expression, and in contrast with PAR(2)AP and Der p 3, Der p 1 was unable to affect calcium signaling via PAR(2) in PAR(2)-expressing KNRK cells. Finally, cleavage by Der p 1 of a synthetic peptide representing the N-terminal activation-cleavage site of PAR(2) did not release a high potency activator of PAR(2) as does Der p 3. We conclude that Der p 1 (but not Der p 3)-induced IL-8 production in A549 epithelial cells is independent of PAR(2) activation.

  2. Cloning and molecular characterization of a human intracellular serine proteinase inhibitor.

    PubMed Central

    Coughlin, P; Sun, J; Cerruti, L; Salem, H H; Bird, P

    1993-01-01

    We describe a cDNA encoding a serine proteinase inhibitor present in placental tissue and the cytosolic fraction of K562 cells. On the basis of its interaction with thrombin, through which it was discovered, the inhibitor has been operationally named the placental thrombin inhibitor (PTI). Amino acid sequence comparisons suggest that its reactive center is located at Arg-341 and Cys-342, that it lacks a classical N-terminal signal sequence, and that it has the highest degree of similarity to intracellular serine proteinase inhibitors (serpins), such as the human monocyte/neutrophil elastase inhibitor and the equine leukocyte elastase inhibitor. PTI also resembles these inhibitors in that it contains oxidation-sensitive residues adjacent to the reactive site. The PTI cDNA was expressed in rabbit reticulocyte lysate and in COS-7 cells and a 42-kDa protein was produced. Recombinant PTI formed a 67-kDa complex when incubated with thrombin. The ability of native PTI to bind thrombin was destroyed by incubation with iodoacetamide. Analysis of human tissue mRNA indicated that PTI is expressed widely with the highest levels in cardiac and skeletal muscle and placenta. We conclude that PTI is a member of an emerging class of intracellular serpins. Images Fig. 2 Fig. 3 Fig. 4 PMID:8415716

  3. Proteinases, their receptors and inflammatory signalling: the Oxford South Parks Road connection*

    PubMed Central

    Hollenberg, M D

    2015-01-01

    In keeping with the aim of the Paton Memorial Lecture to ‘facilitate the historical study of pharmacology’, this overview, which is my distinct honour to write, represents a ‘Janus-like’ personal perspective looking both backwards and forwards at the birth and growth of ‘receptor molecular pharmacology’ with special relevance to inflammatory diseases. The overview begins in the Oxford Department of Pharmacology in the mid-1960s and then goes on to provide a current perspective of signalling by proteinases. Looking backwards, the synopsis describes the fruitful Oxford Pharmacology Department infrastructure that Bill Paton generated in keeping with the blueprint begun by his predecessor, J H Burn. Looking forwards, the overview illustrates the legacy of that environment in generating some of the first receptor ligand-binding data and providing the inspiration and vision for those like me who were training in the department at the same time. With apologies, I mention only in passing a number of individuals who benefitted from the ‘South Parks Road connection’ using myself as one of the ‘outcome study’ examples. It is also by looking forward that I can meet the complementary aim of summarizing the lecture presented at a ‘BPS 2014 Focused Meeting on Cell Signalling’ to provide an overview of the role of proteinases and their signalling mechanisms in the setting of inflammation. PMID:25521749

  4. Purification and characterization of a proteinase inhibitor from field bean, Dolichos lablab perpureus L.

    PubMed

    Devaraj, V R; Manjunatha, N H

    1999-01-01

    A proteinase inhibitor resembling Bowman-Birk family inhibitors has been purified from the seeds of cultivar HA-3 of Dolichos lablab perpureus L. The protein was apparently homogeneous as judged by SDS-PAGE, PAGE, IEF, and immunodiffusion. The inhibitor had 12 mole% 1/2-cystine and a few aromatic amino acids, and lacks tryptophan. Field bean proteinase inhibitor (FBPI) exhibited a pI of 4.3 and an Mr of 18,500 Da. CD spectral studies showed random coiled secondary structure. Conformational changes were detected in the FBPI-trypsin/chymotrypsin complexes by difference spectral studies. Apparent Ka values of complexes of inhibitor with trypsin and chymotrypsin were 2.1x10(7) M(-1) and 3.1x10(7) M(-1), respectively. The binary and ternary complexes of FBPI with trypsin and chymotrypsin have been isolated indicating 1:1 stoichiometry with independent sites for cognate enzymes. Amino acid modification studies showed lysine and tyrosine at the reactive sites of FBPI for trypsin and chymotrypsin, respectively.

  5. A recombinant cysteine proteinase from Leishmania (Leishmania) chagasi suitable for serodiagnosis of American visceral leishmaniasis.

    PubMed

    de Souza Dias, Suzana; da Costa Pinheiro, Paulo Henrique; Katz, Simone; dos Santos, Márcia Regina Machado; Barbiéri, Clara Lúcia

    2005-02-01

    A recombinant protein, rLdccys1, which was produced by expression of the gene encoding a 30 kDa cysteine proteinase from Leishmania (Leishmania) chagasi, was used for detection of antibodies in sera from patients with active visceral leishmaniasis (VL) in enzyme-linked immunosorbent assays. Analysis of the predicted amino acid sequence of rLdccys1 showed that it contains all the characteristics of a cysteine proteinase. The ability of the protein to react with sera from humans with VL was also shown by Western blotting. The sensitivity for detection of specific antibodies to L. (L.) chagasi bodies using rLdccys1, L. (L.) chagasi promastigote lysates, and amastigote lysates was 80%, 98%, and 99%, respectively. No cross-reactivity between rLdccys1 and Chagas disease was observed, and there was little positive reactivity with sera from patients with cutaneous leishmaniasis and tuberculosis, compared with promastigote and amastigote extracts. Our findings indicate that rLdccys1 from L. (L.) chagasi constitutes a potential tool for the diagnosis of American VL.

  6. Primary structure and reactive site of a novel wheat proteinase inhibitor of subtilisin and chymotrypsin.

    PubMed

    Poerio, Elia; Di Gennaro, Simone; Di Maro, Antimo; Farisei, Francesca; Ferranti, Pasquale; Parente, Augusto

    2003-02-01

    The proteinase inhibitor WSCI, active in inhibiting bacterial subtilisin and a number of animal chymotrypsins, was purified from endosperm of exaploid wheat (Triticum aestivum, c.v. San Pastore) by ion exchange chromatography and its complete amino acid sequence was established by automated Edman degradation. WSCI consists of a single polypeptide chain of 72 amino acid residues, has a molecular mass of 8126.3 Da and a pl of 5.8. The inhibition constants (Ki) for Bacillus licheniformis subtilisin and bovine pancreatic alpha-chymotrypsin are 3.92 x 10(-9) M and 7.24 x 10(-9) M, respectively. The inhibitor contains one methionine and of tryptophan residue and has a high content of essential amino acids (41 over a total of 72 residues), but no cysteines. The primary structure of WSCI shows high similarity with barley subtilisin-chymotrypsin isoinhibitors of the Cl-2 type and with maize subtilisinchymotrypsin inhibitor MPI. Significant degrees of similarity were also found between sequences of WSCI and of other members of the potato inhibitor I family of the serine proteinase inhibitors. The wheat inhibitor WSCI has a single reactive site (the peptide bond between methionyl-48 and glutamyl-49 residues) as identified by affinity chromatography and sequence analysis.

  7. Suppression of collagen-induced arthritis with a serine proteinase inhibitor (serpin) derived from myxoma virus.

    PubMed

    Brahn, Ernest; Lee, Sarah; Lucas, Alexandra; McFadden, Grant; Macaulay, Colin

    2014-08-01

    Many viruses encode virulence factors to facilitate their own survival by modulating a host's inflammatory response. One of these factors, secreted from cells infected with myxoma virus, is the serine proteinase inhibitor (serpin) Serp-1. Because Serp-1 had demonstrated anti-inflammatory properties in arterial injury models and viral infections, it was cloned and evaluated for therapeutic efficacy in collagen-induced arthritis (CIA). Clinical severity was significantly lower in the Serp-1 protocols (p<0.0001) and blinded radiographs indicated that the Serp-1 group had significantly less erosions than the controls (p<0.01). Delayed-type hypersensitivity was lower in the Serp-1 group but antibody titers to type II collagen were not significantly altered. Recipients had minimal histopathologic synovial changes and did not develop neutralizing antibodies to Serp-1. These results indicate that Serp-1 impedes the pathogenesis of CIA and suggests that the therapeutic potential of serine proteinase inhibitors in inflammatory joint diseases, such as rheumatoid arthritis, should be investigated further.

  8. Bio-physical evaluation and in vivo delivery of plant proteinase inhibitor immobilized on silica nanospheres.

    PubMed

    Khandelwal, Neha; Doke, Dhananjay S; Khandare, Jayant J; Jawale, Priyanka V; Biradar, Ankush V; Giri, Ashok P

    2015-06-01

    Recombinant expression of Capsicum annuum proteinase inhibitors (CanPI-13) and its application via synthetic carrier for the crop protection is the prime objective of our study. Herein, we explored proteinase inhibitor peptide immobilization on silica based nanospheres and rods followed by its pH mediated release in vitro and in vivo. Initial studies suggested silica nanospheres to be a suitable candidate for peptide immobilization. Furthermore, the interactions were characterized biophysically to ascertain their conformational stability and biological activity. Interestingly, bioactive peptide loading at acidic pH on nanospheres was found to be 62% and showed 56% of peptide release at pH 10, simulating gut milieu of the target pest Helicoverpa armigera. Additionally, in vivo study demonstrated significant reduction in insect body mass (158 mg) as compared to the control insects (265 mg) on 8th day after feeding with CanPI-13 based silica nanospheres. The study confirms that peptide immobilized silica nanosphere is capable of affecting overall growth and development of the feeding insects, which is known to hamper fecundity and fertility of the insects. Our study illustrates the utility and development of peptide-nanocarrier based platform in delivering diverse biologically active complexes specific to gut pH of H. armigera.

  9. Identification and characterization of alpha-I-proteinase inhibitor from common carp sarcoplasmic proteins.

    PubMed

    Siriangkanakun, Siriphon; Li-Chan, Eunice C Y; Yongsawadigul, Jirawat

    2016-02-01

    Purification of proteinase inhibitor from common carp (Cyprinus carpio) sarcoplasmic proteins resulted in 2.8% yield with purification fold of 111. Two inhibitors, namely inhibitor I and II, exhibited molecular mass of 47 and 52 kDa, respectively, based on non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both inhibitors I and II were identified to be alpha-1-proteinase inhibitor (α1-PI) based on LC-MS/MS. They were glycoproteins and molecular mass after peptide-N-glycosidase F treatment was 38 and 45 kDa, respectively. The N-glycosylation sites of both inhibitors were determined to be at N214 and N226. The inhibitors specifically inhibited trypsin. The common carp α1-PI showed high thermal stability with denaturation temperatures of 65.43 and 73.31 °C, which were slightly less than those of ovomucoid. High stability toward NaCl was also evident up to 3M. The common carp α1-PI effectively reduced autolytic degradation of bigeye snapper surimi at the concentration as low as 0.025%.

  10. Specificity of an extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine beta-casein.

    PubMed Central

    Rattray, F P; Fox, P F; Healy, A

    1997-01-01

    The specificity of the extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine beta-casein was studied. Hydrolysis was monitored over time by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and urea-PAGE. The major pH 4.6-soluble peptides were isolated by high-performance liquid chromatography and identified by N-terminal amino acid sequencing and mass spectrometry. The major sites of hydrolysis were Ser-18-Ser-19, Glu-20-Glu-21, Gln-56-Ser-57, Gln-72-Asn-73, Leu-77-Thr-78, Ala-101-Met-102, Phe-119-Thr-120, Leu-139-Leu-140, Ser-142-Trp-143, His-145-Gln-146, Gln-167-Ser-168, Gln-175-Lys-176, Tyr-180-Pro-181, and Phe-190-Leu-191. The proteinase had a broad specificity for the amino acid residues present at the P1 and P'1 positions but showed a preference for hydrophobic residues at the P2, P3, P4, P'2, P'3, and P'4 positions. PMID:9172371

  11. Aspartic proteinases of Candida spp.: role in pathogenicity and antifungal resistance.

    PubMed

    Silva, Naiara C; Nery, Jéssica M; Dias, Amanda L T

    2014-01-01

    Fungal infections represent a serious health risk as they are particularly prevalent in immunocompromised individuals. Candida spp. pathogenicity depends on several factors and secreted aspartic proteinases (Sap) are considered one of the most critical factors as they are associated with adhesion, invasion and tissue damage. The production of proteinases is encoded by a family of 10 genes known as SAP, which are distributed differently among the species. The expression of these genes may be influenced by environmental conditions, which generally result in a higher fungal invasive potential. Non-pathogenic Candida spp. usually have fewer SAP genes, which are not necessarily expressed in the genome. Exposure to subinhibitory concentrations of antifungal agents promotes the development of resistant strains with an increased expression of SAP genes. In general, Candida spp. isolates that are resistant to antifungals show a higher secretion of Sap than the susceptible isolates. The relationship between Sap secretion and the susceptibility profile of the isolates is of great interest, although the role of SAPs in the development of resistance to antifungal agents remains still unclear. This review is the first one to address these issues.

  12. Wound-Inducible Proteinase Inhibitors in Pepper. Differential Regulation upon Wounding, Systemin, and Methyl Jasmonate1

    PubMed Central

    Moura, Daniel S.; Ryan, Clarence A.

    2001-01-01

    Seven small (approximately 6,000 D) wound-inducible proteinase inhibitor proteins were isolated from leaves of pepper (Capsicum annuum) plants that are members of the potato inhibitor II family. N-terminal sequences obtained indicated that the pepper leaf proteinase inhibitors (PLPIs) exhibit homology to two GenBank accessions that code for preproteins containing three isoinhibitors domains each that, when post-translationally processed, can account for the mixture of isoinhibitors that are reported herein from pepper leaves. A constitutive level of PLPI proteins was found in pepper leaves, and these levels increased up to 2.6-fold upon wounding of the lower leaves. Exposing intact plants to methyl jasmonate vapors induced the accumulation of PLPIs. Supplying excised young pepper plants with water through the cut stems induced PLPI proteins to levels higher than those found in intact plants, but with high variability. Supplying the excised plants with systemin did not result in an increase of PLPI levels that were statistically higher than levels found in excised plants. Gel-blot analyses of PLPI induction revealed the presence of two mRNA bands, having slightly different mobilities in agarose gels. Only the low Mr mRNA is present in untreated control plants, and it appears to be responsible for the constitutive levels of PLPI found in leaves. Both mRNA species are wound- and methyl jasmonate-inducible. Only the low- Mr species is weakly induced by systemin, indicating a differential expression of the two PLPI species. PMID:11351092

  13. [Prions and proteinaceous proteinase inhibitors: structural analogs and their consequences. II. Dynamics of prion diseases].

    PubMed

    Verevka, S V

    2000-01-01

    The assumption about pathogenic prions as the proteins supplying the extracellular proteinases transport into intracellular space permits to bring the pathogenesis of prion diseases to order of the known and partially proved process regarding the case of prion diseases. We present the mathematical model of the dynamics of prion pathogenesis explaining the existence of the minimal infectious dose and small influence of its exceeding on the duration of long-term latent period of the disease. According to the model proposed the transformation of the neuronal cell into PrPSc breeder is the result of proteolytic damage of shaperoning system caused by accumulation in the cell of some crucial amount of proteinase-transporting prions. Such an accumulation is considered as the result of successive and centripheral lay-by-lay transformation of compact cellular locus from higher affinity to prions to normal one. The formation in the moveable frontier lays of the wave with high prion consisting and its closing into the locus center leads to dramatic splash of prion concentration even at moderate difference between higher and normal affinity levels. The final concentration of prions depends mainly on the correlation between these affinities whilst on exceeding of some value the dimension of the locus is of no importance.

  14. Structure of the SARS coronavirus main proteinase as an active C{sub 2} crystallographic dimer

    SciTech Connect

    Xu, Ting; Ooi, Amy; Lee, Hooi Chen; Wilmouth, Rupert; Liu, Ding Xiang; Lescar, Julien

    2005-11-01

    An orthorhombic crystal form of the SARS CoV main proteinase diffracting to a resolution of 1.9 Å is reported. The conformation of residues in the catalytic site indicates an active enzyme. The 34 kDa main proteinase (M{sup pro}) from the severe acute respiratory syndrome coronavirus (SARS-CoV) plays an important role in the virus life cycle through the specific processing of viral polyproteins. As such, SARS-CoV M{sup pro} is a key target for the identification of specific inhibitors directed against the SARS virus. With a view to facilitating the development of such compounds, crystals were obtained of the enzyme at pH 6.5 in the orthorhombic space group P2{sub 1}2{sub 1}2 that diffract to a resolution of 1.9 Å. These crystals contain one monomer per asymmetric unit and the biologically active dimer is generated via the crystallographic twofold axis. The conformation of the catalytic site indicates that the enzyme is active in the crystalline form and thus suitable for structure-based inhibition studies.

  15. Effect of the Solvent Temperatures on Dynamics of Serine Protease Proteinase K

    PubMed Central

    Sang, Peng; Yang, Qiong; Du, Xing; Yang, Nan; Yang, Li-Quan; Ji, Xing-Lai; Fu, Yun-Xin; Meng, Zhao-Hui; Liu, Shu-Qun

    2016-01-01

    To obtain detailed information about the effect of the solvent temperatures on protein dynamics, multiple long molecular dynamics (MD) simulations of serine protease proteinase K with the solute and solvent coupled to different temperatures (either 300 or 180 K) have been performed. Comparative analyses demonstrate that the internal flexibility and mobility of proteinase K are strongly dependent on the solvent temperatures but weakly on the protein temperatures. The constructed free energy landscapes (FELs) at the high solvent temperatures exhibit a more rugged surface, broader spanning range, and higher minimum free energy level than do those at the low solvent temperatures. Comparison between the dynamic hydrogen bond (HB) numbers reveals that the high solvent temperatures intensify the competitive HB interactions between water molecules and protein surface atoms, and this in turn exacerbates the competitive HB interactions between protein internal atoms, thus enhancing the conformational flexibility and facilitating the collective motions of the protein. A refined FEL model was proposed to explain the role of the solvent mobility in facilitating the cascade amplification of microscopic motions of atoms and atomic groups into the global collective motions of the protein. PMID:26907253

  16. Proteinases involved in the degradation of trypsin inhibitor in germinating mung beans.

    PubMed

    Wilson, K A; Tan-Wilson, A L

    1983-01-01

    The mung bean (Vigna radiata (L.) Wilczek) trypsin inhibitor (MBTI) is rapidly modified by limited proteolysis during the early stages of seedling growth. Using an electrophoretic assay that separates the unmodified inhibitor (MBTI-F) and the first two modified species (MBTI-E and -C), a pH optimum of approximately 4 was found for the modification reaction. The inhibitor modifying activity is initially low in ungerminated seeds, with the reaction F leads to E being the primary reaction catalyzed. Activity catalyzing the production of MBTI-C appears on the first day of germination. This activity (F leads to E leads to C) increases up to 6 days after inhibition, at which time the cotyledons begin to abscise. The activity converting MBTI-F and -E to MBTI-C was strongly inhibited by phenylmethylsulfonyl fluoride (3.3 mM) but only weakly by iodoacetate (9 mM) and not at all by pepstatin A (9 microM), leupeptin (18 microM), or EDTA (5 mM). These results suggest the involvement of proteinases other than the major endopeptidase of the germinating seed, vicilin peptidohydrolase. This conclusion is further supported by gel filtration of the extracts of cotyledons on Sephacryl S-200. At least three proteinases are present in germinated cotyledons capable of modifying MBTI-F to MBTI-C and/or -E. All are distinguishable from vicilin peptidohydrolase on the basis of their molecular weight and inhibition by low molecular weight organic reagents.

  17. Functional Characterization of Cucumis metuliferus Proteinase Inhibitor Gene (CmSPI) in Potyviruses Resistance

    PubMed Central

    Lin, Chia-Wei; Su, Mei-Hsiu; Lin, Yu-Tsung; Chung, Chien-Hung; Ku, Hsin-Mei

    2015-01-01

    Proteinase inhibitors are ubiquitous proteins that block the active center or interact allosterically with proteinases and are involved in plant physiological processes and defense responses to biotic and abiotic stresses. The CmSPI gene identified from Cucumis metuliferus encodes a serine type PI (8 kDa) that belongs to potato I type family. To evaluate the effect of silencing CmSPI gene on Papaya ringspot virus resistance, RNA interference (RNAi) with an inter-space hairpin RNA (ihpRNA) construct was introduced into a PRSV-resistant C. metuliferus line. CmSPI was down-regulated in CmSPI RNAi transgenic lines in which synchronously PRSV symptoms were evident at 21 day post inoculation. Alternatively, heterogeneous expression of CmSPI in Nicotiana benthamiana was also conducted and showed that CmSPI can provide resistance to Potato virus Y, another member of Potyvirus, in transgenic N. benthamiana lines. This study demonstrated that CmSPI plays an important role in resistant function against potyviruses in C. metuliferus and N. benthamiana. PMID:26184285

  18. Specificity of an extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine beta-casein.

    PubMed

    Rattray, F P; Fox, P F; Healy, A

    1997-06-01

    The specificity of the extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine beta-casein was studied. Hydrolysis was monitored over time by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and urea-PAGE. The major pH 4.6-soluble peptides were isolated by high-performance liquid chromatography and identified by N-terminal amino acid sequencing and mass spectrometry. The major sites of hydrolysis were Ser-18-Ser-19, Glu-20-Glu-21, Gln-56-Ser-57, Gln-72-Asn-73, Leu-77-Thr-78, Ala-101-Met-102, Phe-119-Thr-120, Leu-139-Leu-140, Ser-142-Trp-143, His-145-Gln-146, Gln-167-Ser-168, Gln-175-Lys-176, Tyr-180-Pro-181, and Phe-190-Leu-191. The proteinase had a broad specificity for the amino acid residues present at the P1 and P'1 positions but showed a preference for hydrophobic residues at the P2, P3, P4, P'2, P'3, and P'4 positions.

  19. Candida tropicalis Biofilms: Biomass, Metabolic Activity and Secreted Aspartyl Proteinase Production.

    PubMed

    Negri, Melyssa; Silva, Sónia; Capoci, Isis Regina Grenier; Azeredo, Joana; Henriques, Mariana

    2016-04-01

    According to epidemiological data, Candida tropicalis has been related to urinary tract infections and haematological malignancy. Several virulence factors seem to be responsible for C. tropicalis infections, for example: their ability to adhere and to form biofilms onto different indwelling medical devices; their capacity to adhere, invade and damage host human tissues due to enzymes production such as proteinases. The main aim of this work was to study the behaviour of C. tropicalis biofilms of different ages (24-120 h) formed in artificial urine (AU) and their ability to express aspartyl proteinase (SAPT) genes. The reference strain C. tropicalis ATCC 750 and two C. tropicalis isolates from urine were used. Biofilms were evaluated in terms of culturable cells by colony-forming units enumeration; total biofilm biomass was evaluated using the crystal violet staining method; metabolic activity was evaluated by XTT assay; and SAPT gene expression was determined by real-time PCR. All strains of C. tropicalis were able to form biofilms in AU, although with differences between strains. Candida tropicalis biofilms showed a decrease in terms of the number of culturable cells from 48 to 72 h. Generally, SAPT3 was highly expressed. C. tropicalis strains assayed were able to form biofilms in the presence of AU although in a strain- and time-dependent way, and SAPT genes are expressed during C. tropicalis biofilm formation.

  20. Coordinate expression of the Porphyromonas gingivalis lysine-specific gingipain proteinase, Kgp, arginine-specific gingipain proteinase, RgpA, and the heme/hemoglobin receptor, HmuR.

    PubMed

    Liu, Xinyan; Sroka, Aneta; Potempa, Jan; Genco, Caroline Attardo

    2004-11-01

    Heme utilization in Porphyromonas gingivalis requires the participation of an outer membrane hemin/hemoglobin receptor, HmuR, the lysine-specific gingipain proteinase (Kgp) and arginine-specific gingipain proteinase (Rgp). In this study, the expression of hmuR , kgp and rgpA genes in response to growth with different heme sources was examined by reverse transcription-polymerase chain reaction and enzyme-linked immunoassay. Coordinate regulation of hmuR , kgp and rgpA gene expression was evaluated through utilization of P. gingivalis hmuR and kgp mutants or by selective inactivation of proteinases with Kgp- and Rgp-specific inhibitors. We observed that expression of the kgp and rgpA genes was not tightly regulated by heme, but rather by the growth phase. In contrast, expression of the hmuR gene was negatively regulated by heme, while growth of P. gingivalis with human serum resulted in increased hmuR expression. A P. gingivalis kgp isogenic mutant demonstrated significantly increased hmuR gene expression, and inactivation of Kgp and Rgp activity by specific inhibitors up-regulated hmuR gene transcription. Moreover, inactivation of Kgp up-regulated rgpA transcription. Finally, a P. gingivalis hmuR mutant exhibited repressed kgp gene expression and lysine-specific proteinase activity. Collectively, these results indicate that kgp , rgpA and hmuR gene transcription is coordinately regulated and may facilitate greater efficiency of heme utilization in P. gingivalis .

  1. Carotenoid-cleavage activities of crude enzymes from Pandanous amryllifolius.

    PubMed

    Ningrum, Andriati; Schreiner, Matthias

    2014-11-01

    Carotenoid degradation products, known as norisoprenoids, are aroma-impact compounds in several plants. Pandan wangi is a common name of the shrub Pandanus amaryllifolius. The genus name 'Pandanus' is derived from the Indonesian name of the tree, pandan. In Indonesia, the leaves from the plant are used for several purposes, e.g., as natural colorants and flavor, and as traditional treatments. The aim of this study was to determine the cleavage of β-carotene and β-apo-8'-carotenal by carotenoid-cleavage enzymes isolated from pandan leaves, to investigate dependencies of the enzymatic activities on temperature and pH, to determine the enzymatic reaction products by using Headspace Solid Phase Microextraction Gas Chromatography/Mass Spectrophotometry (HS-SPME GC/MS), and to investigate the influence of heat treatment and addition of crude enzyme on formation of norisoprenoids. Crude enzymes from pandan leaves showed higher activity against β-carotene than β-apo-8'-carotenal. The optimum temperature of crude enzymes was 70°, while the optimum pH value was 6. We identified β-ionone as the major volatile reaction product from the incubations of two different carotenoid substrates, β-carotene and β-apo-8'-carotenal. Several treatments, e.g., heat treatment and addition of crude enzymes in pandan leaves contributed to the norisoprenoid content. Our findings revealed that the crude enzymes from pandan leaves with carotenoid-cleavage activity might provide a potential application, especially for biocatalysis, in natural-flavor industry.

  2. Programmable RNA recognition and cleavage by CRISPR/Cas9.

    PubMed

    O'Connell, Mitchell R; Oakes, Benjamin L; Sternberg, Samuel H; East-Seletsky, Alexandra; Kaplan, Matias; Doudna, Jennifer A

    2014-12-11

    The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA-DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms, but it has been thought to be incapable of targeting RNA. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.

  3. Micromechanisms and Toughness for Cleavage Fracture of Steel,

    DTIC Science & Technology

    1986-06-01

    AD-A69 916 MICRONECHANISNS AND TOUGHNESS FOR CLEAVAGE FRACTURE OF 1/1 STEEL (U) BATTELLE MEMORIAL INST COLUMBUS OH A R ROSENFIELD ET AL. JUN 86 ARO...OF STEEL 6. PERFORMING ORG. REPORT NUMBER N/A 7. AUTHOR(e) 8. CONTRACT OR GRANT NUMBER(e) K A. R. Rosenfield and B. S. Majumdar DAAG29-85jOO35 9...decision, unless so 9. KEY WORDS (Continue on reveree eide It necesey atd Identify by block number) .L Steel , HSLA Fracture toughness . Ductile fracture

  4. Selective cleavage of methoxy protecting groups in carbohydrates.

    PubMed

    Boto, Alicia; Hernández, Dácil; Hernández, Rosendo; Suárez, Ernesto

    2006-03-03

    The selective cleavage of methoxy protecting groups next to hydroxy groups is achieved using a radical hydrogen abstraction reaction as the key step. Under the reaction conditions, the hydroxy group generates an alkoxyl radical that reacts with the sterically accessible adjacent methoxy group, which is transformed into an acetal. In the second step, the acetals are hydrolyzed to give alcohols or diols. A one-pot hydrogen abstraction-hydrolysis procedure was also developed. Good yields were usually achieved, and the mild conditions of this methodology were compatible with different functional groups and sensitive substrates such as carbohydrates.

  5. Agent-based modeling: case study in cleavage furrow models.

    PubMed

    Mogilner, Alex; Manhart, Angelika

    2016-11-07

    The number of studies in cell biology in which quantitative models accompany experiments has been growing steadily. Roughly, mathematical and computational techniques of these models can be classified as "differential equation based" (DE) or "agent based" (AB). Recently AB models have started to outnumber DE models, but understanding of AB philosophy and methodology is much less widespread than familiarity with DE techniques. Here we use the history of modeling a fundamental biological problem-positioning of the cleavage furrow in dividing cells-to explain how and why DE and AB models are used. We discuss differences, advantages, and shortcomings of these two approaches.

  6. Morphological and cytogenetic assessment of cleavage and blastocyst stage embryos.

    PubMed

    Fragouli, E; Alfarawati, S; Spath, K; Wells, D

    2014-02-01

    Morphological assessments are the main way in which fertility clinics select in vitro generated embryo(s) for transfer to the uterus. However, it is widely acknowledged that the microscopic appearance of an embryo is only weakly correlated with its viability. Furthermore, the extent to which morphology is affected by aneuploidy, a genetic defect common in human preimplantation embryos, remains unclear. Aneuploidy is of great relevance to embryo selection as it represents one of the most important causes of implantation failure and miscarriage. The current study aimed to examine whether morphological appearance can assist in identifying embryos at risk of aneuploidy. Additionally, the data produced sheds light on how chromosomal anomalies impact development from the cleavage to the blastocyst stage. A total of 1213 embryos were examined. Comprehensive chromosome analysis was combined with well-established criteria for the assessment of embryo morphology. At the cleavage stage, chromosome abnormalities were common even amongst embryos assigned the best morphological scores, indicating that aneuploidy has little effect on microscopic appearance at fixed time points up until Day 3 of development. However, at the blastocyst stage aneuploidies were found to be significantly less common among embryos of optimal morphological quality, while such abnormalities were overrepresented amongst embryos considered to be of poor morphology. Despite the link between aneuploidy and blastocyst appearance, many chromosomally abnormal embryos were able to achieve the highest morphological scores. In particular, blastocysts affected by forms of aneuploidy with the greatest capacity to produce clinical pregnancies (e.g. trisomy 21) were indistinguishable from euploid embryos. The sex ratio was seen to be equal throughout preimplantation development. Interestingly, however, females were overrepresented amongst the fastest growing cleavage-stage embryos, whereas a sex-related skew in the

  7. Kinetics of acid-catalyzed cleavage of cumene hydroperoxide.

    PubMed

    Levin, M E; Gonzales, N O; Zimmerman, L W; Yang, J

    2006-03-17

    The cleavage of cumene hydroperoxide, in the presence of sulfuric acid, to form phenol and acetone has been examined by adiabatic calorimetry. As expected, acid can catalyze cumene hydroperoxide reaction at temperatures below that of thermally-induced decomposition. At elevated acid concentrations, reactivity is also observed at or below room temperature. The exhibited reactivity behavior is complex and is significantly affected by the presence of other species (including the products). Several reaction models have been explored to explain the behavior and these are discussed.

  8. Agent-based modeling: case study in cleavage furrow models

    PubMed Central

    Mogilner, Alex; Manhart, Angelika

    2016-01-01

    The number of studies in cell biology in which quantitative models accompany experiments has been growing steadily. Roughly, mathematical and computational techniques of these models can be classified as “differential equation based” (DE) or “agent based” (AB). Recently AB models have started to outnumber DE models, but understanding of AB philosophy and methodology is much less widespread than familiarity with DE techniques. Here we use the history of modeling a fundamental biological problem—positioning of the cleavage furrow in dividing cells—to explain how and why DE and AB models are used. We discuss differences, advantages, and shortcomings of these two approaches. PMID:27811328

  9. Biotic and abiotic carbon to sulfur bond cleavage

    SciTech Connect

    Frost, J.W.

    1991-01-01

    Cleavage of aliphatic organosulfonate carbon to sulfur (C-S) bonds, a critical link in the global biogeochemical sulfur cycle, has been identified in Escherichia coli K-12. Enormous quantities of inorganic sulfate are continuously converted (Scheme I) into methanesulfonic acid 1 and acylated 3-(6-sulfo-{alpha}-D-quinovopyranosyl)-L-glycerol 2. Biocatalytic desulfurization (Scheme I) of 1 and 2, which share the structural feature of an aliphatic carbon bonded to a sulfonic acid sulfur, completes the cycle, Discovery of this desulfurization in E. coli provides an invaluable paradigm for study of a biotic process which, via the biogeochemical cycle, significantly influences the atmospheric concentration of sulfur-containing molecules.

  10. Effects of cysteine proteinase inhibitors scN and E-64 on southern corn rootworm larval development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The southern corn rootworm (SCRW) can be a serious pest of peanut pods. A laboratory bioassay was developed to test feeding cysteine proteinase inhibitors soyacystatin N (scN) and E-64 against southern corn rootworm reared on artificial diet to determine the effects on larvae development and mortal...

  11. Purification and characterization of native and recombinant SaPIN2a, a plant sieve element-localized proteinase inhibitor.

    PubMed

    Wang, Zhen-Yu; Ding, Ling-Wen; Ge, Zhi-Juan; Wang, Zhaoyu; Wang, Fanghai; Li, Ning; Xu, Zeng-Fu

    2007-01-01

    SaPIN2a encodes a proteinase inhibitor in nightshade (Solanum americanum), which is specifically localized to the enucleate sieve elements. It has been proposed to play an important role in phloem development by regulating proteolysis in sieve elements. In this study, we purified and characterized native SaPIN2a from nightshade stems and recombinant SaPIN2a expressed in Escherichia coli. Purified native SaPIN2a was found as a charge isomer family of homodimers, and was weakly glycosylated. Native SaPIN2a significantly inhibited serine proteinases such as trypsin, chymotrypsin, and subtilisin, with the most potent inhibitory activity on subtilisin. It did not inhibit cysteine proteinase papain and aspartic proteinase cathepsin D. Recombinant SaPIN2a had a strong inhibitory effect on chymotrypsin, but its inhibitory activities toward trypsin and especially toward subtilisin were greatly reduced. In addition, native SaPIN2a can effectively inhibit midgut trypsin-like activities from Trichoplusia ni and Spodoptera litura larvae, suggesting a potential for the production of insect-resistant transgenic plants.

  12. Granzyme M is a regulatory protease that inactivates proteinase inhibitor 9, an endogenous inhibitor of granzyme B.

    PubMed

    Mahrus, Sami; Kisiel, Walter; Craik, Charles S

    2004-12-24

    Granzyme M is a trypsin-fold serine protease that is specifically found in the granules of natural killer cells. This enzyme has been implicated recently in the induction of target cell death by cytotoxic lymphocytes, but unlike granzymes A and B, the molecular mechanism of action of granzyme M is unknown. We have characterized the extended substrate specificity of human granzyme M by using purified recombinant enzyme, several positional scanning libraries of coumarin substrates, and a panel of individual p-nitroanilide and coumarin substrates. In contrast to previous studies conducted using thiobenzyl ester substrates (Smyth, M. J., O'Connor, M. D., Trapani, J. A., Kershaw, M. H., and Brinkworth, R. I. (1996) J. Immunol. 156, 4174-4181), a strong preference for leucine at P1 over methionine was demonstrated. The extended substrate specificity was determined to be lysine = norleucine at P4, broad at P3, proline > alanine at P2, and leucine > norleucine > methionine at P1. The enzyme activity was found to be highly dependent on the length and sequence of substrates, indicative of a regulatory function for human granzyme M. Finally, the interaction between granzyme M and the serpins alpha(1)-antichymotrypsin, alpha(1)-proteinase inhibitor, and proteinase inhibitor 9 was characterized by using a candidate-based approach to identify potential endogenous inhibitors. Proteinase inhibitor 9 was effectively hydrolyzed and inactivated by human granzyme M, raising the possibility that this orphan granzyme bypasses proteinase inhibitor 9 inhibition of granzyme B.

  13. Highly conserved salt bridge stabilizes a proteinase K subfamily enzyme, Aqualysin I, from Thermus aquaticus YT-1.

    PubMed

    Sakaguchi, Masayoshi; Osaku, Kanae; Maejima, Susumu; Ohno, Nao; Sugahara, Yasusato; Oyama, Fumitaka; Kawakita, Masao

    2014-01-01

    The proteinase K subfamily enzymes, thermophilic Aqualysin I (AQN) from Thermus aquaticus YT-1 and psychrophilic serine protease (VPR) from Vibrio sp. PA-44, have six and seven salt bridges, respectively. To understand the possible significance of salt bridges in the thermal stability of AQN, we prepared mutant proteins in which amino acid residues participating in salt bridges common to proteinase K subfamily members and intrinsic to AQN were replaced to disrupt the bridges one at a time. Disruption of a salt bridge common to proteinase K subfamily enzymes in the D183N mutant resulted in a significant reduction in thermal stability, and a massive change in the content of the secondary structure was observed, even at 70°C, in the circular dichroism (CD) analysis. These results indicate that the common salt bridge Asp183-Arg12 is important in maintaining the conformation of proteinase K subfamily enzymes and suggest the importance of proximity between the regions around Asp183 and the N-terminal region around Arg12. Of the three mutants that lack an AQN intrinsic salt bridge, D212N was more prone to unfolding at 80°C than the wild-type enzyme. Similarly, D17N and E237Q were less thermostable than the wild-type enzyme, although this may be partially due to increased autolysis. The AQN intrinsic salt bridges appear to confer additional thermal stability to this enzyme. These findings will further our understanding of the factors involved in stabilizing protein structure.

  14. DNase I and proteinase K impair Listeria monocytogenes biofilm formation and induce dispersal of pre-existing biofilms.

    PubMed

    Nguyen, Uyen T; Burrows, Lori L

    2014-09-18

    Current sanitation methods in the food industry are not always sufficient for prevention or dispersal of Listeria monocytogenes biofilms. Here, we determined if prevention of adherence or dispersal of existing biofilms could occur if biofilm matrix components were disrupted enzymatically. Addition of DNase during biofilm formation reduced attachment (<50% of control) to polystyrene. Treatment of established 72h biofilms with 100μg/ml of DNase for 24h induced incomplete biofilm dispersal, with <25% biofilm remaining compared to control. In contrast, addition of proteinase K completely inhibited biofilm formation, and 72h biofilms-including those grown under stimulatory conditions-were completely dispersed with 100μg/ml proteinase K. Generally-regarded-as-safe proteases bromelain and papain were less effective dispersants than proteinase K. In a time course assay, complete dispersal of L. monocytogenes biofilms from both polystyrene and type 304H food-grade stainless steel occurred within 5min at proteinase K concentrations above 25μg/ml. These data confirm that both DNA and proteins are required for L. monocytogenes biofilm development and maintenance, and that these components of the biofilm matrix can be targeted for effective prevention and removal of biofilms.

  15. Alpha-2-macroglobulin functions as an inhibitor of fibrinolytic, clotting, and neutrophilic proteinases in sepsis: studies using a baboon model.

    PubMed

    de Boer, J P; Creasey, A A; Chang, A; Abbink, J J; Roem, D; Eerenberg, A J; Hack, C E; Taylor, F B

    1993-12-01

    Alpha-2-macroglobulin (alpha 2M) may function as a proteinase inhibitor in vivo. Levels of this protein are decreased in sepsis, but the reason these levels are low is unknown. Therefore, we analyzed the behavior of alpha 2M in a baboon model for sepsis. Upon challenge with a lethal (4 baboons) or a sublethal (10 baboons) dose of Escherichia coli, levels of inactivated alpha 2M (i alpha 2M) steadily increased, the changes being more pronounced in the animals that received the lethal dose. The rise in i alpha 2M significantly correlated with the increase of thrombin-antithrombin III, plasmin-alpha 2-antiplasmin, and, to a lesser extent, with that of elastase-alpha 1-antitrypsin complexes, raising the question of involvement of fibrinolytic, clotting, and neutrophilic proteinases in the inactivation of alpha 2M. Experiments with chromogenic substrates confirmed that thrombin, plasmin, elastase, and cathepsin G indeed had formed complexes with alpha 2M. Changes in alpha 2M similar to those observed in the animals that received E. coli occurred in baboons challenged with Staphylococcus aureus, indicating that alpha 2M formed complexes with the proteinases just mentioned in gram-positive sepsis as well. We conclude that alpha 2M in this baboon model for sepsis is inactivated by formation of complexes with proteinases, derived from activated neutrophils and from fibrinolytic and coagulation cascades. We suggest that similar mechanisms may account for the decreased alpha 2M levels in clinical sepsis.

  16. Microplate fluorescence protease assays test the inhibition of select North American snake venoms' activities with an anti-proteinase library.

    PubMed

    Price, Joseph A

    2015-09-01

    Snake envenomation is a relatively neglected significant world health problem, designated an orphan disease by the WHO. While often effective, antivenins are insufficient. Could another approach greatly aid inhibition of the venom toxins? New fluorescent substrates for measuring protease activity in microplate assays suitable for high throughput screening were tested and found reproducible with snake venom. Representative North American venoms showed relatively strong proteinase and collagenase, but weaker elastase activities. Caseinolytic activity is inhibited by the nonspecific proteinase inhibitor 1,10-phenanthroline and by EDTA, as is collagenase activity, consistent with the action of metalloproteinases. Both general protease and collagenase assays CV average 3%, and Km measured were above normal working conditions. Using a library of anti -proteinase compounds with multiple venoms revealed high inhibitor activity by three agents with known multiple metalloproteinase inhibitor activity (Actinonin, GM6001, and NNGH), which incidentally supports the concept that much of the degradative activity of certain venoms is due to metalloproteinases with collagenase activity. These results together support the use of microplate proteinase assays, particularly this collagenase assay, in future drug repurposing studies leading to the development of new treatments for those envenomations that have a major proteolytic component in their pathophysiology.

  17. Divalent metals stabilize cellular prion proteins and alter the rate of proteinase-K dependent limited proteolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: The key biochemical event in the pathogenesis of prion diseases is the conversion of normal cellular prion proteins (PrP**c) to the proteinase K (PK) resistant, abnormal form (PrP**sc); however, the cellular mechanisms underlying the conversion remain enigmatic. Binding of divalent ca...

  18. 3C-like protease of rabbit hemorrhagic disease virus: identification of cleavage sites in the ORF1 polyprotein and analysis of cleavage specificity.

    PubMed Central

    Wirblich, C; Sibilia, M; Boniotti, M B; Rossi, C; Thiel, H J; Meyers, G

    1995-01-01

    Rabbit hemorrhagic disease virus, a positive-stranded RNA virus of the family Caliciviridae, encodes a trypsin-like cysteine protease as part of a large polyprotein. Upon expression in Escherichia coli, the protease releases itself from larger precursors by proteolytic cleavages at its N and C termini. Both cleavage sites were determined by N-terminal sequence analysis of the cleavage products. Cleavage at the N terminus of the protease occurred with high efficiency at an EG dipeptide at positions 1108 and 1109. Cleavage at the C terminus of the protease occurred with low efficiency at an ET dipeptide at positions 1251 and 1252. To study the cleavage specificity of the protease, amino acid substitutions were introduced at the P2, P1, and P1' positions at the cleavage site at the N-terminal boundary of the protease. This analysis showed that the amino acid at the P1 position is the most important determinant for substrate recognition. Only glutamic acid, glutamine, and aspartic acid were tolerated at this position. At the P1' position, glycine, serine, and alanine were the preferred substrates of the protease, but a number of amino acids with larger side chains were also tolerated. Substitutions at the P2 position had only little effect on the cleavage efficiency. Cell-free expression of the C-terminal half of the ORF1 polyprotein showed that the protease catalyzes cleavage at the junction of the RNA polymerase and the capsid protein. An EG dipeptide at positions 1767 and 1768 was identified as the putative cleavage site. Our data show that rabbit hemorrhagic disease virus encodes a trypsin-like cysteine protease that is similar to 3C proteases with regard to function and specificity but is more similar to 2A proteases with regard to size. PMID:7474137

  19. Expression of the maize proteinase inhibitor (mpi) gene in rice plants enhances resistance against the striped stem borer (Chilo suppressalis): effects on larval growth and insect gut proteinases.

    PubMed

    Vila, Laura; Quilis, Jordi; Meynard, Donaldo; Breitler, Jean Christophe; Marfà, Victoria; Murillo, Isabel; Vassal, Jean Michel; Messeguer, Joaquima; Guiderdoni, Emmanuel; San Segundo, Blanca

    2005-03-01

    The maize proteinase inhibitor (mpi) gene was introduced into two elite japonica rice varieties. Both constitutive expression of the mpi gene driven by the maize ubiquitin 1 promoter and wound-inducible expression of the mpi gene driven by its own promoter resulted in the accumulation of MPI protein in the transgenic plants. No effect on plant phenotype was observed in mpi-expressing lines. The stability of transgene expression through successive generations of mpi rice lines (up to the T(4) generation) and the production of functional MPI protein were confirmed. Expression of the mpi gene in rice enhanced resistance to the striped stem borer (Chilo suppressalis), one of the most important pests of rice. In addition, transgenic mpi plants were evaluated in terms of their effects on the growth of C. suppressalis larvae and the insect digestive proteolytic system. An important dose-dependent reduction of larval weight of C. suppressalis larvae fed on mpi rice, compared with larvae fed on untransformed rice plants, was observed. Analysis of the digestive proteolytic activity from the gut of C. suppressalis demonstrated that larvae adapted to mpi transgene expression by increasing the complement of digestive proteolytic activity: the serine and cysteine endoproteinases as well as the exopeptidases leucine aminopeptidase and carboxypeptidases A and B. However, the induction of such proteolytic activity did not prevent the deleterious effects of MPI on larval growth. The introduction of the mpi gene into rice plants can thus be considered as a promising strategy to protect rice plants against striped stem borer.

  20. A role for trigger factor and an rgg-like regulator in the transcription, secretion and processing of the cysteine proteinase of Streptococcus pyogenes.

    PubMed Central

    Lyon, W R; Gibson, C M; Caparon, M G

    1998-01-01

    The ability of numerous microorganisms to cause disease relies upon the highly regulated expression of secreted proteinases. In this study, mutagenesis with a novel derivative of Tn4001 was used to identify genes required for the expression of the secreted cysteine proteinase (SCP) of the pathogenic Gram-positive bacterium Streptococcus pyogenes. Designated as Rop loci (regulation of proteinase), ropB is a rgg-like transcriptional activator required for transcription of the gene which encodes the proteinase. In contrast, ropA contributes post-transcriptionally to the secretion and processing of SCP and encodes a homologue of Trigger Factor, a peptidyl-prolyl isomerase and putative chaparone which is highly conserved in most bacterial species, but of unknown function. Analysis of additional ropA mutants demonstrated that RopA acts both to assist in targeting SCP to the secretory pathway and to promote the ability of the proprotein to establish an active conformation upon secretion. This latter function was dependent upon the peptidyl-prolyl isomerase domain of RopA and mutants that lacked this domain exhibited a bipartite deficiency manifested as a kinetic defect in autologous processing of the proprotein to the mature proteinase, and as a catalytic defect in the mature proteinase. These results provide insight into the function of Trigger Factor, the regulation of proteinase activity and the mechanism of secretion in Gram-positive bacteria. PMID:9799235

  1. Distortion of the catalytic domain of tissue-type plasminogen activator by plasminogen activator inhibitor-1 coincides with the formation of stable serpin-proteinase complexes.

    PubMed

    Perron, Michel J; Blouse, Grant E; Shore, Joseph D

    2003-11-28

    Plasminogen activator inhibitor-1 (PAI-1) is a typical member of the serpin family that kinetically traps its target proteinase as a covalent complex by distortion of the proteinase domain. Incorporation of the fluorescently silent 4-fluorotryptophan analog into PAI-1 permitted us to observe changes in the intrinsic tryptophan fluorescence of two-chain tissue-type plasminogen activator (tPA) and the proteinase domain of tPA during the inhibition reaction. We demonstrated three distinct conformational changes of the proteinase that occur during complex formation and distortion. A conformational change occurred during the initial formation of the non-covalent Michaelis complex followed by a large conformational change associated with the distortion of the proteinase catalytic domain that occurs concurrently with the formation of stable proteinase-inhibitor complexes. Following distortion, a very slow structural change occurs that may be involved in the stabilization or regulation of the trapped complex. Furthermore, by comparing the inhibition rates of two-chain tPA and the proteinase domain of tPA by PAI-1, we demonstrate that the accessory domains of tPA play a prominent role in the initial formation of the non-covalent Michaelis complex.

  2. A snake-like serine proteinase (PmSnake) activates prophenoloxidase-activating system in black tiger shrimp Penaeus monodon.

    PubMed

    Monwan, Warunthorn; Amparyup, Piti; Tassanakajon, Anchalee

    2017-02-01

    Clip domain serine proteinases (ClipSPs) play critical roles in the activation of proteolytic cascade in invertebrate immune systems including the prophenoloxidase (proPO) activating system. In this study, we characterized a snake-like serine protease, namely PmSnake, from the shrimp Penaeus monodon which has previously been identified based on the subtractive cDNA library of proPO double-stranded RNA (dsRNA)-treated hemocytes. An open reading frame of PmSnake contains 1068 bp encoding a predicted protein of 355 amino acid residues with a putative signal peptide of 22 amino acids and two conserved domains (N-terminal clip domain and C-terminal trypsin-like serine proteinase domain). Sequence analysis revealed that PmSnake was closest to the AeSnake from ant Acromyrmex echinatior (53% similarity), but was quite relatively distant from other shrimp PmclipSPs. PmSnake transcript was mainly expressed in shrimp hemocytes and up-regulated after systemic Vibrio harveyi infection indicating that it is an immune-responsive gene. Suppression of PmSnake expression by dsRNA interference reduced both transcript and protein levels leading to a reduction of the hemolymph phenoloxidase (PO) activity (36%), compared to the control, suggesting that the PmSnake functions as a clip-SP in shrimp proPO system. Western blot analysis using anti-PmSnake showed that the PmSnake was detected in hemocytes but not in cell-free plasma. In vitro PO activity and serine proteinase activity assays showed that adding rPmSnake into the shrimp hemolymph could increase PO activity as well as serine proteinase activity suggesting that the rPmSnake activates the proPO system via serine proteinase cascade.

  3. Improving the prospects of cleavage-based nanopore sequencing engines

    NASA Astrophysics Data System (ADS)

    Brady, Kyle T.; Reiner, Joseph E.

    2015-08-01

    Recently proposed methods for DNA sequencing involve the use of cleavage-based enzymes attached to the opening of a nanopore. The idea is that DNA interacting with either an exonuclease or polymerase protein will lead to a small molecule being cleaved near the mouth of the nanopore, and subsequent entry into the pore will yield information about the DNA sequence. The prospects for this approach seem promising, but it has been shown that diffusion related effects impose a limit on the capture probability of molecules by the pore, which limits the efficacy of the technique. Here, we revisit the problem with the goal of optimizing the capture probability via a step decrease in the nucleotide diffusion coefficient between the pore and bulk solutions. It is shown through random walk simulations and a simplified analytical model that decreasing the molecule's diffusion coefficient in the bulk relative to its value in the pore increases the nucleotide capture probability. Specifically, we show that at sufficiently high applied transmembrane potentials (≥100 mV), increasing the potential by a factor f is equivalent to decreasing the diffusion coefficient ratio Dbulk/Dpore by the same factor f. This suggests a promising route toward implementation of cleavage-based sequencing protocols. We also discuss the feasibility of forming a step function in the diffusion coefficient across the pore-bulk interface.

  4. Improving the prospects of cleavage-based nanopore sequencing engines.

    PubMed

    Brady, Kyle T; Reiner, Joseph E

    2015-08-21

    Recently proposed methods for DNA sequencing involve the use of cleavage-based enzymes attached to the opening of a nanopore. The idea is that DNA interacting with either an exonuclease or polymerase protein will lead to a small molecule being cleaved near the mouth of the nanopore, and subsequent entry into the pore will yield information about the DNA sequence. The prospects for this approach seem promising, but it has been shown that diffusion related effects impose a limit on the capture probability of molecules by the pore, which limits the efficacy of the technique. Here, we revisit the problem with the goal of optimizing the capture probability via a step decrease in the nucleotide diffusion coefficient between the pore and bulk solutions. It is shown through random walk simulations and a simplified analytical model that decreasing the molecule's diffusion coefficient in the bulk relative to its value in the pore increases the nucleotide capture probability. Specifically, we show that at sufficiently high applied transmembrane potentials (≥100 mV), increasing the potential by a factor f is equivalent to decreasing the diffusion coefficient ratio D(bulk)/D(pore) by the same factor f. This suggests a promising route toward implementation of cleavage-based sequencing protocols. We also discuss the feasibility of forming a step function in the diffusion coefficient across the pore-bulk interface.

  5. Cleavage by MALT1 induces cytosolic release of A20.

    PubMed

    Malinverni, Claire; Unterreiner, Adeline; Staal, Jens; Demeyer, Annelies; Galaup, Marion; Luyten, Marcel; Beyaert, Rudi; Bornancin, Frédéric

    2010-10-01

    The MALT1 paracaspase has arginine-directed proteolytic activity. A20 is a dual ubiquitin-editing enzyme involved in termination of NF-κB signaling. Upon T- or B-cell receptor engagement human (h) A20 is cleaved by MALT1 after arginine 439, yielding an N-terminal fragment (hA20p50) and a C-terminal one (hA20p37). The hA20p50 fragment has never been detected directly, thus limiting insight into the functional consequences of MALT1-mediated cleavage of A20. Here, various antibodies were tested, including newly generated hA20p50 and hA20p37 specific antibodies, leading to detection of the hA20p50 fragment produced after MALT1-mediated cleavage of ectopically expressed as well as endogenous A20 proteins. The properties of both A20 fragments, generated upon co-expression with a constitutively active MALT1 protein, were further studied by sub-cellular fractionation and fluorescence microscopy. In contrast to full-length A20 which is particulate and insoluble, we found hA20p50 to be soluble and readily released into the cytosol whereas hA20p37 was partially soluble, thus suggesting loss of compartmentalization as a possible mechanism for MALT1-mediated dampening of A20 function.

  6. piRNA-directed cleavage of meiotic transcripts regulates spermatogenesis.

    PubMed

    Goh, Wee Siong Sho; Falciatori, Ilaria; Tam, Oliver H; Burgess, Ralph; Meikar, Oliver; Kotaja, Noora; Hammell, Molly; Hannon, Gregory J

    2015-05-15

    MIWI catalytic activity is required for spermatogenesis, indicating that piRNA-guided cleavage is critical for germ cell development. To identify meiotic piRNA targets, we augmented the mouse piRNA repertoire by introducing a human meiotic piRNA cluster. This triggered a spermatogenesis defect by inappropriately targeting the piRNA machinery to mouse mRNAs essential for germ cell development. Analysis of such de novo targets revealed a signature for pachytene piRNA target recognition. This enabled identification of both transposable elements and meiotically expressed protein-coding genes as targets of native piRNAs. Cleavage of genic targets began at the pachytene stage and resulted in progressive repression through meiosis, driven at least in part via the ping-pong cycle. Our data support the idea that meiotic piRNA populations must be strongly selected to enable successful spermatogenesis, both driving the response away from essential genes and directing the pathway toward mRNA targets that are regulated by small RNAs in meiotic cells.

  7. Stress-induced cleavage of Myc promotes cancer cell survival

    PubMed Central

    Conacci-Sorrell, Maralice; Ngouenet, Celine; Anderson, Sarah; Brabletz, Thomas; Eisenman, Robert N.

    2014-01-01

    Evasion of apoptosis is critical in Myc-induced tumor progression. Here we report that cancer cells evade death under stress by activating calpain-mediated proteolysis of Myc. This generates Myc-nick, a cytoplasmic, transcriptionally inactive cleavage product of Myc. We found conversion of Myc into Myc-nick in cell lines and tissues derived from multiple cancers. In colon cancer, the production of Myc-nick is enhanced under stress conditions such as hypoxia and nutrient deprivation. Under these conditions, ectopic expression of Myc-nick promotes anchorage-independent growth and cell survival at least in part by promoting autophagy. Myc-nick also delays colon cancer cell death after treatment with chemotherapeutic drugs such as etoposide, cisplatin, and imatinib. Furthermore, colon cancer cells expressing a cleavage-resistant form of Myc undergo extensive apoptosis but are rescued by overexpression of Myc-nick. We also found that ectopic expression of Myc-nick results in the induction of the actin-bundling protein fascin, formation of filopodia, and increased cell motility—all mediators of tumor metastasis. Myc-nick-induced survival, autophagy, and motility require Myc box II (MBII), a region of Myc-nick that recruits acetyltransferases that in turn modify cytoplasmic proteins, including α-tubulin and ATG3. Our results suggest that Myc-nick-induced survival and motility contribute to colon cancer progression and metastasis. PMID:24696454

  8. Biochemical properties of Bacillus intermedius subtilisin-like proteinase secreted by a Bacillus subtilis recombinant strain in its stationary phase of growth.

    PubMed

    Mikhailova, E O; Mardanova, A M; Balaban, N P; Rudenskaya, G N; Ilyinskaya, O N; Sharipova, M R

    2009-03-01

    Biochemical properties of Bacillus intermedius subtilisin-like proteinase (AprBi) secreted by a B. subtilis recombinant strain in the early and late stationary phases of growth have been determined. Protein structure was analyzed and its stability estimated. It was noted that the enzyme corresponding to different phases of bacterial growth retains activity in the presence of reducing and oxidizing agents (C2H5OH and H2O2). Different effects of bivalent metal ions on activity of two proteinase fractions were found. Calcium ions more efficiently activate proteinase secreted in the late stationary phase. Unlike the first enzyme fraction, the second forms catalytically active dimers.

  9. Binding and biomimetic cleavage of the RNA poly(U) by synthetic polyimidazoles

    PubMed Central

    Cheng, Liang; Abhilash, K.G.; Breslow, Ronald

    2012-01-01

    Four polyimidazoles were used in the binding and cleavage studies with poly(U). The two polydisperse polyvinylimidazoles were previously described by others, while the other two new polymers of polyethyleneimines were prepared by cationic polymerization of oxazolines. The latter had imidazole units attached to each nitrogen of the polymers. They were characterized by gel permeation chromatography and had very low polydispersities. When they were partially protonated they bound to the poly(U) and catalyzed its cleavage by a process analogous to that used by the enzyme ribonuclease A. The kinetics of the cleavage were followed by an assay we had previously described using phosphodiesterase I from Crotalus venom after the cleavage processes. Cleavage of poly(U) with Zn2+ was also examined, with and without the polymers. A scheme is described in which these cleavages could be made sequence selective with various RNAs, particularly with important targets, such as viral RNAs. PMID:22826260

  10. Itraconazole-resistant Candida auris with phospholipase, proteinase and hemolysin activity from a case of vulvovaginitis.

    PubMed

    Kumar, Dharmendra; Banerjee, Tuhina; Pratap, Chandra Bhan; Tilak, Ragini

    2015-04-15

    Since the emergence of pathogenic non-albicans Candida species, a number of new isolates have been added to the list. One such unusual species is Candida auris (C. auris), recently isolated and studied in few reports. In this study, a case of vulvovaginitis caused by Candida auris incidentally identified by molecular methods using internal transcribed spacer polymerase chain reaction (ITS PCR) is described. Antifungal susceptibility testing revealed the isolate to be resistant to itraconazole (MIC ≥ 2 µg/ml) and expressed important virulence factors including phospholipase, proteinase and hemolysin activity. The patient was successfully treated with oral fluconazole and did not have any invasive fungemia. Very few cases of this emerging pathogen have been reported. However, its isolation from clinical specimens reveals the significance of non-albicans candida species over C. albicans and the diversity of Candida spp causing infections.

  11. Inhibitors of acrosin and granulocyte proteinases from human genital tract secretions.

    PubMed

    Schiessler, H; Arnhold, M; Ohlsson, K; Fritz, H

    1976-09-01

    Human seminal plasma contains two acid-stable proteinase inhibitors, HUSI-II (Mr approximately 6500) and HUSI-I, (Mr approximately 11 000) with different inhibition specificities. The inhibitory activity of HUSI-II is strongly limited to trypsin and acrosin; both enzyme-inhibitor complexes are very stable (e.g. bovine trypsin-HUSI-II complex: Ki = 1 x 10(-10)M; human acrosin-HUSI-II complex: Ki = 2.7 x 10(-10)M). The inhibitor from human seminal plasma HUSI-II may therefore be seen as the natural antagonist of the sperm protease acrosin. In addition to pancreatic trypsin and alpha-chymotrypsin, HUSI-I forms strong complexes with neutral proteases of the lysosome-like granules from human granulocytes, for example, the elastase (Ki = 2.5 x 10(-9)M) and cathepsin G, the chymotrypsin like protease (Ki = 7 x 10(-8)M).

  12. Cystein proteinase inhibitor stefin A as an indicator of efficiency of tumor treatment in mice.

    PubMed

    Korolenko, T A; Poteryaeva, O N; Falameeva, O V; Levina, O A

    2003-07-01

    The concentration of stefin A (cystatin A in mice) was measured in animals with experimental tumors (LS lymphosarcoma, HA-1-hepatoma, and Lewis lung carcinoma) during effective antitumor therapy. In mice with these tumors serum concentrations of stefin A increased, while the concentration of cystatin C (extracellular cystein proteinase inhibitor) decreased. The concentration of stefin A in tumor tissue in Lewis lung carcinoma was higher than in LS lymphosarcoma and HA-1-hepatoma ascitic cells, which can be explained by the degree of their malignancy. The content of stefin A in tumor tissue was similar to that in the liver and spleen of tumor-bearing animals, while its concentration in the liver and spleen of tumor-bearing animals was lower than in intact mice. The level of stefin A is an important marker of malignancy and an indicator of the efficiency of antitumor therapy.

  13. Design of dimerization inhibitors of HIV-1 aspartic proteinase: A computer-based combinatorial approach

    NASA Astrophysics Data System (ADS)

    Caflisch, Amedeo; Schramm, Hans J.; Karplus, Martin

    2000-02-01

    Inhibition of dimerization to the active form of the HIV-1 aspartic proteinase (HIV-1 PR) may be a way to decrease the probability of escape mutations for this viral protein. The Multiple Copy Simultaneous Search (MCSS) methodology was used to generate functionality maps for the dimerization interface of HIV-1 PR. The positions of the MCSS minima of 19 organic fragments, once postprocessed to take into account solvation effects, are in good agreement with experimental data on peptides that bind to the interface. The MCSS minima combined with an approach for computational combinatorial ligand design yielded a set of modified HIV-1 PR C-terminal peptides that are similar to known nanomolar inhibitors of HIV-1 PR dimerization. A number of N-substituted 2,5-diketopiperazines are predicted to be potential dimerization inhibitors of HIV-1 PR.

  14. Targeting a Proteinase-Activated Receptor 4 (PAR4) Carboxyl Terminal Motif to Regulate Platelet Function.

    PubMed

    Ramachandran, Rithwik; Mihara, Koichiro; Thibeault, Pierre; Vanderboor, Christina M; Petri, Björn; Saifeddine, Mahmoud; Bouvier, Michel; Hollenberg, Morley D

    2017-04-01

    Thrombin initiates human platelet aggregation by coordinately activating proteinase-activated receptors (PARs) 1 and 4. However, targeting PAR1 with an orthosteric-tethered ligand binding-site antagonist results in bleeding, possibly owing to the important role of PAR1 activation on cells other than platelets. Because of its more restricted tissue expression profile, we have therefore turned to PAR4 as an antiplatelet target. We have identified an intracellular PAR4 C-terminal motif that regulates calcium signaling and β-arrestin interactions. By disrupting this PAR4 calcium/β-arrestin signaling process with a novel cell-penetrating peptide, we were able to inhibit both thrombin-triggered platelet aggregation in vitro and clot consolidation in vivo. We suggest that targeting PAR4 represents an attractive alternative to blocking PAR1 for antiplatelet therapy in humans.

  15. Proteinase 3-antineutrophil cytoplasmic antibody-positive ulcerative colitis presenting with abducens neuropathy

    PubMed Central

    Kirito, Yuki; Yamamoto, Daisuke; Uchiyama, Tsuyoshi

    2017-01-01

    A 72-year-old man with ulcerative colitis (UC) presented with complete left abducens nerve palsy. Although MRI showed no significant changes, cerebrospinal fluid analysis revealed pleocytosis and elevated protein and interleukin (IL)-6 levels. His serum proteinase 3-antineutrophil cytoplasmic antibody (PR3-ANCA) level was also elevated to 31.1 U/mL, but granulomatosis with polyangiitis was not observed. On the basis of the diagnosis of autoimmune cranial neuropathy, he was treated with steroid therapy. While tapering steroid therapy, his serum PR3-ANCA levels; cerebrospinal fluid findings, including IL-6 levels; and symptoms improved. Serum PR3-ANCA could be a useful parameter of neurological disorders associated with ANCA-positive UC. PMID:28069788

  16. [Effect of proteinaceous proteinase inhibitors from potato tubers on the growth and development of phytopathogenic microorganisms].

    PubMed

    Revina, T A; Gerasimova, N G; Kladnitskaia, G V; Chalenko, G I; Valueva, T A

    2008-01-01

    We studied the effect of two proteins, PSPI-21 and PKSI, on the growth and development of phytopathogenic microorganisms (Phytophthora infestans oomycete and Fusarium culmorum fungus). Both proteins were isolated from potato tubers (Solanum tuberosum L., cv. Istrinskii) and served as inhibitors of serine proteinases. These proteins differed in the ability to inhibit growth of Phytophthora infestans oomycete and Fusarium culmorum fungus. PSPI-21 was the most potent in modulating the growth of oomycete mycelium. PKSI primarily affected the growth of the fungal mycelium. The proteins under study induced complete destruction of oomycete zoospores and partial destruction of fungal macroconidia. Our results suggest that these proteins are involved in the protection of potato plants from phytopathogenic microorganisms.

  17. Role of calcium-dependent proteinase in molt-induced claw muscle atrophy

    SciTech Connect

    Mykles, D.L.; Skinner, D.M.

    1984-01-01

    The claw closer muscle of the Bermuda land crab Gecarcinus lateralis undergoes a sequential atrophy and restoration during each intermolt cycle. Muscle protein decreases 40% during proecdysis and is restored following ecdysis. Amino acid incorporation into protein of postecdysial muscle is five times greater than that in anecdysial muscle. Since the rates of protein synthesis in anecdysial and proecdysial muscle are the same it appears that proecdysial muscle atrophy is caused primarily by an increase in protein degradation. A calcium-dependent proteinase (CDP) active at neutral pH has been implicated in the nonlysosomal hydrolysis of myofibrillar proteins. We have examined the role of a CDP in atrophy of the claw closer muscle. The many similarities between crustacean and vertebrate CDPs have established this crustacean system as a simple and convenient model for the role of Ca/sup 2 +/-dependent proteolysis in myofibrillar protein turnover and its manifestation in the structure of the sarcomere. 16 references, 8 figures. (ACR)

  18. Atomic resolution structure of serine protease proteinase K at ambient temperature

    PubMed Central

    Masuda, Tetsuya; Suzuki, Mamoru; Inoue, Shigeyuki; Song, Changyong; Nakane, Takanori; Nango, Eriko; Tanaka, Rie; Tono, Kensuke; Joti, Yasumasa; Kameshima, Takashi; Hatsui, Takaki; Yabashi, Makina; Mikami, Bunzo; Nureki, Osamu; Numata, Keiji; Iwata, So; Sugahara, Michihiro

    2017-01-01

    Atomic resolution structures (beyond 1.20 Å) at ambient temperature, which is usually hampered by the radiation damage in synchrotron X-ray crystallography (SRX), will add to our understanding of the structure-function relationships of enzymes. Serial femtosecond crystallography (SFX) has attracted surging interest by providing a route to bypass such challenges. Yet the progress on atomic resolution analysis with SFX has been rather slow. In this report, we describe the 1.20 Å resolution structure of proteinase K using 13 keV photon energy. Hydrogen atoms, water molecules, and a number of alternative side-chain conformations have been resolved. The increase in the value of B-factor in SFX suggests that the residues and water molecules adjacent to active sites were flexible and exhibited dynamic motions at specific substrate-recognition sites. PMID:28361898

  19. Roles of platelets and proteinase-activated receptors in gastric ulcer healing.

    PubMed

    Perini, Rafael; Wallace, John L; Ma, Li

    2005-10-01

    Proteinase-activated receptors (PARs) are expressed on the surface of many cells, but those on the platelet have been among the most thoroughly characterized. PARs act as key receptors mediating the proaggregatory and pro-secretory effects of thrombin. In addition to contributing to hemostasis, platelets are increasingly being viewed as important contributors to healing and to tumor growth. This is attributable to the many pro- and anti-angiogenic factors that are stored within platelets, which can be released at the sites of injury and new vessel growth. In this paper, we review the importance of the platelet in gastric ulcer healing, the contribution of platelet-contained angiogenic factors to the healing of gastric ulcers, and the role of PARs in regulating the release of angiogenic factors from platelets. Taken together, our results suggest that PARs, including those expressed on platelets, are a rational therapeutic target for modulating healing processes and tumor growth.

  20. High cell density cultivation of Brevibacterium linens and formation of proteinases and lipase.

    PubMed

    Adamitsch, Bernhard F; Karner, Ferdinand; Hampel, Werner A

    2003-05-01

    Brevibacterium linens forms hydrolytic enzymes which can be used to accelerate the ripening of cheese without causing bitterness. B. linens ATCC 9172 was grown to a high cell density (50 g dry wt l-1 after 60 h) in a mineral medium containing lactic acid, soy-peptone and ammonium sulphate by applying a continuous feed of nutrients. The maximal activities of L-leucine aminopeptidase and cell-associated proteinase were 286 U l-1 and 202 U l-1, respectively. The cell-associated lipolytic activity exhibited a strong and sudden increase at 46 h, resulting in a maximum of 9.5 U g-1 dry wt; thus the volumetric productivity of proteolytic and lipolytic activity was 4220 U l-1 h-1 and 7.3 U l-1 h-1, respectively.

  1. The anthelmintic efficacy of natural plant cysteine proteinases against the equine tapeworm, Anoplocephala perfoliata in vitro.

    PubMed

    Mansur, F; Luoga, W; Buttle, D J; Duce, I R; Lowe, A E; Behnke, J M

    2016-09-01

    Papaya latex has been demonstrated to be an efficacious anthelmintic against murine, porcine, ovine and canine nematode parasites, and even those infecting poultry, and it has some efficacy against rodent cestodes. The active ingredients of papaya latex are known to be cysteine proteinases (CPs). The experiments described in this paper indicate that CPs in papaya latex, and also those in pineapples, are highly efficacious against the equine cestode Anoplocephala perfoliata in vitro, by causing a significant reduction in motility leading to death of the worms. The susceptibility of A. perfoliata to damage by CPs was considerably greater than that of the rodent cestodes Hymenolepis diminuta and H. microstoma. Our results are the first to report anthelmintic efficacy of CPs against an economically important equine helminth. Moreover, they provide further evidence that the spectrum of activity of CPs is not restricted to nematodes and support the idea that these plant-derived enzymes can be developed into useful broad-spectrum anthelmintics.

  2. Atomic resolution structure of serine protease proteinase K at ambient temperature.

    PubMed

    Masuda, Tetsuya; Suzuki, Mamoru; Inoue, Shigeyuki; Song, Changyong; Nakane, Takanori; Nango, Eriko; Tanaka, Rie; Tono, Kensuke; Joti, Yasumasa; Kameshima, Takashi; Hatsui, Takaki; Yabashi, Makina; Mikami, Bunzo; Nureki, Osamu; Numata, Keiji; Iwata, So; Sugahara, Michihiro

    2017-03-31

    Atomic resolution structures (beyond 1.20 Å) at ambient temperature, which is usually hampered by the radiation damage in synchrotron X-ray crystallography (SRX), will add to our understanding of the structure-function relationships of enzymes. Serial femtosecond crystallography (SFX) has attracted surging interest by providing a route to bypass such challenges. Yet the progress on atomic resolution analysis with SFX has been rather slow. In this report, we describe the 1.20 Å resolution structure of proteinase K using 13 keV photon energy. Hydrogen atoms, water molecules, and a number of alternative side-chain conformations have been resolved. The increase in the value of B-factor in SFX suggests that the residues and water molecules adjacent to active sites were flexible and exhibited dynamic motions at specific substrate-recognition sites.

  3. Molecular orbital studies of enzyme activity: catalytic mechanism of serine proteinases.

    PubMed Central

    Scheiner, S; Lipscomb, W N

    1976-01-01

    The catalytic activity of the serine proteinases is studied using molecular orbital methods on a model of the enzyme-substrate complex. A mechanism is employed in which Ser-195, upon donating a proton to the His-57-Asp-102 dyad, attacks the substrate to form the tetrahedral intermediate. As His-57 then donates a proton to the leaving group, the intermediate decomposes to the acyl enzyme. An analogous process takes place during deacylation, as a water molecule takes the place of Ser-195 as the nucleophile. The motility of the histidine is found to be an important factor in both steps. An attempt is made to include the effects of those atoms not explicitly included in the calculations and to compare the reaction rate of the proposed mechanism with that of the uncatalyzed hydrolysis. This mechanism is found to be in good agreement with structural and kinetic data. PMID:1061145

  4. Protein degradation in Euglena gracilis: Purification and characterization of the major proteinase

    SciTech Connect

    Yoo, Y.J.

    1988-01-01

    Protolysis in a crude extract of Euglena gracilis was characterized by autolysis and the hydrolysis of {sup 125}I-labeled bovine serum albumin ({sup 125}I-BSA). Both procedures showed similar properties: stimulation by dithiothreitol, inhibition by leupeptin, and the same pH optima. Hydrolysis of {sup 125}I-BSA increased with growth stage and with the depletion of nutrient in the medium. The major proteolytic enzyme was purified to near homogeneity from extracts of dark-grown, stationary-phase Euglena gracilis by acid treatment, and by chromatography on CM-cellulose, DEAE-cellulose, Sephadex G-75, and hydroxyapatite using {sup 125}I-BSA as substrate. The molecular weight of the proteinase was 30,000 when determined by gel filtration on Sephadex G-75 and 15,000 when estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme therefore appears to be composed of two subunits.

  5. A feedback regulatory pathway between LDL and alpha-1 proteinase inhibitor in chronic inflammation and infection.

    PubMed

    Bristow, Cynthia L; Modarresi, Rozbeh; Babayeva, Mariya A; LaBrunda, Michelle; Mukhtarzad, Roya; Trucy, Maylis; Franklin, Aaron; Reeves, Rudy E R; Long, Allegra; Mullen, Michael P; Cortes, Jose; Winston, Ronald

    2013-11-01

    Dietary lipids are transported via lymph to the liver and transformed to lipoproteins which bind to members of the low density lipoprotein receptor family (LDL-RFMs). Certain LDL-RFMs, e.g., very low density lipoprotein receptor (VLDLR), are also bound by inactivated proteinase inhibitors, the most abundant being α1proteinase inhibitor (α1PI, α1antitrypsin). Inflammation/infection, including HIV-1 infection, is accompanied by low levels of CD4+ T cells and active α1PI and high levels of inactivated α1PI. By inducing LDL-RFMs-mediated cellular locomotion, active α1PI regulates the number of CD4+ T cells. We sought to investigate whether CD4+ T cells and α1PI directly impact lipoprotein levels. At the cellular level, we show that active α1PI is required for VLDLR-mediated uptake of receptor-associated cargo, specifically CD4-bound HIV-1. We show that active α1PI levels linearly correlate with LDL levels in HIV-1 infected individuals (P<0.001) and that therapeutic, weekly infusions of active α1PI elevate the number of CD4+ T cells and HDL levels while lowering LDL levels in patients on antiretroviral therapy with controlled HIV-1. Based on the unusual combination of lipodystrophy and low levels of α1PI and CD4+ T cells in HIV-1 disease, we reveal that LDL and α1PI participate in a feedback regulatory pathway. We demonstrate integral roles for sequentially acting active and inactive α1PI in the uptake and recycling of receptors and cargo aggregated with VLDLR including CD4 and chemokine receptors. Evidence supports a role for α1PI as a primary sentinel to deploy the immune system as a consequence of its role in lipoprotein transport.

  6. Unmasking of complements using proteinase-K in formalin fixed paraffin embedded renal biopsies.

    PubMed

    Nada, R; Kumar, A; Kumar, V G; Gupta, K L; Joshi, K

    2016-01-01

    Renal biopsy interpretation requires histopathology, direct immunofluorescence (DIF) and electron microscopy. Formalin-fixed, paraffin-embedded tissue (FFPE) sent for light microscopy can be used for DIF after antigen retrieval. However, complement staining has not been satisfactory. We standardized DIF using proteinase-K for antigen retrieval in FFPE renal biopsies. A pilot study was conducted on known cases of membranous glomerulonephritis (MGN), membranoproliferative type-1 (MPGN-1), immunoglobulin A nephropathy (IgAN), and anti-glomerular basement disease (anti-GBM). Immunofluorescence panel included fluorescein isothiocyanate (FITC) conjugated IgG, IgA, IgM, complements (C3 and C1q), light chains (kappa, lambda) and fibrinogen antibodies. After standardization of the technique, 75 renal biopsies and 43 autopsies cases were stained. Out of 43 autopsy cases, immune-complex mediated glomerulonephritis (GN) was confirmed in 18 cases (Lupus nephritis-11, IgAN-6, MGN-1), complement-mediated dense deposit disease (DDD-1) and monoclonal diseases in 4 cases (amyloidosis-3, cast nephropathy-1). Immune-mediated injury was excluded in 17 cases (focal segmental glomerulosclerosis -3, crescentic GN-6 [pauci-immune-3, anti-GBM-3], thrombotic microangiopathy-5, atherosclerosis-3). Renal biopsies (n-75) where inadequate or no frozen sample was available; this technique classified 52 mesangiocapillary pattern as MPGN type-1-46, DDD-2 and (C3GN-4). Others were diagnosed as IgAN-3, lupus nephritis-2, MGN-4, diffuse proliferative glomerulonephritis (DPGN)-1, Non-IC crescentic GN-1, monoclonal diseases-3. In nine cases, DIF on FFPE tissue could not help in making diagnosis. Proteinase-K enzymatic digestion of FFPE renal biopsies can unmask complements (both C3 and C1q) in immune-complexes mediated and complement-mediated diseases. This method showed good results on autopsy tissues archived for as long as 15 years.

  7. Application of Asian pumpkin (Cucurbita ficifolia) serine proteinase for production of biologically active peptides from casein.

    PubMed

    Dąbrowska, Anna; Szołtysik, Marek; Babij, Konrad; Pokora, Marta; Zambrowicz, Aleksandra; Chrzanowska, Józefa

    2013-01-01

    The main objective of this study was to determine potential application of a serine proteinase derived from Asian pumpkin for obtaining biologically active peptides from casein. The course of casein hydrolysis by three doses of the enzyme (50, 150, 300 U/mg of protein) was monitored for 24 hours by the determinations of: hydrolysis degree DH (%), free amino group content (μmole Gly/g), RP HPLC peptide profiles and by polyacrylamide gel electrophoresis. In all hydrolyzates analyzed antioxidant activities were determined using three tests: the ability to reduce iron ions in FRAP test, the ability to scavenge free radicals in DPPH test, and Fe(2+) chelating activity. The antimicrobial activity of obtained peptide fractions was determined as the ability to inhibit the growth of Escherichia coli, Bacillus cereus and Pseudomonas fluorescens in a diffusion plate test. The deepest degradation, expressed as the DH [%] and the free amino group content (67% and 7528 µmole Gly/mg, respectively), was noted in samples hydrolyzed with 300 U/ml of enzyme for 24 hours, while in other samples the determined values were about three and two times lower. The results were in agreement with the peptide profiles obtained by RP HPLC. The highest antioxidative activities determined in all tests were seen for the casein hydrolysate obtained with 300 U/mg protein of serine proteinase after 24 h of reaction (2.15 µM Trolox/mg, 96.15 µg Fe(3+)/mg, 814.97 µg Fe(2+)/mg). Antimicrobial activity was presented in three preparations. In other samples no antimicrobial activity was detected.

  8. Affinity purification and biochemical characterization of histolysin, the major cysteine proteinase of Entamoeba histolytica.

    PubMed Central

    Luaces, A L; Barrett, A J

    1988-01-01

    We report a one-step method for the purification to homogeneity of a cysteine proteinase of Entamoeba histolytica (histolysin) by affinity chromatography of the soluble extract of the parasite on immobilized phenylalanyl(2-phenyl)aminoacetaldehyde semicarbazone. The enzyme has an apparent Mr of 26,000 by SDS/polyacrylamide-gel electrophoresis and 29,000 by gel chromatography. Its pH optimum varies widely, from 5.5 with azocasein to approx. 7 with other protein substrates and benzyloxycarbonylphenylalanyl-L-citrullylaminomethylcourmarin++ + (Z-Phe-Cit-NHMec), and to 9.5 with benzyloxycarbonylphenylalanylarginylaminomethylcoumarin (Z-Phe-Arg-NHMec) and benzyloxycarbonylarginylarginylaminomethylcourmarin (Z-Arg-Arg-NHMec). Values of Km, kcat. and kcat/Km are 1.5 microM, 130 s-1 and 87 X 10(6) M-1.s-1 for Z-Arg-Arg-NHMec, and 32 microM, 0.4 s-1 and 0.012 x 10(6) M-1.s-1 for Z-Phe-Arg-NHMec, respectively, at pH 7.5 and 37 degrees C. The enzyme is inhibited by leupeptin and such inhibitors of cysteine proteinases as L-transepoxysuccinyl-L-leucylamido-4-(guanidino)butane, peptidyldiazomethanes, iodoacetic acid and chicken cystatin. The tentative N-terminal amino acid sequence of the enzyme closely resembles that of papain. Histolysin does not degrade type I collagen or elastin, but it is active against cartilage proteoglycan and kidney glomerular basement-membrane collagen. It also detaches cells from their substratum in vitro, and could well play a role in tissue invasion. Images Fig. 2. Fig. 4. PMID:2898937

  9. Enzymatic cleavage of myoferlin releases a dual C2-domain module linked to ERK signalling.

    PubMed

    Piper, Ann-Katrin; Ross, Samuel E; Redpath, Gregory M; Lemckert, Frances A; Woolger, Natalie; Bournazos, Adam; Greer, Peter A; Sutton, Roger B; Cooper, Sandra T

    2017-05-01

    Myoferlin and dysferlin are closely related members of the ferlin family of Ca(2+)-regulated vesicle fusion proteins. Dysferlin is proposed to play a role in Ca(2+)-triggered vesicle fusion during membrane repair. Myoferlin regulates endocytosis, recycling of growth factor receptors and adhesion proteins, and is linked to the metastatic potential of cancer cells. Our previous studies establish that dysferlin is cleaved by calpains during membrane injury, with the cleavage motif encoded by alternately-spliced exon 40a. Herein we describe the cleavage of myoferlin, yielding a membrane-associated dual C2 domain 'mini-myoferlin'. Myoferlin bears two enzymatic cleavage sites: a canonical cleavage site encoded by exon 38 within the C2DE domain; and a second cleavage site in the linker adjacent to C2DE, encoded by alternately-spliced exon 38a, homologous to dysferlin exon 40a. Both myoferlin cleavage sites, when introduced into dysferlin, can functionally substitute for exon 40a to confer Ca(2+)-triggered calpain cleavage in response to membrane injury. However, enzymatic cleavage of myoferlin is complex, showing both constitutive or Ca(2+)-enhanced cleavage in different cell lines, that is not solely dependent on calpains-1 or -2. The functional impact of myoferlin cleavage was explored through signalling protein phospho-protein arrays revealing specific activation of ERK1/2 by ectopic expression of cleavable myoferlin, but not an uncleavable isoform. In summary, we molecularly define two enzymatic cleavage sites within myoferlin and demonstrate 'mini-myoferlin' can be detected in human breast cancer tumour samples and cell lines. These data further illustrate that enzymatic cleavage of ferlins is an evolutionarily preserved mechanism to release functionally specialized mini-modules.

  10. Conserved functions of the trigger loop and Gre factors in RNA cleavage by bacterial RNA polymerases.

    PubMed

    Miropolskaya, Nataliya; Esyunina, Daria; Kulbachinskiy, Andrey

    2017-02-27

    RNA cleavage by RNA polymerase (RNAP) is the central step in co-transcriptional RNA proofreading. Bacterial RNAPs were proposed to rely on the same mobile element of the active site, the trigger loop (TL), for both nucleotide addition and RNA cleavage. RNA cleavage can also be stimulated by universal Gre factors, which should replace the TL to get access to the RNAP active site. The contributions of the TL and Gre factors to RNA cleavage reportedly vary between RNAPs from different bacterial species and, probably, different types of transcription complexes. Here, by comparing RNAPs from Escherichia coli (Eco), Deinococcus radiodurans (Dra) and Thermus aquaticus (Taq) we show that the functions of the TL and Gre factors in RNA cleavage are conserved in various species, with important variations which may be related to extremophilic adaptation. Deletions of the TL strongly impair intrinsic RNA cleavage by all three RNAPs and eliminate the inter-species differences in the reaction rates. GreA factors activate RNA cleavage by wild-type RNAPs to similar levels. The rates of GreA-dependent cleavage are lower for ΔTL RNAP variants, suggesting that the TL contributes to the Gre function. Finally, neither the TL nor GreA can efficiently activate RNA cleavage in certain types of backtracked transcription complexes suggesting that these complexes adopt a catalytically inactive conformation probably important for transcription regulation.

  11. Predicting caspase substrate cleavage sites based on a hybrid SVM-PSSM method.

    PubMed

    Li, Dandan; Jiang, Zhenran; Yu, Weiming; Du, Lei

    2010-12-01

    Caspases play an important role in many critical non-apoptosis processes by cleaving relevant substrates at cleavage sites. Identification of caspase substrate cleavage sites is the key to understand these processes. This paper proposes a hybrid method using support vector machine (SVM) in conjunction with position specific scoring matrices (PSSM) for caspase substrate cleavage sites prediction. Three encoding schemes including orthonormal binary encoding, BLOSUM62 matrix profile and PSSM profile of neighborhood surrounding the substrate cleavage sites were regarded as the input of SVM. The 10-fold cross validation results demonstrate that the SVM-PSSM method performs well with an overall accuracy of 97.619% on a larger dataset.

  12. γ-Secretase Catalyzes Sequential Cleavages of the AβPP Transmembrane Domain

    PubMed Central

    Xu, Xuemin

    2009-01-01

    The biogenesis of the amyloid-β peptide (Aβ) is a central issue in Alzheimer's disease (AD) research. Aβ is produced by β- and γ-secretases from the amyloid-β protein precursor (AβPP). These proteases are targets for the development of therapeutic compounds to downregulate Aβ production. γ-secretase has received more attention 1) because it generates the C-terminus of Aβ, which is important in the pathogenesis of AD because the longer Aβ species are more amyloidogenic, and 2) because it cleaves AβPP within its transmembrane domain. In the understanding the mechanism of γ-secretase cleavage, three major cleavage sites have been identified, namely, γ-cleavage site at Aβ40/42, ζ-cleavage site at Aβ46, and ε-cleavage site at Aβ49. Moreover, the novel finding that some of the known γ-secretase inhibitors inhibit the formation of secreted Aβ40 and Aβ42, but cause an intracellular accumulation of long Aβ46, provided information extremely important for the development of strategies aimed at the design of γ-secretase inhibitors to prevent and treat AD. In addition, it has been established that the C-terminus of Aβ is generated by a series of sequential cleavages: first, ε-cleavage, followed by ζ-cleavage and finally by γ-cleavage, commencing from the membrane boundary to the middle of the AβPP membrane domain. PMID:19221413

  13. Hydrolytic cleavage of DNA-model substrates promoted by polyoxovanadates.

    PubMed

    Steens, Nele; Ramadan, Ahmed M; Absillis, Gregory; Parac-Vogt, Tatjana N

    2010-01-14

    Hydrolysis of 4-nitrophenyl phosphate (NPP) and bis-4-nitrophenyl phosphate (BNPP), two commonly used DNA model substrates, was examined in vanadate solutions by means of (1)H, (31)P and (51)V NMR spectroscopy. The hydrolysis of the phosphoester bond in NPP at 50 degrees C and pH 5.0 proceeds with a rate constant of 1.74 x 10(-5) s(-1). The cleavage of the phosphoester bond in BNPP at 70 degrees C and pH 5.0 proceeds with a rate constant of 3.32 x 10(-6) s(-1), representing an acceleration of four orders of magnitude compared to the uncatalyzed cleavage. Inorganic phosphate and nitrophenol (NP) were the only products of hydrolysis. The NMR spectra did not show evidence of any paramagnetic species, excluding the possibility of V(V) reduction to V(IV), indicating that the cleavage of the phosphoester bond is purely hydrolytic. The pH dependence of k(obs) revealed that the hydrolysis proceeds fastest in solutions of pH 5.5. Comparison of the rate profile with the concentration profile of polyoxovanadates shows a striking overlap of the k(obs) profile with the concentration of decavanadate (V(10)). Kinetic experiments at 37 degrees C using a fixed amount of NPP and increasing amounts of V(10) permitted the calculation of catalytic (k(c) = 5.67 x 10(-6) s(-1)) and formation constants for the NPP-V(10) complex (K(f) = 71.53 M(-1)). Variable temperature (31)P NMR spectra of a reaction mixture revealed broadening and shifting of the (31)P resonance upon addition of increasing amounts of decavanadate and upon increasing temperature, implying the dynamic exchange process between free and bound NPP at higher temperatures. The origin of the hydrolytic activity of V(10) is most likely due its high lability and its dissociation into smaller fragments which may allow the attachment of NPP and BNPP into the polyoxovanadate framework.

  14. Embryo apoptosis identification: Oocyte grade or cleavage stage?

    PubMed Central

    Bakri, Noraina Mohd; Ibrahim, Siti Fatimah; Osman, Nurul Atikah; Hasan, Nurhaslina; Jaffar, Farah Hanan Fathihah; Rahman, Zulaiha Abdul; Osman, Khairul

    2015-01-01

    Apoptosis is a programed cell death that is vital for tissue homeostasis. However, embryo apoptosis had been known to be related to embryo fragmentation which should be avoided in in vitro fertilization (IVF). The purpose of this study was to evaluate the relationship of embryo apoptosis with the grade of immature oocytes and cleavage stage of in vitro produced (IVP) cattle embryos. This study consisted of 345 oocytes collected through ovary slicing. Immature oocytes were graded as A, B and C. This grading was based on cumulus cell thickness and compactness. All oocytes then underwent an in vitro maturation (IVM) procedure. An IVF was done 24 h after IVM culture. Prior to staining, stage of cleaved embryos was determined and classified as either 2, 4, 8 or >8-cell embryo stage. Apoptosis status of cleaved IVP embryos was determined by using annexin V-FITC staining technique at 48 and 72 h post insemination (hpi). Apoptosis status for each embryo was classified as either early or late. The result showed that there was no significant difference (p > 0.05) of apoptosis status among grade A, B and C embryos. All grades of oocytes showed embryo apoptosis where 1.5% late apoptosis for grade A, 4.5% and 10.4% of early and late apoptosis for grade B and grade C. Early apoptosis was not seen in grade A embryo. We also noted no significant difference (p > 0.05) of apoptosis status between 2, 4, 8 and >8-cell embryo stage. Early apoptosis was also not seen in >8-cell stage. Even though there were no differences in apoptosis expression between the three classes, the cleavage rate of grade A oocytes was significantly higher (p < 0.01) than grade B and grade C. In conclusion, the apoptosis expression in the embryo can occur regardless of the oocyte quality and the cleavage stage of the embryo produced. PMID:26858565

  15. Enzymic Pathways for Formation of Carotenoid Cleavage Products

    NASA Astrophysics Data System (ADS)

    Fleischmann, Peter; Zorn, Holger

    Degraded carotenoids (apocarotenoids, norisoprenoids) have been a subject of intensive research for several decades. From the perspective of human physiology and nutrition, the retinoids, acting as vitamins, signalling molecules, and visual pigments, attracted the greatest attention (Chapters 15 and 16). Plant scientists, however, detected a wealth of different apocarotenoids, presumably derived by the excentric cleavage of carotenoids in various species, the plant hormone abscisic acid (1, Scheme 6) being the best-investigated example. With the onset of fruit ripening, flower opening or senescence of green tissues, carotenoids are degraded oxidatively to smaller, volatile compounds. The natural biological functions of the reaction products are outlined in Chapter 15. As many of these apocarotenoids act as potent flavour compounds, food chemists and flavourists worldwide have investigated meticulously their structural and sensory properties. Many aspects of carotenoid metabolites and breakdown products as aroma compounds are presented in a comprehensive book [1].

  16. Assessment of HDACi-Induced Protein Cleavage by Caspases.

    PubMed

    Treude, Fabian; Gladbach, Tobias; Plaster, Jacqueline; Hartkamp, Jörg

    2017-01-01

    Aberrant histone deacetylase (HDAC) activity often correlates with neoplastic transformation and inhibition of HDACs by small molecules has emerged as a promising strategy to treat hematological malignancies in particular. Treatment with HDAC inhibitors (HDACis) often prompts tumor cells to undergo apoptosis, thereby causing a caspase-dependent cleavage of target proteins. An unexpectedly large number of proteins are in vivo caspase substrates and defining caspase-mediated substrate specificity is a major challenge. In this chapter we demonstrate that the hematopoietic transcription factor PU.1 becomes cleaved after treatment of acute myeloid leukemia (AML) cells with the HDACis LBH589 (panobinostat) or MS-275 (entinostat). To define caspase specificity for PU.1, an in vitro caspase assay including caspases 1-10 with in vitro-translated PU.1 is described in detail.

  17. Identification of Plastoglobules as a Site of Carotenoid Cleavage

    PubMed Central

    Rottet, Sarah; Devillers, Julie; Glauser, Gaétan; Douet, Véronique; Besagni, Céline; Kessler, Felix

    2016-01-01

    Carotenoids play an essential role in light harvesting and protection from excess light. During chloroplast senescence carotenoids are released from their binding proteins and are eventually metabolized. Carotenoid cleavage dioxygenase 4 (CCD4) is involved in carotenoid breakdown in senescing leaf and desiccating seed, and is part of the proteome of plastoglobules (PG), which are thylakoid-associated lipid droplets. Here, we demonstrate that CCD4 is functionally active in PG. Leaves of Arabidopsis thaliana ccd4 mutants constitutively expressing CCD4 fused to yellow fluorescent protein showed strong fluorescence in PG and reduced carotenoid levels upon dark-induced senescence. Lipidome-wide analysis indicated that β-carotene, lutein, and violaxanthin were the principle substrates of CCD4 in vivo and were cleaved in senescing chloroplasts. Moreover, carotenoids were shown to accumulate in PG of ccd4 mutant plants during senescence, indicating translocation of carotenoids to PG prior to degradation. PMID:28018391

  18. Cleavage fracture of austenite induced by nitrogen supersaturation

    SciTech Connect

    Vogt, J.B.; Messai, A.; Foct, J. . Lab. de Metallurgie Physique)

    1994-09-01

    Austenitic stainless steels and more generally FCC structure materials are good candidates for cryogenic applications because they remain ductile at low temperatures. In some cases, brittleness may occasionally occur in severe and specific conditions such as hydrogen embrittlement or during stress corrosion cracking at low strain rates. The present study shows that the brittleness observed in the P900 austenitic stainless steel is associated with the presence of a high amount of nitrogen atoms. Brittle fracture occurs both intergranularly and transgranularly. Cleavage mostly on [111] planes is associated with marked slip but with the absence of rivers. The occurrence of a DBTT is explained by the converse variations of brittle rupture stress and flow stress against nitrogen content. The flow stress increases and is mainly controlled by a short range which leads the stress for brittle rupture to be reached before the plastic flow stress.

  19. Cleavage of an amide bond by a ribozyme

    NASA Technical Reports Server (NTRS)

    Dai, X.; De Mesmaeker, A.; Joyce, G. F.; Miller, S. L. (Principal Investigator)

    1995-01-01

    A variant form of a group I ribozyme, optimized by in vitro evolution for its ability to catalyze magnesium-dependent phosphoester transfer reactions involving DNA substrates, also catalyzes the cleavage of an unactivated alkyl amide when that linkage is presented in the context of an oligodeoxynucleotide analog. Substrates containing an amide bond that joins either two DNA oligos, or a DNA oligo and a short peptide, are cleaved in a magnesium-dependent fashion to generate the expected products. The first-order rate constant, kcat, is 0.1 x 10(-5) min-1 to 1 x 10(-5) min-1 for the DNA-flanked substrates, which corresponds to a rate acceleration of more than 10(3) as compared with the uncatalyzed reaction.

  20. Repetitive cleavage of elastomeric membrane via controlled interfacial fracture.

    PubMed

    Kim, Jeong Hun; Choi, Yong Whan; Kim, Min Sung; Um, Hyung Sik; Lee, Sung Hoon; Kim, Pilnam; Suh, Kahp-Yang

    2014-07-23

    Here, we report a method of fabricating thin layer of polydimethylsiloxane (PDMS), with a thickness in the range of 60-80 nm, which can be repeatedly generated (more than 10 times) from the same block of PDMS via controlled interfacial fracture. The thin layers can be transferred to various substrates by peeling off from the bulk PDMS. The cleavage is attributed to the built-in stress at the fracture interface due to plasma treatment, resulting in the repetitive formation of the thin membranes, with no residue from processing, and with a surface roughness of ∼5 nm. We were able to demonstrate transferred patterns with controlled thickness by varying the oxygen plasma treatment conditions and the composition of bulk PDMS stamp. Using the method, we achieved residual-free patterns with submicrometer resolution for applications in biomolecule array templates.

  1. Regulation of DNA Replication in Early Embryonic Cleavages

    PubMed Central

    Kermi, Chames; Lo Furno, Elena; Maiorano, Domenico

    2017-01-01

    Early embryonic cleavages are characterized by short and highly synchronous cell cycles made of alternating S- and M-phases with virtually absent gap phases. In this contracted cell cycle, the duration of DNA synthesis can be extraordinarily short. Depending on the organism, the whole genome of an embryo is replicated at a speed that is between 20 to 60 times faster than that of a somatic cell. Because transcription in the early embryo is repressed, DNA synthesis relies on a large stockpile of maternally supplied proteins stored in the egg representing most, if not all, cellular genes. In addition, in early embryonic cell cycles, both replication and DNA damage checkpoints are inefficient. In this article, we will review current knowledge on how DNA synthesis is regulated in early embryos and discuss possible consequences of replicating chromosomes with little or no quality control. PMID:28106858

  2. Catalyzed hydrolytic cleavage reaction of carbon-carbon bond

    SciTech Connect

    Ioffe, I.I.; Rubinskaya, E.V.

    1986-12-01

    The authors split the carbon-carbon bond for a series of simple and complex organic compounds in neutral aqueous solutions on a heterogeneous metal-containing catalyst, palladium on carbon. The experimental results are given. In each case, the catalytic effect was controlled by a blank experiment, without a catalyst, where there was no decomposition of the substrate. The occurrence of the heterogeneous-catalytic cleavage reaction of the carbon-carbon bonds in the molecules is indicated not only by their extensive conversion, but also by the almost complete depletion of the content of organic carbon, confirmed by a similar decrease in the chemical consumption of oxygen coefficient in the system, which is possible only in the complete decomposition of the organic compounds to gaseous products or with the formation of inappreciable amounts of low-molecular-weight water-soluble compounds.

  3. Dinitrogen cleavage and hydrogenation by a trinuclear titanium polyhydride complex.

    PubMed

    Shima, Takanori; Hu, Shaowei; Luo, Gen; Kang, Xiaohui; Luo, Yi; Hou, Zhaomin

    2013-06-28

    Both the Haber-Bosch and biological ammonia syntheses are thought to rely on the cooperation of multiple metals in breaking the strong N≡N triple bond and forming an N-H bond. This has spurred investigations of the reactivity of molecular multimetallic hydrides with dinitrogen. We report here the reaction of a trinuclear titanium polyhydride complex with dinitrogen, which induces dinitrogen cleavage and partial hydrogenation at ambient temperature and pressure. By (1)H and (15)N nuclear magnetic resonance, x-ray crystallographic, and computational studies of some key reaction steps and products, we have determined that the dinitrogen (N2) reduction proceeds sequentially through scission of a N2 molecule bonded to three Ti atoms in a μ-η(1):η(2):η(2)-end-on-side-on fashion to give a μ2-N/μ3-N dinitrido species, followed by intramolecular hydrogen migration from Ti to the μ2-N nitrido unit.

  4. Catalysts of DNA Strand Cleavage at Apurinic/Apyrimidinic Sites

    PubMed Central

    Minko, Irina G.; Jacobs, Aaron C.; de Leon, Arnie R.; Gruppi, Francesca; Donley, Nathan; Harris, Thomas M.; Rizzo, Carmelo J.; McCullough, Amanda K.; Lloyd, R. Stephen

    2016-01-01

    Apurinic/apyrimidinic (AP) sites are constantly formed in cellular DNA due to instability of the glycosidic bond, particularly at purines and various oxidized, alkylated, or otherwise damaged nucleobases. AP sites are also generated by DNA glycosylases that initiate DNA base excision repair. These lesions represent a significant block to DNA replication and are extremely mutagenic. Some DNA glycosylases possess AP lyase activities that nick the DNA strand at the deoxyribose moiety via a β- or β,δ-elimination reaction. Various amines can incise AP sites via a similar mechanism, but this non-enzymatic cleavage typically requires high reagent concentrations. Herein, we describe a new class of small molecules that function at low micromolar concentrations as both β- and β,δ-elimination catalysts at AP sites. Structure-activity relationships have established several characteristics that appear to be necessary for the formation of an iminium ion intermediate that self-catalyzes the elimination at the deoxyribose ring. PMID:27363485

  5. Structural determinants of RNA recognition and cleavage by Dicer.

    PubMed

    MacRae, Ian J; Zhou, Kaihong; Doudna, Jennifer A

    2007-10-01

    A hallmark of RNA interference is the production of short double-stranded RNA (dsRNA) molecules 21-28 nucleotides in length by the specialized RNase III protein Dicer. Dicer enzymes uniquely generate RNA products of specific lengths by mechanisms that have not been fully elucidated. Here we show that the PAZ domain responsible for dsRNA end recognition confers this measuring ability through both its structural position and RNA-binding specificity. Point mutations define the dsRNA-binding surface and reveal a protein loop important for cleavage of substrates containing perfect or imperfect base pairing. On the basis of these results, we reengineered Dicer with a U1A RNA-binding domain in place of the PAZ domain to create an enzyme with altered end-recognition specificity and RNA product length. These results explain how Dicer functions as a molecular ruler and provide a structural basis for modifying its activity in cells.

  6. [Molecular cloning and analysis of cDNA sequences encoding serine proteinase and Kunitz type inhibitor in venom gland of Vipera nikolskii viper].

    PubMed

    Ramazanova, A S; Fil'kin, S Iu; Starkov, V G; Utkin, Iu N

    2011-01-01

    Serine proteinases and Kunitz type inhibitors are widely represented in venoms of snakes from different genera. During the study of the venoms from snakes inhabiting Russia we have cloned cDNAs encoding new proteins belonging to these protein families. Thus, a new serine proteinase called nikobin was identified in the venom gland of Vipera nikolskii viper. By amino acid sequence deduced from the cDNA sequence, nikobin differs from serine proteinases identified in other snake species. Nikobin amino acid sequence contains 15 unique substitutions. This is the first serine proteinase of viper from Vipera genus for which a complete amino acid sequence established. The cDNA encoding Kunitz type inhibitor was also cloned. The deduced amino acid sequence of inhibitor is homologous to those of other proteins from that snakes of Vipera genus. However there are several unusual amino acid substitutions that might result in the change of biological activity of inhibitor.

  7. Effects of antirheumatic drugs on the interleukin-1 alpha induced synthesis and activation of proteinases in articular cartilage explants in culture.

    PubMed

    Arsenis, C; McDonnell, J

    1989-06-01

    Three human cytokines (interleukin-1 alpha, interleukin-1 beta and tumor necrosis factor-alpha), added into the medium of bovine or rabbit articular cartilage explant cultures, stimulated the synthesis and activation of various proteinases. Proteoglycan degradation, measured by assaying for sulfated glycosaminoglycans released into the medium, was correlated with the proteinase stimulation. Several antirheumatic drugs were tested in a similar tissue culture system as potential inhibitors of the interleukin-1 alpha mediated stimulation of proteinase and PGE2 syntheses. Arteparon, Dexamethasone, Ibuprofen, Indomethacin, Levamisole, Naproxen, Phenylbutazone, Prednisolone, Piroxicam, Rumalon, Tamoxifen and Diclofenac were essentially ineffective in inhibiting the interleukin-1 alpha mediated induction of proteinase synthesis and sulfated glycosaminoglycan release, although some of them inhibited PGE2 synthesis. Two antimalarial drugs showed some inhibition, but only at higher concentrations.

  8. Ultrarapid mutation detection by multiplex, solid-phase chemical cleavage

    SciTech Connect

    Rowley, G.; Saad, S.; Giannelli, F.; Green, P.M.

    1995-12-10

    The chemical cleavage of mismatches in heteroduplexes formed by probe and test DNA detects and locates any sequence change in long DNA segments ({approximately}1.8 kb), and its efficiency has been well tested in the analysis of both average (e.g., coagulation factor IX) and large, complex genes (e.g., coagulation factor VIII and dystrophin). In the latter application RT/PCR products allow the examination of all essential sequences of the gene in a minimum number of reactions. We use two specific chemical reactants (hydroxylamine and osmium tetroxide) and piperidine cleavage of the above procedure to develop a very fast mutation screening method. This is based on: (1) 5{prime} or internal fluorescent labeling to allow concurrent screening of three to four DNA fragments and (2) solid-phase chemistry to use a microliter format and reduce the time required for the procedure, from amplification of sequence to gel loading inclusive, to one person-working-day. We test the two variations of the method, one entailing 5{prime} labeling of probe DNA and the other uniform labeling of both probe and target DNA, by detecting 114 known hemophilia B (coagulation factor IX) mutations and by analyzing 129 new patients. Uniform labeling of both probe and target DNA prior to formation of the heteroduplexes leads to almost twofold redundancy in the ability to detect mutations. Alternatively, the latter procedure may offer very efficient though less than 100% screening for sequence changes with only hydroxylamine. The full method with two chemical reactions (hydroxylamine and osmium tetroxide) should allow one person to screen with virtually 100% accuracy more than 300 kb of sequence in three ABI 373 gels in 1 day. 26 refs., 7 figs., 1 tab.

  9. Identification of TMPRSS6 cleavage sites of hemojuvelin

    PubMed Central

    Rausa, Marco; Ghitti, Michela; Pagani, Alessia; Nai, Antonella; Campanella, Alessandro; Musco, Giovanna; Camaschella, Clara; Silvestri, Laura

    2015-01-01

    Hemojuvelin (HJV), the coreceptor of the BMP-SMAD pathway that up-regulates hepcidin transcription, is a repulsive guidance molecule (RGMc) which undergoes a complex intracellular processing. Following autoproteolysis, it is exported to the cell surface both as a full-length and a heterodimeric protein. In vitro membrane HJV (m-HJV) is cleaved by the transmembrane serine protease TMPRSS6 to attenuate signalling and to inhibit hepcidin expression. In this study, we investigated the number and position of HJV cleavage sites by mutagenizing arginine residues (R), potential TMPRSS6 targets, to alanine (A). We analysed translation and membrane expression of HJV R mutants and the pattern of fragments they release in the culture media in the presence of TMPRSS6. Abnormal fragments were observed for mutants at arginine 121, 176, 218, 288 and 326. Considering that all variants, except HJVR121A, lack autoproteolytic activity and some (HJVR176A and HJVR288A) are expressed at reduced levels on cell surface, we identified the fragments originating from either full-length or heterodimeric proteins and defined the residues 121 and 326 as the TMPRSS6 cleavage sites in both isoforms. Using the N-terminal FLAG-tagged HJV, we showed that residue 121 is critical also in the rearrangement of the N-terminal heterodimeric HJV. Exploiting the recently reported RGMb crystallographic structure, we generated a model of HJV that was used as input structure for all-atoms molecular dynamics simulation in explicit solvent. As assessed by in silico studies, we concluded that some arginines in the von Willebrand domain appear TMPRSS6 insensitive, likely because of partial protein structure destabilization. PMID:25704252

  10. Mechanism of endonuclease cleavage by the HigB toxin

    PubMed Central

    Schureck, Marc A.; Repack, Adrienne; Miles, Stacey J.; Marquez, Jhomar; Dunham, Christine M.

    2016-01-01

    Bacteria encode multiple type II toxin–antitoxin modules that cleave ribosome-bound mRNAs in response to stress. All ribosome-dependent toxin family members structurally characterized to date adopt similar microbial RNase architectures despite possessing low sequence identities. Therefore, determining which residues are catalytically important in this specialized RNase family has been a challenge in the field. Structural studies of RelE and YoeB toxins bound to the ribosome provided significant insights but biochemical experiments with RelE were required to clearly demonstrate which residues are critical for acid-base catalysis of mRNA cleavage. Here, we solved an X-ray crystal structure of the wild-type, ribosome-dependent toxin HigB bound to the ribosome revealing potential catalytic residues proximal to the mRNA substrate. Using cell-based and biochemical assays, we further determined that HigB residues His54, Asp90, Tyr91 and His92 are critical for activity in vivo, while HigB H54A and Y91A variants have the largest effect on mRNA cleavage in vitro. Comparison of X-ray crystal structures of two catalytically inactive HigB variants with 70S-HigB bound structures reveal that HigB active site residues undergo conformational rearrangements likely required for recognition of its mRNA substrate. These data support the emerging concept that ribosome-dependent toxins have diverse modes of mRNA recognition. PMID:27378776

  11. The families of papain- and legumain-like cysteine proteinases from embryonic axes and cotyledons of Vicia seeds: developmental patterns, intracellular localization and functions in globulin proteolysis.

    PubMed

    Fischer, J; Becker, C; Hillmer, S; Horstmann, C; Neubohn, B; Schlereth, A; Senyuk, V; Shutov, A; Müntz, K

    2000-05-01

    Families of papain- and legumain-like cysteine proteinases (CPR) were found in Vicia seeds. cDNAs and antibodies were used to follow organ specificity and the developmental course of CPR-specific mRNAs and polypeptides. Four papain-like cysteine proteinases (CPR1, CPR2, proteinase A and CPR4) from vetch seeds (Vicia sativa L.) were analysed. CPR2 and its mRNA were already found in dry embryonic axes. CPR1 was only detected there during early germination. Both CPR1 and CPR2 strongly increased later during germination. In cotyledons, both CPR1 and CPR2 were only observed one to two days later than in the axis. Proteinase A was not found in axes. In cotyledons it could only be detected several days after seeds had germinated. CPR4 mRNA and polypeptide were already present in embryonic axes and cotyledons during seed maturation and decreased in both organs during germination. Purified CPR1, CPR2 and proteinase A exhibited partially different patterns of globulin degradation products in vitro. Although the cDNA-deduced amino acid sequence of the precursor of proteinase A has an N-terminal signal peptide, the enzyme was not found in vacuoles whereas the other papain-like CPRs showed vacuolar localization. Four different legumain-like cysteine proteinases (VsPB2, proteinase B, VnPB1 and VnPB2) of Vicia species were analysed. Proteinase B and VnPB1 mRNAs were detected in cotyledons and seedling organs after seeds had germinated. Proteinase B degraded globulins isolated from mature vetch seeds in vitro. VsPB2 and proteinase B are localized to protein bodies of maturing seeds and seedlings, respectively, of V. sativa. Like VsPB2 from V sativa, also VnPB2 of V. narbonensis corresponds to vacuolar processing enzymes (betaVPE). Based on these results different functions in molecular maturation and mobilization of storage proteins could be attributed to the various members of the CPR families.

  12. Sterol Regulatory Element-binding Protein (SREBP) Cleavage Regulates Golgi-to-Endoplasmic Reticulum Recycling of SREBP Cleavage-activating Protein (SCAP)*

    PubMed Central

    Shao, Wei; Espenshade, Peter J.

    2014-01-01

    Sterol regulatory element-binding protein (SREBP) transcription factors are central regulators of cellular lipogenesis. Release of membrane-bound SREBP requires SREBP cleavage-activating protein (SCAP) to escort SREBP from the endoplasmic reticulum (ER) to the Golgi for cleavage by site-1 and site-2 proteases. SCAP then recycles to the ER for additional rounds of SREBP binding and transport. Mechanisms regulating ER-to-Golgi transport of SCAP-SREBP are understood in molecular detail, but little is known about SCAP recycling. Here, we have demonstrated that SCAP Golgi-to-ER transport requires cleavage of SREBP at site-1. Reductions in SREBP cleavage lead to SCAP degradation in lysosomes, providing additional negative feedback control to the SREBP pathway. Current models suggest that SREBP plays a passive role prior to cleavage. However, we show that SREBP actively prevents premature recycling of SCAP-SREBP until initiation of SREBP cleavage. SREBP regulates SCAP in human cells and yeast, indicating that this is an ancient regulatory mechanism. PMID:24478315

  13. Molecular cloning of a cysteine proteinase cDNA from adult Ancylostoma ceylanicum by the method of rapid amplification of cDNA ends using polymerase chain reaction.

    PubMed

    Mieszczanek, J; Kofta, W; Wedrychowicz, H

    2000-12-01

    The hookworm Ancylostoma ceylanicum is a parasite of great importance in human and veterinary medicine. The most promising vaccination trials against hookworm infections are based on antigens belonging to the proteinase family. The aim of the present research was to isolate a cysteine proteinase gene from A. ceylanicum. This was achieved by rapid amplification of cDNA ends using polymerase chain reaction (RACE-PCR). A set of consensus oligonucleotide primers was designed to anneal to the conserved coding regions of cysteine proteinase. The PCR products were cloned and sequenced. The novel sequence displayed a high degree of homology with genes of cysteine proteinases known from other hookworm species. In the coding region the nucleotide identity with accp-1, the cysteine proteinase gene of A. caninum, reaches 84.3%. Analysis of the expression of acey-1. the cysteine proteinase gene of A. ceylanicum, suggests that it is produced exclusively in the gland cells of either adult worms or blood-feeding stages of A. ceylanicum.

  14. MicroRNA-34a Mediates the Autocrine Signaling of PAR2-Activating Proteinase and Its Role in Colonic Cancer Cell Proliferation

    PubMed Central

    Ma, Yiming; Bao-Han, Wuyun; Lv, Xue; Su, Yuntao; Zhao, Xinhua; Yin, Yongmei; Zhang, Xingmao; Zhou, Zhixiang; MacNaughton, Wallace K.; Wang, Hongying

    2013-01-01

    The tumor microenvironment is replete with proteinases. As a sensor of proteinases, proteinase activated receptor 2 (PAR2) plays critical roles in tumorigenesis. We showed that PAR2 and its activating proteinase were coexpressed in different colon cancer cell lines, including HT29. Inactivating proteinase or knockdown of PAR2 significantly not only reduced cell proliferation in vitro but also inhibited tumorigenicity of HT29 in vivo. In addition, activation of PAR2 promoted DNA synthesis and upregulated Cyclin D1 activity at both transcriptional and post-transcriptional levels. Further studies showed that miRNA-34a mediated PAR2-induced Cyclin D1 upregulation. Inhibition of miR-34a partially abolished the suppression of Cyclin D1 induced by PAR2 deficiency. In addition, we showed that TGF-β contributed to the regulation of miR-34a by PAR2. Finally, in colorectal carcinoma samples, upregulation of PAR2 and downregulation of miR-34a were significantly correlated with grade and lymphomatic metastasis. Our findings provide the first evidence that miRNA mediates autocrine proteinase signaling-mediated cancer cell proliferation. PMID:23991105

  15. 13C- and 1H-NMR studies of oxyanion and tetrahedral intermediate stabilization by the serine proteinases: optimizing inhibitor warhead specificity and potency by studying the inhibition of the serine proteinases by peptide-derived chloromethane and glyoxal inhibitors.

    PubMed

    Malthouse, J P G

    2007-06-01

    Catalysis by the serine proteinases proceeds via a tetrahedral intermediate whose oxyanion is stabilized by hydrogen-bonding in the oxyanion hole. There have been extensive (13)C-NMR studies of oxyanion and tetrahedral intermediate stabilization in trypsin, subtilisin and chymotrypsin using substrate-derived chloromethane inhibitors. One of the limitations of these inhibitors is that they irreversibly alkylate the active-site histidine residue which results in the oxyanion not being in the optimal position in the oxyanion hole. Substrate-derived glyoxal inhibitors are reversible inhibitors which, if they form tetrahedral adducts in the same way as substrates form tetrahedral intermediates, will overcome this limitation. Therefore we have synthesized (13)C-enriched substrate-derived glyoxal inhibitors which have allowed us to use (13)C-NMR and (1)H-NMR to determine how they interact with proteinases. It is hoped that these studies will help in the design of specific and highly potent warheads for serine proteinase inhibitors.

  16. Purification, crystallization and preliminary X-ray analysis of CMS1MS2: a cysteine proteinase from Carica candamarcensis latex

    PubMed Central

    Gomes, Marco Túlio Ribeiro; Teixeira, Raphael Dias; Ribeiro, Henrique de Assis Lopes; Turchetti, Andréia Pereira; Junqueira, Caroline Furtado; Lopes, Míriam Tereza Paz; Salas, Carlos Edmundo; Nagem, Ronaldo Alves Pinto

    2008-01-01

    Cysteine proteinases from the latex of plants of the family Caricaceae are widely used industrially as well as in pharmaceutical preparations. In the present work, a 23 kDa cysteine proteinase from Carica candamarcensis latex (designated CMS1MS2) was purified for crystallization using three chromatography steps. The enzyme shows about fourfold higher activity than papain with BAPNA as substrate. Crystals suitable for X-ray diffraction experiments were obtained by the hanging-drop method in the presence of PEG and ammonium sulfate as precipitants. The crystals are monoclinic (space group P21), with unit-cell parameters a = 53.26, b = 75.71, c = 53.23 Å, β = 96.81°, and diffract X-rays to 1.8 Å resolution. PMID:18540057

  17. Effect of the oxidizing agents chloramine-T and cigarette smoke on dog serum proteinase inhibitor(s)

    SciTech Connect

    Abrams, W.R.; Eliraz, A.; Kimbel, P.; Weinbaum, G.

    1980-08-01

    Dog serum treated with the oxidant chloramine-T is rapidly and selectively depleted of its ability to inhibit porcine pancreatic elastase or dog neutrophil elastase. Trypsin inhibitory capacity of serum is not affected. Purified dog alpha-1-proteinase inhibitor (alpha-1-PI) is similarly oxidized with an apparent rate constant of 1.1 x 10(3) M-1 sec-1. Reversal of the oxidative inactivation using dithiothreitol was demonstrated. Cigarette smoke also directly affects the inhibitory capacity of both serum and pure alpha-1-PI. These studies form a basis for developing a model of functionally deficient alpha-1-PI by taking advantage of oxidative inactivation of normal proteinase inhibitor levels.

  18. Peptides containing acylated C-terminal gem diamines: novel irreversible inactivators of the cysteine and serine proteinases.

    PubMed

    Gilmore, B F; Lynas, J F; Harriott, P; Healy, A; Walker, B

    2006-05-01

    This study reports on the synthesis of peptides containing C-terminal acylated gem-diamines and their utilization for the preparation of irreversible inactivators of the serine and cysteine proteinases. We have succeeded in obtaining an inhibitor Acetyl-Val-Pro-g-Val-CO-O-C(6)H(4)-NO(2) of neutrophil and pancreatic elastases that functions in a time-dependent manner, indicative of the action of an irreversible inactivator, functioning, most probably, through the formation of a long-lived acyl enzyme intermediate. In addition, we have demonstrated the irreversible inhibition of the cysteine proteinase bovine cathepsin B, by chloroacetyl and bromoacetyl derivatives of a dipeptide gem-diamine, Cbz-Phe-g-Ala-CO-CH(2)Hal (Hal = Br, Cl).

  19. Juvenile and adult metachromatic leukodystrophy: partial restoration of arylsulfatase A (cerebroside sulfatase) activity by inhibitors of thiol proteinases.

    PubMed Central

    von Figura, K; Steckel, F; Hasilik, A

    1983-01-01

    Arylsulfatase A polypeptides were examined in cultured fibroblasts from a patient with juvenile metachromatic leukodystrophy and three patients with the adult form of the disease, with the aid of metabolic labeling and immunoprecipitation. The mutant cells were severely deficient in the arylsulfatase polypeptides. The apparent rate of synthesis, however, as estimated from the secretion of polypeptides or activity by cells incubated in the presence of 10 mM NH4Cl was 20-50% of control. In the absence of NH4Cl, the mutant enzyme was rapidly degraded upon transport into lysosomes. In the presence of inhibitors of thiol proteinases arylsulfatase A polypeptides were partially protected from degradation, and the catalytic activity of arylsulfatase A was increased. In addition, the treatment partially corrected the capacity of the cells to degrade cerebroside sulfates. Inhibitors of thiol proteinases may be of therapeutic value in variants of metachromatic leukodystrophy, in which an unstable arylsulfatase A is synthesized. Images PMID:6136972

  20. Modification of L-triiodothyronine binding sites from rat erythrocyte membrane by heating and by proteinase treatments.

    PubMed

    Angel, R C; Botta, J A; Farías, R N

    1987-03-12

    The number of binding sites for L-triiodothyronine in rat erythrocyte membranes was increased 2-fold by incubation at 37 degrees C for 60 min. An increase of approximately 3-fold was found when the incubation was carried out at 50 degrees C. The proteinase inhibitor phenylmethylsulfonyl fluoride abolished the effect. Similar increments in the number of binding sites were obtained by treatment of the membranes with proteinases. The Kd values (0.09 X 10(-10) M and 3.6 X 10(-10) M for the high-affinity and the low-affinity binding sites, respectively) remained unchanged after the treatment, as did the free-SH group requirements, storage stability and stereospecificity. Our results suggest that endogenous proteolytic activity could be involved in the increase of the number of membrane latent sites for L-triiodothyronine.

  1. Identification and characterization of the genomic termini and cleavage/packaging signals of gallid herpesvirus 2

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Herpesvirus replication within host cells produces concatameric genomic DNA which is cleaved into unit-length genomes and packaged into the capsid by a complex of proteins. The sites of cleavage have been identified for many herpesviruses and conserved signaling sequences involved in cleavage and p...

  2. Use of Divalent Metal Ions in the DNA Cleavage Reaction of Human Type II Topoisomerases†

    PubMed Central

    Deweese, Joseph E.; Burch, Amber M.; Burgin, Alex B.; Osheroff, Neil

    2009-01-01

    All type II topoisomerases require divalent metal ions in order to cleave and ligate DNA. In order to further elucidate the mechanistic basis for these critical enzyme-mediated events, the role of the metal ion in the DNA cleavage reaction of human topoisomerase IIβ was characterized and compared to that of topoisomerase IIα. The present study utilized divalent metal ions with varying thiophilicities in conjunction with DNA cleavage substrates that substituted a sulfur atom for the 3′-bridging oxygen or the non-bridging oxygens of the scissile phosphate. Based on time courses of DNA cleavage, cation titrations, and metal ion mixing experiments, we propose the following model for the use of divalent metal ions by human type II topoisomerases. First, both enzymes employ a two-metal-ion mechanism to support DNA cleavage. Second, an interaction between one divalent metal ion and the 3′-bridging atom of the scissile phosphate greatly enhances enzyme-mediated DNA cleavage, most likely by stabilizing the leaving 3′-oxygen. Third, there is an important interaction between a divalent second metal ion and a non-bridging atom of the scissile phosphate that stimulates DNA cleavage mediated by topoisomerase IIβ. If this interaction exists in topoisomerase IIα, its effects on DNA cleavage are equivocal. This last aspect of the model highlights a difference in metal ion utilization during DNA cleavage mediated by human topoisomerase IIα and IIβ. PMID:19222228

  3. Comparative and phylogenetic perspectives of the cleavage process in tailed amphibians.

    PubMed

    Desnitskiy, Alexey G; Litvinchuk, Spartak N

    2015-10-01

    The order Caudata includes about 660 species and displays a variety of important developmental traits such as cleavage pattern and egg size. However, the cleavage process of tailed amphibians has never been analyzed within a phylogenetic framework. We use published data on the embryos of 36 species concerning the character of the third cleavage furrow (latitudinal, longitudinal or variable) and the magnitude of synchronous cleavage period (up to 3-4 synchronous cell divisions in the animal hemisphere or a considerably longer series of synchronous divisions followed by midblastula transition). Several species from basal caudate families Cryptobranchidae (Andrias davidianus and Cryptobranchus alleganiensis) and Hynobiidae (Onychodactylus japonicus) as well as several representatives from derived families Plethodontidae (Desmognathus fuscus and Ensatina eschscholtzii) and Proteidae (Necturus maculosus) are characterized by longitudinal furrows of the third cleavage and the loss of synchrony as early as the 8-cell stage. By contrast, many representatives of derived families Ambystomatidae and Salamandridae have latitudinal furrows of the third cleavage and extensive period of synchronous divisions. Our analysis of these ontogenetic characters mapped onto a phylogenetic tree shows that the cleavage pattern of large, yolky eggs with short series of synchronous divisions is an ancestral trait for the tailed amphibians, while the data on the orientation of third cleavage furrows seem to be ambiguous with respect to phylogeny. Nevertheless, the midblastula transition, which is characteristic of the model species Ambystoma mexicanum (Caudata) and Xenopus laevis (Anura), might have evolved convergently in these two amphibian orders.

  4. GPS-CCD: A Novel Computational Program for the Prediction of Calpain Cleavage Sites

    PubMed Central

    Gao, Xinjiao; Ma, Qian; Ren, Jian; Xue, Yu

    2011-01-01

    As one of the most essential post-translational modifications (PTMs) of proteins, proteolysis, especially calpain-mediated cleavage, plays an important role in many biological processes, including cell death/apoptosis, cytoskeletal remodeling, and the cell cycle. Experimental identification of calpain targets with bona fide cleavage sites is fundamental for dissecting the molecular mechanisms and biological roles of calpain cleavage. In contrast to time-consuming and labor-intensive experimental approaches, computational prediction of calpain cleavage sites might more cheaply and readily provide useful information for further experimental investigation. In this work, we constructed a novel software package of GPS-CCD (Calpain Cleavage Detector) for the prediction of calpain cleavage sites, with an accuracy of 89.98%, sensitivity of 60.87% and specificity of 90.07%. With this software, we annotated potential calpain cleavage sites for hundreds of calpain substrates, for which the exact cleavage sites had not been previously determined. In this regard, GPS-CCD 1.0 is considered to be a useful tool for experimentalists. The online service and local packages of GPS-CCD 1.0 were implemented in JAVA and are freely available at: http://ccd.biocuckoo.org/. PMID:21533053

  5. Notch receptor cleavage depends on but is not directly executed by presenilins

    PubMed Central

    Taniguchi, Yoshihito; Karlström, Helena; Lundkvist, Johan; Mizutani, Tomohiko; Otaka, Akira; Vestling, Monica; Bernstein, Alan; Donoviel, Dorit; Lendahl, Urban; Honjo, Tasuku

    2002-01-01

    Notch receptors undergo three distinct proteolytic cleavages during maturation and activation. The third cleavage occurs within the plasma membrane and results in the release and translocation of the intracellular domain into the nucleus to execute Notch signaling. This so-called γ-secretase cleavage is under the control of presenilins, but it is not known whether presenilins themselves carry out the cleavage or whether they act by means of yet-unidentified γ-secretase(s). In this article, we show that Notch intracellular cleavage in intact cells completely depends on presenilins. In contrast, partial purification of the Notch cleavage activity reveals an activity, which is present only in protein extracts from presenilin-containing cells, and which does not comigrate with presenilin. This finding provides evidence for the existence of a specific Notch-processing activity, which is physically distinct from presenilins. We conclude from these experiments that presenilins are critically required for Notch intracellular cleavage but are not themselves directly mediating the cleavage. PMID:11891288

  6. Proteolytic Characteristics of Cathepsin D Related to the Recognition and Cleavage of Its Target Proteins

    PubMed Central

    Sun, Huiying; Lou, Xiaomin; Shan, Qiang; Zhang, Ju; Zhu, Xu; Zhang, Jia; Wang, Yang; Xie, Yingying; Xu, Ningzhi; Liu, Siqi

    2013-01-01

    Cathepsin D (CD) plays an important role in both biological and pathological processes, although the cleavage characteristics and substrate selection of CD have yet to be fully explored. We employed liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the CD cleavage sites in bovine serum albumin (BSA). We found that the hydrophobic residues at P1 were not only a preferential factor for CD cleavage but that the hydrophobicity at P1’ also contributed to CD recognition. The concept of hydrophobic scores of neighbors (HSN) was proposed to describe the hydrophobic microenvironment of CD recognition sites. The survey of CD cleavage characteristics in several proteins suggested that the HSN was a sensitive indicator for judging the favorable sites in peptides for CD cleavage, with HSN values of 0.5–1.0 representing a likely threshold. Ovalbumin (OVA), a protein resistant to CD cleavage in its native state, was easily cleaved by CD after denaturation, and the features of the cleaved peptides were quite similar to those found in BSA, where a higher HSN value indicated greater cleavability. We further conducted two-dimensional gel electrophoresis (2DE) to find more proteins that were insensitive to CD cleavage in CD-knockdown cells. Based on an analysis of secondary and three-dimensional structures, we postulated that intact proteins with a structure consisting of all α-helices would be relatively accessible to CD cleavage. PMID:23840360

  7. Hepatocyte growth factor activator is a potential target proteinase for Kazal-type inhibitor in turkey (Meleagris gallopavo) seminal plasma.

    PubMed

    Słowińska, Mariola; Bukowska, Joanna; Hejmej, Anna; Bilińska, Barbara; Kozłowski, Krzysztof; Jankowski, Jan; Ciereszko, Andrzej

    2015-08-01

    A peculiar characteristic of turkey seminal plasma is the increased activity of serine proteinases. It is of interest if the single-domain Kazal-type inhibitor controls the activity of turkey seminal plasma proteinases. Pure preparations of the Kazal-type inhibitor and anti-Kazal-type inhibitor monospecific immunoglobulin Gs were used as ligands in affinity chromatography for proteinase isolation from turkey seminal plasma. Gene expression and the immunohistochemical detection of the single-domain Kazal-type inhibitor in the reproductive tract of turkey toms are described. The hepatocyte growth factor activator (HGFA) was identified in the binding fraction in affinity chromatography. Hepatocyte growth factor activator activity was inhibited by the Kazal-type inhibitor in a dose-dependent manner. This protease was a primary physiological target for the single-domain Kazal-type inhibitor. Numerous proteoforms of HGFA were present in turkey seminal plasma, and phosphorylation was the primary posttranslational modification of HGFA. In addition to HGFA, acrosin was a target proteinase for the single-domain Kazal-type inhibitor. In seminal plasma, acrosin was present only in complexes with the Kazal-type inhibitor and was not present as a free enzyme. The single-domain Kazal-type inhibitor was specific for the reproductive tract. The germ cell-specific expression of Kazal-type inhibitors in the testis indicated an important function in spermatogenesis; secretion by the epithelial cells of the epididymis and the ductus deferens indicated that the Kazal-type inhibitor was an important factor involved in the changes in sperm membranes during maturation and in the maintenance of the microenvironment in which sperm maturation occurred and sperm was stored. The role of HGFA in these processes remains to be established.

  8. N-terminal extension of the yeast IA3 aspartic proteinase inhibitor relaxes the strict intrinsic selectivity.

    PubMed

    Winterburn, Tim J; Phylip, Lowri H; Bur, Daniel; Wyatt, David M; Berry, Colin; Kay, John

    2007-07-01

    Yeast IA(3) aspartic proteinase inhibitor operates through an unprecedented mechanism and exhibits a remarkable specificity for one target enzyme, saccharopepsin. Even aspartic proteinases that are very closely similar to saccharopepsin (e.g. the vacuolar enzyme from Pichia pastoris) are not susceptible to significant inhibition. The Pichia proteinase was selected as the target for initial attempts to engineer IA(3) to re-design the specificity. The IA(3) polypeptides from Saccharomyces cerevisiae and Saccharomyces castellii differ considerably in sequence. Alterations made by deletion or exchange of the residues in the C-terminal segment of these polypeptides had only minor effects. By contrast, extension of each of these wild-type and chimaeric polypeptides at its N-terminus by an MK(H)(7)MQ sequence generated inhibitors that displayed subnanomolar potency towards the Pichia enzyme. This gain-in-function was completely reversed upon removal of the extension sequence by exopeptidase trimming. Capture of the potentially positively charged aromatic histidine residues of the extension by remote, negatively charged side-chains, which were identified in the Pichia enzyme by modelling, may increase the local IA(3) concentration and create an anchor that enables the N-terminal segment residues to be harboured in closer proximity to the enzyme active site, thus promoting their interaction. In saccharopepsin, some of the counterpart residues are different and, consistent with this, the N-terminal extension of each IA(3) polypeptide was without major effect on the potency of interaction with saccharopepsin. In this way, it is possible to convert IA(3) polypeptides that display little affinity for the Pichia enzyme into potent inhibitors of this proteinase and thus broaden the target selectivity of this remarkable small protein.

  9. Bond cleavage of lignin model compounds into aromatic monomers using supported metal catalysts in supercritical water

    PubMed Central

    Yamaguchi, Aritomo; Mimura, Naoki; Shirai, Masayuki; Sato, Osamu

    2017-01-01

    More efficient use of lignin carbon is necessary for carbon-efficient utilization of lignocellulosic biomass. Conversion of lignin into valuable aromatic compounds requires the cleavage of C–O ether bonds and C–C bonds between lignin monomer units. The catalytic cleavage of C–O bonds is still challenging, and cleavage of C–C bonds is even more difficult. Here, we report cleavage of the aromatic C–O bonds in lignin model compounds using supported metal catalysts in supercritical water without adding hydrogen gas and without causing hydrogenation of the aromatic rings. The cleavage of the C–C bond in bibenzyl was also achieved with Rh/C as a catalyst. Use of this technique may greatly facilitate the conversion of lignin into valuable aromatic compounds. PMID:28387304

  10. Engineering a ribozyme cleavage-induced split fluorescent aptamer complementation assay

    PubMed Central

    Ausländer, Simon; Fuchs, David; Hürlemann, Samuel; Ausländer, David; Fussenegger, Martin

    2016-01-01

    Hammerhead ribozymes are self-cleaving RNA molecules capable of regulating gene expression in living cells. Their cleavage performance is strongly influenced by intra-molecular loop–loop interactions, a feature not readily accessible through modern prediction algorithms. Ribozyme engineering and efficient implementation of ribozyme-based genetic switches requires detailed knowledge of individual self-cleavage performances. By rational design, we devised fluorescent aptamer-ribozyme RNA architectures that allow for the real-time measurement of ribozyme self-cleavage activity in vitro. The engineered nucleic acid molecules implement a split Spinach aptamer sequence that is made accessible for strand displacement upon ribozyme self-cleavage, thereby complementing the fluorescent Spinach aptamer. This fully RNA-based ribozyme performance assay correlates ribozyme cleavage activity with Spinach fluorescence to provide a rapid and straightforward technology for the validation of loop–loop interactions in hammerhead ribozymes. PMID:26939886

  11. An in-built proteinase inhibitor system for the protection of recombinant proteins recovered from transgenic plants.

    PubMed

    Rivard, Daniel; Anguenot, Raphaël; Brunelle, France; Le, Van Quy; Vézina, Louis-Philippe; Trépanier, Sonia; Michaud, Dominique

    2006-05-01

    Proteolytic degradation represents a significant barrier to the efficient production of several recombinant proteins in plants, both in vivo during their expression and in vitro during their recovery from source tissues. Here, we describe a strategy to protect recombinant proteins during the recovery process, based on the coexpression of a heterologous proteinase inhibitor acting as a 'mouse trap' against the host proteases during extraction. After confirming the importance of trypsin- and chymotrypsin-like activities in crude protein extracts of potato (Solanum tuberosum L.) leaves, transgenic lines of potato expressing either tomato cathepsin D inhibitor (CDI) or bovine aprotinin, both active against trypsin and chymotrypsin, were generated by Agrobacterium tumefaciens-mediated genetic transformation. Leaf crude protein extracts from CDI-expressing lines, showing decreased levels of cathepsin D-like and ribulose 1,5-bisphosphate carboxylase/oxygenase hydrolysing activities in vitro, conducted decreased turnover rates of the selection marker protein neomycin phosphotransferase II (NPTII) relative to the turnover rates measured for transgenic lines expressing only the marker protein. A similar stabilizing effect on NPTII was observed in leaf protein extracts from plant lines coexpressing bovine aprotinin, confirming the ability of ectopically expressed broad-spectrum serine proteinase inhibitors to reproduce the protein-stabilizing effect of low-molecular-weight proteinase inhibitors generally added to protein extraction media.

  12. Secreted Frizzled Related Protein 2 is a procollagen C proteinase enhancer with a role in myocardial infarction-associated fibrosis

    PubMed Central

    Kobayashi, Koichi; Luo, Min; Zhang, Yue; Wilkes, David C.; Ge, Gaoxiang; Grieskamp, Thomas; Yamada, Chikaomi; Liu, Ting-Chun; Huang, Guorui; Basson, Craig T.; Kispert, Andreas; Greenspan, Daniel S.; Sato, Thomas N.

    2009-01-01

    Secreted frizzled related proteins (sFRPs) have emerged as key regulators of a wide range of developmental and disease processes, with virtually all known functions of mammalian sFRPs attributed to their ability to antagonize Wnt signaling. Recently however, the Xenopus and zebrafish sFRP, Sizzled, was shown to function as an antagonist of Chordin processing by Tolloid-like metalloproteinases, leading to the proposal that sFRPs may function as evolutionarily-conserved antagonists of the chordinase activities of this class of proteinases. Herein, in contrast to this proposal, we show that the mammalian sFRP, sFRP2, does not affect Chordin processing, but instead can serve as a direct enhancer of the procollagen C-proteinase activity of Tolloid-like metalloproteinases. We further show that the level of fibrosis, in which procollagen processing by Tolloid-like proteinases plays a rate-limiting role, is markedl