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Sample records for 3c protease complexed

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

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

    2016-01-01

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

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

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

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

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

    2016-01-01

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

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

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

    2016-01-01

    stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious...... different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested...

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

    Raheem Ullah

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

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

    Tufail, Soban; Ismat, Fouzia; Imran, Muhammad; Iqbal, Mazhar; Mirza, Osman; Rhaman, Moazur

    2016-01-01

    Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested. Though number of fusion proteins tested is limited, we expect that these finding will facilitate the use of the human rhinovirus 3C protease in recombinant protein production for pharmaceutical and biotechnological applications. PMID:27093053

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

    Erin J Walker

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

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

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

    2016-07-15

    Human rhinovirus (HRV) is a primary cause of common cold and is linked to exacerbation of underlying respiratory diseases such as asthma and COPD. HRV 3C protease, which is responsible for cleavage of viral polyprotein in to proteins essential for viral life-cycle, represents an important target. We have designed proline- and azetidine-based analogues of Rupintrivir that target the P2 pocket of the binding site. Potency optimization, aided with X-ray crystallography and quantum mechanical calculations, led to compounds with activity against a broad spectrum of HRV serotypes. Altogether, these compounds represent alternative starting points to identify promising leads in our continual efforts to treat HRV infections. PMID:27265257

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

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

    2016-01-01

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

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

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

    2016-01-01

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

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

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

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

    Kuo-Feng Weng

    2009-09-01

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

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

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

    2016-04-01

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

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

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

    2016-03-01

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

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

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

    2010-01-01

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

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

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

    2016-01-01

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

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

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

    2016-09-14

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

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

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

    2008-04-01

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

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

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

    2016-01-01

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

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

    Chia-Nan Chen

    2005-01-01

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

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

    Gullberg, Maria; Polacek, Charlotta; Belsham, Graham

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

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

    Gullberg, Maria; Polacek, Charlotta; Belsham, Graham

    2014-01-01

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

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

    Porta, Claudine; Xu, Xiaodong; Loureiro, Silvia;

    2013-01-01

    Foot-and-mouth disease virus (FMDV) is a significant economically and distributed globally pathogen of Artiodactyla. Current vaccines are chemically inactivated whole virus particles that require large-scale virus growth in strict bio-containment with the associated risks of accidental release...... precursor P1-2A by the action of the virus-encoded 3C protease. To date recombinant empty capsid assembly has been limited by poor expression levels, restricting the development of empty capsids as a viable vaccine. Here expression of the FMDV structural protein precursor P1-2A in insect cells is shown...... assembled into empty capsids. Expression was independent of the insect host cell background and leads to capsids that are recognised as authentic by a range of anti-FMDV bovine sera suggesting their feasibility as an alternate vaccine....

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

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

    2013-01-01

    The foot-and-mouth disease virus (FMDV) capsid protein precursor (P1-2A) is processed by the virus-encoded 3C protease (3Cpro) to produce VP0, VP3, VP1 and 2A. Within the virus-encoded polyprotein, the P1-2A and 3Cpro can be expected to be produced at equivalent concentrations. However, using...... with that achieved with a single P1-2A-3C polyprotein. Expression of the FMDV 3Cpro is poorly tolerated by mammalian cells and higher levels of the 3Cpro greatly inhibit protein expression. In addition, it is demonstrated that both the intact P1-2A precursor and the processed capsid proteins can be efficiently...... detected by FMDV antigen detection assays. Furthermore, the P1-2A and the processed forms each bind to the integrin αvβ6, the major FMDV receptor. These results contribute to the development of systems which efficiently express the components of empty capsid particles and may represent the basis for safer...

  5. Molecular dynamics simulations of HIV-1 protease complexed with saquinavir

    Watson, S. J.

    2009-01-01

    Inhibition of the Human Immunode�ficiency virus type-1 (HIV-1) protease enzyme blocks HIV-1 replication. Protease inhibitor drugs have successfully been used as a therapy for HIV-infected individuals to reduce their viral loads and slow the progression to Acquired Immune Defi�ciency Syndrome (AIDS). However, mutations readily and rapidly accrue in the protease gene resulting in a reduced sensitivity of the protein to the inhibitor. In this thesis, molecular dynamics simulations (MDS)...

  6. Conserved hydrogen bonds and water molecules in MDR HIV-1 protease substrate complexes

    Liu, Zhigang [Wayne State Univ., Detroit, MI (United States); Case Western Reserve Univ., Cleveland, OH (United States); Harbor Hospital Baltimore, MD (United States); Wang, Yong [Wayne State Univ., Detroit, MI (United States); Yedidi, Ravikiran S. [Wayne State Univ., Detroit, MI (United States); National Institutes of Health, Bethesda, MD (United States); Dewdney, Tamaria G. [Wayne State Univ., Detroit, MI (United States); Reiter, Samuel J. [Wayne State Univ., Detroit, MI (United States); Brunzelle, Joseph S. [Northwestern Univ. Feinberg School of Medicine, Chicago, IL (United States); Kovari, Iulia A. [Wayne State Univ., Detroit, MI (United States); Kovari, Ladislau C. [Wayne State Univ., Detroit, MI (United States)

    2012-12-19

    Success of highly active antiretroviral therapy (HAART) in anti-HIV therapy is severely compromised by the rapidly developing drug resistance. HIV-1 protease inhibitors, part of HAART, are losing their potency and efficacy in inhibiting the target. Multi-drug resistant (MDR) 769 HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, 90) was selected for the present study to understand the binding to its natural substrates. The nine crystal structures of MDR769 HIV-1 protease substrate hepta-peptide complexes were analyzed in order to reveal the conserved structural elements for the purpose of drug design against MDR HIV-1 protease. Our structural studies demonstrated that highly conserved hydrogen bonds between the protease and substrate peptides, together with the conserved crystallographic water molecules, played a crucial role in the substrate recognition, substrate stabilization and protease stabilization. Additionally, the absence of the key flap-ligand bridging water molecule might imply a different catalytic mechanism of MDR769 HIV-1 protease compared to that of wild type (WT) HIV-1 protease.

  7. Structure of a HIV-1 Protease-Inhibitor Complex determined at 1.1A resolution

    Brynda, Jiří; Řezáčová, Pavlína; Štouračová, Renata; Fábry, Milan; Hradilek, Martin; Souček, Milan; Konvalinka, Jan; Sedláček, Juraj

    Jena : Jena, 2002, s. -. [International Conference on the crystal lization of Biological Macromolecules /9./. Jena, Německo (DE), 23.03.2002-28.03.2002] Institutional research plan: CEZ:AV0Z4055905; CEZ:AV0Z5052915 Keywords : Structure of a HIV-1 * protease * Inhibitor complex Subject RIV: EB - Genetics ; Molecular Biology

  8. Comparative study of antithrombin III. Protease complex metabolism by fibroblasts and vascular endothelial cells

    125I-labeled human antithrombin III (125I-AT III).protease complexes are specifically bound to both cultured human skin fibroblast (HSF) cells and adult bovine aortic endothelial (ABAE) cells; however, there is a significant difference in the rate and degree of metabolism of the complexes by these two cell types. HSF cells appear to internalize the complexes at a rate of about 2.5 pmole/1 X 10(6) cells/h and subsequently degrade them at a rate of 0.6 pmole/1 X 10(6) cells/h. ABAE cells internalize and degrade the complexes at rates approximately 100 and 30 times lower, respectively. Neither cell type interacts with free 125I-AT III but only with its combined form with either thrombin or trypsin. These data indicate the major role of HSF cells in the removal of AT III.protease complexes from extravascular spaces in the body, in contrast to the inert vascular surface with regard to AT III.protease complexes provided by the vascular endothelium

  9. Crystal structures of inhibitor complexes of human T-cell leukemia virus (HTLV-1) protease

    Satoh, Tadashi; Li, Mi; Nguyen, Jeffrey-Tri; Kiso, Yoshiaki; Gustchina, Alla; Wlodawer, Alexander (NCI); (Kyoto)

    2010-09-28

    Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus associated with several serious diseases, such as adult T-cell leukemia and tropical spastic paraparesis/myelopathy. For a number of years, the protease (PR) encoded by HTLV-1 has been a target for designing antiviral drugs, but that effort was hampered by limited available structural information. We report a high-resolution crystal structure of HTLV-1 PR complexed with a statine-containing inhibitor, a significant improvement over the previously available moderate-resolution structure. We also report crystal structures of the complexes of HTLV-1 PR with five different inhibitors that are more compact and more potent. A detailed study of structure-activity relationships was performed to interpret in detail the influence of the polar and hydrophobic interactions between the inhibitors and the protease.

  10. Crystal Structures of Inhibitir Complexes of Human T-Cell Leukemia Virus (HTLV-1) Protease

    Satoh, Tadashi; Li, Mi; Nguyen, Jeffrey-Tri; Kiso, Yoshiaki; Gustchina, Alla; Wlodawer, Alexander (NCI); (Kyoto)

    2010-09-17

    Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus associated with several serious diseases, such as adult T-cell leukemia and tropical spastic paraparesis/myelopathy. For a number of years, the protease (PR) encoded by HTLV-1 has been a target for designing antiviral drugs, but that effort was hampered by limited available structural information. We report a high-resolution crystal structure of HTLV-1 PR complexed with a statine-containing inhibitor, a significant improvement over the previously available moderate-resolution structure. We also report crystal structures of the complexes of HTLV-1 PR with five different inhibitors that are more compact and more potent. A detailed study of structure-activity relationships was performed to interpret in detail the influence of the polar and hydrophobic interactions between the inhibitors and the protease.

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

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

    2012-03-30

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

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

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

    2010-03-04

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

  13. NMR Analysis of a Novel Enzymatically Active Unlinked Dengue NS2B-NS3 Protease Complex*

    Kim, Young Mee; Gayen, Shovanlal; Kang, CongBao; Joy, Joma; Huang, Qiwei; Chen, Angela Shuyi; Wee, John Liang Kuan; Ang, Melgious Jin Yan; Lim, Huichang Annie; Hung, Alvin W.; Li, Rong; Noble, Christian G.; Lee, Le Tian; Yip, Andy; Wang, Qing-Yin; Chia, Cheng San Brian; Hill, Jeffrey; Shi, Pei-Yong; Keller, Thomas H.

    2013-01-01

    The dengue virus (DENV) is a mosquito-borne pathogen responsible for an estimated 100 million human infections annually. The viral genome encodes a two-component trypsin-like protease that contains the cofactor region from the nonstructural protein NS2B and the protease domain from NS3 (NS3pro). The NS2B-NS3pro complex plays a crucial role in viral maturation and has been identified as a potential drug target. Using a DENV protease construct containing NS2B covalently linked to NS3pro via a Gly4-Ser-Gly4 linker (“linked protease”), previous x-ray crystal structures show that the C-terminal fragment of NS2B is remote from NS3pro and exists in an open state in the absence of an inhibitor; however, in the presence of an inhibitor, NS2B complexes with NS3pro to form a closed state. This linked enzyme produced NMR spectra with severe signal overlap and line broadening. To obtain a protease construct with a resolved NMR spectrum, we expressed and purified an unlinked protease complex containing a 50-residue segment of the NS2B cofactor region and NS3pro without the glycine linker using a coexpression system. This unlinked protease complex was catalytically active at neutral pH in the absence of glycerol and produced dispersed cross-peaks in a 1H-15N heteronuclear single quantum correlation spectrum that enabled us to conduct backbone assignments using conventional techniques. In addition, titration with an active-site peptide aldehyde inhibitor and paramagnetic relaxation enhancement studies demonstrated that the unlinked DENV protease exists predominantly in a closed conformation in solution. This protease complex can serve as a useful tool for drug discovery against DENV. PMID:23511634

  14. Crystal structure of Zika virus NS2B-NS3 protease in complex with a boronate inhibitor.

    Lei, Jian; Hansen, Guido; Nitsche, Christoph; Klein, Christian D; Zhang, Linlin; Hilgenfeld, Rolf

    2016-07-29

    The ongoing Zika virus (ZIKV) outbreak is linked to severe neurological disorders. ZIKV relies on its NS2B/NS3 protease for polyprotein processing; hence, this enzyme is an attractive drug target. The 2.7 angstrom; crystal structure of ZIKV protease in complex with a peptidomimetic boronic acid inhibitor reveals a cyclic diester between the boronic acid and glycerol. The P2 4-aminomethylphenylalanine moiety of the inhibitor forms a salt-bridge with the nonconserved Asp(83) of NS2B; ion-pairing between Asp(83) and the P2 residue of the substrate likely accounts for the enzyme's high catalytic efficiency. The unusual dimer of the ZIKV protease:inhibitor complex seen in the crystal may provide a model for assemblies formed at high local concentrations of protease at the endoplasmatic reticulum membrane, the site of polyprotein processing. PMID:27386922

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

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

    2012-01-01

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

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

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

    2016-01-01

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

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

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

    2014-01-15

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

  18. NMR study of xenotropic murine leukemia virus-related virus protease in a complex with amprenavir

    Highlights: ► Protease (PR) of XMR virus (XMRV) was successfully synthesized with cell-free system. ► Interface of XMRV PR with an inhibitor, amprenavir (APV), was identified with NMR. ► Structural heterogeneity is induced for two PR protomers in the APV:PR = 1:2 complex. ► Structural heterogeneity is transmitted even to distant regions from the interface. ► Long-range transmission of structural change may be utilized for drug discovery. -- Abstract: Xenotropic murine leukemia virus-related virus (XMRV) is a virus created through recombination of two murine leukemia proviruses under artificial conditions during the passage of human prostate cancer cells in athymic nude mice. The homodimeric protease (PR) of XMRV plays a critical role in the production of functional viral proteins and is a prerequisite for viral replication. We synthesized XMRV PR using the wheat germ cell-free expression system and carried out structural analysis of XMRV PR in a complex with an inhibitor, amprenavir (APV), by means of NMR. Five different combinatorially 15N-labeled samples were prepared and backbone resonance assignments were made by applying Otting’s method, with which the amino acid types of the [1H, 15N] HSQC resonances were automatically identified using the five samples (Wu et al., 2006) . A titration experiment involving APV revealed that one APV molecule binds to one XMRV PR dimer. For many residues, two distinct resonances were observed, which is thought to be due to the structural heterogeneity between the two protomers in the APV:XMRV PR = 1:2 complex. PR residues at the interface with APV have been identified on the basis of chemical shift perturbation and identification of the intermolecular NOEs by means of filtered NOE experiments. Interestingly, chemical shift heterogeneity between the two protomers of XMRV PR has been observed not only at the interface with APV but also in regions apart from the interface. This indicates that the structural heterogeneity

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

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

  20. Insights into affinity and specificity in the complexes of α-lytic protease and its inhibitor proteins: binding free energy from molecular dynamics simulation†

    Deng, Nan-Jie; Cieplak, Piotr

    2009-01-01

    We report the binding free energy calculation and its decomposition for the complexes of α-lytic protease and its protein inhibitors using molecular dynamics simulation. Standard mechanism serine protease inhibitors eglin C and OMTKY3 are known to have strong binding affinity for many serine proteases. Their binding loops have significant similarities, including a common P1 Leu as the main anchor in the binding interface. However, recent experiments demonstrate that the two inhibitors have va...

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

    Little Tom J

    2009-06-01

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

  2. Substrate binding and specificity of rhomboid intramembrane protease revealed by substrate-peptide complex structures

    Zoll, Sebastian; Stanchev, Stancho; Began, Jakub; Škerle, Jan; Lepšík, Martin; Peclinovská, Lucie; Majer, Pavel; Stříšovský, Kvido

    2014-01-01

    Roč. 33, č. 20 (2014), s. 2408-2421. ISSN 0261-4189 R&D Projects: GA ČR GAP305/11/1886; GA MŠk(CZ) LK11206; GA MŠk LO1302; GA ČR GBP208/12/G016 Institutional support: RVO:61388963 Keywords : intramembrane protease * rhomboid family * rhomboid protease * structure * substrate recognition Subject RIV: CE - Biochemistry Impact factor: 10.434, year: 2014

  3. Crystallization, high-resolution data collection and preliminary crystallographic analysis of Aura virus capsid protease and its complex with dioxane

    A 17 kDa capsid protease domain from Aura virus was purified, crystallized together with its complex with dioxane and characterized by the X-ray diffraction method. The C-terminal protease domain of capsid protein from Aura virus expressed in a bacterial expression system has been purified to homogeneity and crystallized. Crystals suitable for X-ray diffraction analysis were obtained by the vapour-diffusion method using 0.1 M bis-tris and polyethylene glycol monomethyl ether 2000. Crystals of the C-terminal protease domain of capsid protein in complex with dioxane were also produced and crystal data were obtained. Both crystals belonged to space group C2, with unit-cell parameters a = 79.6, b = 35.2, c = 49.5 Å. High-resolution data sets were collected to a resolution of 1.81 Å for the native protein and 1.98 Å for the complex. Preliminary crystallographic studies suggested the presence of a single molecule in the crystallographic asymmetric unit, with a solvent content of 38.5%

  4. Structure of Compstatin in Complex with Complement Component C3c Reveals a New Mechanism of Complement Inhibition

    Janssen, B.J.C.; Halff, E.F.; Lambris, J.D.; Gros, P.

    2007-01-01

    Undesired complement activation is a major cause of tissue injury in various pathological conditions and contributes to several immune complex diseases. Compstatin, a 13-residue peptide, is an effective inhibitor of the activation of complement component C3 and thus blocks a central and crucial step

  5. Structure of Compstatin in Complex with Complement Component C3c Reveals a New Mechanism of Complement Inhibition

    Janssen, B.J.C.; Halff, E.F.; LAMBRIS, J. D.; Gros, P

    2007-01-01

    Undesired complement activation is a major cause of tissue injury in various pathological conditions and contributes to several immune complex diseases. Compstatin, a 13-residue peptide, is an effective inhibitor of the activation of complement component C3 and thus blocks a central and crucial step in the complement cascade. The precise binding site on C3, the structure in the bound form, and the exact mode of action of compstatin are unknown. Here we present the crystal structure of compsta...

  6. Nitrogen-15 NMR spectroscopy of the catalytic-triad histidine of a serine protease in peptide boronic acid inhibitor complexes

    15N NMR spectroscopy was used to examine the active-site histidyl residue of α-lytic protease in peptide boronic acid inhibitor complexes. Two distinct types of complexes were observed: (1) Boronic acids that are analogues of substrates form complexes in which the active-site imidazole ring is protonated and both imidazole N-H protons are strongly hydrogen bonded. (2) Boronic acids that are not substrate analogues form complexes in which Nε2 of the active-site histidine is covalently bonded to the boron atom of the inhibitor. The proton bound to Nδ1 of the histidine in these histidine-boronate adducts remains strongly hydrogen bonded, presumably to the active-site aspartate. In both types of complexes the N-H protons of His-57 exchange unusually slowly as evidenced by the room temperature visibility of the low-field 1H resonances and the 15N-H spin couplings. These results indicate that occupancy of the specificity subsites may be required to fully form the transition-state binding site. The significance of these findings for understanding inhibitor binding and the catalytic mechanism of serine proteases is discussed

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

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

    2010-09-01

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

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

    Machalski, J; Stawarz, L; Wezgowiec, M

    2016-01-01

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

  9. Crystal structure of HIV-1 protease in situ product complex and observation of a low-barrier hydrogen bond between catalytic aspartates

    Das, Amit; Prashar, Vishal; Mahale, Smita; Serre, L; Ferrer, J.-L.; Hosur, M. V.

    2006-01-01

    HIV-1 protease is an effective target for designing drugs against AIDS, and structural information about the true transition state and the correct mechanism can provide important inputs. We present here the three-dimensional structure of a bi-product complex between HIV-1 protease and the two cleavage product peptides AETF and YVDGAA. The structure, refined against synchrotron data to 1.65 Å resolution, shows the occurrence of the cleavage reaction in the crystal, with the product peptides st...

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

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

    2012-10-23

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

  11. A Genomic Analysis of Rat Proteases and Protease Inhibitors

    Puente, Xose S.; López-Otín, Carlos

    2004-01-01

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

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

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

    2010-01-01

    Foot-and-mouth disease (FMD) is a serious, widespread viral disease of cloven-hoofed animals, including important agricultural species such as cattle, sheep, pigs and goats (19, 45). The virus spreads rapidly and, although endemic and epidemic situations can be controlled using vaccines that are ...... of the molecular basis of viral replication. In this paper we focus on the structural basis of the cleavage activity of FMDV 3Cpro; as a highly conserved viral enzyme (11), FMDV 3Cpro is a potential drug target....

  13. MBL-associated serine protease-3 circulates in high serum concentrations predominantly in complex with Ficolin-3 and regulates Ficolin-3 mediated complement activation

    Skjoedt, Mikkel-Ole; Palarasah, Yaseelan; Munthe-Fog, Lea;

    2010-01-01

    The human lectin complement pathway (LCP) involves circulating complexes consisting of mannose-binding lectin (MBL) or ficolins in association with serine proteases named MASP-1, -2 and -3 and a non-enzymatic protein, sMAP. MASP-3 originates from the MASP1 gene through differential splicing and...

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

    Hansen, Cecilie Bo; Csuka, Dorottya; Munthe-Fog, Lea; Varga, Lilian; Farkas, Henriette; Hansen, Karin Møller; Koch, Claus; Skjødt, Karsten; Garred, Peter; Skjoedt, Mikkel-Ole

    2015-01-01

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

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

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

    2008-01-01

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

  16. High-resolution complex of papain with remnants of a cysteine protease inhibitor derived from Trypanosoma brucei

    Alphey, Magnus S.; Hunter, William N.

    2006-01-01

    Attempts to cocrystallize the cysteine protease papain derived from the latex of Carica papaya with an inhibitor of cysteine proteases (ICP) from Trypanosoma brucei were unsuccessful. However, crystals of papain that diffracted to higher resolution, 1.5 Å, than other crystals of this archetypal cysteine protease were obtained, so the analysis was continued. Surprisingly, the substrate-binding cleft was occupied by two short peptide fragments which have been assigned as remnants of ICP. Compar...

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

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

    2007-01-01

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

  18. Extended intermolecular interactions in a serine protease-canonical inhibitor complex account for strong and highly specific inhibition.

    Fodor, Krisztián; Harmat, Veronika; Hetényi, Csaba; Kardos, József; Antal, József; Perczel, András; Patthy, András; Katona, Gergely; Gráf, László

    2005-07-01

    We have previously shown that a trypsin inhibitor from desert locust Schistocerca gregaria (SGTI) is a taxon-specific inhibitor that inhibits arthropod trypsins, such as crayfish trypsin, five orders of magnitude more effectively than mammalian trypsins. Thermal denaturation experiments, presented here, confirm the inhibition kinetics studies; upon addition of SGTI the melting temperatures of crayfish and bovine trypsins increased 27 degrees C and 4.5 degrees C, respectively. To explore the structural features responsible for this taxon specificity we crystallized natural crayfish trypsin in complex with chemically synthesized SGTI. This is the first X-ray structure of an arthropod trypsin and also the highest resolution (1.2A) structure of a trypsin-protein inhibitor complex reported so far. Structural data show that in addition to the primary binding loop, residues P3-P3' of SGTI, the interactions between SGTI and the crayfish enzyme are also extended over the P12-P4 and P4'-P5' regions. This is partly due to a structural change of region P10-P4 in the SGTI structure induced by binding of the inhibitor to crayfish trypsin. The comparison of SGTI-crayfish trypsin and SGTI-bovine trypsin complexes by structure-based calculations revealed a significant interaction energy surplus for the SGTI-crayfish trypsin complex distributed over the entire binding region. The new regions that account for stronger and more specific binding of SGTI to crayfish than to bovine trypsin offer new inhibitor sites to engineer in order to develop efficient and specific protease inhibitors for practical use. PMID:15922357

  19. Crystal structures of yellowtail ascites virus VP4 protease: trapping an internal cleavage site trans acyl-enzyme complex in a native Ser/Lys dyad active site.

    Chung, Ivy Yeuk Wah; Paetzel, Mark

    2013-05-01

    Yellowtail ascites virus (YAV) is an aquabirnavirus that causes ascites in yellowtail, a fish often used in sushi. Segment A of the YAV genome codes for a polyprotein (pVP2-VP4-VP3), where processing by its own VP4 protease yields the capsid protein precursor pVP2, the ribonucleoprotein-forming VP3, and free VP4. VP4 protease utilizes the rarely observed serine-lysine catalytic dyad mechanism. Here we have confirmed the existence of an internal cleavage site, preceding the VP4/VP3 cleavage site. The resulting C-terminally truncated enzyme (ending at Ala(716)) is active, as shown by a trans full-length VP4 cleavage assay and a fluorometric peptide cleavage assay. We present a crystal structure of a native active site YAV VP4 with the internal cleavage site trapped as trans product complexes and trans acyl-enzyme complexes. The acyl-enzyme complexes confirm directly the role of Ser(633) as the nucleophile. A crystal structure of the lysine general base mutant (K674A) reveals the acyl-enzyme and empty binding site states of VP4, which allows for the observation of structural changes upon substrate or product binding. These snapshots of three different stages in the VP4 protease reaction mechanism will aid in the design of anti-birnavirus compounds, provide insight into previous site-directed mutagenesis results, and contribute to understanding of the serine-lysine dyad protease mechanism. In addition, we have discovered that this protease contains a channel that leads from the enzyme surface (adjacent to the substrate binding groove) to the active site and the deacylating water. PMID:23511637

  20. Characteristic Ligand-Induced Crystal Forms of HIV-1 Protease Complexes: A Novel Discovery of X-Ray Crystallography

    Mixtures of saquinavir (SQV) and ritonavir (RTV) were cocrystallized with HIV-1 protease (PR) in an attempt to compare their relative potencies using a crystallographic approach and factors responsible for the respective crystal forms obtained were examined. The mixture ratio of the SQV/RTV was in the range of 1:1 to 1:50 with increasing concentration of dimethyl sulphoxide (DMSO) used. Two crystal forms of PR complexes were obtained. At concentrations of 0.8 and 1.2 % DMSO using 1:1 and 1:15 ratios of SQV/RTV, the crystal form was monoclinic while increasing the concentration of DMSO to 3.2 and 5.0% using 1:15 and 1:50 ratios of SQV/RTV, the orthorhombic crystal form was obtained. The high resolution X-ray crystal structures of the PR/ inhibitor complexes reveal that crystal forms with respective space groups are dependent on the occupancy of either SQV or RTV in the active site of the PR. The occupancy of either of the PR inhibitors in the active site of PR has interestingly demonstrated unique cooperativity effects in crystallization of protein-ligand complexes. The crystal forms obtained were also related to the concentration of DMSO and ammonium sulphate in crystallization, and storage conditions of purified PR. Surprisingly, the relative occupancies of these inhibitors in the active site suggested a competition between the two inhibitors which were not inhibition constants related. Analysis of the structures in both crystal forms show no difference in DMSO content but at higher concentration of DMSO (3.2 - 5.0%) in the orthorhombic crystal forms, there were protein-sulphate interactions which were absent in the monoclinic forms with lower concentration (0.8 - 1.2%) of DMSO. This work has clearly demonstrated that there is cooperativity in crystallization and the conditions of crystallization influence specific intermolecular contacts in crystal packing (crystal form). (author)

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

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

    1987-01-01

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

  2. Protease inhibitors as possible pitfalls in proteomic analyses of complex biological samples

    Clifton, James; Huang, Feilei; Rucevic, Marijana; Cao, Lulu; Hixson, Douglas; Josic, Djuro

    2011-01-01

    Sample preparation, especially protein and peptide fractionation prior to identification by mass spectrometry (MS) are typically applied to reduce sample complexity. The second key element in this process is proteolytic digestion that is performed mostly by trypsin. Optimization of this step is an important factor in order to achieve both speed and better performance of proteomic analysis, and tryptic digestion prior to the MS analysis is topic of many studies. To date, only few studies pay a...

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

    Little Tom J; Welch John J; Obbard Darren J

    2009-01-01

    Abstract Background Mosquitoes of the Anopheles gambiae species complex are the primary vectors of human malaria in sub-Saharan Africa. Many host genes have been shown to affect Plasmodium development in the mosquito, and so are expected to engage in an evolutionary arms race with the pathogen. However, there is little conclusive evidence that any of these mosquito genes evolve rapidly, or show other signatures of adaptive evolution. Methods Three serine protease inhibitors have previously be...

  4. Protease inhibitor

    2009-01-01

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

  5. Processing Proteases

    Ødum, Anders Sebastian Rosenkrans

    Processing proteases are proteases which proteolytically activate proteins and peptides into their biologically active form. Processing proteases play an important role in biotechnology as tools in protein fusion technology. Fusion strategies where helper proteins or peptide tags are fused to the...... protein of interest are an elaborate method to optimize expression or purification systems. It is however critical that fusion proteins can be removed and processing proteases can facilitate this in a highly specific manner. The commonly used proteases all have substrate specificities to the N-terminal of...... the scissile bond, leaving C-terminal fusions to have non-native C-termini after processing. A solution yielding native C-termini would allow novel expression and purification systems for therapeutic proteins and peptides.The peptidyl-Lys metallopeptidase (LysN) of the fungus Armillaria mellea (Am) is...

  6. Clustering of OB-fold domains of the partner protease complexed with trimeric stomatin from Thermococcales.

    Yokoyama, Hideshi; Matsui, Eriko; Hiramoto, Kana; Forterre, Patrick; Matsui, Ikuo

    2013-07-01

    The C-terminal soluble domain of stomatin operon partner protein (STOPP) of the hyperthermophilic archaeon Pyrococcus horikoshii has an oligonucleotide binding-fold (OB-fold). STOPP lacks the conserved surface residues necessary for binding to DNA/RNA. A tryptophan (W) residue is conserved instead at the molecular surface. Solvent-accessible W residues are often found at interfaces of protein-protein complexes, which suggested the possibility of self-assembling of STOPP. Protein-protein interactions among the C-terminal soluble domains of STOPP PH1510 (1510-C) were then analyzed by chemical linking and blue native polyacrylamide gel electrophoresis (BN-PAGE) methods. These results suggest that the soluble domains of STOPP could assemble into homo-oligomers. Since hexameric subcomplex I from archaeal proteasome consists of coiled-coil segments and OB-fold domains, molecular modeling of 1510-C was performed using hexameric subcomplex I as a template. Although 1510-C is a comparatively small polypeptide consisting of approximately 60 residues, numerous salt bridges and hydrophobic interactions were observed in the predicted hexamer of 1510-C, suggesting the stability of the homo-oligomeric structure. This oligomeric property of STOPP may be favorable for triplicate proteolysis of the trimer of prokaryotic stomatin. PMID:23587725

  7. [Studies on human alpha-2 macroglobulin structure and its complexes with proteases, using polyacrylamide gel electrophoresis].

    Fine, J M; Lambin, P; Steinbuch, M

    1975-09-01

    Pure alpha2M is prepared with fresh plasma as starting material, to prevent the interaction of alpha2M from proteolytic enzymes of plasma such as thrombin, plasmin and kallikrein. During the purification steps, polybrene and aprotin are used as inhibitors and plasminogen is absorbed onto bentonite. When alpha 2M is submitted to polyacrylamide gel electrophoresis (PAA) containing 0.1% SDS, a complete dissociation in two half-molecules of MW 380,000 occurs. When alpha2M is incubated in 1% SDS and 1% beta-mercaptoethanol as reducing agent, only one component of MW 190,000 is observed in PAA-SDS. This experiments show that the alpha2M molecule consist of two symetric halves of same MW (380,000) linked by non covalent bonds. Each two-half-molecules is made of two polypeptides chains MW 190,000 linked by disulfide bonds. Thus alpha2M molecule contains four polypeptides chains having a same MW. The same techniques were applied to the study of alaph2M proteinases complexes. Three different proteinases (plasmin, trypsin and papain) were used in these experiments. Trypsin and papain are commercialy available. Plasminogen was obtained by affinity chromatography and activated into plasmin by insoluble streptokinase fixed on PAB cellulose. PMID:59941

  8. Insights into affinity and specificity in the complexes of α-lytic protease and its inhibitor proteins: binding free energy from molecular dynamics simulation†

    Cieplak, Piotr

    2014-01-01

    We report the binding free energy calculation and its decomposition for the complexes of α-lytic protease and its protein inhibitors using molecular dynamics simulation. Standard mechanism serine protease inhibitors eglin C and OMTKY3 are known to have strong binding affinity for many serine proteases. Their binding loops have significant similarities, including a common P1 Leu as the main anchor in the binding interface. However, recent experiments demonstrate that the two inhibitors have vastly different affinity towards α-lytic protease (ALP), a bacterial serine protease. OMTKY3 inhibits the enzyme much more weakly (by ~106 times) than eglin C. Moreover, a variant of OMTKY3 with five mutations, OMTKY3M, has been shown to inhibit 104 times more strongly than the wild-type inhibitor. The underlying mechanisms for the unusually large difference in binding affinities and the effect of mutation are not well understood. Here we use molecular dynamics simulation with molecular mechanics–Poisson Boltzmann/surface area method (MM-PB/SA) to investigate quantitatively the binding specificity. The calculated absolute binding free energies correctly differentiate the thermodynamic stabilities of these protein complexes, but the magnitudes of the binding affinities are systematically overestimated. Analysis of the binding free energy components provides insights into the molecular mechanism of binding specificity. The large ΔΔGbind between eglin C and wild type OMTKY3 towards ALP is mainly attributable to the stronger nonpolar interactions in the ALP-eglin C complex, arising from a higher degree of structural complementarity. Here the electrostatic interaction contributes to a lesser extent. The enhanced inhibition in the penta-mutant OMTKY3M over its wild type is entirely due to an overall improvement in the solvent-mediated electrostatic interactions in the ALP-OMTKY3M complex. The results suggest that for these protein-complexes and similar enzyme-inhibitor systems

  9. Insights into affinity and specificity in the complexes of alpha-lytic protease and its inhibitor proteins: binding free energy from molecular dynamics simulation.

    Deng, Nan-Jie; Cieplak, Piotr

    2009-07-01

    We report the binding free energy calculation and its decomposition for the complexes of alpha-lytic protease and its protein inhibitors using molecular dynamics simulation. Standard mechanism serine protease inhibitors eglin C and OMTKY3 are known to have strong binding affinity for many serine proteases. Their binding loops have significant similarities, including a common P1 Leu as the main anchor in the binding interface. However, recent experiments demonstrate that the two inhibitors have vastly different affinity towards alpha-lytic protease (ALP), a bacterial serine protease. OMTKY3 inhibits the enzyme much more weakly (by approximately 10(6) times) than eglin C. Moreover, a variant of OMTKY3 with five mutations, OMTKY3M, has been shown to inhibit 10(4) times more strongly than the wild-type inhibitor. The underlying mechanisms for the unusually large difference in binding affinities and the effect of mutation are not well understood. Here we use molecular dynamics simulation with molecular mechanics-Poisson Boltzmann/surface area method (MM-PB/SA) to investigate quantitatively the binding specificity. The calculated absolute binding free energies correctly differentiate the thermodynamic stabilities of these protein complexes, but the magnitudes of the binding affinities are systematically overestimated. Analysis of the binding free energy components provides insights into the molecular mechanism of binding specificity. The large DeltaDeltaG(bind) between eglin C and wild type OMTKY3 towards ALP is mainly attributable to the stronger nonpolar interactions in the ALP-eglin C complex, arising from a higher degree of structural complementarity. Here the electrostatic interaction contributes to a lesser extent. The enhanced inhibition in the penta-mutant OMTKY3M over its wild type is entirely due to an overall improvement in the solvent-mediated electrostatic interactions in the ALP-OMTKY3M complex. The results suggest that for these protein-complexes and

  10. Synthesis and Characterization of Bis-benzyl and Bis-allyl Complexes of Titanium(III) and Vanadium(III); Catalytic Isomerization of Alkenes with CpV(η3-C3H5)2

    Nieman, J.; Pattiasina, J.W.; Teuben, J.H.

    1984-01-01

    Reactions of CpTiCl2 (Cp = η5-C5H5) with RMgX (X = Cl, Br) yield the complexes CpTiR2 (R = CH2Ph, η3-C3H5). The complex Cp*Ti(η3-C3H5)2 (Cp* = η5-C5Me5) was prepared analogously from Cp*TiCl2(THF). CpVCl2(PEt3)2 and Cp´VCl2(PEt3)2 (Cp´ = η5-C5H4Me) were used for the preparation of CpV(CH2Ph)2, CpV(η3-C3H5)2, CpV(η3-1-MeC3H4)2 and Cp´V(η3-C3H5)2, respectively. The corresponding Cp*V derivatives could not be obtained. The reaction of CpCrCl2(thf) with C3H5MgCl gives a dimeric complex [CpCr(C3H5...

  11. Supermarket Proteases.

    Hagar, William G.; Bullerwell, Lornie D.

    2003-01-01

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

  12. Earthworm Protease

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

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

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

    2012-05-01

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

  14. Bacterial proteases and virulence

    Frees, Dorte; Brøndsted, Lone; Ingmer, Hanne

    Bacterial pathogens rely on proteolysis for variety of purposes during the infection process. In the cytosol, the main proteolytic players are the conserved Clp and Lon proteases that directly contribute to virulence through the timely degradation of virulence regulators and indirectly by providing...... tolerance to adverse conditions such as those experienced in the host. In the membrane, HtrA performs similar functions whereas the extracellular proteases, in close contact with host components, pave the way for spreading infections by degrading host matrix components or interfering with host cell...... cell. These extracellular proteases are activated in complex cascades involving auto-processing and proteolytic maturation. Thus, proteolysis has been adopted by bacterial pathogens at multiple levels to ensure the success of the pathogen in contact with the human host....

  15. Revisiting the mechanism of the autoactivation of the complement protease C1r in the C1 complex: structure of the active catalytic region of C1r.

    Kardos, József; Harmat, Veronika; Palló, Anna; Barabás, Orsolya; Szilágyi, Katalin; Gráf, László; Náray-Szabó, Gábor; Goto, Yuji; Závodszky, Péter; Gál, Péter

    2008-03-01

    C1r is a modular serine protease which is the autoactivating component of the C1 complex of the classical pathway of the complement system. We have determined the first crystal structure of the entire active catalytic region of human C1r. This fragment contains the C-terminal serine protease (SP) domain and the preceding two complement control protein (CCP) modules. The activated CCP1-CCP2-SP fragment makes up a dimer in a head-to-tail fashion similarly to the previously characterized zymogen. The present structure shows an increased number of stabilizing interactions. Moreover, in the crystal lattice there is an enzyme-product relationship between the C1r molecules of neighboring dimers. This enzyme-product complex exhibits the crucial S1-P1 salt bridge between Asp631 and Arg446 residues, and intermolecular interaction between the CCP2 module and the SP domain. Based on these novel structural information we propose a new split-and-reassembly model for the autoactivation of the C1r. This model is consistent with experimental results that have not been explained adequately by previous models. It allows autoactivation of C1r without large-scale, directed movement of C1q arms. The model is concordant with the stability of the C1 complex during activation of the next complement components. PMID:17996945

  16. The trimeric serine protease HtrA1 forms a cage-like inhibition complex with an anti-HtrA1 antibody.

    Ciferri, Claudio; Lipari, Michael T; Liang, Wei-Ching; Estevez, Alberto; Hang, Julie; Stawicki, Scott; Wu, Yan; Moran, Paul; Elliott, Mike; Eigenbrot, Charles; Katschke, Kenneth J; van Lookeren Campagne, Menno; Kirchhofer, Daniel

    2015-12-01

    High temperature requirement A1 (HtrA1) is a trypsin-fold serine protease implicated in the progression of age-related macular degeneration (AMD). Our interest in an antibody therapy to neutralize HtrA1 faces the complication that the target adopts a trimeric arrangement, with three active sites in close proximity. In the present study, we describe antibody 94, obtained from a human antibody phage display library, which forms a distinct macromolecular complex with HtrA1 and inhibits the enzymatic activity of recombinant and native HtrA1 forms. Using biochemical methods and negative-staining EM we were able to elucidate the molecular composition of the IgG94 and Fab94 complexes and the associated inhibition mechanism. The 246-kDa complex between the HtrA1 catalytic domain trimer (HtrA1_Cat) and Fab94 had a propeller-like organization with one Fab bound peripherally to each protomer. Low-resolution EM structures and epitope mapping indicated that the antibody binds to the surface-exposed loops B and C of the catalytic domain, suggesting an allosteric inhibition mechanism. The HtrA1_Cat-IgG94 complex (636 kDa) is a cage-like structure with three centrally located IgG94 molecules co-ordinating two HtrA1_Cat trimers and the six active sites pointing into the cavity of the cage. In both complexes, all antigen-recognition regions (paratopes) are found to bind one HtrA1 protomer and all protomers are bound by a paratope, consistent with the complete inhibition of enzyme activity. Therefore, in addition to its potential therapeutic usefulness, antibody 94 establishes a new paradigm of multimeric serine protease inhibition. PMID:26385991

  17. Developpement of a photoaffinity probe for the sensitive detection of matrix metallo-protease active forms from complex biological systems

    A new activity-based probe able to covalently modify the active site of proteases belonging to the matrix metallo-protease family (MMPs) has been developed in this thesis project. The probe was shown to behave as potent inhibitor of several MMPs, with nanomolar Ki values. This probe was also able to modify specifically only the free active site of MMPs, with particular high yields of cross-linking varying from 50 % to 11 %, depending of the MMPs tested. Using radioactivity as means of detection, this probe was able to detect active form of MMPs with a threshold of 1 femto-mole. Applied to the study of bronchoalveolar fluids (BAL) from mice exposed to nanoparticles by a lung aspiration protocol, this probe revealed the presence of the catalytic domain of MMP-12 under its active form, but not in control animals. When used to detect active form of MMPs from extracts obtained from human arteries of patient suffering from atherosclerosis, the probe was not able to detect such MMP active forms. Despite this negative result, the detection of active form of MMP in pathological fluid like BAL has never been reported before this work. Having validated this novel MMP activity-based probe, it will be possible to use it now for detecting MMPs from other pathological fluids or tissues extracts in which MMPs can be good markers of the pathology. (author)

  18. Insights into the mechanism of drug resistance: X-ray structure analysis of G48V/C95F tethered HIV-1 protease dimer/saquinavir complex

    The mutation G48V in HIV-1 protease is a major resistance mutation against the drug saquinavir. Recently, G48V mutation is found to co-exist with the mutation C95F in AIDS patients treated with saquinavir. We report here the three-dimensional crystal structure of G48V/C95F tethered HIV-1 protease/saquinavir complex. The structure indicates following as the possible causes of drug resistance: (1) loss of direct van der Waals interactions between saquinavir and enzyme residues PHE-53 and PRO-1081, (2) loss of water-mediated hydrogen bonds between the carbonyl oxygen atoms in saquinavir and amide nitrogen atoms of flap residues 50 and 1050, (3) changes in inter-monomer interactions, which could affect the energetics of domain movements associated with inhibitor-binding, and (4) significant reduction in the stability of the mutant dimer. The present structure also provides a rationale for the clinical observation that the resistance mutations C95F/G48V/V82A occur as a cluster in AIDS patients.

  19. Insights into the mechanism of drug resistance: X-ray structure analysis of G48V/C95F tethered HIV-1 protease dimer/saquinavir complex

    Prashar, Vishal; Bihani, Subhash C.; Das, Amit [Solid State Physics Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085 (India); Rao, D.R. [Manufacturing and Research Division, CIPLA Ltd., L.B.S. Marg, Vikhroli, Mumbai 400083 (India); Hosur, M.V., E-mail: hosur@barc.gov.in [Solid State Physics Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085 (India)

    2010-06-11

    The mutation G48V in HIV-1 protease is a major resistance mutation against the drug saquinavir. Recently, G48V mutation is found to co-exist with the mutation C95F in AIDS patients treated with saquinavir. We report here the three-dimensional crystal structure of G48V/C95F tethered HIV-1 protease/saquinavir complex. The structure indicates following as the possible causes of drug resistance: (1) loss of direct van der Waals interactions between saquinavir and enzyme residues PHE-53 and PRO-1081, (2) loss of water-mediated hydrogen bonds between the carbonyl oxygen atoms in saquinavir and amide nitrogen atoms of flap residues 50 and 1050, (3) changes in inter-monomer interactions, which could affect the energetics of domain movements associated with inhibitor-binding, and (4) significant reduction in the stability of the mutant dimer. The present structure also provides a rationale for the clinical observation that the resistance mutations C95F/G48V/V82A occur as a cluster in AIDS patients.

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

    Hansen, Cecilie Bo; Csuka, Dorottya; Munthe-Fog, Lea; Varga, Lilian; Farkas, Henriette; Hansen, Karin Møller; Koch, Claus; Skjødt, Karsten; Garred, Peter; Skjoedt, Mikkel-Ole

    2015-10-15

    C1 inhibitor (C1-INH) is known to form complexes with the lectin complement pathway serine proteases MASP-1 and MASP-2. Deficiency of C1-INH is associated with hereditary angioedema (HAE), an autosomal inherited disease characterized by swelling attacks caused by elevated levels of bradykinin. MASP-1 was shown to cleave high m.w. kininogen into bradykinin; therefore, we hypothesized that MASP-1 levels and the quantity of MASP-1/C1-INH complexes might be associated with different paraclinical and clinical outcomes of HAE. We measured MASP-1 serum concentrations and endogenous MASP-1/C1-INH complex levels in 128 HAE patients and 100 controls. Relatively high levels of pre-existing MASP-1/C1-INH complexes were observed in normal serum, and we found that both the serum levels of MASP-1 and the complex formation between MASP-1 and C1-INH were significantly reduced in HAE patients compared with matched controls (p < 0.0001). The level of MASP-1 and MASP-1/C1-INH complexes in HE patients correlated with the level of C1-INH (p = 0.0009 and p = 0.0047, respectively), the level of C4 (p = 0.0084 and p < 0.0001, respectively), and the number of attacks in the year of blood sampling (p = 0.0075 and p = 0.0058, respectively). In conclusion, we show that MASP-1/C1-INH complexes circulate in normal human blood. The levels of MASP-1 and MASP-1/C1-INH complexes are reduced in HAE patients compared with controls. Both MASP-1 and MASP-1/C1-INH complexes are related to the degree of complement C4 consumption, as well as the severity of disease. These results suggest that MASP-1 may exert a previously unrecognized role in the pathophysiology of HAE. PMID:26371246

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

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

    2013-06-01

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

  2. Protease gene families in Populus and Arabidopsis

    Jansson Stefan

    2006-12-01

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

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

    Mukhopadhayay Bishnu P

    2001-09-01

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

  4. Instability of metal 1,3-benzodi(thiophosphinoyl)methandiide complexes: formation of hafnium, tin and zirconium complexes of 1,3-benzodi(thiophosphinoyl)thioketone dianionic ligand [1,3-C6H4(PhPS)2CS](2(-)).

    Yang, Ya-Xiu; Li, Yongxin; Ganguly, Rakesh; So, Cheuk-Wai

    2015-07-28

    The reaction of [LCH2] (1, L = 1,3-C6H4(PhPS)2) and M(NMe2)4 (M = Hf, Zr) in toluene at 110 °C afforded a mixture of group 4 metal complexes [{LC(S)}2M] [M = Hf (2), Zr (3)] and [1,3-C6H4(PhPS)(PhP)CH2]. The reactions appear to proceed through the formation of metal bis(carbene) complexes, [LC=M=CL], which then undergo an intermolecular sulphur transfer reaction with the P=S bond of [LCH2] to form 2 and 3, and the byproduct is [1,3-C6H4(PhPS)(PhP)CH2]. In addition, the reaction of 1, [CH2(PPh2S)2] (4) and M(NMe2)4 in refluxing toluene gave a mixture of [{LC(S)}M(NHMe2){C(PPh2S)2}] [M = Hf (5), Zr (6)], [1,3-C6H4(PhPS)(PhP)CH2] and [CH2(PPh2S)(PPh2)]. Moreover, the intermolecular sulfur transfer reaction is evidenced by the reaction of the tin(ii) 1,3-benzodi(thiophosphinoyl)methandiide complex [{μ-1,3-C6H4(PhPS)2C}Sn]2 (7) with two equivalents of elemental sulfur in CH2Cl2 at ambient temperature to give [{1,3-C6H4(PhPS)2CS}2Sn] (8). Compounds 2, 3, 5, 6, and 8 were characterized by NMR spectroscopy and X-ray crystallography. PMID:26079794

  5. Proteases as Insecticidal Agents

    Harrison, Robert L.; Bonning, Bryony C.

    2010-01-01

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

  6. Proteolytic crosstalk in multi-protease networks

    Ogle, Curtis T.; Mather, William H.

    2016-04-01

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

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

    Yuichi Endo

    2012-01-01

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

  8. Control of natural transformation in salivarius Streptococci through specific degradation of σX by the MecA-ClpCP protease complex.

    Wahl, Astrid; Servais, Florence; Drucbert, Anne-Sophie; Foulon, Catherine; Fontaine, Laetitia; Hols, Pascal

    2014-08-01

    Competence for natural DNA transformation is a tightly controlled developmental process in streptococci. In mutans and salivarius species, the abundance of the central competence regulator σ(X) is regulated at two levels: transcriptional, by the ComRS signaling system via the σ(X)/ComX/SigX-inducing peptide (XIP), and posttranscriptional, by the adaptor protein MecA and its associated Clp ATPase, ClpC. In this study, we further investigated the mechanism and function of the MecA-ClpC control system in the salivarius species Streptococcus thermophilus. Using in vitro approaches, we showed that MecA specifically interacts with both σ(X) and ClpC, suggesting the formation of a ternary σ(X)-MecA-ClpC complex. Moreover, we demonstrated that MecA ultimately targets σ(X) for its degradation by the ClpCP protease in an ATP-dependent manner. We also identify a short sequence (18 amino acids) in the N-terminal domain of σ(X) as essential for the interaction with MecA and subsequent σ(X) degradation. Finally, increased transformability of a MecA-deficient strain in the presence of subinducing XIP concentrations suggests that the MecA-ClpCP proteolytic complex acts as an additional locking device to prevent competence under inappropriate conditions. A model of the interplay between ComRS and MecA-ClpCP in the control of σ(X) activity is proposed. PMID:24837292

  9. Complement component 3 (C3)

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

  10. Targeting Proteases in Cardiovascular Diseases by Mass Spectrometry-Based Proteomics

    Klingler, Diana; Hardt, Markus

    2012-01-01

    Proteases hydrolyze peptide bonds, thereby controlling the function of proteins and peptides on the posttranslational level. In the cardiovascular system, proteases play pivotal roles in the regulation of blood pressure, coagulation and other essential physiological processes. Accordingly, proteases are prime targets for therapeutic interventions and diagnostics. Proteases are part of complex proteolytic networks comprised of enzymes, inhibitors, activators, substrates and cleavage products. ...

  11. Proteases of neutrophilic granulocytes

    Wiesława Roszkowska-Jakimiec

    2002-06-01

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

  12. Proteases of neutrophilic granulocytes

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

    2002-01-01

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

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

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

  14. Lung protease/anti-protease network and modulation of mucus production and surfactant activity.

    Garcia-Verdugo, Ignacio; Descamps, Delphyne; Chignard, Michel; Touqui, Lhousseine; Sallenave, Jean-Michel

    2010-11-01

    Lung epithelium guarantees gas-exchange (performed in the alveoli) and protects from external insults (pathogens, pollutants…) present within inhaled air. Both functions are facilitated by secretions lining airway surface liquid, mucus (in the upper airways) and pulmonary surfactant (in the alveoli). Mucins, the main glycoproteins present within the mucus, are responsible for its rheologic properties and participate in lung defense mechanisms. In parallel, lung collectins are pattern recognition molecules present in pulmonary surfactant that also modulate lung defense. During chronic airways diseases, excessive protease activity can promote mucus hypersecretion and degradation of lung collectins and therefore contribute to the pathophysiology of these diseases. Importantly, secretion of local and systemic anti-proteases might be crucial to equilibrate the protease/anti-protease unbalance and therefore preserve the function of lung host defense compounds and airway surface liquid homeostasis. In this review we will present information relative to proteases able to modulate mucin production and lung collectin integrity, two important compounds of innate immune defense. One strategy to preserve physiological mucus production and collectin integrity during chronic airways diseases might be the over-expression of local 'alarm' anti-proteases such as SLPI and elafin. Interestingly, a cross-talk between lung collectins and anti-protease activity has recently been described, implicating the presence within the lung of a complex network between proteases, anti-proteases and pattern recognition molecules, which aims to keep or restore homeostasis in resting or inflamed lungs. PMID:20493919

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

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

    2011-01-01

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

  16. Structural basis of rhomboid intramembrane protease specificity and mechanism revealed by X-ray crystallographic analysis of rhomboid-substrate peptide complexes

    Zoll, Sebastian; Stanchev, Stancho; Began, Jakub; Škerle, Jan; Peclinovská, Lucie; Lepšík, Martin; Majer, Pavel; Stříšovský, Kvido

    Praha : Ústav organické chemie a biochemie AV ČR, v. v. i, 2014. s. 24. ISBN 978-80-86241-51-7. [Prague Protein Spring Meeting 2014: Proteins and their interactions /3./. 09.05.2014-11.05.2014, Praha] Institutional support: RVO:61388963 Keywords : intramembrane proteases * rhomboids Subject RIV: CE - Biochemistry

  17. Synthesis of amino heterocycle aspartyl protease inhibitors.

    Chambers, Rachel K; Khan, Tanweer A; Olsen, David B; Sleebs, Brad E

    2016-06-14

    Aspartyl proteases are important pharmacological targets. Historically aspartyl proteases have been commonly targeted with transition state derived peptidomimetics. The strategy to develop aspartyl protease inhibitors has undertaken a dramatic paradigm shift in the last 10 years. The pharmaceutical industry in 2005 disclosed several scaffolds or "head groups" that prompted the field to move beyond peptidomimetic derived inhibitors. Since the discovery of the first amino heterocycle aspartyl protease inhibitor, the amino hydantoin, industry and academia have positioned themselves for a foothold on the new molecular space, designing a variety of related "head groups". Both the design and synthetic efforts involved in constructing these scaffolds are varied and complex. Here we highlight the synthetic strategies used to access these amino heterocycle scaffolds. PMID:27143279

  18. Expression, purification and preliminary X-ray crystallographic studies of the human immunodeficiency virus 1 subtype C protease

    Crystals of the human immunodeficiency virus 1 subtype C protease complexed with indinavir and nelfinavir have been grown in the monoclinic space group P21 and shown to diffract X-rays to 2.3 Å resolution. Crystals of the human immunodeficiency virus 1 (HIV-1) subtype C protease (PR) complexed with the clinically used inhibitors indinavir (IDV) and nelfinavir (NFV) have been grown in the monoclinic space group P21, with mean unit-cell parameters a = 46.7 (±0.1), b = 59.8 (±0.3), c = 87.0 (±0.4) Å, β = 95.2 (±0.5)°. The crystals of both complexes have been shown to diffract X-rays to 2.3 Å resolution. The diffraction data for the subtype C PR complexes with IDV and NFV were subsequently processed and reduced, with overall Rsym values of 8.4 and 11.4%, respectively. Based on the unit-cell volumes, molecular-replacement results and packing considerations, there are two protease homodimers per crystallographic asymmetric unit in each of the complexes. The data were initially phased using a model based on the crystal structure of HIV-1 subtype B PR; the structures have been determined and further refinement and analysis are in progress. These structures and subsequent studies with other inhibitors will greatly aid in correlating the amino-acid variation between the different HIV PRs and understanding their differential sensitivity and resistance to current drug therapy

  19. On the reliability of the corrected semiempirical quantum chemical method (PM6-DH2) for assigning the protonation states in HIV-1 protease/inhibitor complexes

    Pecina, Adam; Přenosil, Ondřej; Fanfrlík, Jindřich; Řezáč, Jan; Granatier, Jaroslav; Hobza, Pavel; Lepšík, Martin

    2011-01-01

    Roč. 76, č. 5 (2011), s. 457-479. ISSN 0010-0765 R&D Projects: GA MŠk LC512; GA MŠk 1M0508; GA ČR GAP208/11/0295 Institutional research plan: CEZ:AV0Z40550506 Keywords : HIV-1 protease inhibition * protonation * QM/MM calculations Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 1.283, year: 2011

  20. Autocatalytic Processing of m-AAA Protease Subunits in Mitochondria

    Koppen, Mirko; Bonn, Florian; Ehses, Sarah; Langer, Thomas

    2009-01-01

    m-AAA proteases are ATP-dependent proteolytic machines in the inner membrane of mitochondria which are crucial for the maintenance of mitochondrial activities. Conserved nuclear-encoded subunits, termed paraplegin, Afg3l1, and Afg3l2, form various isoenzymes differing in their subunit composition in mammalian mitochondria. Mutations in different m-AAA protease subunits are associated with distinct neuronal disorders in human. However, the biogenesis of m-AAA protease complexes or of individua...

  1. The peculiar radio galaxy 3C 433

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

    1983-01-01

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

  2. Native Elution of Yeast Protein Complexes Obtained by Affinity Capture.

    LaCava, John; Fernandez-Martinez, Javier; Rout, Michael P

    2016-01-01

    This protocol describes two options for the native (nondenaturing) elution of protein complexes obtained by affinity capture. The first approach involves the elution of complexes purified through a tag that includes a human rhinovirus 3C protease (PreScission protease) cleavage site sequence between the protein of interest and the tag. Incubation with the protease cleaves immobilized complexes from the affinity medium. The second approach involves the release of protein A-tagged protein complexes using a competitive elution reagent called PEGylOx. The degree of purity of the native assemblies eluted is sample dependent and strongly influenced by the affinity capture. It should be noted that the efficiency of native elution is commonly lower than that of elution by a denaturing agent (e.g., SDS) and the release of the complex will be limited by the activity of the protease or the inhibition constant (Ki) of the competitive release agent. However, an advantage of native release is that some nonspecifically bound materials tend to stay adsorbed to the affinity medium, providing an eluted fraction of higher purity. Finally, keep in mind that the presence of the protease or elution peptide could potentially affect downstream applications; thus, their removal should be considered. PMID:27371597

  3. Secretory Granule Proteases in Rat Mast Cells. Cloning of 10 Different Serine Proteases and a Carboxypeptidase A from Various Rat Mast Cell Populations

    Lützelschwab, Claudia; Pejler, Gunnar; Aveskogh, Maria; Hellman, Lars

    1997-01-01

    Two of the major rat mast cell proteases, rat mast cell protease 1 (RMCP-1) and RMCP-2, have for many years served as important phenotypic markers for studies of various aspects of mast cell (MC) biology. However, except for these proteases only fragmentary information has been available on the structure and complexity of proteases expressed by different subpopulations of rat MCs. To address these questions, cDNA libraries were constructed from freshly isolated rat peritoneal MCs and from the...

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

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

    2014-01-01

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

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

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

    2016-07-01

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

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

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

    2012-01-01

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

  7. Commercial proteases: present and future.

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

    2013-04-17

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

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

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

    2014-10-02

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

  9. Regulator of G protein signaling 8 inhibits protease-activated receptor 1/Gi/o signaling by forming a distinct G protein-dependent complex in live cells.

    Lee, Jinyong; Ghil, Sungho

    2016-05-01

    Activation of seven-transmembrane-domain-possessing G protein-coupled receptors (GPCRs) by extracellular stimuli elicits intracellular responses. One class of GPCRs-protease-activated receptors (PARs)-is activated by endogenous proteases, such as thrombin and trypsin. Members of the regulator of G protein signaling (RGS) family stimulate GTP hydrolysis of G protein alpha (Gα) subunits, thereby inhibiting GPCR/Gα-mediated signaling. We previously reported that RGS2 and RGS4 inhibit PAR1/Gα-mediated signaling by interacting with PAR1 in a Gα-dependent manner. Here, employing the bioluminescence resonance energy transfer (BRET) technique, we identified RGS8 as a novel PAR1-interacting protein. Very little BRET activity was observed between PAR1-Venus (PAR1-Ven) and RGS8-Luciferase (RGS8-Luc) in the absence of Gα. However, in the presence of Gαo, BRET activity was specifically and significantly increased. This interaction was confirmed by biochemical and immunofluorescence assays. Notably, RGS8 inhibited PAR1/Gαi/o-mediated adenylyl cyclase and ERK activation, and prevented Gαo-induced neurite outgrowth and activation of Necdin protein, a downstream target of Gαo. Our findings suggest a novel function of RGS8 and reveal cellular mechanisms by which RGS8 mediates PAR1 inhibition. PMID:26829215

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

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

    2011-09-15

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

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

    Hans, F.; Sanfacon, H.

    1995-01-01

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

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

    Ladislau C. Kovari

    2012-05-01

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

  13. Subtilisin-like proteases in nematodes.

    Poole, Catherine B; Jin, Jingmin; McReynolds, Larry A

    2007-09-01

    Cleavage by subtilisin-like proteases (subtilases) is an essential step in post-translational processing of proteins found in organisms ranging from yeast to mammals. Our knowledge of the diversity of this protease family in nematodes is aided by the rapid increase in sequence information, especially from the Brugia malayi genome project. Genetic studies of the subtilases in Caenorhabitis elegans give valuable insight into the biological function of these proteases in other nematode species. In this review, we focus on the subtilases in filarial nematodes as well as other parasitic and free-living nematodes in comparison to what is known in C. elegans. Topics to be addressed include expansion and diversity of the subtilase gene family during evolution, enhanced complexity created by alternative RNA splicing, molecular and biochemical characterization of the different subtilases and the challenges of designing subtilase-specific inhibitors for parasitic nematodes. PMID:17570539

  14. 3C 273 - half a century later

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

    2013-01-01

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

  15. Structural basis for HTLV-1 protease inhibition by the HIV-1 protease inhibitor indinavir.

    Kuhnert, Maren; Steuber, Holger; Diederich, Wibke E

    2014-07-24

    HTLV-1 protease (HTLV-1 PR) is an aspartic protease which represents a promising drug target for the discovery of novel anti-HTLV-1 drugs. The X-ray structure of HTLV-1 PR in complex with the well-known and approved HIV-1 PR inhibitor Indinavir was determined at 2.40 Å resolution. In this contribution, we describe the first crystal structure in complex with a nonpeptidic inhibitor that accounts for rationalizing the rather moderate affinity of Indinavir against HTLV-1 PR and provides the basis for further structure-guided optimization strategies. PMID:25006983

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

    Marian Dorcas Quain

    2013-08-01

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

  17. Structure-Function of Falcipains: Malarial Cysteine Proteases

    Kailash C. Pandey

    2012-01-01

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

  18. Nucleic Acid Aptamers Against Proteases

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

    2011-01-01

    Proteases are potential or realized therapeutic targets in a wide variety of pathological conditions. Moreover, proteases are classical subjects for studies of enzymatic and regulatory mechanisms. We here review the literature on nucleic acid aptamers selected with proteases as targets. Designing small molecule protease inhibitors of sufficient specificity has proved a daunting task. Aptamers seem to represent a promising alternative. In our review, we concentrate on biochemical mechanisms of...

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

    van Wijk, Klaas

    2009-01-12

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

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

    Schulze, Stefanie; Westhoff, Peter; Gowik, Udo

    2016-06-01

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

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

    Li, Lidong

    2014-01-01

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

  2. Death proteases come alive

    Woltering, E.J.

    2004-01-01

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

  3. Proteases in Periodontal Disease

    Ana Rita Sokolonski ANTON

    2006-09-01

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

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

    Westerbeck Jason W

    2011-10-01

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

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

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

  6. Novel 2-oxoimidazolidine-4-carboxylic acid derivatives as Hepatitis C virus NS3-4A serine protease inhibitors: synthesis, activity, and X-ray crystal structure of an enzyme inhibitor complex

    Arasappan, Ashok; Njoroge, F. George; Parekh, Tejal N.; Yang, Xiaozheng; Pichardo, John; Butkiewicz, Nancy; Prongay, Andrew; Yao, Nanhua; Girijavallabhan, Viyyoor (SPRI)

    2008-06-30

    Synthesis and HCV NS3 serine protease inhibitory activity of some novel 2-oxoimidazolidine-4-carboxylic acid derivatives are reported. Inhibitors derived from this new P2 core exhibited activity in the low {micro}M range. X-ray structure of an inhibitor, 15c bound to the protease is presented.

  7. A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior

    Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu;

    2016-01-01

    -ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its CDR-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to...

  8. NATO-3C/Delta launch

    1978-01-01

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

  9. BATSE observations of 3C273

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

    1994-01-01

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

  10. Substrate modulation of enzyme activity in the herpesvirus protease family

    Lazic, Ana; Goetz, David H.; Nomura, Anson M.; Marnett, Alan B.; Craik, Charles S.

    2007-01-01

    The herpesvirus proteases are an example in which allosteric regulation of an enzyme activity is achieved through the formation of quaternary structure. Here, we report a 1.7 Å resolution structure of Kaposi’s Sarcoma herpesvirus protease in complex with a hexapeptide transition state analogue that stabilizes the dimeric state of the enzyme. Extended substrate binding sites are induced upon peptide binding. In particular, 104 Å2 of surface are buried in the newly formed S4 pocket when tyrosin...

  11. Identification of an Archaeal Presenilin-Like Intramembrane Protease

    Torres-Arancivia, Celia; Ross, Carolyn M.; Chavez, Jose; Assur, Zahra; Dolios, Georgia; Mancia, Filippo; Ubarretxena-Belandia, Iban

    2010-01-01

    Background The GXGD-type diaspartyl intramembrane protease, presenilin, constitutes the catalytic core of the γ-secretase multi-protein complex responsible for activating critical signaling cascades during development and for the production of β-amyloid peptides (Aβ) implicated in Alzheimer's disease. The only other known GXGD-type diaspartyl intramembrane proteases are the eukaryotic signal peptide peptidases (SPPs). The presence of presenilin-like enzymes outside eukaryots has not been demo...

  12. Analyzing polarization swings in 3C 279

    Kiehlmann S.

    2013-12-01

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

  13. The Bright Quasar 3C 273

    Courvoisier, Thierry J. -L.

    1998-01-01

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

  14. The spectrum of 3C 58

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

  15. Analyzing polarization swings in 3C 279

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

    2013-01-01

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

  16. L3+C air shower array

    Laurent Guiraud

    2000-01-01

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

  17. Radio jet of 3C273

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

  18. IRIS photometry of 3C273

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

  19. IRIS photometry of 3C273

    Lanning, H.H.

    1976-04-01

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

  20. Cathepsin proteases in Toxoplasma gondii

    Dou, Zhicheng; Carruthers, Vern B.

    2011-01-01

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

  1. Nucleic Acid Aptamers Against Proteases

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

    2011-01-01

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

  2. Protease-mediated drug delivery

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

    2003-12-01

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

  3. Superluminal expansion of quasar 3C273

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

  4. The Papain-Like Protease from the Severe Acute Respiratory Syndrome Coronavirus Is a Deubiquitinating Enzyme

    Lindner, Holger A.; Fotouhi-Ardakani, Nasser; Lytvyn, Viktoria; Lachance, Paule; Sulea, Traian; Ménard, Robert

    2005-01-01

    The severe acute respiratory syndrome coronavirus papain-like protease (SARS-CoV PLpro) is involved in the processing of the viral polyprotein and, thereby, contributes to the biogenesis of the virus replication complex. Structural bioinformatics has revealed a relationship for the SARS-CoV PLpro to herpesvirus-associated ubiquitin-specific protease (HAUSP), a ubiquitin-specific protease, indicating potential deubiquitinating activity in addition to its function in polyprotein processing (T. ...

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

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

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

    Yuan, Yuhai; Fan, Junhui

    2015-06-01

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

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

    姜军; 杨晓达; 王夔

    2007-01-01

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

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

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

    2013-11-01

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

  9. The radio jet of 3C273

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

  10. Protease-Sensitive Synthetic Prions

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

    2010-01-01

    Prions arise when the cellular prion protein (PrP(C)) undergoes a self-propagating conformational change; the resulting infectious conformer is designated PrP(Sc). Frequently, PrP(Sc) is protease-resistant but protease-sensitive (s) prions have been isolated in humans and other animals. We report here that protease-sensitive, synthetic prions were generated in vitro during polymerization of recombinant (rec) PrP into amyloid fibers. In 22 independent experiments, recPrP amyloid preparations, ...

  11. Double Faraday rotation toward 3C 27

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

    1984-08-01

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

  12. Inner radio jet of 3C273

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

    1988-08-04

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

  13. Computational analysis of HIV-1 protease protein binding pockets.

    Ko, Gene M; Reddy, A Srinivas; Kumar, Sunil; Bailey, Barbara A; Garg, Rajni

    2010-10-25

    Mutations that arise in HIV-1 protease after exposure to various HIV-1 protease inhibitors have proved to be a difficult aspect in the treatment of HIV. Mutations in the binding pocket of the protease can prevent the protease inhibitor from binding to the protein effectively. In the present study, the crystal structures of 68 HIV-1 proteases complexed with one of the nine FDA approved protease inhibitors from the Protein Data Bank (PDB) were analyzed by (a) identifying the mutational changes with the aid of a developed mutation map and (b) correlating the structure of the binding pockets with the complexed inhibitors. The mutations of each crystal structure were identified by comparing the amino acid sequence of each structure against the HIV-1 wild-type strain HXB2. These mutations were visually presented in the form of a mutation map to analyze mutation patterns corresponding to each protease inhibitor. The crystal structure mutation patterns of each inhibitor (in vitro) were compared against the mutation patterns observed in in vivo data. The in vitro mutation patterns were found to be representative of most of the major in vivo mutations. We then performed a data mining analysis of the binding pockets from each crystal structure in terms of their chemical descriptors to identify important structural features of the HIV-1 protease protein with respect to the binding conformation of the HIV-1 protease inhibitors. Data mining analysis is performed using several classification techniques: Random Forest (RF), linear discriminant analysis (LDA), and logistic regression (LR). We developed two hybrid models, RF-LDA and RF-LR. Random Forest is used as a feature selection proxy, reducing the descriptor space to a few of the most relevant descriptors determined by the classifier. These descriptors are then used to develop the subsequent LDA, LR, and hierarchical classification models. Clustering analysis of the binding pockets using the selected descriptors used to

  14. Serine proteases of parasitic helminths.

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

    2015-02-01

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

  15. Serine Proteases of Parasitic Helminths

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

    2015-01-01

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

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

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

    1992-01-01

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

  17. 3C236 Radio Source, Interrupted?

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

    2001-01-01

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

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

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

    2012-03-27

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

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

    Jong W Yu

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

  20. CLIP proteases and Plasmodium melanization in Anopheles gambiae.

    Barillas-Mury, Carolina

    2007-07-01

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

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

    Jiang, Longguang; Svane, Anna S P; Sørensen, Hans Peter; Jensen, Jan K; Hosseini, Masood; Chen, Zhuo; Weydert, Caroline; Nielsen, Jakob T; Christensen, Anni; Yuan, Cai; Jensen, Knud Jørgen; Nielsen, Niels Chr; Malmendal, Anders; Huang, Mingdong; Andreasen, Peter

    2011-01-01

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

  2. Current trends and challenges in proteomic identification of protease substrates.

    Vizovišek, Matej; Vidmar, Robert; Fonović, Marko; Turk, Boris

    2016-03-01

    Proteolytic cleavage is a ubiquitous, irreversible, posttranslational modification that changes protein structure and function and plays an important role in numerous physiological and pathological processes. Over the last decade, proteases have become increasingly important clinical targets because many of their inhibitors are already used in the clinic or in various stages of clinical testing. Therefore, a better understanding of protease action and their repertoires of physiological substrates can not only provide an important insight into their mechanisms of action but also open a path toward novel drug design. Historically, proteases and their substrates were mainly studied on a case-by-case basis, but recent advancements in mass spectrometry-based proteomics have enabled proteolysis studies on a global scale. Because there are many different types of proteases that can operate in various cellular contexts, multiple experimental approaches for their degradomic characterization had to be developed. The present paper reviews the mass spectrometry-based approaches for determining the proteolytic events in complex biological samples. The methodologies for substrate identification and the determination of protease specificity are discussed, with a special focus on terminomic strategies, which combine peptide labeling and enrichment. PMID:26514758

  3. Microbial inhibitors of cysteine proteases.

    Kędzior, Mateusz; Seredyński, Rafał; Gutowicz, Jan

    2016-08-01

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

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

    Biretta, J.A.

    1986-01-01

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

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

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

  6. The Resolved Outflow from 3C 48

    Shih, Hsin-Yi; Stockton, Alan

    2014-10-01

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

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

    LI Ping; WANG WeiHua; BI SiWei; SONG Rui; BU YuXiang

    2009-01-01

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

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

    2009-01-01

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

  9. Cytomegalovirus protease targeted prodrug development.

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

    2013-04-01

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

  10. Gastrointestinal absorption and biological activities of serine and cysteine proteases of animal and plant origin: review on absorption of serine and cysteine proteases

    Lorkowski, Gerhard

    2012-01-01

    Research has confirmed that peptides and larger protein molecules pass through the mucosal barrier of the gastrointestinal tract. Orally administered serine and cysteine proteases of plant and animal origin also reach blood and lymph as intact, high molecular weight and physiologically active protein molecules. Their absorption may be supported by a self-enhanced paracellular transport mechanism resulting in sub-nanomolar concentration of transiently free protease molecules or, in a complex w...

  11. Enzymatic Degradation of Ovalbumin by Various Proteases

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

    1997-01-01

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

  12. Curcumin derivatives as HIV-1 protease inhibitors

    Sui, Z.; Li, J.; Craik, C.S.; Ortiz de Montellano, P.R. [Univ. of California, San Francisco, CA (United States)

    1993-12-31

    Curcumin, a non-toxic natural compound from Curcuma longa, has been found to be an HIV-1 protease inhibitor. Some of its derivatives were synthesized and their inhibitory activity against the HIV-1 protease was tested. Curcumin analogues containing boron enhanced the inhibitory activity. At least of the the synthesized compounds irreversibly inhibits the HIV-1 protease.

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

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

    2012-10-23

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

  14. Full quantum mechanical study of binding of HIV-1 protease drugs

    Zhang, Da W.; Zhang, John Z. H.

    Fully quantum mechanical studies of detailed binding interactions between HIV-1 protease and six FDA (Food and Drug Administration)-approved drugs (saquinavir, indinavir, ritonavir, nelfinavir, amprenavir, and lopinavir) are carried out using a recently developed MFCC (molecular fractionation with conjugate caps) method. The MFCC calculation produces a quantum mechanical interaction spectrum for any protease drug binding complex. Detailed quantitative analysis on binding of lopinavir to specific residues of the protease is given from the current study. The present calculation shows that the dominant binding of lopinavir to the protease is through the formation of a strong hydrogen bond between the central hydroxyl group of the drug to the aspartate oxygen of Asp25 in one of the two chains of the protease (A chain). This is closely followed by hydrogen binding of the drug to Asp29 in the B chain and somewhat weak hydrogen bonding to Asp30, Gly27, Gly48, and Ile50 in both chains. By partitioning all six drugs into four building blocks besides the central component containing the hydroxyl group, MFCC calculation finds that block III has essentially no binding interaction with the protease and the major binding interactions of these drugs are from blocks II and IV, in addition to the dominant central hydroxyl group. This detailed quantitative information on drug binding to the protease is very useful in rational design of new and improved inhibitors of HIV-1 protease and its mutants.

  15. Exogenous proteases for meat tenderization.

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

    2014-01-01

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

  16. Comparative Studies on Retroviral Proteases: Substrate Specificity

    József Tözsér

    2010-01-01

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

  17. Cycloaddition Reactions of the Diphosphenyl Complex (η5-C5Me5)(CO)2Fe-P=P-Mes* (Mes* = 2,4,6-tBu3C6H2) with Hexafluoroacetone. X-Ray Structure Analyses of (η5-C5Me5)(CO) Fe P(=PMes*)OC(CF3)2CO and (η5-C5Me5)(CO)2FePP(Mes*)OC(CF3)2

    Weber, Lothar; Buchwald, Susanne; Lentz, Dieter; Preugschat, Dagmar; Stammler, Hans-Georg; Neumann, Beate

    1992-01-01

    The diphosphenyl complex (eta-5-C5Me5)-(CO)2Fe-P=P-Mes* (Mes* = 2,4,6-tBu3C6H2) undergoes a [3 + 2] dipolar cycloaddition with hexafluoroacetone to give the metalla heterocycle (eta-5-C5Me5)(CO)-Fe-P(=PMes*)OC(CF3)2C(O) with a remarkably short Fe-P bond (2.084 (4) angstrom) and an exocyclic P=P bond. When stored in solution at -40-degrees-C, this complex partly rearranges to the metalated 1-oxa-2,3-diphosphetane (eta-5-C5Me5)(CO)2Fe-P-P(Mes*)OC(CF3)2. The molecular structures of both isomers ...

  18. ATP-dependent protease in maize mitochondria

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

  19. Biotechnology of Cold-Active Proteases

    Tulasi Satyanarayana

    2013-05-01

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

  20. On the Y-chromosome haplogroup C3c classification.

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

    2012-10-01

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

  1. Reaction mechanism of -acylhydroxamate with cysteine proteases

    R Shankar; P Kolandaivel

    2007-09-01

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

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

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

    2016-07-01

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

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

    Attridge, J M

    2001-01-01

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

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

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

    2000-01-01

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

  5. Inhibitors of lysosomal cysteine proteases

    Lyanna O. L.; Chorna V. I.

    2011-01-01

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

  6. Inhibitors of lysosomal cysteine proteases

    Lyanna O. L.

    2011-04-01

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

  7. Protease-sensitive synthetic prions.

    David W Colby

    2010-01-01

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

  8. 27 CFR 21.37 - Formula No. 3-C.

    2010-04-01

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

  9. Role and efforts of T3C in corrosion economics

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

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

    Liu, Lei

    2006-01-01

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

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

    Marie-Eve eDumez

    2014-03-01

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

  12. Advances in protease engineering for laundry detergents.

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

    2015-12-25

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

  13. Extracellular proteases as targets for drug development.

    Cudic, Mare; Fields, Gregg B

    2009-08-01

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

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

    Palarasah, Yaseelan; Skjødt, Karsten; Brandt, Jette;

    2010-01-01

    complex (C5b-C9) and quantification of complement split products by precipitation-in-gel techniques (e.g. C3d). We have developed a mouse monoclonal antibody (mAb) that is able to detect fluid phase C3c without interference from other products generated from the complement component C3. The C3c specific m......Ab was tested in different ELISA combinations with various types of in vitro activated sera and with plasma or serum samples from factor I deficient patients. The specificity of the mAb was evaluated in immunoprecipitation techniques and by analysis of eluted fragments of C3 after immunoaffinity...... chromatography. The C3c mAb was confirmed to be C3c specific, as it showed no cross-reactivity with native (un-cleaved) C3, with C3b, iC3b, or with C3d. Also, no significant reaction was observed with C3 fragments in factor I deficient sera or plasma. This antibody forms the basis for the generation of a robust...

  15. 3C 33: another case of photoionized soft X-ray emission in radio galaxies

    Torresi, E.; Grandi, P; Guainazzi, M.; Palumbo, G. G. C.; Ponti, G; Bianchi, S.

    2009-01-01

    All the observations available in the Chandra and XMM-Newton archives have been used to investigate the X-ray spectral properties of 3C 33. In this paper is presented a complete X-ray analysis of the nuclear emission of this narrow line radio galaxy. The broad band spectrum of 3C 33 is complex. The hard part resembles that of Seyfert 2 galaxies, with a heavily obscured nuclear continuum (N_H~10^23 cm^-2) and a prominent Fe Kalpha line. This represents the nuclear radiation directly observed i...

  16. Synthesis of macrocyclic trypanosomal cysteine protease inhibitors

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

    2008-01-01

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

  17. A biotechnology perspective of fungal proteases

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

    2015-01-01

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

  18. Extracellular proteases as targets for drug development

    Cudic, Mare; Fields, Gregg B.

    2009-01-01

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

  19. Thioamide-Based Fluorescent Protease Sensors

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

    2014-01-01

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

  20. Biased Signaling of Protease-activated Receptors

    PeishenZhao; NigelWilliamBunnett

    2014-01-01

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

  1. Protein targeting to ATP-dependent proteases

    Inobe, Tomonao; Matouschek, Andreas

    2008-01-01

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

  2. Effect of the Active Site D25N Mutation on the Structure, Stability and Ligand Binding of the Mature HIV-1 Protease

    Sayer, Jane M.; Liu, Fengling; Ishima, Rieko; Weber, Irene T.; Louis, John M. (Pitt); (GSU)

    2008-09-03

    All aspartic proteases, including retroviral proteases, share the triplet DTG critical for the active site geometry and catalytic function. These residues interact closely in the active, dimeric structure of HIV-1 protease (PR). We have systematically assessed the effect of the D25N mutation on the structure and stability of the mature PR monomer and dimer. The D25N mutation (PR{sub D25N}) increases the equilibrium dimer dissociation constant by a factor >100-fold (1.3 {+-} 0.09 {mu}m) relative to PR. In the absence of inhibitor, NMR studies reveal clear structural differences between PR and PR{sub D25N} in the relatively mobile P1 loop (residues 79-83) and flap regions, and differential scanning calorimetric analyses show that the mutation lowers the stabilities of both the monomer and dimer folds by 5 and 7.3 C, respectively. Only minimal differences are observed in high resolution crystal structures of PR{sub D25N} complexed to darunavir (DRV), a potent clinical inhibitor, or a non-hydrolyzable substrate analogue, Ac-Thr-Ile-Nle-r-Nle-Gln-Arg-NH{sub 2} (RPB), as compared with PR{center_dot}DRV and PR{center_dot}RPB complexes. Although complexation with RPB stabilizes both dimers, the effect on their T{sub m} is smaller for PR{sub D25N} (6.2 C) than for PR (8.7 C). The T{sub m} of PR{sub D25N}{center_dot}DRV increases by only 3 C relative to free PR{sub D25N}, as compared with a 22 C increase for PR{center_dot}DRV, and the mutation increases the ligand dissociation constant of PR{sub D25N}{center_dot}DRV by a factor of {approx}10{sup 6} relative to PR{center_dot}DRV. These results suggest that interactions mediated by the catalytic Asp residues make a major contribution to the tight binding of DRV to PR.

  3. Complexity

    Gershenson, Carlos

    2011-01-01

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

  4. Privacy Issues of the W3C Geolocation API

    Doty, Nick; Wilde, Erik

    2010-01-01

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

  5. Privacy Issues of the W3C Geolocation API

    Doty, Nick; Mulligan, Deirdre K.; Wilde, Erik

    2010-01-01

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

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

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

    1990-07-01

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

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

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

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

    Whitcomb, David C

    2014-01-01

    About half of all patients with chronic pancreatitis (CP) develop diabetes mellitus (DM) due to the loss of islet cell mass, not just beta cells as in Type 1 DM (T1DM), or due to insulin resistance, as in Type 2 DM (T2DM). Patients with DM from loss of islets due to pancreatic disease or resection are diagnosed with pancreatogenic or Type 3c DM (T3cDM). Patients with T3cDM also lose counter-regulatory hormones, such as glucagon and pancreatic polypeptide, and experience maldigestion associate...

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

    Yu-Yin Li

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

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

    Sungho Ghil

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

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

    Lim, J; Combes, F

    2000-01-01

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

  12. Structure, electronic, mechanical and optical properties of ternary YAl3C3 carbide

    Hussain, Altaf; Javed, Athar; Mehmood, Salman; Rasool, M. Nasir; Khan, Muhammad Azhar; Iqbal, Faisal

    2016-05-01

    The electronic structure, mechanical and optical properties of ternary yttrium aluminum carbide (YAl3C3) has been studied by first principles approach. The crystal structure and elastic properties are studied by using Vienna ab initio simulation package (VASP). An orthogonalized linear combination of atomic orbitals (OLCAO) method based on the density functional theory (DFT) is implemented to elucidate the electronic structure and optical properties of ternary YAl3C3 carbide. The YAl3C3 carbide exhibits a narrow indirect band gap, Eg=0.12 eV which shows its poor metallic and/or semiconductor behavior. The effective charge (Q*) calculation reveals more charge transfer from Al-sites as compared to Y-sites which indicates dominant ionic character of Al-sites. The analysis of structure and bond order (BO) calculations show that the Al-C bonds in the basal plane are much stronger as compared to Al-C bonds along the c-axis. The Al-C bonds lying in the basal plane have main contribution into the overall stiffness of YAl3C3 carbide. The effective mass of charge carriers (electrons and holes) and inter-band optical properties (complex dielectric function and optical conductivity) are also studied which show high degree of anisotropy in YAl3C3.

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

    Mäntsälä, P; Zalkin, H

    1980-01-01

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

  14. Radio jet of the quasar 3C273

    Flatters, C.; Conway, R.G.

    1985-04-04

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

  15. A Modified Stratified Model for 3C 273 Jet

    Liu, Wen-Po; Shen, Zhi-Qiang

    2008-01-01

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

  16. Optical Periodicity Analysis of 3C 446 using Period04

    Fei Guo; Hao Jing Zhang

    2014-09-01

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

  17. Matriptase Complexes and Prostasin Complexes with HAI-1 and HAI-2 in Human Milk: Significant Proteolysis in Lactation

    Lai, Chih-Hsin; Lai, Ying-Jung J.; Chou, Feng-Pai; Chang, Hsiang-Hua D.; Tseng, Chun-Che; Johnson, Michael D.; Wang, Jehng-Kang; Lin, Chen-Yong

    2016-01-01

    Significant proteolysis may occur during milk synthesis and secretion, as evidenced by the presence of protease-protease inhibitor complex containing the activated form of the type 2 transmembrane serine protease matriptase and the transmembrane Kunitz-type serine protease inhibitor HAI-1. In order to identify other proteolysis events that may occur during lactation, human milk was analyzed for species containing HAI-1 and HAI-2 which is closely related to HAI-1. In addition to the previously...

  18. Modelling of potentially promising SARS protease inhibitors

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

    2007-07-18

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

  19. Immobilization to prevent enzyme incompatibility with proteases

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

    2011-01-01

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

  20. Production of alkaline protease from Cellulosimicrobium cellulans

    Luciana Ferracini-Santos; Sato, Hélia H

    2009-01-01

    Cellulosimicrobium cellulans is one of the microorganisms that produces a wide variety of yeast cell wall-degrading enzymes, β-1,3-glucanase, protease and chitinase. Dried cells of Saccharomyces cerevisiae were used as carbon and nitrogen source for cell growth and protease production. The medium components KH2PO4, KOH and dried yeast cells showed a significant effect (p

  1. Fine Structure in 3C 120 and 3C 84. Ph.D. Thesis - Maryland Univ., 24 Aug. 1976

    Hutton, L. K.

    1976-01-01

    Seven epochs of very long baseline radio interferometric observations of the Seyfert galaxies 3C 120 and 3C 84, at 3.8-cm wave length using stations at Westford, Massachusetts, Goldstone, California, Green Bank, West Virginia, and Onsala, Sweden, have been analyzed for source structure. An algorithm for reconstructing the brightness distribution of a spatially confined source from fringe amplitude and so called closure phase data has been developed and successfully applied to artificially generated test data and to data on the above mentioned sources. Over the two year time period of observation, 3C 120 was observed to consist of a double source showing apparent super relativistic expansion and separation velocities. The total flux changes comprising one outburst can be attributed to one of these components. 3C 84 showed much slower changes, evidently involving flux density changes in individual stationary components rather than relative motion.

  2. Progress and prospects on DENV protease inhibitors.

    Timiri, Ajay Kumar; Sinha, Barij Nayan; Jayaprakash, Venkatesan

    2016-07-19

    New treatments are desperately required to combat increasing rate of dengue fever cases reported in tropical and sub-tropical parts of the world. Among the ten proteins (structural and non-structural) encoded by dengue viral genome, NS2B-NS3 protease is an ideal target for drug discovery. It is responsible for the processing of poly protein that is required for genome replication of the virus. Moreover, inhibitors designed against proteases were found successful in Human Immuno-deficiency Virus (HIV) and Hepatitis C Virus (HCV). Complete molecular mechanism and a survey of inhibitors reported against dengue protease will be helpful in designing effective and potent inhibitors. This review provides an insight on molecular mechanism of dengue virus protease and covers up-to-date information on different inhibitors reported against dengue proteases with medicinal chemistry perspective. PMID:27092412

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

    Attridge, Joanne M.

    2001-01-01

    86 GHz Very Long Baseline Polarimetry probes magnetic field structures within the cores of Active Galactic Nuclei at higher angular resolutions and a spectral octave higher than previously achievable. Observations of 3C273 and 3C279 taken in April 2000 with the Coordinated Millimeter VLBI Array have resulted in the first total intensity (Stokes I) and linear polarization VLBI images reported of any source at 86 GHz. These results reveal the 86 GHz electric vector position angles within the je...

  4. Epstein-Barr Virus Nuclear Antigen 3C Augments Mdm2-Mediated p53 Ubiquitination and Degradation by Deubiquitinating Mdm2▿

    Saha, Abhik; Murakami, Masanao; Kumar, Pankaj; Bajaj, Bharat; Sims, Karen; Erle S Robertson

    2009-01-01

    Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is one of the essential latent antigens for primary B-cell transformation. Previous studies established that EBNA3C facilitates degradation of several vital cell cycle regulators, including the retinoblastoma (pRb) and p27KIP proteins, by recruitment of the SCFSkp2 E3 ubiquitin ligase complex. EBNA3C was also shown to be ubiquitinated at its N-terminal residues. Furthermore, EBNA3C can bind to and be degraded in vitro by purified 20S protea...

  5. Kvn Source-Frequency Phase-Referencing Observation of 3c 66A and 3c 66B

    Zhao, Guang-Yao; Jung, Taehyun; Dodson, Richard; Rioja, Maria; Sohn, Bong Won

    2015-09-01

    In this proceedings, preliminary results of the KVN Source-Frequency Phase-Referencing (SFPR) observation of 3C 66A and 3C 66B are presented. The motivation of this work is to measure the core-shift of these 2 sources and study the temporal evolution of the jet opacity. Two more sources were observed as secondary reference calibrators and each source was observed at 22, 43, and 86 GHz simultaneously. Our preliminary results show that after using the observations at the lower frequency to calibrate those at the higher frequency of the same source, the residual visibility phases for each source at the higher frequencies became more aligned, and the coherence time became much longer; also, the residual phases for different sources, within 10 degrees angular separations, follow similar trends. After reference to the nearby calibrator, the SFPRed maps were obtained as well as the astrometric measurements, i.e. the combined coreshift. The measurements were found to be affected by structural blending effects because of the large beamsize of KVN, but this can be corrected with higher resolution maps (e.g. KAVA maps). *%K Astrometry, radio continuum: galaxies, galaxies: active, galaxy: individual(3C 66A, 3C 66B), techniques: interferometric *%O 3C 66A, 3C 66B

  6. HIV-1 protease mutations and protease inhibitor cross-resistance.

    Rhee, Soo-Yon; Taylor, Jonathan; Fessel, W Jeffrey; Kaufman, David; Towner, William; Troia, Paolo; Ruane, Peter; Hellinger, James; Shirvani, Vivian; Zolopa, Andrew; Shafer, Robert W

    2010-10-01

    The effects of many protease inhibitor (PI)-selected mutations on the susceptibility to individual PIs are unknown. We analyzed in vitro susceptibility test results on 2,725 HIV-1 protease isolates. More than 2,400 isolates had been tested for susceptibility to fosamprenavir, indinavir, nelfinavir, and saquinavir; 2,130 isolates had been tested for susceptibility to lopinavir; 1,644 isolates had been tested for susceptibility to atazanavir; 1,265 isolates had been tested for susceptibility to tipranavir; and 642 isolates had been tested for susceptibility to darunavir. We applied least-angle regression (LARS) to the 200 most common mutations in the data set and identified a set of 46 mutations associated with decreased PI susceptibility of which 40 were not polymorphic in the eight most common HIV-1 group M subtypes. We then used least-squares regression to ascertain the relative contribution of each of these 46 mutations. The median number of mutations associated with decreased susceptibility to each PI was 28 (range, 19 to 32), and the median number of mutations associated with increased susceptibility to each PI was 2.5 (range, 1 to 8). Of the mutations with the greatest effect on PI susceptibility, I84AV was associated with decreased susceptibility to eight PIs; V32I, G48V, I54ALMSTV, V82F, and L90M were associated with decreased susceptibility to six to seven PIs; I47A, G48M, I50V, L76V, V82ST, and N88S were associated with decreased susceptibility to four to five PIs; and D30N, I50L, and V82AL were associated with decreased susceptibility to fewer than four PIs. This study underscores the greater impact of nonpolymorphic mutations compared with polymorphic mutations on decreased PI susceptibility and provides a comprehensive quantitative assessment of the effects of individual mutations on susceptibility to the eight clinically available PIs. PMID:20660676

  7. Polarization-maintaining amplifier based on 3C fiber structures

    Enokidani, Jun; Ito, Rumi; Sakurai, Tsutomu; Shin, Sumida; Tei, Kazuyoku

    2015-03-01

    Chirally-Coupled-Core (3C) fiber structure can preserve a single mode quality and even a linear polarization for a large core size. A principal advantage of fiber laser is its compatibility with monolithic integration and robust system. But so far, devices such as a combiner using the 3C fibers have not been reported. Here we report the first demonstration of such monolithic amplifier structure which contains an active fiber and a combiner based on 3C fibers. A single-stage amplifier is seeded by an EO Q-switched micro-laser and pumped by two high power fiber pigtailed 976-nm laser diodes via an in-house fabricated (2 + 1) × 1 pump signal combiner. The active fiber is based on a 3-m-long, 3C Yb-doped fiber (33 μm/250 μm core/cladding diameter with 0.06/0.46 NA). The amplifier demonstrates scaling up to 30W average power and 150 kW peak power in 0.3mJ, 2ns pulses. The beam profiles and beam qualities were characterized as its output power was varied up to 30W. The beam profile was maintained at a high beam quality of around M2=1.2. The spectral properties of the 3C fiber were also characterized as its output peak power was varied.

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

    Thiel, S; Jensen, L; Degn, Søren Egedal; Nielsen, Hans Jørgen; Gál, Peter; Dobó, Joseph; Jensenius, J C

    2012-01-01

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

  9. Mast cell proteases as pharmacological targets.

    Caughey, George H

    2016-05-01

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

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

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

    2008-06-03

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

  11. Six alternative proteases for mass spectrometry-based proteomics beyond trypsin.

    Giansanti, Piero; Tsiatsiani, Liana; Low, Teck Yew; Heck, Albert J R

    2016-05-01

    Protein digestion using a dedicated protease represents a key element in a typical mass spectrometry (MS)-based shotgun proteomics experiment. Up to now, digestion has been predominantly performed with trypsin, mainly because of its high specificity, widespread availability and ease of use. Lately, it has become apparent that the sole use of trypsin in bottom-up proteomics may impose certain limits in our ability to grasp the full proteome, missing out particular sites of post-translational modifications, protein segments or even subsets of proteins. To overcome this problem, the proteomics community has begun to explore alternative proteases to complement trypsin. However, protocols, as well as expected results generated from these alternative proteases, have not been systematically documented. Therefore, here we provide an optimized protocol for six alternative proteases that have already shown promise in their applicability in proteomics, namely chymotrypsin, LysC, LysN, AspN, GluC and ArgC. This protocol is formulated to promote ease of use and robustness, which enable parallel digestion with each of the six tested proteases. We present data on protease availability and usage including recommendations for reagent preparation. We additionally describe the appropriate MS data analysis methods and the anticipated results in the case of the analysis of a single protein (BSA) and a more complex cellular lysate (Escherichia coli). The digestion protocol presented here is convenient and robust and can be completed in ∼2 d. PMID:27123950

  12. Peculiar variations in the structure of the quasar 3C454.3

    Following a major outburst at radio wavelengths in the quasar 3C454.3, observations by means of very long baseline interferometry have revealed a superluminal increase in the size of the compact 'core' region of the source and the appearance of a complex structure within it. Some features within this structure remained stationary with respect to each other, but others showed superluminal motion. This behaviour is distinctly different from that generally seen in superluminal sources. (author)

  13. Peculiar variations in the structure of the quasar 3C454. 3

    Pauliny-Toth, I.I.K.; Porcas, R.W.; Zensus, J.A.; Kellermann, K.I.; Wu, S.Y.; Nicolson, G.D.; Mantovani, F.

    1987-08-27

    Following a major outburst at radio wavelengths in the quasar 3C454.3, observations by means of very long baseline interferometry have revealed a superluminal increase in the size of the compact 'core' region of the source and the appearance of a complex structure within it. Some features within this structure remained stationary with respect to each other, but others showed superluminal motion. This behaviour is distinctly different from that generally seen in superluminal sources.

  14. An optically stimulated superconducting-like phase in K3C60 far above equilibrium Tc

    Mitrano, M.; Cantaluppi, A.; Nicoletti, D.; Kaiser, S.; Perucchi, A.; Lupi, S.; Di Pietro, P.; Pontiroli, D.; Riccò, M.; A. Subedi; Clark, S. R.; Jaksch, D.; A. Cavalleri

    2015-01-01

    The control of non-equilibrium phenomena in complex solids is an important research frontier, encompassing new effects like light induced superconductivity. Here, we show that coherent optical excitation of molecular vibrations in the organic conductor K3C60 can induce a non-equilibrium state with the optical properties of a superconductor. A transient gap in the real part of the optical conductivity and a low-frequency divergence of the imaginary part are measured for base temperatures far a...

  15. Suzaku Observations of the Radio Galaxy 3C 33

    Evans, Daniel A; Hardcastle, Martin J; Kraft, Ralph P; Lee, Julia C; Virani, Shanil N

    2009-01-01

    We present results from a new 100-ks Suzaku observation of the nearby radio galaxy 3C 33, and investigate the nature of absorption, reflection, and jet production in this source. We model the 2-70 keV nuclear continuum with a power law that is absorbed either through one or more layers of pc-scale neutral material, or through a modestly ionized pc-scale obscurer. The expected signatures of reflection from a neutral accretion disk are absent in 3C 33: there is no evidence of a relativistically blurred Fe K$\\alpha$ emission line, and no Compton reflection hump above 10 keV. We discuss the implications of this for the nature of jet production in 3C 33.

  16. A Modified Stratified Model for 3C 273 Jet

    Liu, Wen-Po

    2008-01-01

    We present a modified stratified jet model to interpret the observed spectral energy distributions of knots in 3C 273 jet. Based on the hypothesis of the single index of the particle energy spectrum at injection and identical emission processes among all the knots, the observed difference of spectral shape among different 3C273 knots can be understood as a manifestation of deviation of the equivalent Doppler factor of stratified emission regions in individual knot from a characteristic one. The summed spectral energy distribution of all the ten knots in 3C 273 jet can be well fitted by two components, low-energy (radio to optical) component dominated by the synchrotron radiation and high-energy component (UV, X-ray and $\\gamma$-ray) dominated by the inverse Compton scattering of the cosmic microwave background. This gives a consistent spectral index of $\\alpha=0.88$ ($S_\

  17. Optical variability of PHL 1811 and 3C 273

    Fan, J. H.; Kurtanidze, O.; Liu, Y.; Yuan, Y. H.; Hao, J. M.; Cai, W.; Xiao, H. B.; Pei, Z. Y.

    2015-03-01

    In this work, we reported the optical photometry monitoring results for two brightest nearby quasars, PHL 1811 and 3C 273 using the ST-6 camera at Abastumani Observatory, Georgia. For PHL 1811, we found 3 microvariability events with time scale of ΔT = 6.0 min. For 3C273, we found that the largest variations are ΔV = 0.369 +/- 0.028 mag, ΔR = 0.495 +/- 0.076 mag, and ΔI = 0.355 +/- 0.009 mag. When periodicity analysis methods are adopted to the available data, a period of p = 5.80 +/- 1.12 years is obtained for PHL 1811, and p = 21.10 +/- 0.14, 10.00 +/- 0.14, 7.30 +/- 0.09, 13.20 +/- 0.09, 2.10 +/- 0.06, and 0.68 +/- 0.05 years are obtained for 3C 273.

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

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

    2016-01-01

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

  19. Evolutionary analysis of novel serine proteases in the venom gland transcriptome of Bitis gabonica rhinoceros.

    Sakthivel Vaiyapuri

    Full Text Available BACKGROUND: Serine proteases are major components of viper venom and target various stages of the blood coagulation system in victims and prey. A better understanding of the diversity of serine proteases and other enzymes present in snake venom will help to understand how the complexity of snake venom has evolved and will aid the development of novel therapeutics for treating snake bites. METHODOLOGY AND PRINCIPAL FINDINGS: Four serine protease-encoding genes from the venom gland transcriptome of Bitis gabonica rhinoceros were amplified and sequenced. Mass spectrometry suggests the four enzymes corresponding to these genes are present in the venom of B. g. rhinoceros. Two of the enzymes, rhinocerases 2 and 3 have substitutions to two of the serine protease catalytic triad residues and are thus unlikely to be catalytically active, though they may have evolved other toxic functions. The other two enzymes, rhinocerases 4 and 5, have classical serine protease catalytic triad residues and thus are likely to be catalytically active, however they have glycine rather than the more typical aspartic acid at the base of the primary specificity pocket (position 189. Based on a detailed analysis of these sequences we suggest that alternative splicing together with individual amino acid mutations may have been involved in their evolution. Changes within amino acid segments which were previously proposed to undergo accelerated change in venom serine proteases have also been observed. CONCLUSIONS AND SIGNIFICANCE: Our study provides further insight into the diversity of serine protease isoforms present within snake venom and discusses their possible functions and how they may have evolved. These multiple serine protease isoforms with different substrate specificities may enhance the envenomation effects and help the snake to adapt to new habitats and diets. Our findings have potential for helping the future development of improved therapeutics for snake bites.

  20. Caspase Family Proteases and Apoptosis

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

    2005-01-01

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

  1. Antibacterial Activity of Ti3C2Tx MXene.

    Rasool, Kashif; Helal, Mohamed; Ali, Adnan; Ren, Chang E; Gogotsi, Yury; Mahmoud, Khaled A

    2016-03-22

    MXenes are a family of atomically thin, two-dimensional (2D) transition metal carbides and carbonitrides with many attractive properties. Two-dimensional Ti3C2Tx (MXene) has been recently explored for applications in water desalination/purification membranes. A major success indicator for any water treatment membrane is the resistance to biofouling. To validate this and to understand better the health and environmental impacts of the new 2D carbides, we investigated the antibacterial properties of single- and few-layer Ti3C2Tx MXene flakes in colloidal solution. The antibacterial properties of Ti3C2Tx were tested against Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis) by using bacterial growth curves based on optical densities (OD) and colonies growth on agar nutritive plates. Ti3C2Tx shows a higher antibacterial efficiency toward both Gram-negative E. coli and Gram-positive B. subtilis compared with graphene oxide (GO), which has been widely reported as an antibacterial agent. Concentration dependent antibacterial activity was observed and more than 98% bacterial cell viability loss was found at 200 μg/mL Ti3C2Tx for both bacterial cells within 4 h of exposure, as confirmed by colony forming unit (CFU) and regrowth curve. Antibacterial mechanism investigation by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) coupled with lactate dehydrogenase (LDH) release assay indicated the damage to the cell membrane, which resulted in release of cytoplasmic materials from the bacterial cells. Reactive oxygen species (ROS) dependent and independent stress induction by Ti3C2Tx was investigated in two separate abiotic assays. MXenes are expected to be resistant to biofouling and offer bactericidal properties. PMID:26909865

  2. Natural inhibitors of tumor-associated proteases

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

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

    Ghose, R.

    2012-01-01

    A digital 3C array seismic cone penetrometer has been developed for multidisciplinary geophysical and geotechnical applications. Seven digital triaxial microelectromechanical system accelerometers are installed at 0.25-m intervals to make a 1.5-m-long downhole seismic array. The accelerometers have a flat response up to 2 kHz. The seismic array is attached to a class 1 digital seismic cone, which measures cone tip resistance, sleeve friction, pore-pressure, and inclination. The downhole 3C ar...

  4. Suzaku Observations of the Radio Galaxy 3C 33

    Evans, A. Daniel; Reeves, James N.; Hardcastle, Martin J.; Kraft, Ralph P.; Lee, Julia C.; Virani, Shanil N.

    2010-01-01

    We present results from a new 100 - ks Suzaku observation of the nearby radio galaxy 3C 33, and investigate the nature of absorption, reflection, and jet production in this source. We model the 2 - 70 keV nuclear continuum with a power law that is absorbed either through one or more layers of pc-scale neutral material, or through a modestly ionized pc-scale obscurer. The expected signatures of reflection from a neutral accretion disk are absent in 3C 33 : there is no evidence of a relativisti...

  5. Suzaku Observations of the Radio Galaxy 3C 33

    Evans, Daniel A.; Reeves, James N.; Hardcastle, Martin J.; Kraft, Ralph P.; Lee, Julia C.; Virani, Shanil N.

    2009-01-01

    We present results from a new 100-ks Suzaku observation of the nearby radio galaxy 3C 33, and investigate the nature of absorption, reflection, and jet production in this source. We model the 2-70 keV nuclear continuum with a power law that is absorbed either through one or more layers of pc-scale neutral material, or through a modestly ionized pc-scale obscurer. The expected signatures of reflection from a neutral accretion disk are absent in 3C 33: there is no evidence of a relativistically...

  6. New infrared spectral component of the quasar 3C273

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

    1986-09-11

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

  7. On the Jet Activity in 3C 273

    Stawarz, L.

    2004-01-01

    In this paper we comment on the possibility for intermittent jet activity in quasar 3C 273 on different time-scales. We propose, that striking morphology of the large-scale radio jet in this source, as well as the apparent lack of its counterpart on the opposite side of the active center, may be explained in a framework of a restarting jet model. In particular, we propose that 3C 273 radio source is intrinsically two-sided, and represents an analogue of double-double radio galaxies, but only ...

  8. The high energy spectrum of 3C 273

    Esposito, V; R. Walter(ISDC); Jean, P.; Tramacere, A.; M. Türler; A. Lähteenmäki(Aalto University Metsähovi Radio Observatory, Kylmälä, Finland); Tornikoski, M.

    2015-01-01

    Aims. The high energy spectrum of 3C 273 is usually understood in terms of inverse-Compton emission in a relativistic leptonic jet. This model predicts variability patterns and delays that could be tested with simultaneous observations from the radio to the GeV range. Methods. The instruments IBIS, SPI, JEM-X on board INTEGRAL, PCA on board RXTE, and LAT on board Fermi have enough sensitivity to follow the spectral variability of 3C 273 from the keV to the GeV. We looked for correlations betw...

  9. 基于S3C2410上U-Boot的移植与实现%U-Boot’s Transplantation and Implementation Based on S3C2410

    张伟; 刘斌; 董群锋

    2014-01-01

    The operating system kernel transplantation is the premise and foundation of the embedded system development. In view of the complexity and diversity of the U-boot transplantation, this paper analyzed the file structure and starting process of U-boot, it has chosen S3C2410 of SanSung company for development board, cross-compilation environment construction and U-boot transplantation and the kernel of the burning process were introduced in detail. We combined the function of U-boot with the characteristics of Linux in the transplantation process, this method has the characters of fast transplant, simple modify of kernel and strong commonality. Through compile testing, U-boot transplantation is implemented successful on the S3C2410, provides a reference for other U-boot transplantation.%操作系统内核移植是嵌入式系统开发的前提和基础,针对U-boot移植的复杂性和多样性,在分析了U-boot的文件结构和启动过程的基础上,选取了以SanSung公司的S3C2410为处理器的开发板,详细介绍了交叉编译环境的搭建、U-boot的移植、内核的烧写等过程。移植过程中将U-boot的功能与Linux的特点相结合,此方法具有移植速度快、内核修改简单、通用性强的特点。通过编译测试,成功实现了U-boot在S3C2410的移植,为其他U-boot的移植提供了一种参考。

  10. 基于S3C2410上U-Boot的移植与实现%U-Boot’s Transplantation and Implementation Based on S3C2410

    张伟; 刘斌; 董群锋

    2014-01-01

    操作系统内核移植是嵌入式系统开发的前提和基础,针对U-boot移植的复杂性和多样性,在分析了U-boot的文件结构和启动过程的基础上,选取了以SanSung公司的S3C2410为处理器的开发板,详细介绍了交叉编译环境的搭建、U-boot的移植、内核的烧写等过程。移植过程中将U-boot的功能与Linux的特点相结合,此方法具有移植速度快、内核修改简单、通用性强的特点。通过编译测试,成功实现了U-boot在S3C2410的移植,为其他U-boot的移植提供了一种参考。%The operating system kernel transplantation is the premise and foundation of the embedded system development. In view of the complexity and diversity of the U-boot transplantation, this paper analyzed the file structure and starting process of U-boot, it has chosen S3C2410 of SanSung company for development board, cross-compilation environment construction and U-boot transplantation and the kernel of the burning process were introduced in detail. We combined the function of U-boot with the characteristics of Linux in the transplantation process, this method has the characters of fast transplant, simple modify of kernel and strong commonality. Through compile testing, U-boot transplantation is implemented successful on the S3C2410, provides a reference for other U-boot transplantation.

  11. Evolutionary implications of C3 -C4 intermediates in the grass Alloteropsis semialata.

    Lundgren, Marjorie R; Christin, Pascal-Antoine; Escobar, Emmanuel Gonzalez; Ripley, Brad S; Besnard, Guillaume; Long, Christine M; Hattersley, Paul W; Ellis, Roger P; Leegood, Richard C; Osborne, Colin P

    2016-09-01

    C4 photosynthesis is a complex trait resulting from a series of anatomical and biochemical modifications to the ancestral C3 pathway. It is thought to evolve in a stepwise manner, creating intermediates with different combinations of C4 -like components. Determining the adaptive value of these components is key to understanding how C4 photosynthesis can gradually assemble through natural selection. Here, we decompose the photosynthetic phenotypes of numerous individuals of the grass Alloteropsis semialata, the only species known to include both C3 and C4 genotypes. Analyses of δ(13) C, physiology and leaf anatomy demonstrate for the first time the existence of physiological C3 -C4 intermediate individuals in the species. Based on previous phylogenetic analyses, the C3 -C4 individuals are not hybrids between the C3 and C4 genotypes analysed, but instead belong to a distinct genetic lineage, and might have given rise to C4 descendants. C3 A. semialata, present in colder climates, likely represents a reversal from a C3 -C4 intermediate state, indicating that, unlike C4 photosynthesis, evolution of the C3 -C4 phenotype is not irreversible. PMID:26524631

  12. Mammalian m-AAA Proteases as Key Regulators of Mitochondrial Function - Analysis of Dominant Negative Mutant Variants

    Raschke, Ines

    2009-01-01

    To ensure the removal of excess and non-assembled proteins, mitochondria require a protein quality control system which is constituted by several proteases located in different compartments of mitochondria. m-AAA proteases, oligomeric ATP-dependent metallopeptidases, are key components of this system active at the matrix side of the inner membrane. Human m-AAA proteases build up homo- and hetero-oligomeric complexes composed of AFG3L2 and SPG7. Mice express a third subunit, Afg3l1, resulting ...

  13. Revisiting correlations between broad-line and jet emission variations for AGNs: 3C 120 and 3C 273

    Liu, H. T.; Bai, J. M.; Feng, H. C.; Li, S. K.

    2015-06-01

    We restudy the issue of cross-correlations between broad-line and jet emission variations, and aim to locate the position of a radio (and gamma-ray) emitting region in a jet of active galactic nuclei. Considering the radial profiles of the radius and number density of clouds in a spherical broad-line region (BLR), we derive new formulae connecting the jet-emitting position Rjet to the time lag τob between broad-line and jet emission variations, and the BLR radius. Also, formulae are derived for a disc-like BLR and a spherical shell BLR. The model-independent flux randomization/random subset selection method is used to estimate τob. For 3C 120, positive lags of about 0.3 yr are found between the 15 GHz emission and the Hβ, Hγ and He II λ4686 lines, including broad-line data in a newly published paper, indicating that the line variations lead the 15 GHz ones. Each of the broad-line light curves corresponds to a radio outburst. Rjet = 1.1-1.5 parsec (pc) is obtained for 3C 120. For 3C 273, a common feature of negative time lags is found in the cross-correlation functions between light curves of radio emission and the Balmer lines, as well as Lyα λ1216 and C IV λ1549 lines. Rjet = 1.0-2.6 pc is obtained for 3C 273. The estimated Rjet is comparable for 3C 120 and 3C 273, and the gamma-ray-emitting positions will be within ˜1-3 pc from the central engines. Comparisons show that the cloud number density and radius radial distributions and the BLR structures have only negligible effects on Rjet.

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

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

    2011-03-01

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

  15. Microwave Radiometer – 3 Channel (MWR3C) Handbook

    Cadeddu, MP

    2012-05-04

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

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

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

    2009-01-01

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

  17. Optical polarization in the jet of 3C273

    A linear polarization map of the optical jet of 3C273 is presented. Along the whole length of the visible jet significant levels of polarization are detected with an orientation approximately perpendicular to the jet axis. The results remove the need to invoke a non-synchrotron contribution to the optical emission from the jet. (author)

  18. Is the 3C273 quasar mach nearer?

    Effect of ''superlight'' expansion of some quasars resulting from cosmologic interpretation of red shift is considered. Possibility of determining distance to 3C273 quasar, taking account of its expansion angular velocity in frames of Doppler interpretation of quasar red shift value, is discussed

  19. Optical polarization in the jet of 3C273

    Scarrott, S.M.; Warren-Smith, R.F.

    1987-10-01

    A linear polarization map of the optical jet of 3C273 is presented. Along the whole length of the visible jet significant levels of polarization are detected with an orientation approximately perpendicular to the jet axis. The results remove the need to invoke a non-synchrotron contribution to the optical emission from the jet.

  20. The jet in the quasar 3C273

    The quasar 3C273 has been mapped with the six telescope array, MERLIN, at both 408 MHz and 1666 MHz, yielding maps with resolutions of 0.9 and 0.35 arc s. Both maps show evidence for quasi-sinusoidal wiggles. Two models explaining this, are shortly discussed. (Auth.)

  1. The Double–Double Radio Galaxy 3C293

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

    2011-12-01

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

  2. W3C head Berners-Lee to be knighted

    Gross, G

    2004-01-01

    "Tim Berners-Lee, credited with inventing the World Wide Web and now director of the World Wide Web Consortium, will be named a knight commander, Order of the British Empire, by Queen Elizabeth II, the W3C announced Wednesday" (1 page)

  3. A 3C-path for Glauberman-Norton theory

    Griess, Robert L

    2009-01-01

    One would like an explanation of the provocative McKay and Glauberman-Norton observations connecting the extended $E_8$-diagram with pairs of 2A involutions in the Monster sporadic simple group. We propose a down-to-earth model for the 3C-case which exhibits a logic to these connections.

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

    Goupil, Louise S.; Ivry, Sam L.; Hsieh, Ivy; Suzuki, Brian M.; Craik, Charles S.; O’Donoghue, Anthony J.; McKerrow, James H.

    2016-01-01

    Proteases perform numerous vital functions in flatworms, many of which are likely to be conserved throughout the phylum Platyhelminthes. Within this phylum are several parasitic worms that are often poorly characterized due to their complex life-cycles and lack of responsiveness to genetic manipulation. The flatworm Schmidtea mediterranea, or planaria, is an ideal model organism to study the complex role of protein digestion due to its simple life cycle and amenability to techniques like RNA interference (RNAi). In this study, we were interested in deconvoluting the digestive protease system that exists in the planarian gut. To do this, we developed an alcohol-induced regurgitation technique to enrich for the gut enzymes in S. mediterranea. Using a panel of fluorescent substrates, we show that this treatment produces a sharp increase in proteolytic activity. These enzymes have broad yet diverse substrate specificity profiles. Proteomic analysis of the gut contents revealed the presence of cysteine and metallo-proteases. However, treatment with class-specific inhibitors showed that aspartyl and cysteine proteases are responsible for the majority of protein digestion. Specific RNAi knockdown of the cathepsin B-like cysteine protease (SmedCB) reduced protein degradation in vivo. Immunohistochemistry and whole-mount in situ hybridization (WISH) confirmed that the full-length and active forms of SmedCB are found in secretory cells surrounding the planaria intestinal lumen. Finally, we show that the knockdown of SmedCB reduces the speed of tissue regeneration. Defining the roles of proteases in planaria can provide insight to functions of conserved proteases in parasitic flatworms, potentially uncovering drug targets in parasites. PMID:27501047

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

    Goupil, Louise S; Ivry, Sam L; Hsieh, Ivy; Suzuki, Brian M; Craik, Charles S; O'Donoghue, Anthony J; McKerrow, James H

    2016-08-01

    Proteases perform numerous vital functions in flatworms, many of which are likely to be conserved throughout the phylum Platyhelminthes. Within this phylum are several parasitic worms that are often poorly characterized due to their complex life-cycles and lack of responsiveness to genetic manipulation. The flatworm Schmidtea mediterranea, or planaria, is an ideal model organism to study the complex role of protein digestion due to its simple life cycle and amenability to techniques like RNA interference (RNAi). In this study, we were interested in deconvoluting the digestive protease system that exists in the planarian gut. To do this, we developed an alcohol-induced regurgitation technique to enrich for the gut enzymes in S. mediterranea. Using a panel of fluorescent substrates, we show that this treatment produces a sharp increase in proteolytic activity. These enzymes have broad yet diverse substrate specificity profiles. Proteomic analysis of the gut contents revealed the presence of cysteine and metallo-proteases. However, treatment with class-specific inhibitors showed that aspartyl and cysteine proteases are responsible for the majority of protein digestion. Specific RNAi knockdown of the cathepsin B-like cysteine protease (SmedCB) reduced protein degradation in vivo. Immunohistochemistry and whole-mount in situ hybridization (WISH) confirmed that the full-length and active forms of SmedCB are found in secretory cells surrounding the planaria intestinal lumen. Finally, we show that the knockdown of SmedCB reduces the speed of tissue regeneration. Defining the roles of proteases in planaria can provide insight to functions of conserved proteases in parasitic flatworms, potentially uncovering drug targets in parasites. PMID:27501047

  6. Dysregulation of Protease and Protease Inhibitors in a Mouse Model of Human Pelvic Organ Prolapse

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

    2013-01-01

    Mice deficient for the fibulin-5 gene (Fbln5(-/-)) develop pelvic organ prolapse (POP) due to compromised elastic fibers and upregulation of matrix metalloprotease (MMP)-9. Here, we used casein zymography, inhibitor profiling, affinity pull-down, and mass spectrometry to discover additional protease upregulated in the vaginal wall of Fbln5(-/-) mice, herein named V1 (25 kDa). V1 was a serine protease with trypsin-like activity similar to protease, serine (PRSS) 3, a major extrapancreatic tryp...

  7. Nidovirus papain-like proteases: multifunctional enzymes with protease, deubiquitinating and deISGylating activities

    Mielech, Anna M.; Chen, Yafang; MESECAR, Andrew D.; Baker, Susan C.

    2014-01-01

    Coronaviruses and arteriviruses, members of the order Nidovirales, are positive strand RNA viruses that encode large replicase polyproteins that are processed by viral proteases to generate the nonstructural proteins which mediate viral RNA synthesis. The viral papain-like proteases (PLPs) are critical for processing the amino-terminal end of the replicase and are attractive targets for antiviral therapies. With the analysis of the papain-like protease of Severe Acute Respiratory Syndrome cor...

  8. Solid-state characterization of the HIV protease inhibitor

    Kim, Y A

    2002-01-01

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

  9. Antimalarial Synergy of Cysteine and Aspartic Protease Inhibitors

    Semenov, Andrey; Olson, Jed E.; Rosenthal, Philip J.

    1998-01-01

    It has been proposed that the Plasmodium falciparum cysteine protease falcipain and aspartic proteases plasmepsin I and plasmepsin II act cooperatively to hydrolyze hemoglobin as a source of amino acids for erythrocytic parasites. Inhibitors of each of these proteases have potent antimalarial effects. We have now evaluated the antimalarial effects of combinations of cysteine and aspartic protease inhibitors. When incubated with cultured P. falciparum parasites, cysteine and aspartic protease ...

  10. Structure-Function of Falcipains: Malarial Cysteine Proteases

    Pandey, Kailash C.; Rajnikant Dixit

    2012-01-01

    Evidence indicates that cysteine proteases play essential role in malaria parasites; therefore an obvious area of investigation is the inhibition of these enzymes to treat malaria. Studies with cysteine protease inhibitors and manipulating cysteine proteases genes have suggested a role for cysteine proteases in hemoglobin hydrolysis. The best characterized Plasmodium cysteine proteases are falcipains, which are papain family enzymes. Falcipain-2 and falcipain-3 are major hemoglobinases of P. ...

  11. Targeting exosites on blood coagulation proteases

    Robson Q. Monteiro

    2005-06-01

    Full Text Available The high specificity of blood coagulation proteases has been attributed not only to residues surrounding the active site but also to other surface domains that are involved in recognizing and interacting with macromolecular substrates and inhibitors. Specific blood coagulation inhibitors obtained from exogenous sources such as blood sucking salivary glands and snake venoms have been identified. Some of these inhibitors interact with exosites on coagulation enzymes. Two examples are discussed in this short revision. Bothrojaracin is a snake venom-derived protein that binds to thrombin exosites 1 and 2. Complex formation impairs several exosite-dependent activities of thrombin including fibrinogen cleavage and platelet activation. Bothrojaracin also interacts with proexosite 1 on prothrombin thus decreasing the zymogen activation by the prothrombinase complex (FXa/FVa. Ixolaris is a two Kunitz tick salivary gland inhibitor, that is homologous to tissue factor pathway inhibitor. Recently it was demonstrated that ixolaris binds to heparin-binding exosite of FXa, thus impairing the recognition of prothrombin by the enzyme. In addition, ixolaris interacts with FX possibly through the heparin-binding proexosite. Differently from FX, the ixolaris-FX complex is not recognized as substrate by the intrinsic tenase complex (FIXa/FVIIIa. We conclude that these inhibitors may serve as tools for the study of coagulation exosites as well as prototypes for new anticoagulant drugs.A alta especificidade das proteases da coagulação tem sido atribuída não somente aos resíduos que cercam o sítio ativo, mas também a outros domínios de superfície que estão envolvidos no reconhecimento e interação com substratos macromoleculares e inibidores. Inibidores específicos da coagulação sanguínea obtidos de fontes exógenas como glândulas salivares de animais hematófagos e venenos de serpentes têm sido identificados. Alguns desses inibidores interagem com os

  12. Cysteine Proteases from Bloodfeeding Arthropod Ectoparasites

    Sojka, Daniel; FRANCISCHETTI, IVO M. B.; Calvo, Eric; KOTSYFAKIS, MICHALIS

    2011-01-01

    Cysteine proteases have been discovered in various bloodfeeding ectoparasites. Here, we assemble the available information about the function of these peptidases and reveal their role in hematophagy and parasite development. While most of the data shed light on key proteolytic events that play a role in arthropod physiology, we also report on the association of cysteine proteases with arthropod vectorial capacity. With emphasis on ticks, specifically Ixodes ricinus, we finally propose a model...

  13. Protease Inhibitors Targeting Coronavirus and Filovirus Entry

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

    2015-01-01

    In order to gain entry into cells, diverse viruses, including Ebola virus, SARS-coronavirus and the emerging MERS-coronavirus, depend on activation of their envelope glycoproteins by host cell proteases. The respective enzymes are thus excellent targets for antiviral intervention. In cell culture, activation of Ebola virus, as well as SARS- and MERS-coronavirus can be accomplished by the endosomal cysteine proteases, cathepsin L (CTSL) and cathepsin B (CTSB). In addition, SARS- and MERS-coron...

  14. Electrically sensing protease activity with nanopores

    Kukwikila, Mikiembo; Howorka, Stefan

    2010-11-01

    The enzymatic activity of a protease was electrically detected using nanopore recordings. A peptide substrate was tethered to microscale beads, and cleavage by the enzyme trypsin released a soluble fragment that was electrophoretically driven through the α-hemolysin protein pore, leading to detectable blockades in the ionic current. Owing to its simplicity, this approach to sense enzymatic activity may be applied to other proteases.

  15. Extracellular acid proteases produced by Saccharomycopsis lipolytica.

    T. Yamada; Ogrydziak, D M

    1983-01-01

    Saccharomycopsis lipolytica CX161-1B produced at least three extracellular acid proteases during exponential growth in medium containing glycerol, Difco Proteose Peptone, and mineral salts at pH 3.4 (Difco Laboratories, Detroit, Mich.). Little extracellular acid protease activity was produced with glutamic acid as the sole nitrogen source, somewhat higher levels were obtained with peptone, and much higher levels were obtained with Difco Proteose Peptone. The relative amounts of the three prot...

  16. A radiometric assay for HIV-1 protease

    A rapid, high-throughput radiometric assay for HIV-1 protease has been developed using ion-exchange chromatography performed in 96-well filtration plates. The assay monitors the activity of the HIV-1 protease on the radiolabeled form of a heptapeptide substrate, [tyrosyl-3,5-3H]Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, which is based on the p17-p24 cleavage site found in the viral polyprotein substrate Pr55gag. Specific cleavage of this uncharged heptapeptide substrate by HIV-1 protease releases the anionic product [tyrosyl-3,5-3H]Ac-Ser-Gln-Asn-Tyr, which is retained upon minicolumns of the anion-exchange resin AG1-X8. Protease activity is determined from the recovery of this radiolabeled product following elution with formic acid. This facile and highly sensitive assay may be utilized for steady-state kinetic analysis of the protease, for measurements of enzyme activity during its purification, and as a routine assay for the evaluation of protease inhibitors from natural product or synthetic sources

  17. ADAM Proteases and Gastrointestinal Function.

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

    2016-01-01

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

  18. BeppoSAX Observations of 3C 279

    We report on BeppoSAX AO1 Core Program observations of 3C 279, performed in January 1997. 3C 279 was found in a low state, with constant X-ray flux in the 5 observations. The spectra obtained with the LECS and MECS instruments combining the 5 observations are well fitted by a single power law with energy spectral index α = 0.64 ± 0.03 and Galactic absorption. The source is weakly detected by the PDS instrument. Comparison with simultaneous γ-ray data obtained by EGRET and with previous multifrequency measurements shows that the X-ray emission is well correlated with the γ-ray emission over long timescales

  19. A Dust Lane in the Radio galaxy 3C270

    Mahabal, Ashish; Kembhavi, Ajit; Singh, K. P.; Bhat, P.N.; Prabhu, T. P.

    1995-01-01

    We present broad band surface photometry of the radio galaxy 3C270 (NGC~4261). We find a distinct dust lane in the $V-R$ image of the galaxy, and determine its orientation and size. We use the major axis profile of the galaxy to estimate the optical depth of the dust lane, and discuss the significance of the lane to the shape of the galaxy.

  20. The Global Life Long Learning Communities (GL3C) Project

    Castelein, Folkert; Ratna, Vivek

    2007-01-01

    In our opinion learning is moving from pushing content to an individual into the integration of formal & informal learning, just in time help and coaching, and is highly adaptive. There is a way to improve how people, organizations, and institutions are learning and working together. Therefore the Global Learning Institute and RSM Erasmus University launched the Global Life Long Learning Communities (GL3C) project and invites you to join. We started this project by first determining the cause...

  1. The blue-bump of 3C 273

    Paltani, S.; Courvoisier, T. J-L.; R. Walter(ISDC)

    1998-01-01

    We present optical and ultraviolet observations of 3C273 covering the whole life of the IUE satellite. We analyze the variability properties of the light curves, and find that two variable components, written B and R respectively, must contribute to the blue-bump emission in this object. The B component produces most of the variability in the ultraviolet domain. A maximum time scale of variability of about 2 yr identical at all wavelengths is found. If discrete events produce this component, ...

  2. Pion elastic scattering from polarized sup 1 sup 3 C

    Lee, D H; Kim, B T

    1999-01-01

    The elastic scattering cross sections and the analyzing powers for pi sup + and pi sup - from a sup 1 sup 3 C target are calculated by solving the Schroedinger equation reduced from the Klein-Gordon equation. In doing so, kinematical variables are redefined, local potentials are assumed , and the potential parameters are fixed to reproduce the experimental data. The calculated cross sections and the analyzing powers are compared with the observed data.

  3. Carbohydrate protease conjugates: Stabilized proteases for peptide synthesis

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

    1995-12-31

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

  4. A warm hot intergalactic medium towards 3C 120?

    McKernan, B; Mushotzky, R; George, I M; Turner, T J

    2003-01-01

    We observed the Seyfert I active galaxy/broad line radio galaxy 3C120 with the Chandra high energy transmission gratings and present an analysis of the soft X-ray spectrum. We identify the strongest absorption feature (detected at >99.9% confidence) with O VIII Lya (FWHM=1010^{+295}_{-265} km/s), blueshifted by -5500 +/- 140 km/s from systemic velocity. The absorption may be due to missing baryons in warm/hot intergalactic medium (WHIGM) along the line-of-sight to 3C 120 at z=0.0147 +/- 0.0005, or it could be intrinsic to the jet of 3C 120. Assuming metallicities of 0.1 solar, we estimate an ionic column density of N_{O VIII}>3.4 \\times 10^{16} cm^{-2} for WHIGM and a filament depth of 56 relative to the critical density of a $\\Lambda$-dominated cold dark matter universe, which is in reasonable agreement with WHIGM simulations. We detect, at marginal significance, absorption of O VIII Lya at z\\sim 0 due to a hot medium in the Local Group. We also detect an unidentified absorption feature at \\sim 0.71 keV. Abs...

  5. Synchrotron flaring in the jet of 3C 279

    Lindfors, E J; Valtaoja, E; Aller, H; Aller, M; Mazin, D; Raiteri, C M; Stevens, J A; Tornikoski, M; Tosti, G; Villata, M

    2006-01-01

    We study the synchrotron flaring behaviour of the blazar 3C279 based on an extensive dataset covering 10 years of monitoring at 19 different frequencies in the radio-to-optical range. The properties of a typical outburst are derived from the observations by decomposing the 19 lightcurves into a series of self-similar events. This analysis is achieved by fitting all data simultaneously to a succession of outbursts defined according to the shock-in-jet model of Marscher & Gear (1985). We compare the derived properties of the synchrotron outbursts in 3C279 to those obtained with a similar method for the quasar 3C273. It is argued that differences in the flaring behaviour of these two sources are intrinsic to the sources themselves rather than being due to orientation effects. We also compare the start times and flux densities of our modelled outbursts with those measured from radio components identified in Very Long Baseline Interferometry (VLBI) images. We find VLBI counterparts for most of our model outbur...

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

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

    2007-01-01

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

  7. The milliarcsecond structure of 3C 273 at 22 GHz

    The first VLBI images at 22 GHz of the jet in the quasar 3C 273 are presented. In addition to the compact core region, two emission regions can be identified with features seen at lower frequencies; they separate from the core with constant speeds of 0.65 + or - 0.09 and 0.92 + or - 0.11 mas/yr, corresponding to apparent superluminal motion of 4.3 + or - 0.3c and 6.1 + or - 0.3c (for Ho = 100 km/s Mpc, qo = 0.5). The core region brightened at about the estimated epoch of zero separation for the latest superluminal component, suggesting a causal relationship. The curved ridge line of the jet smoothly extends inward towards the core, although no pronounced bends in the range of core distance 0.5-2.5 mas are seen. No significant evidence is found against a common path of subsequent superluminal features. An apparent frequency dependence in the position of one superluminal feature tentatively suggests that opacity effects across the jet direction are present. The results are consistent with an interpretation of the superluminal features as shocks in an underlying relativistic flow, although alternative explanations cannot be ruled out. 43 refs

  8. The milliarcsecond structure of 3C 273 at 22 GHz

    Zensus, J.A.; Biretta, J.A.; Unwin, S.C.; Cohen, M.H. (National Radio Astronomy Observatory, Socorro, NM (USA) Owens Valley Radio Observatory, Pasadena, CA (USA))

    1990-12-01

    The first VLBI images at 22 GHz of the jet in the quasar 3C 273 are presented. In addition to the compact core region, two emission regions can be identified with features seen at lower frequencies; they separate from the core with constant speeds of 0.65 + or - 0.09 and 0.92 + or - 0.11 mas/yr, corresponding to apparent superluminal motion of 4.3 + or - 0.3c and 6.1 + or - 0.3c (for Ho = 100 km/s Mpc, qo = 0.5). The core region brightened at about the estimated epoch of zero separation for the latest superluminal component, suggesting a causal relationship. The curved ridge line of the jet smoothly extends inward towards the core, although no pronounced bends in the range of core distance 0.5-2.5 mas are seen. No significant evidence is found against a common path of subsequent superluminal features. An apparent frequency dependence in the position of one superluminal feature tentatively suggests that opacity effects across the jet direction are present. The results are consistent with an interpretation of the superluminal features as shocks in an underlying relativistic flow, although alternative explanations cannot be ruled out. 43 refs.

  9. Protease inhibitors targeting coronavirus and filovirus entry.

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

    2015-04-01

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

  10. New soluble ATP-dependent protease, Ti, in Escherichia coli that is distinct from protease La

    E. coli must contain other ATP-requiring proteolytic systems in addition to protease La (the lon gene product). A new ATP-dependent protease was purified from lon cells which lack protease La, as shown by immuno-blotting. This enzyme hydrolyzes [3H]casein to acid-soluble products in the presence of ATP (or dATP) and Mg2+. Nonhydrolyzable ATP analogs, other nucleoside triphosphates and AMP can not replace ATP. Therefore, ATP hydrolysis appears necessary for proteolysis. The enzyme appears to be a serine protease, but also contains essential thiol residues. Unlike protease La, it is not inhibited by vanadate, heparin, or the defective R9 subunit of protease La. On gel filtration, this enzyme has an apparent Mr of 340,000 and is comprised of two components of 190,000D and 130,000D, which can be separated by phosphocellulose chromatography. By themselves, these components do not show ATP-dependent proteolysis, but when mixed, full activity is restored. These finding and similar ones of Maurizi and Gottesman indicate that E. coli contain two soluble ATP-dependent proteases, which function by different mechanisms. This new enzyme may contribute to the rapid breakdown of abnormal polypeptides or of normal proteins during starvation. The authors propose to name it protease Ti

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

    The energy requirement for protein breakdown in Escherichia coli has generally been attributed to the ATP-dependence of protease La, the lon gene product. The authors have partially purified another ATP-dependent protease from lon- cells that lack protease La (as shown by immunoblotting). This enzyme hydrolyzes [3H]methyl-casein to acid-soluble products in the presence of ATP and Mg2+. ATP hydrolysis appears necessary for proteolytic activity. Since this enzyme is inhibited by diisopropyl fluorophosphate, it appears to be a serine protease, but it also contains essential thiol residues. They propose to name this enzyme protease Ti. It differs from protease La in nucleotide specificity, inhibitor sensitivity, and subunit composition. On gel filtration, protease Ti has an apparent molecular weight of 370,000. It can be fractionated by phosphocellulose chromatography or by DEAE chromatography into two components with apparent molecular weights of 260,000 and 140,000. When separated, they do not show preteolytic activity. One of these components, by itself, has ATPase activity and is labile in the absence of ATP. The other contains the diisopropyl fluorophosphate-sensitive proteolytic site. These results and the similar findings of Katayama-Fujimura et al. indicate that E. coli contains two ATP-hydrolyzing proteases, which differ in many biochemical features and probably in their physiological roles

  12. Crystal Structures of a Multidrug-Resistant Human Immunodeficiency Virus Type 1 Protease Reveal an Expanded Active-Site Cavity

    Logsdon, Bradley C.; Vickrey, John F.; Martin, Philip; Proteasa, Gheorghe; Koepke, Jay I.; Terlecky, Stanley R.; Wawrzak, Zdzislaw; Winters, Mark A.; Merigan, Thomas C.; Kovari, Ladislau C. (Stanford); (WSU-MED); (NWU); (Stanford-MED)

    2010-03-08

    The goal of this study was to use X-ray crystallography to investigate the structural basis of resistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors. We overexpressed, purified, and crystallized a multidrug-resistant (MDR) HIV-1 protease enzyme derived from a patient failing on several protease inhibitor-containing regimens. This HIV-1 variant contained codon mutations at positions 10, 36, 46, 54, 63, 71, 82, 84, and 90 that confer drug resistance to protease inhibitors. The 1.8-{angstrom} crystal structure of this MDR patient isolate reveals an expanded active-site cavity. The active-site expansion includes position 82 and 84 mutations due to the alterations in the amino acid side chains from longer to shorter (e.g., V82A and I84V). The MDR isolate 769 protease 'flaps' stay open wider, and the difference in the flap tip distances in the MDR 769 variant is 12 {angstrom}. The MDR 769 protease crystal complexes with lopinavir and DMP450 reveal completely different binding modes. The network of interactions between the ligands and the MDR 769 protease is completely different from that seen with the wild-type protease-ligand complexes. The water molecule-forming hydrogen bonds bridging between the two flaps and either the substrate or the peptide-based inhibitor are lacking in the MDR 769 clinical isolate. The S1, S1', S3, and S3' pockets show expansion and conformational change. Surface plasmon resonance measurements with the MDR 769 protease indicate higher k{sub off} rates, resulting in a change of binding affinity. Surface plasmon resonance measurements provide k{sub on} and k{sub off} data (K{sub d} = k{sub off}/k{sub on}) to measure binding of the multidrug-resistant protease to various ligands. This MDR 769 protease represents a new antiviral target, presenting the possibility of designing novel inhibitors with activity against the open and expanded protease forms.

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

    Koh-Stenta, Xiaoying; Joy, Joma; Wang, Si Fang; Kwek, Perlyn Zekui; Wee, John Liang Kuan; Wan, Kah Fei; Gayen, Shovanlal; Chen, Angela Shuyi; Kang, CongBao; Lee, May Ann; Poulsen, Anders; Vasudevan, Subhash G; Hill, Jeffrey; Nacro, Kassoum

    2015-01-01

    Dengue virus (DENV) protease is an attractive target for drug development; however, no compounds have reached clinical development to date. In this study, we utilized a potent West Nile virus protease inhibitor of the pyrazole ester derivative class as a chemical starting point for DENV protease drug development. Compound potency and selectivity for DENV protease were improved through structure-guided small molecule optimization, and protease-inhibitor binding interactions were validated biophysically using nuclear magnetic resonance. Our work strongly suggests that this class of compounds inhibits flavivirus protease through targeted covalent modification of active site serine, contrary to an allosteric binding mechanism as previously described. PMID:26677315

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

    Koh-Stenta, Xiaoying; Joy, Joma; Wang, Si Fang; Kwek, Perlyn Zekui; Wee, John Liang Kuan; Wan, Kah Fei; Gayen, Shovanlal; Chen, Angela Shuyi; Kang, CongBao; Lee, May Ann; Poulsen, Anders; Vasudevan, Subhash G; Hill, Jeffrey; Nacro, Kassoum

    2015-01-01

    Dengue virus (DENV) protease is an attractive target for drug development; however, no compounds have reached clinical development to date. In this study, we utilized a potent West Nile virus protease inhibitor of the pyrazole ester derivative class as a chemical starting point for DENV protease drug development. Compound potency and selectivity for DENV protease were improved through structure-guided small molecule optimization, and protease-inhibitor binding interactions were validated biophysically using nuclear magnetic resonance. Our work strongly suggests that this class of compounds inhibits flavivirus protease through targeted covalent modification of active site serine, contrary to an allosteric binding mechanism as previously described. PMID:26677315

  15. Immobilization of bacterial proteases on water-solved polymer by means of electron beam

    Possibility of electron beam usage for proteases' immobilization on 1,4-polyalkylene oxide (1,4-PAO) was studied to obtain biologically active complex for multi-purpose usage. It is shown that immobilization of Bacillus Subtilis protease takes place due to free-radical linking of enzyme and carrier with formation of mycellium-like structures. Immobilization improves heat resistance of enzyme up to 60oC without substrate and up to 80oC in presence of substrate, widens range of pH activity in comparison with non-immobilized forms. Immobilized proteases do not contain peroxides or long-live radicals. Our results permitted to create technologies for production of medical and veterinary preparations, active components for wool washing agents and leather fabrication technology. (Author)

  16. Activation of Bacteroides fragilis toxin by a novel bacterial protease contributes to anaerobic sepsis in mice.

    Choi, Vivian M; Herrou, Julien; Hecht, Aaron L; Teoh, Wei Ping; Turner, Jerrold R; Crosson, Sean; Bubeck Wardenburg, Juliane

    2016-05-01

    Bacteroides fragilis is the leading cause of anaerobic bacteremia and sepsis. Enterotoxigenic strains that produce B. fragilis toxin (BFT, fragilysin) contribute to colitis and intestinal malignancy, yet are also isolated in bloodstream infection. It is not known whether these strains harbor unique genetic determinants that confer virulence in extra-intestinal disease. We demonstrate that BFT contributes to sepsis in mice, and we identify a B. fragilis protease called fragipain (Fpn) that is required for the endogenous activation of BFT through the removal of its auto-inhibitory prodomain. Structural analysis of Fpn reveals a His-Cys catalytic dyad that is characteristic of C11-family cysteine proteases that are conserved in multiple pathogenic Bacteroides spp. and Clostridium spp. Fpn-deficient, enterotoxigenic B. fragilis has an attenuated ability to induce sepsis in mice; however, Fpn is dispensable in B. fragilis colitis, wherein host proteases mediate BFT activation. Our findings define a role for B. fragilis enterotoxin and its activating protease in the pathogenesis of bloodstream infection, which indicates a greater complexity of cellular targeting and activity of BFT than previously recognized. The expression of fpn by both toxigenic and nontoxigenic strains suggests that this protease may contribute to anaerobic sepsis in ways that extend beyond its role in toxin activation. It could thus potentially serve as a target for disease modification. PMID:27089515

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

    Adrian Velazquez-Campoy

    2013-06-01

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

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

    R.PRABAVATHI

    2012-07-01

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

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

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

    2007-01-01

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

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

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

    2016-03-01

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

  1. Peculiar radio structure in the quasar 3C380

    Wilkinson, P.N.; Booth, R.S.; Cornwell, T.J.; Clark, R.R.

    1984-04-12

    The authors present radio maps which hint at an explanation for the small size of at least some of the steep-spectrum compact sources (SSCSs). The maps, of the quasar 3C380, reveal a peculiar radio structure which is difficult to interpret other than by a powerful interaction between the radio-emitting plasma and its environment. Thus, the sub-galactic (projected) dimensions of this and other SSCSs may be a result of the beams being disrupted before they have propagated beyond the parent object.

  2. Peculiar radio structure in the quasar 3C380

    The authors present radio maps which hint at an explanation for the small size of at least some of the steep-spectrum compact sources (SSCSs). The maps, of the quasar 3C380, reveal a peculiar radio structure which is difficult to interpret other than by a powerful interaction between the radio-emitting plasma and its environment. Thus, the sub-galactic (projected) dimensions of this and other SSCSs may be a result of the beams being disrupted before they have propagated beyond the parent object. (author)

  3. Experience with Server Self Service Center (S3C)

    Sucik, J

    2009-01-01

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

  4. First results from the L3+C experiment at CERN

    Ladrón de Guevara, P

    2001-01-01

    The L3+C experiment combines the high precision spectrometer of the L3 detector at LEP, CERN, with a small air shower array. The momenta of the cosmic ray induced muons can be measured from 20 to 2000 GeV /c. During the 1999 data taking period 5 billion muon events were recorded in the spectrometer. From April to November, 2000, an additional 6.8 billion muon events have been recorded as well as 33 million air shower events. Here the first results on the muon momentum spectrum and charge ratio will be presented. (9 refs).

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

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

    2014-09-01

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

  6. MERLIN radio observations of the quasar 3C 273

    MERLIN observations of the radio jet of the quasar 3C 273 at 408 and 1666 Mhz are presented. Most of the important features previously seen at 408 MHz are confirmed. The jet extends from 12 to 23 arcsec from the quasar, and has a single bright head at 408 MHz. At the higher resolution of the 1666-MHz map the head is seen to consist of at least three subcomponents, the brightest of which is set back from the outermost point of the jet. The ridgeline of emission shows oscillations from side to side ('wiggles'), the wavelength of which decreases markedly as the bright head is approached. (author)

  7. HST/STIS Spectroscopy of 3C 273

    Heap, S. R.; Williger, G. M.; Dave, R.; Weymann, R. J.; Jenkins, E. B.; Tripp, T. M.

    2001-01-01

    We present preliminary results on the low-redshift Lyman alpha forest as based on STIS spectra of 3C 273. A total of 121 intergalactic Lyman alpha-absorbing systems were detected, of which 60 are above the 3.5 sigma completness limit, log N(HI)~12.3. The median Doppler parameter, b=27 km/s, is similar to that seen at high redshift. However the distribution of HI column densities (dN/dN(HI) propto N(HI)^-beta) has a steeper slope, beta = 2.02 +- 0.21, than is seen at high redshift. Overall, th...

  8. XMM-Newton observations of 3C 273

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

    2003-01-01

    A series of nine XMM-Newton observations of the radio-loud quasar 3C 273 are presented, concentrating mainly on the soft excess. Although most of the individual observations do not show evidence for iron emission, co-adding them reveals a weak, broad line (EW ~ 56 eV). The soft excess component is found to vary, confirming previous work, and can be well fitted with multiple blackbody components, with temperatures ranging between ~40 and ~330 eV, together with a power-law. Alternatively, a Com...

  9. The jet of 3C 273 observed with ROSAT HRI

    Roeser, Hermann-Josef; Meisenheimer, Klaus; Neumann, Martin; Conway, R. G.; Perley, R.A.

    2000-01-01

    ROSAT HRI observations of 3C 273 reveal X-ray emission all along the optically visible jet with the peak of emission at the inner end. Whereas the X-ray emission from the innermost knot A is consistent with a continuation of the radio-to-optical synchrotron continuum, a second population of particles with higher maximum energy has to be invoked to explain the X-ray emission from knots B, C and D in terms of synchrotron radiation. Inverse Compton emission could account for the X-ray flux from ...

  10. The radio jet of the quasar 3C273

    The authors present here new MERLIN observations of the brightness and polarization of the radio jet of the quasar 3C273 at a resolution of 0.35 arc s. One of the most marked features of the map, the high polarization found within the head of the source, is hard to explain. If the motion is indeed fast, then relativistic aberration should be taken into account; it is suggested that this leads to a natural explanation of the high observed polarization. (author)

  11. The mass of the black hole in 3C 273

    Paltani, S.; Turler, M.

    2005-01-01

    In this paper we apply the reverberation method to determine the mass of the black hole in 3C273 from the Ly a and C iv emission lines using archival IUE observations. Following the standard assumptions of the method, we find a maximum-likelihood estimate of the mass of 6.59 10^9 Mo, with a 1 sigma confidence interval 5.69-8.27 10^9 Mo. This estimate is more than one order of magnitude larger than that obtained in a previous study using Balmer lines. We reanalyze the optical data and show tha...

  12. Rapid variability of 3C273 at 300 GHz

    With a broadband bolometer mounted on the 3.6 m ESO telescope the authors have measured 3C273 at 300 GHz 6 times in 6 days. During this series of measurements they observed a sudden drop in flux density by 11.6 +- 2.9 Jy (spectral index assumed to be zero, and all known errors included). In an interpretation with the model of bulk relativistic motion the brightness temperature inferred can be combined with the observed superluminal motion, and strict limits on the model parameters can be derived. (Auth.)

  13. The high-energy spectrum of 3C 273

    Esposito, Valentino; Walter, Roland; Jean, Pierre; Tramacere, Andrea

    2013-01-01

    The high energy spectral shape of 3C 273 is usually understood in terms of Inverse-Compton emission in a relativistic leptonic jet. This model predicts variability patterns and delays which could be tested if simultaneous observations are available from the infrared to the GeV range. The instruments IBIS, SPI, JEM-X on board INTEGRAL, PCA on board RXTE and LAT on board Fermi have enough sensitivity to follow the spectral variability from the keV to the GeV and to compare them with model predi...

  14. Ultraviolet spectrum of quasi-stellar object 3C273

    Davidsen, A. F.; Hartig, G. F.; Fastie, W. G.

    1977-01-01

    The first direct observation of the ultraviolet spectrum of a quasi-stellar object (QSO) has been made with a rocket-borne telescope. The emission line spectrum of 3C273 is similar to the spectra of high-redshift QSOs, but no absorption is observed. The results provide important new constraints on theoretical models of QSOs, place a severe limit on the density of neutral hydrogen in the intergalactic medium, and suggest a cosmological origin for much of the absorption seen in high-redshift QSOs. Comparison of the ultraviolet spectrophotometry of low- and high-redshift QSOs suggests that the universe is closed, with a deceleration parameter of about 1.

  15. Evolution of 3C 273 at 10.7 GHz

    The quasar 3C 273 has been observed at 10.7 GHz at three epochs spanning 1984.1-1985.6. Two new superluminal components, C5 and C7a, are separating from the core with apparent transverse velocity v/c = (8.0 + or - 0.2)/h and (5.1 + or - 0.3)/h. The old components C3 and C4 may still be recognizable, with C4 having moved from 2.5 to perhaps 10 mas from the core in 7 yr. Nonmonotonic curvature near the core is confirmed. 8 references

  16. Superluminal motion in the quasar 3C 279

    Unwin, S.C.; Cohen, M.H.; Hodges, M.W.; Zensus, J.A.; Biretta, J.A.

    1989-05-01

    VLBI maps of the quasar 3C 279 have been made at 5, 11, and 22 GHz, at several epochs between 1981 and 1985, to study the varying structure of the compact radio source. Spectra derived from the maps show that the NE component has the highest turnover frequency, and thus probably represents the core of the source. By comparing the predicted inverse-Compton X-ray emission with the measured X-ray flux, lower limits to the Doppler factor are derived for the compact components. A simple model of a jet which is mildly relativistic explains both the superluminal motion and the X-ray flux. 48 refs.

  17. Evolution of 3C 273 at 10. 7 GHz

    Cohen, M.H.; Zensus, J.A.; Biretta, J.A.; Comoretto, G.; Kaufmann, P.

    1987-04-01

    The quasar 3C 273 has been observed at 10.7 GHz at three epochs spanning 1984.1-1985.6. Two new superluminal components, C5 and C7a, are separating from the core with apparent transverse velocity v/c = (8.0 + or - 0.2)/h and (5.1 + or - 0.3)/h. The old components C3 and C4 may still be recognizable, with C4 having moved from 2.5 to perhaps 10 mas from the core in 7 yr. Nonmonotonic curvature near the core is confirmed. 8 references.

  18. Superluminal motion in the quasar 3C 279

    VLBI maps of the quasar 3C 279 have been made at 5, 11, and 22 GHz, at several epochs between 1981 and 1985, to study the varying structure of the compact radio source. Spectra derived from the maps show that the NE component has the highest turnover frequency, and thus probably represents the core of the source. By comparing the predicted inverse-Compton X-ray emission with the measured X-ray flux, lower limits to the Doppler factor are derived for the compact components. A simple model of a jet which is mildly relativistic explains both the superluminal motion and the X-ray flux. 48 refs

  19. Electron transport and phonon coupling in K3C60

    The temperature dependence of the resistivity of single-crystal K3C60 below 260 K of Crespi et al. is analyzed utilizing the results of Raman scattering. It is found that coupling to the two lowest intramolecular Hg modes gives an excellent fit to the temperature variation of the resistivity with the inclusion of weak lower frequency acoustic or librational scattering. The relative coupling strength of Hg(1) to Hg(2) is found to be similar to that estimated from Raman studies of thin films. The weaker low-frequency contribution is found to have a value consistent with neutron-scattering studies

  20. Photopolarimetry of Blazar 3C454.3 from MIRO

    Baliyan, Ks; Ganesh, S.; Chandra, Sunil; Joshi, Uc

    2009-12-01

    The Blazar 3C 454.3 has been active in Gamma-rays, optical and X- rays since Sept. 2009 ( Atel #2181, #2200, #2201). Very recently, it has been reported to be flaring up in the optical, X-ray and gamma-ray energy regimes(ATel #2322; #2325; #2326; #2328; #2329; #2330; #2332). In Atel #2333, Sasada et al report optical behaviour of this source on Dec 1.9 with brightness (V=14.06+/-0.02 and degree of polarization 6.0+/-0.1% on the same epoch.

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

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

    2015-04-09

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

  2. Particle acceleration in the hotspot of the jet of quasar 3C273

    Recent radio, optical and near-infrared observations of the dominant hotspot at the outer end of the quasar jet 3C273A have revealed a complex spectrum, which provides a unique probe of the acceleration process supplying the synchrotron-emitting electrons. The authors present theoretical calculations of the electron spectrum produced by non-relativistic shock acceleration which, include both synchrotron losses and a finite emission region. The resultant synchrotron spectrum fits the observations, providing a natural explanation for the steep spectrum at radio to infrared frequencies, the observed flattening at ν 14 Hz. The model yields a new magnetic field estimate of 70 nT. (author)

  3. The Trails of Superluminal Jet Components in 3C111

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

    2008-01-01

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

  4. Molecular hydrogen emission from the bright quasar 3C273

    The discovery of broad emission lines in polarized light from the type 2 Seyfert galaxy NGCl068 led to postulation of the existence of a type 1 Seyfert nucleus shrouded from our direct view by a torus of molecular gas. Theoretical development of this idea included the suggestion that low-angular-momentum clouds in the torus are captured by the central source, fuelling the observed activity. The difficulty in applying this model to all active galactic nuclei (AGNs) is the lack of convincing evidence for molecular gas in 'bare' nucleus objects such as the quasar 3C273, which exhibits a simple power-law continuum and no excess of thermal dust emission. Here we present observations, made during the course of a survey for rotation and vibration lines of H2 emission from type 1 Seyferts and quasars, of molecular hydrogen emission from 3C273. This is the first time such emission has been seen from a radio-loud quasar. (author)

  5. The mass of the black hole in 3C 273

    Paltani, S

    2005-01-01

    In this paper we apply the reverberation method to determine the mass of the black hole in 3C273 from the Ly a and C iv emission lines using archival IUE observations. Following the standard assumptions of the method, we find a maximum-likelihood estimate of the mass of 6.59 10^9 Mo, with a 1 sigma confidence interval 5.69-8.27 10^9 Mo. This estimate is more than one order of magnitude larger than that obtained in a previous study using Balmer lines. We reanalyze the optical data and show that the method applied to the Ha, Hb, and Hg Balmer lines produce mass estimates lower by a factor 2.5, but already much larger than the previous estimate derived from the same lines. The finding of such a high mass in a face-on object is a strong indication that the gas motion is not confined to the accretion disk. The new mass estimate makes 3C273 accreting with an accretion rate about six times lower than the Eddington rate. We discuss the implications of our result for the broad-line-region size and black-hole mass vs l...

  6. Molecular hydrogen emission from the bright quasar 3C273

    Kawara, Kimiaki (National Astronomical Observatory, Mitaka, Tokyo (Japan)); Nishida, Minoru (Kyoto Univ. (Japan). Dept. of Physics); Gregory, Brooke (Cerro Tololo Inter-American Observatory, La Serena (Chile) Royal Observatory, Edinburgh (UK))

    1989-09-21

    The discovery of broad emission lines in polarized light from the type 2 Seyfert galaxy NGCl068 led to postulation of the existence of a type 1 Seyfert nucleus shrouded from our direct view by a torus of molecular gas. Theoretical development of this idea included the suggestion that low-angular-momentum clouds in the torus are captured by the central source, fuelling the observed activity. The difficulty in applying this model to all active galactic nuclei (AGNs) is the lack of convincing evidence for molecular gas in 'bare' nucleus objects such as the quasar 3C273, which exhibits a simple power-law continuum and no excess of thermal dust emission. Here we present observations, made during the course of a survey for rotation and vibration lines of H{sub 2} emission from type 1 Seyferts and quasars, of molecular hydrogen emission from 3C273. This is the first time such emission has been seen from a radio-loud quasar. (author).

  7. Polyendocrine syndrome type 3C in a family from Pakistan.

    Gessoni, G; Antico, F; Caroli, D; Nogara, A; Valverde, S; Fezzi, M; Zucchelli, M; Boscolo-Bariga, A

    2013-09-01

    In type 3 polyendocrine syndrome (PAS3), autoimmune thyroiditis occurs with other organ-specific autoimmune disease, but not with autoimmune adrenalitis. In this report we described a family from Pakistan in which mother and three daughters were affected by a PAS3. We studied a family from Pakistan: Father MMu age 44, mother KN aged 44, three daughters MM age 20, MH age 16 and MA age 14 and a son MU age 18. These subjects were tested for thyroids function, metabolic function, adrenal function, autoimmune disease. In this family the four females were shown hypothyroidism with presence of anti thyroid autoantibodies (AA) and high TSH serum concentration in association with the presence of anti transglutaminase AA. Moreover KN, MM and MH were positive for anti nuclear AA (granular pattern) and for antibodies against Saccaromyces cerevisiae. MM was positive for AA against nuclear extractable antigens (SSA and SSB) too. No diabetes or pernicious anemia were observed. Adrenal and Pituitary function were normal. PAS 3C is an uncommon disease. In this family from Pakistan we observed a PAS3C in the four female members: mother and three daughters while father and son were unaffected. PMID:24126553

  8. VLA observations of the multiple jet galaxy 3C 75

    VLA observations of the central radio source in Abell 400, 3C 75, at 6 and 20 cm are presented. The VLA maps show that this radio source consists of a pair of twin jets originating in the apparently double nucleus of the central galaxy in this cluster. On larger scales the jets merge into two tails resembling the wide angle tail class of radio sources. Just as for the wide angle tail radio source, 3C 465, it is found that the standard models for bending this source fail quantitatively. The problem becomes even harder because of the low velocity dispersion and temperature for Abell 400 and the fact that the jets from both nuclei bend in the same direction. Models with jet velocities less than 1000 km/s at the first bends seem necessary if the sources are bent by the motion of the galaxy through the ICM. Particle acceleration seems necessary in the most diffuse parts of the source with the energy source likely to be the ICM itself. 10 references

  9. Serine Proteases an Ab Initio Molecular Dynamics Study

    De Santis, L

    1999-01-01

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

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

    Roy DM

    2014-07-01

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

  11. Cysteine proteases as potential antigens in antiparasitic DNA vaccines

    Jørgensen, Louise von Gersdorff; Buchmann, Kurt

    2011-01-01

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

  12. Activation of ADAM 12 protease by copper

    Loechel, F; Wewer, Ulla M.

    2001-01-01

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

  13. Hydrolysis of Fish Protein by Analkaline Protease

    2001-01-01

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

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

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

    2013-01-29

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

  15. Importance of lysosomal cysteine proteases in lung disease

    Chapman Harold A; Wolters Paul J

    2000-01-01

    Abstract The human lysosomal cysteine proteases are a family of 11 proteases whose members include cathepsins B, C, H, L, and S. The biology of these proteases was largely ignored for decades because of their lysosomal location and the belief that their function was limited to the terminal degradation of proteins. In the past 10 years, this view has changed as these proteases have been found to have specific functions within cells. This review highlights some of these functions, specifically ...

  16. Centenary celebrations article: Cysteine proteases of human malaria parasites

    Pandey, Kailash C.

    2011-01-01

    There is an urgent need for new drugs against malaria, which takes millions of lives annually. Cysteine proteases are potential new drug targets, especially when current drugs are showing resistance. Falcipains and vivapains are well characterized cysteine proteases of P. falciparum and P. vivax, respectively. Studies with cysteine protease inhibitors and manipulating cysteine proteases specific genes have suggested their roles in hemoglobin hydrolysis. In P. falciparum, falcipain-2 and falci...

  17. A Novel Bioluminescent Protease Assay Using Engineered Firefly Luciferase

    Wigdal, Susan S.; Anderson, Jessica L; Vidugiris, Gediminas J; Shultz, John; Wood, Keith V.; Fan, Frank

    2008-01-01

    Proteases play important roles in a variety of disease processes. Understanding their biological functions underpins the efforts of drug discovery. We have developed a bioluminescent protease assay using a circularly permuted form of firefly luciferase, wherein the native enzyme termini were joined by a peptide containing a protease site of interest. Protease cleavage of these mutant luciferases greatly activates the enzyme, typically over 100 fold. The mutant luciferase substrates are easily...

  18. High Throughput Substrate Phage Display for Protease Profiling

    Ratnikov, Boris; Cieplak, Piotr; Smith, Jeffrey W.

    2009-01-01

    The interplay between a protease and its substrates is controlled at many different levels, including coexpression, colocalization, binding driven by ancillary contacts, and the presence of natural inhibitors. Here we focus on the most basic parameter that guides substrate recognition by a protease, the recognition specificity at the catalytic cleft. An understanding of this substrate specificity can be used to predict the putative substrates of a protease, to design protease activated imagin...

  19. The Role of Protease Activity in ErbB Biology

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

    2008-01-01

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

  20. Comparative Study of Dermatophytic Fungi for Extra Cellular Proteases Efficacy

    Sanchita Chaturvedi; Sonal Pathak; Ruchi Upadhyay; Shweta Dubey

    2013-01-01

    Fungi are known to produce proteases of different kind. The dermatphytic fungal strains were isolated from human skin tissues for extra cellular proteases efficacy. The present study deals with purification, estimation and comparison of extracellular proteases from five fungal species. (Fusarium sp., Curvularia sp. , Fumigatus Sp. , Aspergillus Sp. and Mucor Sp.). All the five fungal strains showed good amount of extra cellular protease activity in terms of unit total protein content. By test...

  1. Predicting protein function from structure: Unique structural features of proteases

    Stawiski, Eric W.; Baucom, Albion E.; Lohr, Scott C.; Gregoret, Lydia M.

    2000-01-01

    We have noted consistent structural similarities among unrelated proteases. In comparison with other proteins of similar size, proteases have smaller than average surface areas, smaller radii of gyration, and higher Cα densities. These findings imply that proteases are, as a group, more tightly packed than other proteins. There are also notable differences in secondary structure content between these two groups of proteins: proteases have fewer helices and more loops. We speculate that both h...

  2. Structure-based design and synthesis of triazole-based macrocyclic inhibitors of norovirus protease: Structural, biochemical, spectroscopic, and antiviral studies.

    Weerawarna, Pathum M; Kim, Yunjeong; Galasiti Kankanamalage, Anushka C; Damalanka, Vishnu C; Lushington, Gerald H; Alliston, Kevin R; Mehzabeen, Nurjahan; Battaile, Kevin P; Lovell, Scott; Chang, Kyeong-Ok; Groutas, William C

    2016-08-25

    Outbreaks of acute gastroenteritis caused by noroviruses constitute a public health concern worldwide. To date, there are no approved drugs or vaccines for the management and prophylaxis of norovirus infections. A potentially effective strategy for the development of norovirus therapeutics entails the discovery of inhibitors of norovirus 3CL protease, an enzyme essential for noroviral replication. We describe herein the structure-based design of the first class of permeable, triazole-based macrocyclic inhibitors of norovirus 3C-like protease, as well as pertinent X-ray crystallographic, biochemical, spectroscopic, and antiviral studies. PMID:27235842

  3. Milliarcsecond polarization structure of the superluminal quasar 3C 273

    Roberts, D.H.; Kollgaard, R.I.; Brown, L.F.; Gabuzda, D.C.; Wardle, J.F. C. (Brandeis Univ., Waltham, MA (USA))

    1990-09-01

    A 2 x 10 marcsec-resolution determination is presented for the total intensity and linear polarization structures of the superluminal quasar 3C 273 at 5 GHz. Substantial polarized flux was detected from several superluminal components of the jet, whose fractional polarization increased symmetrically with distance from the core; the most distant component is highly polarized and exhibits a highly ordered magnetic field. Within a few marcsec of the core, the inferred magnetic field orientation varies rapidly with position along the jet. The primarily longitudinal magnetic field orientation is concluded to become established within 20 marcsec of the core. A highly disorganized magnetic field is the most plausible explanation for the low degree of polarization in the innermost regions of the jet. 48 refs.

  4. Milliarcsecond polarization structure of the superluminal quasar 3C 273

    A 2 x 10 marcsec-resolution determination is presented for the total intensity and linear polarization structures of the superluminal quasar 3C 273 at 5 GHz. Substantial polarized flux was detected from several superluminal components of the jet, whose fractional polarization increased symmetrically with distance from the core; the most distant component is highly polarized and exhibits a highly ordered magnetic field. Within a few marcsec of the core, the inferred magnetic field orientation varies rapidly with position along the jet. The primarily longitudinal magnetic field orientation is concluded to become established within 20 marcsec of the core. A highly disorganized magnetic field is the most plausible explanation for the low degree of polarization in the innermost regions of the jet. 48 refs

  5. Extended Ly-alpha emission associated with 3C 294

    Mccarthy, Patrick J.; Spinrad, Hyron; Dickinson, Mark; Van Breugel, Wil; Liebert, James; Djorgovski, S.; Eisenhardt, Peter

    1990-01-01

    Optical, IR, and radio observations of the powerful radio source 3C 294, which is surrounded by a large cloud of ionized gas, are presented. The galaxy is faint in the rest-frame UV, yet has a near-IR luminosity that is typical of radio galaxies at redshifts of order two. In contrast to the large extent of the ionized gas, the K-band image is quite compact. The emission-line cloud is closely aligned with the radio source axis and has an ionization state indicative of ionization by a nonstellar source. The velocity field of the gas has both large ordered motions and large turbulent components. The total mass required to keep the gas bound to the system is comparable to present-day massive galaxies and their halos. The velocity fields of the high-ionization lines are systematically different from Ly-alpha in a manner that is not easily understood.

  6. Structural variability of 3C 111 on parsec scales

    Großberger, C; Wilms, J; Müller, C; Beuchert, T; Ros, E; Ojha, R; Aller, M; Aller, H; Angelakis, E; Fuhrmann, L; Nestoras, I; Schmidt, R; Zensus, J A; Krichbaum, T P; Ungerechts, H; Sievers, A; Riquelme, D

    2011-01-01

    We discuss the parsec-scale structural variability of the extragalactic jet 3C 111 related to a major radio flux density outburst in 2007. The data analyzed were taken within the scope of the MOJAVE, UMRAO, and F-GAMMA programs, which monitor a large sample of the radio brightest compact extragalactic jets with the VLBA, the University of Michigan 26 m, the Effelsberg 100 m, and the IRAM 30 m radio telescopes. The analysis of the VLBA data is performed by fitting Gaussian model components in the visibility domain. We associate the ejection of bright features in the radio jet with a major flux-density outburst in 2007. The evolution of these features suggests the formation of a leading component and multiple trailing components.

  7. Synthesis of the C3-C12 Fragment of Laulimalide

    Lee, Hyo Won; Hong, Joong Yeoun [Chungbuk National University, Cheongju (Korea, Republic of)

    2003-11-15

    We have reported the successful synthesis of the C3-C12 fragment of laulimalide using the introduction of a dihydropyran ring after the elongation of a side chain. Our on-going synthetic effort toward the synthesis of laulimalide will be reported in the near future. The antimiototic 20-membered macrolide laulimalide also known as fijanolide B, isolated from sea sponges such as Cacospongia mycofijiensis, Hyatella sp. Spongia mycofijiensis, and Fasciospongia rimosa, shows a potent biological activity against cell lines resistant to paclitaxel or epothilones since laulimalide binds to a site of microtubules in a mode distinct from taxoids. The stabilization of polymerization by laulimalide leads to the inhibition of miototic spindle and apoptosis of cells.

  8. Ultraviolet spectrum of quasi-stellar object 3C273

    The first direct observation of the ultraviolet spectrum of a quasi-stellar object has been made with a rocket-borne telescope. The emission line spectrum of 3C273 is similar to the spectra of high-redshift QSOs, but no absorption is observed. The results provide important new constraints on theoretical models of QSOs, place a severe limit on the density of neutral hydrogen in the intergalactic medium, and suggest a cosmological origin for much of the absorption seen in high-redshift QSOs. Comparison of the ultraviolet spectrophotometry of low- and high-redshift QSOs suggests that the universe is closed, with the deceleration parameter q0 approximately 1. (author)

  9. The X-ray emission of 3C273

    The X-ray emission of 3C273 in the 0.2-35 keV band has been studied, with EXOSAT and Ginga, over the period 1983 December to 1988 December. The overall flux is variable on a time-scale of weeks by a factor of 2. The spectral index in the 2-10 keV band (α ∼ 0.5) is significantly different from the 'canonical' AGN index of α = 0.7. There are small but significant variations with time in the spectral index which are not statistically correlated with the overall X-ray flux level. Iron emission with an equivalent width of ∼ 50 eV is detected in one of the Ginga observations. (author)

  10. Ultraviolet emission-line variability in 3C273

    Evidence is presented for correlated variability between the ultraviolet continuum and the Lyα emission line in the luminous quasar 3C273. Using IUE data covering a period of ∼ 1000 d, Lyα varied by 15 per cent whilst the continuum varied by a factor of 2. A time series analysis gives a lag for Lyα relative to the ultraviolet continuum of 74 ± 33 d. The broad flat-topped shape of the cross-correlation function, and the small amplitude of the Lyα variability, indicate that the derived lag is a measure of the inner radius of a geometrically thick broad-line region. (author)

  11. Ultraviolet continuum variability of the quasar 3C273

    The authors report the first observations of variability in the ultraviolet spectrum of the quasar 3C273 (redshift, z=0.158) as observed by the International Ultraviolet Explorer in the period 1978-84. The flux at lambdasub(observed)=1,675 A increased by 1.25 between April and June 1982, then decreased by a factor 2 between June 1982 and April 1983. The amplitude of these variations and the constancy of the intensity of the Lyα emission line during the same period are indications that the ultraviolet variations are caused by variations of the black body and/or non-thermal components. The flux variations are accompanied by variations of the spectral shape. An interpretation is presented of the observed variability in terms of a discontinuous and variable distribution of the temperature on the photosphere emitting the ultraviolet continuum. (author)

  12. The X-ray emission of 3C273

    Turner, M.J.L.; Williams, O.R. (Leicester Univ. (UK). Dept. of Astronomy); Courvoisier, T.J.-L. (Geneva Observatory (Switzerland)) (and others)

    1990-05-15

    The X-ray emission of 3C273 in the 0.2-35 keV band has been studied, with EXOSAT and Ginga, over the period 1983 December to 1988 December. The overall flux is variable on a time-scale of weeks by a factor of 2. The spectral index in the 2-10 keV band ({alpha} {approx} 0.5) is significantly different from the 'canonical' AGN index of {alpha} = 0.7. There are small but significant variations with time in the spectral index which are not statistically correlated with the overall X-ray flux level. Iron emission with an equivalent width of {approx} 50 eV is detected in one of the Ginga observations. (author).

  13. Ultraviolet emission-line variability in 3C273

    O' Brien, P.T.; Harries, T.J. (University Coll., London (UK). Dept. of Physics and Astronomy)

    1991-05-01

    Evidence is presented for correlated variability between the ultraviolet continuum and the Ly{alpha} emission line in the luminous quasar 3C273. Using IUE data covering a period of {approx} 1000 d, Ly{alpha} varied by 15 per cent whilst the continuum varied by a factor of 2. A time series analysis gives a lag for Ly{alpha} relative to the ultraviolet continuum of 74 {+-} 33 d. The broad flat-topped shape of the cross-correlation function, and the small amplitude of the Ly{alpha} variability, indicate that the derived lag is a measure of the inner radius of a geometrically thick broad-line region. (author).

  14. Single ferromagnetic behaviour of nanopowders with Fe3C

    David, Bohumil; Zbořil, R.; Mashlan, M.; Grygar, Tomáš; Dumitrache, F.; Schneeweiss, Oldřich

    2006-01-01

    Roč. 304, č. 2 (2006), e787-e789. ISSN 0304-8853. [International Symposium on Soft Magnetic Materials /17./. Bratislava, 07.09.2005-09.09.2005] R&D Projects: GA ČR GA202/04/0221; GA MŠk 1M0512 Institutional research plan: CEZ:AV0Z20410507; CEZ:AV0Z40320502 Keywords : synthesis * nanopowder * Fe3C Subject RIV: BM - Solid Matter Physics ; Magnetism Impact factor: 1.212, year: 2006 http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TJJ-4JJ27YM-5&_user=625109&_coverDate=09%2F30%2F2006&_alid=501264124&_rdoc=2&_fmt=summary&_orig=search&_cdi=5312&_sort=d&_st=4&_docanchor=&_acct=C000031719&_version=1&_urlVersion=0&_userid=625109&md5=30fafcdb895bbb548857aa25426ae131

  15. The inner radio jet of 3C273

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

  16. The Parsec-Scale Jet of Quasar 3C345

    Zensus, J. A.; Rabaca, C. R.

    1993-12-01

    We discuss the parsec-scale structure of the superluminal quasar 3C345. Monitoring of the structure with VLBI at cm and mm wavelengths has shown apparent superluminal motion of at least four distinct emission features, over a distance of more than 40 pc from the stationary core (Zensus, Cohen, and Unwin, submitted to APJ). Near the core, the projected trajectories are curved and different for individual components, indicative of more complicated flow patterns than previously suspected, and consistent with motion along helical paths. The motion accelerates with increasing separation from the core, as the jet curves towards the extended kiloparsec structure. The flux evolution of individual components can be described using a generalized shock model. We apply this to component C4 and discuss the impact of orientation effects and implications for specific shock models.

  17. Structural Variability of 3C 111 on Parsec Scales

    C. Grossberger

    2012-01-01

    Full Text Available We discuss the parsec-scale structural variability of the extragalactic jet 3C111 related to a major radio flux density outburst in 2007. The data analyzed were taken within the scope of the MOJAVE, UMRAO, and F-GAMMA programs, which monitor a large sample of the radio brightest compact extragalactic jets with the VLBA, the University of Michigan 26 m, the Effelsberg 100 m, and the IRAM 30m radio telescopes. The analysis of the VLBA data is performed by fitting Gaussian model components in the visibility domain. We associate the ejection of bright features in the radio jet with a major flux-density outburst in 2007. The evolution of these features suggests the formation of a leading component andmultiple trailing components

  18. Structural Variability of 3C 111 on Parsec Scales

    Grossberger, C.; Kadler, M.; Wilms, J.; Muller, C.; Beuchert, T.; Ros, E.; Ojha, R.; Aller, M.; Aller, H.; Angelakis, E.; Fuhrmann, L.; Nestoras, I.; Schmidt, R.; Zensus, J. A.; Krichbaum, T. P.; Ungerechts, H.; Sievers, A.; Riquelme, D.

    2011-01-01

    We discuss the parsec-scale structural variability of the extragalactic jet 3C 111 related to a major radio flux density outburst in 2007, The data analyzed were taken within the scope of the MOJAVE, UMRAO, and F-GAMMA programs, which monitor a large sample of the radio brightest compact extragalactic jets with the VLBA, the University of Michigan 26 m, the Effelsberg 100 m, and the IRAM 30 m radio telescopes. The analysis of the VLBA data is performed by fitting Gaussian model components in the visibility domain, We associate the ejection of bright features in the radio jet with a major flux-density outburst in 2007, The evolution of these features suggests the formation of a leading component and multiple trailing components

  19. Fe3C nanopowder prepared by laser-induced pyrolysis

    The Fe3C-based nanocrystalline composite was prepared by the laser pyrolysis method, using a cross-flow reactor in which the laser orthogonally irradiates the gas mixture of Fe(CO)5 and C2H4. Vapours of iron pentacarbonyl served as a source of Fe atoms. Ethylene absorbed CO2 laser radiation but was also a source of C atoms (after its decomposition on hot surfaces of freshly formed Fe particles). The synthesized nanopowder was in-situ passivated with air. The as-synthesized powder was characterized by HRTEM, XRD, Moessbauer spectroscopy, and low temperature magnetic measurements. The nanopowder consisted of aggregated nanoparticles (around 20 nm large) having carbonaceous shells. In our contribution we present the results of the above mentioned characterization techniques. We concentrate especially on the interpretation of the Moessbauer spectra measured at high temperatures (up to 300 grad C) and low temperatures (down to 4 K). (authors)

  20. Overexpression of Elastin Affects the Protease to Anti-Protease Balance and Protects Mice from Colitis. : Elafin protects from colitis

    Motta*, Jean-Paul; Magne, Laurent; Descamps, Delphyne; Rolland, Corinne; Squarzoni-Dale, Camila; Rousset, Perrine; Balloy, Viviane; Huerre, Michel; Jenne, Dieter; Wartelle, Julien; Belaaouaj, Azzaq; Mas, Emmanuel; Vinel, Jean-Pierre; Alric, Laurent; Chignard, Michel

    2010-01-01

    BACKGROUND & AIMS:: Colon tissues of patients with inflammatory bowel disease (IBD) have been reported to have increased proteolytic activity, but no studies have clearly addressed the protease to anti-protease balance in the pathogenesis of colitis. We investigated the role of Elafin, a serine protease inhibitor expressed by skin and mucosal surfaces in human inflammatory conditions, and the proteases neutrophil elastase (NE) and proteinase-3 (PR-3), in mice with colitis. METHODS:: We studie...

  1. MICROSPHERE-BASED FLOW CYTOMETRY PROTEASE ASSAYS FOR USE IN PROTEASE ACTIVITY DETECTION AND HIGH-THROUGHPUT SCREENING

    Saunders, Matthew J.; Edwards, Bruce S.; Zhu, Jingshu; Sklar, Larry A.; Graves, Steven W.

    2010-01-01

    This protocol describes microsphere-based protease assays for use in flow cytometry and high-throughput screening. This platform measures a loss of fluorescence from the surface of a microsphere due to the cleavage of an attached fluorescent protease substrate by a suitable protease enzyme. The assay format can be adapted to any site or protein specific protease of interest and results can be measured in both real time and as end point fluorescence assays on a flow cytometer. End point assays...

  2. Active site conformational changes of prostasin provide a new mechanism of protease regulation by divalent cations

    Prostasin or human channel-activating protease 1 has been reported to play a critical role in the regulation of extracellular sodium ion transport via its activation of the epithelial cell sodium channel. Here, the structure of the extracellular portion of the membrane associated serine protease has been solved to high resolution in complex with a nonselective d-FFR chloromethyl ketone inhibitor, in an apo form, in a form where the apo crystal has been soaked with the covalent inhibitor camostat and in complex with the protein inhibitor aprotinin. It was also crystallized in the presence of the divalent cation Ca+2. Comparison of the structures with each other and with other members of the trypsin-like serine protease family reveals unique structural features of prostasin and a large degree of conformational variation within specificity determining loops. Of particular interest is the S1 subsite loop which opens and closes in response to basic residues or divalent ions, directly binding Ca+2 cations. This induced fit active site provides a new possible mode of regulation of trypsin-like proteases adapted in particular to extracellular regions with variable ionic concentrations such as the outer membrane layer of the epithelial cell.

  3. Preliminary crystallographic analysis of avian infectious bronchitis virus main protease

    The avian infectious bronchitis virus main protease has been crystallized; crystals diffract to 2.7 Å resolution. Infectious bronchitis virus (IBV) is the prototype of the genus Coronavirus. It causes a highly contagious disease which affects the respiratory, reproductive, neurological and renal systems of chickens, resulting great economic losses in the poultry industry worldwide. The coronavirus (CoV) main protease (Mpro), which plays a pivotal role in viral gene expression and replication through a highly complex cascade involving the proteolytic processing of replicase polyproteins, is an attractive target for antiviral drug design. In this study, IBV Mpro was overexpressed in Escherichia coli. Crystals suitable for X-ray crystallography have been obtained using microseeding techniques and belong to space group P6122. X-ray diffraction data were collected in-house to 2.7 Å resolution from a single crystal. The unit-cell parameters were a = b = 119.1, c = 270.7 Å, α = β = 90, γ = 120°. Three molecules were predicted to be present in the asymmetric unit from a calculated self-rotation function

  4. Preliminary crystallographic study of two cuticle-degrading proteases from the nematophagous fungi Lecanicillium psalliotae and Paecilomyces lilacinus

    Two cuticle-degrading proteases Ver112 and PL646 were purified from the nematophagous fungi L. psalliotae and P. lilacinus, respectively. The protease Ver112 and a complex between PL646 and a tetrapeptide inhibitor were crystallized. Diffraction data were collected to 1.65 and 2.2 Å resolution, respectively. Cuticle-degrading proteases are extracellular subtilisin-like serine proteases that are secreted by entomopathogenic and nematophagous fungi. These proteases can digest the host cuticle during invasion of an insect or nematode and serve as a group of important virulence factors during the infection of nematodes by nematophagous fungi. To elucidate the mechanism of interaction between the proteases and the nematode cuticle, two cuticle-degrading proteases, Ver112 from Lecanicillium psalliotae (syn. Verticillium psalliotae) and PL646 from Paecilomyces lilacinus, were studied. The Ver112 protein and the complex between PL646 and the substrate-like tetrapeptide inhibitor methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone (MSU-AAPV) were crystallized using the hanging-drop vapour-diffusion method at 289 K. The crystals were analyzed by X-ray diffraction to resolutions of 1.65 and 2.2 Å, respectively. These analyses identified that crystals of Ver112 belonged to space group P212121, with unit-cell parameters a = 43.7, b = 67.8, c = 76.3 Å, α = β = γ = 90°. In contrast, crystals of the PL646–MSU-AAPV complex belonged to space group P21, with unit-cell parameters a = 65.1, b = 62.5, c = 67.6 Å, β = 92.8°

  5. Associated C IV absorption in radio-loud QSOs - The 3C mini-survey

    A spectroscopic survey at 1 A resolution of twelve 3C and 3CR QSOs reveals a very high incidence of C IV absorption complexes within + or - 5000 km/s of the C IV 1548, 1550-A emission-line redshift. Such associated C IV absorption is found to be strong (rest-frame equivalent width equal to or greater than 1.5 A) in six of these powerful, steep-spectrum radio sources, while four others show weaker associated absorption. Such strong associated C IV complexes with z(abs) approximaely equal to z(em) are comparatively rare in a sample of radio-quiet QSOs investigated previously. The evidence for possible correlations with various radio properties is briefly discussed, although no correlations are thus far confirmed to be statistically significant. 29 references

  6. HIV-1 protease-induced apoptosis

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

    2014-01-01

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

  7. Proteases decode the extracellular matrix cryptome.

    Ricard-Blum, Sylvie; Vallet, Sylvain D

    2016-03-01

    The extracellular matrix is comprised of 1100 core-matrisome and matrisome-associated proteins and of glycosaminoglycans. This structural scaffold contributes to the organization and mechanical properties of tissues and modulates cell behavior. The extracellular matrix is dynamic and undergoes constant remodeling, which leads to diseases if uncontrolled. Bioactive fragments, called matricryptins, are released from the extracellular proteins by limited proteolysis and have biological activities on their own. They regulate numerous physiological and pathological processes such as angiogenesis, cancer, diabetes, wound healing, fibrosis and infectious diseases and either improve or worsen the course of diseases depending on the matricryptins and on the molecular and biological contexts. Several protease families release matricryptins from core-matrisome and matrisome-associated proteins both in vitro and in vivo. The major proteases, which decrypt the extracellular matrix, are zinc metalloproteinases of the metzincin superfamily (matrixins, adamalysins and astacins), cysteine proteinases and serine proteases. Some matricryptins act as enzyme inhibitors, further connecting protease and matricryptin fates and providing intricate regulation of major physiopathological processes such as angiogenesis and tumorigenesis. They strengthen the role of the extracellular matrix as a key player in tissue failure and core-matrisome and matrisome-associated proteins as important therapeutic targets. PMID:26382969

  8. Intracellular aspartic protease of Candida albicans

    Bauerová, Václava; Pichová, Iva; Hrušková-Heidingsfeldová, Olga

    Mátraháza : -, 2007. s. 43. [Alexander Von Humboldt Workshop on Structure Based Approaches Towards Disease Control. 22.05.2007-27.05.2007, Mátraháza] Institutional research plan: CEZ:AV0Z40550506 Keywords : Candida parapsilosis * intracellular * aspartic protease Subject RIV: CE - Biochemistry

  9. A new class of SUMO proteases

    Gillies, Jennifer; Hochstrasser, Mark

    2012-01-01

    A new class of SUMO protease, DeSUMOylating enzyme (DeSI)—that has different substrates and localization to SENP SUMO proteases—is characterized in this issue of EMBO reports. The implications for the field are discussed here.

  10. Crystal Structures of the Histo-Aspartic Protease (HAP) from Plasmodium falciparum

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

    2009-08-07

    The structures of recombinant histo-aspartic protease (HAP) from malaria-causing parasite Plasmodium falciparum as apoenzyme and in complex with two inhibitors, pepstatin A and KNI-10006, were solved at 2.5-, 3.3-, and 3.05-{angstrom} resolutions, respectively. In the apoenzyme crystals, HAP forms a tight dimer not seen previously in any aspartic protease. The interactions between the monomers affect the conformation of two flexible loops, the functionally important 'flap' (residues 70-83) and its structural equivalent in the C-terminal domain (residues 238-245), as well as the orientation of helix 225-235. The flap is found in an open conformation in the apoenzyme. Unexpectedly, the active site of the apoenzyme contains a zinc ion tightly bound to His32 and Asp215 from one monomer and to Glu278A from the other monomer, with the coordination of Zn resembling that seen in metalloproteases. The flap is closed in the structure of the pepstatin A complex, whereas it is open in the complex with KNI-10006. Although the binding mode of pepstatin A is significantly different from that in other pepsin-like aspartic proteases, its location in the active site makes unlikely the previously proposed hypothesis that HAP is a serine protease. The binding mode of KNI-10006 is unusual compared with the binding of other inhibitors from the KNI series to aspartic proteases. The novel features of the HAP active site could facilitate design of specific inhibitors used in the development of antimalarial drugs.

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

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

  12. Proteases and Protease Inhibitors of Urinary Extracellular Vesicles in Diabetic Nephropathy

    Luca Musante; Dorota Tataruch; Dongfeng Gu; Xinyu Liu; Carol Forsblom; Per-Henrik Groop; Harry Holthofer

    2015-01-01

    Diabetic nephropathy (DN) is one of the major complications of diabetes mellitus (DM), leads to chronic kidney disease (CKD), and, ultimately, is the main cause for end-stage kidney disease (ESKD). Beyond urinary albumin, no reliable biomarkers are available for accurate early diagnostics. Urinary extracellular vesicles (UEVs) have recently emerged as an interesting source of diagnostic and prognostic disease biomarkers. Here we used a protease and respective protease inhibitor array to profi...

  13. A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior.

    Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu; Ghassabeh, Gholamreza H; Muyldermans, Serge; Declerck, Paul J; Huang, Mingdong; Andreasen, Peter A; Ngo, Jacky Chi Ki

    2016-07-15

    A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30-40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors. PMID:27226628

  14. A novel protease activity assay using a protease-responsive chaperone protein

    Protease activity assays are important for elucidating protease function and for developing new therapeutic agents. In this study, a novel turbidimetric method for determining the protease activity using a protease-responsive chaperone protein is described. For this purpose, a recombinant small heat-shock protein (sHSP) with an introduced Factor Xa protease recognition site was synthesized in bacteria. This recombinant mutant, FXa-HSP, exhibited chaperone-like activity at high temperatures in cell lysates. However, the chaperone-like activity of FXa-HSP decreased dramatically following treatment with Factor Xa. Protein precipitation was subsequently observed in the cell lysates. The reaction was Factor Xa concentration-dependent and was quantitatively suppressed by a specific inhibitor for Factor Xa. Protein aggregation was detected by a simple method based on turbidimetry. The results clearly demonstrate that this assay is an effective, easy-to-use method for determining protease activities without the requirement of labeling procedures and the use of radioisotopes.

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

    Nathalie Dautin

    2010-05-01

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

  16. Bacterial retropepsin-like proteases : the evidence from Legionella pneumophila

    Teixeira, Paulo Alexandre Gonçalves

    2013-01-01

    A familia A2 de proteases aspárticas é constituida maioritariamente por proteases encontradas em retrovírus – as retropepsinas. As teorias evolutivas inerentes a estas proteases normalmente referem que estarão relacionadas com proteases do tipo pepsina pertencentes à familia A1 de proteases aspárticas. Pela primeira teoria (geralmente a mais aceite), durante a infeção de uma célula eucariota por um retrovírus, o gene da retropepsina terá sofrido duplicação e fusão dando orig...

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

    Highlights: •Inhibitors against MDR HIV-1 protease were designed, synthesized and evaluated. •Lead peptide (6a) showed potent inhibition (IC50: 4.4 nM) of MDR HIV-1 protease. •(6a) Showed favorable binding isotherms against NL4-3 and MDR proteases. •(6a) Induced perturbations in the 15N-HSQC spectrum of MDR HIV-1 protease. •Molecular modeling suggested that (6a) may induce total flap closure inMDR protease. -- Abstract: Multidrug-resistant (MDR) clinical isolate-769, human immunodeficiency virus type-1 (HIV-1) protease (PDB ID: (1TW7)), was shown to exhibit wide-open flaps and an expanded active site cavity, causing loss of contacts with protease inhibitors. In the current study, the expanded active site cavity of MDR769 HIV-1 protease was screened with a series of peptide-inhibitors that were designed to mimic the natural substrate cleavage site, capsid/p2. Scanning Ala/Phe chemical mutagenesis approach was incorporated into the design of the peptide series to mimic the substrate co-evolution. Among the peptides synthesized and evaluated, a lead peptide (6a) with potent activity (IC50: 4.4 nM) was identified against the MDR769 HIV-1 protease. Isothermal titration calorimetry data showed favorable binding profile for 6aagainst both wild type and MDR769 HIV-1 protease variants. Nuclear magnetic resonance spectrum of 15N-labeled MDR769 HIV-1 protease in complex with 6a showed some major perturbations in chemical shift, supporting the peptide induced conformational changes in protease. Modeling analysis revealed multiple contacts between 6a and MDR769 HIV-1 protease. The lead peptide-inhibitor, 6a, with high potency and good binding profile can be used as the basis for developing potent small molecule inhibitors against MDR variants of HIV

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

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

    2013-09-06

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

  19. New directions for protease inhibitors directed drug discovery.

    Hamada, Yoshio; Kiso, Yoshiaki

    2016-11-01

    Proteases play crucial roles in various biological processes, and their activities are essential for all living organisms-from viruses to humans. Since their functions are closely associated with many pathogenic mechanisms, their inhibitors or activators are important molecular targets for developing treatments for various diseases. Here, we describe drugs/drug candidates that target proteases, such as malarial plasmepsins, β-secretase, virus proteases, and dipeptidyl peptidase-4. Previously, we reported inhibitors of aspartic proteases, such as renin, human immunodeficiency virus type 1 protease, human T-lymphotropic virus type I protease, plasmepsins, and β-secretase, as drug candidates for hypertension, adult T-cell leukaemia, human T-lymphotropic virus type I-associated myelopathy, malaria, and Alzheimer's disease. Our inhibitors are also described in this review article as examples of drugs that target proteases. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 563-579, 2016. PMID:26584340

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

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

    2015-03-01

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

  1. Correlated variability in the blazar 3C 454.3

    Bonning, E W; Urry, C M; Buxton, M; Fossati, G; Maraschi, L; Coppi, P; Scalzo, R; Isler, J; Kaptur, A

    2008-01-01

    The blazar 3C 454.3 was revealed by the Fermi Gamma-ray Space Telescope to be in an exceptionally high flux state in July 2008. Accordingly, we performed a multi-wavelength monitoring campaign on this blazar using IR and optical observations from the SMARTS telescopes, optical, UV and X-ray data from the Swift satellite, and public-release gamma-ray data from Fermi. We find an excellent correlation between the IR, optical, UV and gamma-ray light curves, with a time lag of less than one day. The amplitude of the infrared variability is comparable to that in gamma-rays, and larger than at optical or UV wavelengths. The X-ray flux is not strongly correlated with either the gamma-rays or longer wavelength data. These variability characteristics find a natural explanation in the external Compton model, in which electrons with Lorentz factor gamma~10^(3-4) radiate synchrotron emission in the infrared-optical and also scatter accretion disk or emission line photons to gamma-ray energies, while much cooler electrons ...

  2. Anisotropies in the HI gas distribution toward 3C196

    Kalberla, P M W

    2016-01-01

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

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

    Márquez, I; Durret, F; Petitjean, P

    2000-01-01

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

  4. Organic functionalization of 3C-SiC surfaces.

    Schoell, Sebastian J; Sachsenhauser, Matthias; Oliveros, Alexandra; Howgate, John; Stutzmann, Martin; Brandt, Martin S; Frewin, Christopher L; Saddow, Stephen E; Sharp, Ian D

    2013-02-01

    We demonstrate the functionalization of n-type (100) and (111) 3C-SiC surfaces with organosilanes. Self-assembled monolayers (SAMs) of amino-propyldiethoxymethylsilane (APDEMS) and octadecyltrimethoxysilane (ODTMS) are formed via wet chemical processing techniques. Their structural, chemical, and electrical properties are investigated using static water contact angle measurements, atomic force microscopy, and X-ray photoelectron spectroscopy, revealing that the organic layers are smooth and densely packed. Furthermore, combined contact potential difference and surface photovoltage measurements demonstrate that the heterostructure functionality and surface potential can be tuned by utilizing different organosilane precursor molecules. Molecular dipoles are observed to significantly affect the work functions of the modified surfaces. Furthermore, the magnitude of the surface band bending is reduced following reaction of the hydroxylated surfaces with organosilanes, indicating that partial passivation of electrically active surface states is achieved. Micropatterning of organic layers is demonstrated by lithographically defined oxidation of organosilane-derived monolayers in an oxygen plasma, followed by visualization of resulting changes of the local wettability, as well as fluorescence microscopy following immobilization of fluorescently labeled BSA protein. PMID:23357505

  5. The disc-jet-spin connection: 3C273

    Done, C.

    2014-07-01

    Black hole spin is difficult to measure as it leaves an imprint only close to the horizon, but it may be required to produce most dramatic relativistic jets. For stellar mass black holes there are two established methods to measure spin, from the disc continuum peak temperature in disc dominated states, and from the iron line profile in states with more hard X-ray flux. However, these two methods do not always agree! In AGN the higher black hole mass means a lower disc temperature, so the peak is in the unobservable EUV region, and only the iron line method can be used, but in very high mass AGN, the disc temperature is so low that the peak starts to be visible in the far UV. We use archival data from 3C273 where the observed far UV emission clearly requires a disc around a high spin black hole. The accretion flow dissipates all the available accretion energy, yet the jet in this system is known to be as powerful as the observed accretion flow. Since there is no accretion power left, the jet must be powered by another source of energy - and the only one remaining is black hole spin.

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

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

    2009-01-01

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

  7. The Extended Line Region of 3C 299

    Feinstein, C; Martel, A R; Sparks, W B; McCarthy, P J; Feinstein, Carlos; Martel, Andre R.; Sparks, William B.; Carthy, Patrick J. Mc

    1999-01-01

    We present results of HST observations of the radio galaxy 3C 299. The broad-band F702W (R) and F555W (V) images (WFPC2/PC) show an elliptical galaxy, with a comet-like structure extending to the NE in the radio jet direction. The [OIII]$\\lambda$5007 emission line map, shows a bi-conical structure centered on the nucleus, that overlaps the structure found in the broad-band filters. The radio core coincides with the center of the bi-conical structure and the radio axes are aligned with the direction of the cones. These data show clear evidence of a strong interaction between the radio jet and the NE morphology of the galaxy. We show evidence that this NE region is an ENLR; the line-ratio diagnostics show that models involving gas shocked by the radio-jet plus ionization from a precursor HII region, produced itself by the ionizing photons of the postshocked gas on the preshocked gas provide a good match to the observations. We investigate the spatial behavior of the ionizing parameter $U$, by determining the [O...

  8. The blue-bump of 3C 273

    Paltani, S; Walter, R

    1998-01-01

    We present optical and ultraviolet observations of 3C273 covering the whole life of the IUE satellite. We analyze the variability properties of the light curves, and find that two variable components, written B and R respectively, must contribute to the blue-bump emission in this object. The B component produces most of the variability in the ultraviolet domain. A maximum time scale of variability of about 2 yr identical at all wavelengths is found. If discrete events produce this component, the event rate is ~3-30 yr^-1. Assuming an isotropic emission, each event must liberate about 10^51 erg in the form of optical-to-ultraviolet radiation. The spectral properties of the B component suggest that reprocessing on a truncated disk, or partially- thick bremsstrahlung may be the emission mechanism. We find evidence of a lag of a few days between the light curves of the B component at optical and ultraviolet wavelengths. Neither the variability properties, nor the spectral properties of the R component can be accu...

  9. Identification of Genotypic Changes in Human Immunodeficiency Virus Protease That Correlate with Reduced Susceptibility to the Protease Inhibitor Lopinavir among Viral Isolates from Protease Inhibitor-Experienced Patients

    Kempf, Dale J.; Isaacson, Jeffrey D.; King, Martin S.; Brun, Scott C.; Xu, Yi; Real, Kathryn; Bernstein, Barry M.; Japour, Anthony J.; Sun, Eugene; Rode, Richard A

    2001-01-01

    The association of genotypic changes in human immunodeficiency virus (HIV) protease with reduced in vitro susceptibility to the new protease inhibitor lopinavir (previously ABT-378) was explored using a panel of viral isolates from subjects failing therapy with other protease inhibitors. Two statistical tests showed that specific mutations at 11 amino acid positions in protease (L10F/I/R/V, K20M/R, L24I, M46I/L, F53L, I54L/T/V, L63P, A71I/L/T/V, V82A/F/T, I84V, and L90M) were associated with ...

  10. A Self-compartmentalizing Hexamer Serine Protease from Pyrococcus Horikoshii

    Menyhárd, Dóra K.; Kiss-Szemán, Anna; Tichy-Rács, Éva; Hornung, Balázs; Rádi, Krisztina; Szeltner, Zoltán; Domokos, Klarissza; Szamosi, Ilona; Náray-Szabó, Gábor; Polgár, László; Harmat, Veronika

    2013-01-01

    Oligopeptidases impose a size limitation on their substrates, the mechanism of which has long been under debate. Here we present the structure of a hexameric serine protease, an oligopeptidase from Pyrococcus horikoshii (PhAAP), revealing a complex, self-compartmentalized inner space, where substrates may access the monomer active sites passing through a double-gated “check-in” system, first passing through a pore on the hexamer surface and then turning to enter through an even smaller opening at the monomers' domain interface. This substrate screening strategy is unique within the family. We found that among oligopeptidases, a residue of the catalytic apparatus is positioned near an amylogenic β-edge, which needs to be protected to prevent aggregation, and we found that different oligopeptidases use different strategies to achieve such an end. We propose that self-assembly within the family results in characteristically different substrate selection mechanisms coupled to different multimerization states. PMID:23632025

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

    Ergin, Tülün; Yamazaki, Ryo

    2016-01-01

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

  12. Dose determination in interventional procedures at a hospital, using Al2O3:C detectors

    Full text: Interventional practices have increased in most countries over the past 20 years, combining diagnostic and therapeutic procedures. However, the determination of staff doses in fluoroscopy is difficult, because the examinations are dynamic in nature. Such procedures are complex and they involve prolonged irradiations, providing high radiation doses to patients and to the staff. The effective dose of the staff can be estimated by direct methods, using the optically stimulated luminescence (OSL) technique. In the present work inLight dosimeters and an inLight microstar reader, Landauer, were utilized. These dosimeters are formed by four aluminium oxide (Al2O3:C) detectors and there are aluminium, copper and plastic filters and an open window in each badge. In this work, the dosimeters were utilized for the dose determinations in interventional procedures. Initially the calibration factors and the energy dependence of Al2O3:C dosimeters were obtained in the range of 60 to 120 kV. The doses were evaluated at different positions on the staff during the routine procedures at the Hospital Sao Paulo. The results will be compared with those previously obtained using thermoluminescent CaSO4:Dy

  13. 3C 33: another case of photoionized soft X-ray emission in radio galaxies

    Torresi, E; Guainazzi, M; Palumbo, G G C; Ponti, G; Bianchi, S

    2009-01-01

    All the observations available in the Chandra and XMM-Newton archives have been used to investigate the X-ray spectral properties of 3C 33. In this paper is presented a complete X-ray analysis of the nuclear emission of this narrow line radio galaxy. The broad band spectrum of 3C 33 is complex. The hard part resembles that of Seyfert 2 galaxies, with a heavily obscured nuclear continuum (N_H~10^23 cm^-2) and a prominent Fe Kalpha line. This represents the nuclear radiation directly observed in transmission through a cold circumnuclear gas. On the other hand an unabsorbed continuum plus emission lines seem to fit well the soft part of the spectrum (0.5-2 keV) suggesting that the jet does not significantly contribute to the X-ray emission. We discuss the possible collisional or photoionized origin of the gas that emits the soft X-ray lines. Our results, strengthened by optical spectroscopy favor the photoionization scenario.

  14. The long-term evolution of the parsec scale jet of the quasar 3C345

    The quasar 3C 345 is one of the best examples of an active galactic nucleus (AGN) showing structural and flux variability on parsec scales around a compact unresolved radio core. Over the past 30 years, it has been followed up closely from radio to gamma-ray wavebands with a special focus on very long baseline interferometry (VLBI) observations in the range of 1-100 GHz. The complex parsec-scale jet of 3C 345 exemplifies an archetypical 'superluminal' jet with an apparent helical morphology. Here we present first results from a study of the long-term jet evolution, especially focusing on the evolution of trajectories, kinematics, and emission in more than 20 enhanced emission regions embedded in the jet. A 'closeup' on physical properties of individual features implies that the outer jet is most likely dominated by Kelvin-Helmholtz instability. Studies of general trends in properties of those features provide certain evidence for their apparent trajectories to result from an underlying (slowly evolving) pattern lit up by passages of plasma condensations ejected during the nuclear flares. The long-term evolution of this pattern indicates its possible relation either to the elliptical mode of Kelvin-Helmholtz instability or precession of the jet direction.

  15. The Clp Chaperones and Proteases of the Human Malaria Parasite Plasmodium falciparum

    M El Bakkouri; A Pow; A Mulichak; K Cheung; J Artz; M Amani; S Fell; T de Koning-Ward; C Goodman; et al.

    2011-12-31

    The Clpchaperones and proteases play an important role in protein homeostasis in the cell. They are highly conserved across prokaryotes and found also in the mitochondria of eukaryotes and the chloroplasts of plants. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins. Here, we provide a comprehensive analysis of the Clpchaperones and proteases in the humanmalariaparasitePlasmodiumfalciparum. The parasite contains four Clp ATPases, which we term PfClpB1, PfClpB2, PfClpC and PfClpM. One PfClpP, the proteolytic subunit, and one PfClpR, which is an inactive version of the protease, were also identified. Expression of all Clpchaperones and proteases was confirmed in blood-stage parasites. The proteins were localized to the apicoplast, a non-photosynthetic organelle that accommodates several important metabolic pathways in P. falciparum, with the exception of PfClpB2 (also known as Hsp101), which was found in the parasitophorous vacuole. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by size-exclusion chromatography, analytical ultracentrifugation and electron microscopy. The X-ray structure of PfClpP showed the protein as a compacted tetradecamer similar to that observed for Streptococcus pneumoniae and Mycobacterium tuberculosis ClpPs. Our data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.

  16. The Clp Chaperones and Proteases of the Human Malaria Parasite Plasmodium falciparum

    Bakkouri, Majida El; Pow, Andre; Mulichak, Anne; Cheung, Kevin L.Y.; Artz, Jennifer D.; Amani, Mehrnaz; Fell, Stuart; de Koning-Ward, Tania F.; Goodman, C. Dean; McFadden, Geoffrey I.; Ortega, Joaquin; Hui, Raymond; Houry, Walid A. (McMaster U.); (Melbourne); (Toronto); (Deakin); (HWMRI)

    2015-02-09

    The Clp chaperones and proteases play an important role in protein homeostasis in the cell. They are highly conserved across prokaryotes and found also in the mitochondria of eukaryotes and the chloroplasts of plants. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins. Here, we provide a comprehensive analysis of the Clp chaperones and proteases in the human malaria parasite Plasmodium falciparum. The parasite contains four Clp ATPases, which we term PfClpB1, PfClpB2, PfClpC and PfClpM. One PfClpP, the proteolytic subunit, and one PfClpR, which is an inactive version of the protease, were also identified. Expression of all Clp chaperones and proteases was confirmed in blood-stage parasites. The proteins were localized to the apicoplast, a non-photosynthetic organelle that accommodates several important metabolic pathways in P. falciparum, with the exception of PfClpB2 (also known as Hsp101), which was found in the parasitophorous vacuole. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by size-exclusion chromatography, analytical ultracentrifugation and electron microscopy. The X-ray structure of PfClpP showed the protein as a compacted tetradecamer similar to that observed for Streptococcus pneumoniae and Mycobacterium tuberculosis ClpPs. Our data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.

  17. The Race against Protease Activation Defines the Role of ESCRTs in HIV Budding

    Bendjennat, Mourad; Saffarian, Saveez

    2016-01-01

    HIV virions assemble on the plasma membrane and bud out of infected cells using interactions with endosomal sorting complexes required for transport (ESCRTs). HIV protease activation is essential for maturation and infectivity of progeny virions, however, the precise timing of protease activation and its relationship to budding has not been well defined. We show that compromised interactions with ESCRTs result in delayed budding of virions from host cells. Specifically, we show that Gag mutants with compromised interactions with ALIX and Tsg101, two early ESCRT factors, have an average budding delay of ~75 minutes and ~10 hours, respectively. Virions with inactive proteases incorporated the full Gag-Pol and had ~60 minutes delay in budding. We demonstrate that during budding delay, activated proteases release critical HIV enzymes back to host cytosol leading to production of non-infectious progeny virions. To explain the molecular mechanism of the observed budding delay, we modulated the Pol size artificially and show that virion release delays are size-dependent and also show size-dependency in requirements for Tsg101 and ALIX. We highlight the sensitivity of HIV to budding “on-time” and suggest that budding delay is a potent mechanism for inhibition of infectious retroviral release. PMID:27280284

  18. Novel peptide-based protease inhibitors

    Roodbeen, Renée

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

  19. Lipase and protease extraction from activated sludge

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

    2003-01-01

    gentle and efficient enzyme extraction methods from environmental samples is very important. In this study we present a method for the extraction of lipases and proteases from activated sludge using the non-ionic detergent Triton X-100, EDTA, and cation exchange resin (CER), alone or in combination for......In the process of wastewater treatment hydrolysis of polymeric substances is the first and rate-limiting step. A closer study of the enzymes catalysing these reactions is essential for a better understanding of the microbial activity in the wastewater treatment process. Therefore, development of...... the extraction of lipases and proteases from activated sludge. The sludge was continuously stirred in the presence of either buffer alone or in the presence of detergent and/or chelating agents. In all cases, a marked reduction in floc size was observed upon continuous stirring. However, no lipase...

  20. Protease gene shuffling and expression in Pichia pastoris

    Gang Yang

    2015-06-01

    Full Text Available Four kinds of neutral and alkaline protease genes from Aspergillus oryzae and Bacillus subtilis were isolated and shuffled. The shuffled genes were selected, inserted into pGAPZαA plasmid and transformed into Escherichia coli. The gene which could express high-activity protease was selected by screening the sizes of transparent zones around the colonies on casein plates. After an ideal protease gene was selected, it was sequenced and then transformed into Pichia pastoris X33. The result showed that the base in 1022th position of shuffled protease gene was changed from thymine to cytosine, inferring that cysteine was changed to arginine in the mutant protease. After 48 h incubation for the transformed P. pastoris with the mutant or native protease genes, the mutant protease activity was 36.4% higher than the native protease (P<0.05. The optimal pH and temperature of the mutant protease were 6.5-8.0 and 30-70°C, respectively, which indicated better stability than the native protease (P<0.05.

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

    R. Satheeskumar

    2013-10-01

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

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

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

    2013-01-01

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

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

    Madhusudhan Budatha

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

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

    Mi Kyung Park

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

  5. Protease Activation and Inflammation in Acute Pancreatitis

    Regnér, Sara

    2008-01-01

    Approximately 10—20 % of patients with acute pancreatitis (AP) develop a severe disease with high mortality and morbidity. Activation of pancreatic proteases, the inflammatory response and impaired pancreatic circulation are pathophysiological events that are important in order for the disease to develop. There is no specific treatment for severe AP, and no useful marker for predicting the severity of the disease upon admission to the hospital. In this thesis, markers of early pathophysio...

  6. Targeting exosites on blood coagulation proteases

    Monteiro, Robson Q.

    2005-01-01

    The high specificity of blood coagulation proteases has been attributed not only to residues surrounding the active site but also to other surface domains that are involved in recognizing and interacting with macromolecular substrates and inhibitors. Specific blood coagulation inhibitors obtained from exogenous sources such as blood sucking salivary glands and snake venoms have been identified. Some of these inhibitors interact with exosites on coagulation enzymes. Two examples are discussed ...

  7. Luminometric method for screening retroviral protease inhibitors

    Horáková, D.; Rumlová, Michaela; Pichová, Iva; Ruml, Tomáš

    2005-01-01

    Roč. 345, č. 1 (2005), s. 96-101. ISSN 0003-2697 R&D Projects: GA AV ČR(CZ) IAA4055304; GA MŠk(CZ) 1M0508; GA MŠk(CZ) 1M0520 Institutional research plan: CEZ:AV0Z40550506 Keywords : retroviral protease * inhibitors * luminescent assay Subject RIV: CE - Biochemistry Impact factor: 2.670, year: 2005

  8. Corruption of Innate Immunity by Bacterial Proteases

    Potempa, Jan; Pike, Robert N.

    2009-01-01

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

  9. Mitochondrial Proteases as Emerging Pharmacological Targets.

    Gibellini, Lara; De Biasi, Sara; Nasi, Milena; Iannone, Anna; Cossarizza, Andrea; Pinti, Marcello

    2016-01-01

    The preservation of mitochondrial function and integrity is critical for cell viability. Under stress conditions, unfolded, misfolded or damaged proteins accumulate in a certain compartment of the organelle, interfering with oxidative phosphorylation and normal mitochondrial functions. In stress conditions, several mechanisms, including mitochondrial unfolded protease response (UPRmt), fusion and fission, and mitophagy are engaged to restore normal proteostasis of the organelle. Mitochondrial proteases are a family of more than 20 enzymes that not only are involved in the UPRmt, but actively participate at multiple levels in the stress-response system. Alterations in their expression levels, or mutations that determine loss or gain of function of these proteases deeply impair mitochondrial functionality and can be associated with the onset of inherited diseases, with the development of neurodegenerative disorders and with the process of carcinogenesis. In this review, we focus our attention on six of them, namely CLPP, HTRA2 and LONP1, by analysing the current knowledge about their functions, their involvement in the pathogenesis of human diseases, and the compounds currently available for inhibiting their functions. PMID:26831646

  10. Characterization of HC58cDNA, a putative cysteine protease from the parasite Haemonchus contortus

    Muleke, Charles I.; Ruofeng, Yan; Lixin, Xu; Yanming, Sun; Xiangrui, Li

    2006-01-01

    Because of the complexity of the cathepsin B-like (CBL) family, an information on the biological and biochemical characteristics of individual CBL genes is lacking. In this study, we investigated the degradative effects of the recombinant HC58 protein isolated from Haemonchus contortus parasites on protein substrates over a broad pH range in vitro. This protein, which hydrolyzed the synthetic peptide substrates Z-FR-AMC and Z-RR-AMC, had characteristics of the cysteine protease class of prote...

  11. Theranostic Imaging of the Kinases and Proteases that Modulate Cell Death and Survival

    Chen, Howard H.; Yuan, Hushan; Josephson, Lee; Sosnovik, David E.

    2012-01-01

    Several signaling cascades are involved in cell death, with a significant amount of crosstalk between them. Despite the complexity of these cascades several key pro-survival and pro-death players have been identified. These include PI3-kinase, AKT and caspase-3. Here we review the approaches used to date to perform molecular imaging of these important targets. We focus in particular on approaches that include the possibility of modulating the activity of these kinases and proteases in a thera...

  12. Particle acceleration in the hotspot of the jet of quasar 3C273

    Meisenhaimer, K.; Heavens, A.F.

    1986-10-02

    Recent radio, optical and near-infrared observations of the dominant hotspot at the outer end of the quasar jet 3C273A have revealed a complex spectrum, which provides a unique probe of the acceleration process supplying the synchrotron-emitting electrons. The authors present theoretical calculations of the electron spectrum produced by non-relativistic shock acceleration which, include both synchrotron losses and a finite emission region. The resultant synchrotron spectrum fits the observations, providing a natural explanation for the steep spectrum at radio to infrared frequencies, the observed flattening at ..nu.. < ..nu..sub(b) = 1.5 GHz, and the cutoff above ..nu..sub(c) = 2 x 10/sup 14/ Hz. The model yields a new magnetic field estimate of 70 nT.

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

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

    2005-01-01

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

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

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

    2014-09-01

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

  15. On the sintering behaviour of steel bonded TiC-Cr3C2 and TiC-Cr3C2-WC mixed carbides

    Powder mixtures of TiC+Cr3C2 and TiC+Cr3C2 + WC were hot pressed to nearly full density. The lattice parameter of the resulting cubic mixed crystal decreases linearly with increasing additions of Cr3C2 and (Cr3C2+WC 1:1). Microhardness increases with Cr3C2 content up to 20 wt.%. By addition of WC, microhardness is increased further and reaches a maximum value of approx. 38 000 MN/m2 for 20 wt.% Cr3C2 and 20 wt.% WC. From these solid solutions powder compositions of Ferro-TiC type were produced by milling with 55 wt.% Fe and 0.4 wt.% C. The sintering behaviour of these powders was studied in a vacuum dilatometer. The pronounced increase of shrinkage by Cr3C2 and higher amounts of Cr3C2+WC dissolved in TiC previous to binder phase melting is attributed to the increased solubility of the carbide in solid iron. Presintering at 7000C in hydrogen has a negative influence on sintering activity and requires much higher temperatures for complete densification during subsequent vacuum sintering. (orig.)

  16. High throughput substrate phage display for protease profiling.

    Ratnikov, Boris; Cieplak, Piotr; Smith, Jeffrey W

    2009-01-01

    The interplay between a protease and its substrates is controlled at many different levels, including coexpression, colocalization, binding driven by ancillary contacts, and the presence of natural inhibitors. Here we focus on the most basic parameter that guides substrate recognition by a protease, the recognition specificity at the catalytic cleft. An understanding of this substrate specificity can be used to predict the putative substrates of a protease, to design protease activated imaging agents, and to initiate the design of active site inhibitors. Our group has characterized protease specificities of several matrix metalloproteinases using substrate phage display. Recently, we have adapted this method to a semiautomated platform that includes several high-throughput steps. The semiautomated platform allows one to obtain an order of magnitude more data, thus permitting precise comparisons among related proteases to define their functional distinctions. PMID:19377968

  17. Molecular characterization of protease activity in Serratia sp. strain SCBI and its importance in cytotoxicity and virulence.

    Petersen, Lauren M; Tisa, Louis S

    2014-11-01

    A newly recognized Serratia species, termed South African Caenorhabditis briggsae isolate (SCBI), is both a mutualist of the nematode Caenorhabditis briggsae KT0001 and a pathogen of lepidopteran insects. Serratia sp. strain SCBI displays high proteolytic activity, and because secreted proteases are known virulence factors for many pathogens, the purpose of this study was to identify genes essential for extracellular protease activity in Serratia sp. strain SCBI and to determine what role proteases play in insect pathogenesis and cytotoxicity. A bank of 2,100 transposon mutants was generated, and six SCBI mutants with defective proteolytic activity were identified. These mutants were also defective in cytotoxicity. The mutants were found defective in genes encoding the following proteins: alkaline metalloprotease secretion protein AprE, a BglB family transcriptional antiterminator, an inosine/xanthosine triphosphatase, GidA, a methyl-accepting chemotaxis protein, and a PIN domain protein. Gene expression analysis on these six mutants showed significant downregulation in mRNA levels of several different types of predicted protease genes. In addition, transcriptome sequencing (RNA-seq) analysis provided insight into how inactivation of AprE, GidA, and a PIN domain protein influences motility and virulence, as well as protease activity. Using quantitative reverse transcription-PCR (qRT-PCR) to further characterize expression of predicted protease genes in wild-type Serratia sp. SCBI, the highest mRNA levels for the alkaline metalloprotease genes (termed prtA1 to prtA4) occurred following the death of an insect host, while two serine protease and two metalloprotease genes had their highest mRNA levels during active infection. Overall, these results indicate that proteolytic activity is essential for cytotoxicity in Serratia sp. SCBI and that its regulation appears to be highly complex. PMID:25182493

  18. Temporal dependence of cysteine protease activation following excitotoxic hippocampal injury

    Berry, Jennifer N.; Sharrett-Field, Lynda; Butler, Tracy R.; Prendergast, Mark A.

    2012-01-01

    Excitotoxic insults can lead to intracellular signaling cascades that contribute to cell death, in part by activation of proteases, phospholipases, and endonucleases. Cysteine proteases, such as calpains, are calcium-activated enzymes which degrade cytoskeletal proteins, including microtubule-associated proteins, tubulin, and spectrin, among others. The current study used the organotypic hippocampal slice culture model to examine whether pharmacologic inhibition of cysteine protease activity ...

  19. Cold-Adapted Proteases as an Emerging Class of Therapeutics

    Fornbacke, Marcus; Clarsund, Mats

    2013-01-01

    Proteases have been used in medicine for several decades and are an established and well tolerated class of therapeutic agent. These proteases were sourced from mammals or bacteria that exist or have adapted to moderate temperatures (mesophilic organisms); however, proteases derived from organisms from cold environments—cold-adapted or psychrophilic proteases—generally have high specific activity, low substrate affinity, and high catalytic rates at low and moderate temperatures. Made possible...

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

    Nilesh P. Nirmal; R. Seeta Laxman

    2014-01-01

    A fungal strain (Conidiobolus brefeldianus MTCC 5184) isolated from plant detritus secreted a high activity alkaline protease. Thermostability studies of the fungal alkaline protease (FAP) revealed that the protease is stable up to 50°C with 40% residual activity after one hour. Effect of various additives such as sugars, sugar alcohols, polyols, and salts, on the thermostability of FAP was evaluated. Among the additives tested, glycerol, mannitol, xylitol, sorbitol, and trehalose were found ...

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

    Kyöstiö, Sirkka R. M.

    1990-01-01

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

  2. Hydrophobic core flexibility modulates enzyme activity in HIV-1 protease

    Mittal, Seema; Cai, Yufeng; Nalam, Madhavi N.; Bolon, Daniel N. A.; Schiffer, Celia A.

    2012-01-01

    Human immunodeficiency virus Type-1 (HIV-1) protease is crucial for viral maturation and infectivity. Studies of protease dynamics suggest that the rearrangement of the hydrophobic core is essential for enzyme activity. Many mutations in the hydrophobic core are also associated with drug resistance and may modulate the core flexibility. To test the role of flexibility in protease activity, pairs of cysteines were introduced at the interfaces of flexible regions remote from the active site. Di...

  3. Human Immunodeficiency Virus Reverse Transcriptase and Protease Sequence Database

    Shafer, Robert W.; Jung, Duane R.; Betts, Bradley J.; Xi, Yinong; Gonzales, Matthew J.

    2000-01-01

    The HIV RT and Protease Sequence Database is an online relational database that catalogs evolutionary and drug-related human immunodeficiency virus (HIV) reverse transcriptase (RT) and protease sequence variation (http://hivdb.stanford.edu ). The database contains a compilation of nearly all published HIV RT and protease sequences including International Collaboration database submissions (e.g., GenBank) and sequences published in journal articles. Sequences are linked to data about the sourc...

  4. Protein protease inhibitors in insects and comparison with mammalian inhibitors.

    Eguchi, M

    1993-01-01

    1. Studies on insect protein protease inhibitors are summarized. Biochemical, genetic and physiological investigations of the silkworm are performed. 2. In addition, the properties and characteristics of fungal protease inhibitors from the silkworm (Bombyx mori) are described and their importance as defensive functions is emphasized. 3. This review also concerns comparative and evolutionary studies of protease inhibitors from various sources. 4. The biological significance of inhibitors is discussed in view of the extensive experimental results. PMID:8365101

  5. Characterization of Fibrinolytic Proteases from Gloydius blomhoffii siniticus Venom

    Suk Ho Choi

    2011-01-01

    Objectives : This study was undertaken to identify fibrinolytic proteases from Gloydius blomhoffii siniticus venom and to characterize a major fibrinolytic protease purified from the venom. Methods: The venom was subjected to chromatography using columns of Q-Sepharose and Sephadex G-75. The molecular weights of fibrinolytic proteases showing fibrinolytic zone in fibrin plate assay were determined in SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) The effects of inh...

  6. A preliminary neutron diffraction analysis of Achromobacter protease I

    Ohnishi, Yuki; Masaki, Takeharu; Yamada, Taro; Kurihara, Kazuo; Tanaka, Ichiro; Niimura, Nobuo

    2010-11-01

    Achromobacter protease I (API, E.C. 3.4.21.50) is one of the serine proteases produced by Achromobacter lyticus M497-1. API is distinct from the other tripsin type protease in its lysine specificity. The neutron structure analysis of catalytic triad with Trp169 and His210 was presented. His57 was double protonated and formed hydrogen bonds to Ser194Oγ and Asp113Oδ1, Oδ2.

  7. A preliminary neutron diffraction analysis of Achromobacter protease I

    Achromobacter protease I (API, E.C. 3.4.21.50) is one of the serine proteases produced by Achromobacter lyticus M497-1. API is distinct from the other tripsin type protease in its lysine specificity. The neutron structure analysis of catalytic triad with Trp169 and His210 was presented. His57 was double protonated and formed hydrogen bonds to Ser194Oγ and Asp113Oδ1, Oδ2.

  8. A functional proteomics screen of proteases in colorectal carcinoma.

    McKerrow, J H; Bhargava, V.; Hansell, E.; Huling, S.; Kuwahara, T.; Matley, M.; Coussens, L; Warren, R

    2000-01-01

    BACKGROUND: Proteases facilitate several steps in cancer progression. To identify proteases most suitable for drug targeting, actual enzyme activity and not messenger RNA levels or immunoassay of protein is the ideal assay readout. MATERIALS AND METHODS: An automated microtiter plate assay format was modified to allow detection of all four major classes of proteases in tissue samples. Fifteen sets of colorectal carcinoma biopsies representing primary tumor, adjacent normal colon, and liver me...

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

    Lefkovits, I

    1986-01-01

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

  10. 18 CFR 3c.3 - Reporting fraud, waste, abuse, and corruption and cooperation with official inquiries.

    2010-04-01

    ... OF CONDUCT § 3c.3 Reporting fraud, waste, abuse, and corruption and cooperation with official inquiries. (a) Employees shall, in fulfilling the obligation of 5 CFR 2635.101(b)(11), report fraud, waste..., abuse, and corruption and cooperation with official inquiries. 3c.3 Section 3c.3 Conservation of...

  11. Characterization of a Kunitz-type serine protease inhibitor from Solanum tuberosum having lectin activity.

    Shah, Kunal R; Patel, Dhaval K; Pappachan, Anju; Prabha, C Ratna; Singh, Desh Deepak

    2016-02-01

    Plant lectins and protease inhibitors constitute a class of proteins which plays a crucial role in plant defense. In our continuing investigations on lectins from plants, we have isolated, purified and characterized a protein of about 20 kDa, named PotHg, showing hemagglutination activity from tubers of Indian potato, Solanum tuberosum. De novo sequencing and MS/MS analysis confirmed that the purified protein was a Kunitz-type serine protease inhibitor having two chains (15 kDa and 5 kDa). SDS and native PAGE analysis showed that the protein was glycosylated and was a heterodimer of about 15 and 5 kDa subunits. PotHg agglutinated rabbit erythrocytes with specific activity of 640 H.U./mg which was inhibited by complex sugars like fetuin. PotHg retained hemagglutination activity over a pH range 4-9 and up to 80°C. Mannose and galactose interacted with the PotHg with a dissociation constant (Kd) of 1.5×10(-3) M and 2.8×10(-3) M, respectively as determined through fluorescence studies. Fluorescence studies suggested the involvement of a tryptophan in sugar binding which was further confirmed through modification of tryptophan residues using N-bromosuccinimide. Circular dichroism (CD) studies showed that PotHg contains mostly β sheets (∼45%) and loops which is in line with previously characterized protease inhibitors and modeling studies. There are previous reports of Kunitz-type protease inhibitors showing lectin like activity from Peltophorum dubium and Labramia bojeri. This is the first report of a Kunitz-type protease inhibitor showing lectin like activity from a major crop plant and this makes PotHg an interesting candidate for further investigation. PMID:26645142

  12. Role of Protease-Inhibitors in Ocular Diseases

    Nicola Pescosolido

    2014-12-01

    Full Text Available It has been demonstrated that the balance between proteases and protease-inhibitors system plays a key role in maintaining cellular and tissue homeostasis. Indeed, its alteration has been involved in many ocular and systemic diseases. In particular, research has focused on keratoconus, corneal wounds and ulcers, keratitis, endophthalmitis, age-related macular degeneration, Sorsby fundus dystrophy, loss of nerve cells and photoreceptors during optic neuritis both in vivo and in vitro models. Protease-inhibitors have been extensively studied, rather than proteases, because they may represent a therapeutic approach for some ocular diseases. The protease-inhibitors mainly involved in the onset of the above-mentioned ocular pathologies are: α2-macroglobulin, α1-proteinase inhibitor (α1-PI, metalloproteinase inhibitor (TIMP, maspin, SERPINA3K, SERPINB13, secretory leukocyte protease inhibitor (SLPI, and calpeptin. This review is focused on the several characteristics of dysregulation of this system and, particularly, on a possible role of proteases and protease-inhibitors in molecular remodeling that may lead to some ocular diseases. Recently, researchers have even hypothesized a possible therapeutic effect of the protease-inhibitors in the treatment of injured eye in animal models.

  13. Induction of Protease Activity in Vibrio anguillarum by Gastrointestinal Mucus

    Denkin, Steven M.; Nelson, David R.

    1999-01-01

    The effect of gastrointestinal mucus on protease activity in Vibrio anguillarum was investigated. Protease activity was measured by using an azocasein hydrolysis assay. Cells grown to stationary phase in mucus (200 μg of mucus protein/ml) exhibited ninefold-greater protease activity than cells grown in Luria-Bertani broth plus 2% NaCl (LB20). Protease induction was examined with cells grown in LB20 and resuspended in mucus, LB20, nine-salts solution (NSS [a carbon-, nitrogen-, and phosphorus-...

  14. Characterization of Fibrinolytic Proteases from Gloydius blomhoffii siniticus Venom

    Suk Ho Choi

    2011-09-01

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

  15. Hordeum vulgare cysteine protease heterologous expressed in yeast

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

    During germination of barley seeds, the mobilization of protein is essential and Cysteine Proteases accounts for more than 90 % of the total proteolytic activity in the degradation of barley seed storage proteins [1]. Cysteine proteases exist as pro-enzyme until activated through reduction of the...... active site cysteines and via removal of the pro-domain. The complement of cysteine proteases is comprehensive and for detailed studies of the individual components of this complement, a fast and efficient eukaryotic expression platform is highly desirable. One of the key cysteine proteases in Barley...

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

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

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

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

    2012-09-17

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

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

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

    2013-01-01

    The structure and properties of the three smallest nonface-spiral (NS) fullerenes NS-T-C380, NS-D3-C384, NS-D3-C440, and the first isolated pentagon NS-fullerene, NS-D3-C672, are investigated in detail. They are constructed by either a generalized face-spiral algorithm or by vertex insertions...... followed by a force-field optimization using the recently introduced program Fullerene. The obtained structures were then further optimized at the density functional level of theory and their stability analyzed with reference to Ih-C60. The large number of hexagons results in a higher stability of the NS-fullerenes...... compared to C60, but, as expected, in a lower stability than most stable isomers. None of the many investigated halma transforms on nonspiral fullerenes, NS-T-C380, NS-D3-C384, NS-D3-C440, and NS-D3-C672, admit any spirals, and we conjecture that all halma transforms of NS-fullerenes belong to the class of...

  19. The limit set of discrete subgroups of $PSL(3,\\C)$

    Barrera, Waldemar; Navarrete, Juan Pablo

    2010-01-01

    If $\\Gamma$ is a discrete subgroup of $PSL(3,\\Bbb{C})$, it is determined the equicontinuity region $Eq(\\Gamma)$ of the natural action of $\\Gamma$ on $\\Bbb{P}^2_\\Bbb{C}$. It is also proved that the action restricted to $Eq(\\Gamma)$ is discontinuous, and $Eq(\\Gamma)$ agrees with the discontinuity set in the sense of Kulkarni whenever the limit set of $\\Gamma$ in the sense of Kulkarni, $\\Lambda(\\Gamma)$, contains at least three lines in general position. Under some additional hypothesis, it turns out to be the largest open set on which $\\Gamma$ acts discontinuously. Moreover, if $\\Lambda(\\Gamma)$ contains at least four complex lines and $\\Gamma$ acts on $\\Bbb{P}^2_\\Bbb{C}$ without fixed points nor invariant lines, then each connected component of $Eq(\\Gamma)$ is a holomorphy domain and a complete Kobayashi hyperbolic space.

  20. The SARS-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds.

    Báez-Santos, Yahira M; St John, Sarah E; Mesecar, Andrew D

    2015-03-01

    Over 10 years have passed since the deadly human coronavirus that causes severe acute respiratory syndrome (SARS-CoV) emerged from the Guangdong Province of China. Despite the fact that the SARS-CoV pandemic infected over 8500 individuals, claimed over 800 lives and cost billions of dollars in economic loss worldwide, there still are no clinically approved antiviral drugs, vaccines or monoclonal antibody therapies to treat SARS-CoV infections. The recent emergence of the deadly human coronavirus that causes Middle East respiratory syndrome (MERS-CoV) is a sobering reminder that new and deadly coronaviruses can emerge at any time with the potential to become pandemics. Therefore, the continued development of therapeutic and prophylactic countermeasures to potentially deadly coronaviruses is warranted. The coronaviral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro), are attractive antiviral drug targets because they are essential for coronaviral replication. Although the primary function of PLpro and 3CLpro are to process the viral polyprotein in a coordinated manner, PLpro has the additional function of stripping ubiquitin and ISG15 from host-cell proteins to aid coronaviruses in their evasion of the host innate immune responses. Therefore, targeting PLpro with antiviral drugs may have an advantage in not only inhibiting viral replication but also inhibiting the dysregulation of signaling cascades in infected cells that may lead to cell death in surrounding, uninfected cells. This review provides an up-to-date discussion on the SARS-CoV papain-like protease including a brief overview of the SARS-CoV genome and replication followed by a more in-depth discussion on the structure and catalytic mechanism of SARS-CoV PLpro, the multiple cellular functions of SARS-CoV PLpro, the inhibition of SARS-CoV PLpro by small molecule inhibitors, and the prospect of inhibiting papain-like protease from other coronaviruses. This paper forms part of a series of

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

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

  2. Hepatitis C Virus NS3/4A Protease Inhibitors: A Light at the End of the Tunnel

    Laurent Chatel-Chaix

    2010-08-01

    Full Text Available Hepatitis C virus (HCV infection is a serious and growing threat to human health. The current treatment provides limited efficacy and is poorly tolerated, highlighting the urgent medical need for novel therapeutics. The membrane-targeted NS3 protein in complex with the NS4A comprises a serine protease domain (NS3/4A protease that is essential for viral polyprotein maturation and contributes to the evasion of the host innate antiviral immunity by HCV. Therefore, the NS3/4A protease represents an attractive target for drug discovery, which is tied in with the challenge to develop selective small-molecule inhibitors. A rational drug design approach, based on the discovery of N-terminus product inhibition, led to the identification of potent and orally bioavailable NS3 inhibitors that target the highly conserved protease active site. This review summarizes the NS3 protease inhibitors currently challenged in clinical trials as one of the most promising antiviral drug class, and possibly among the first anti-HCV agents to be approved for the treatment of HCV infection.

  3. Multifrequency VLBI follow up study of strong γ-ray flares in the blazars 3C273 and 3C279

    Lisakov, Mikhail M.; Kovalev, Yuri Y.

    2015-03-01

    We present multifrequency VLBI observations of the blazars 3C273 and 3C279 after detecting strong γ-ray flares in both of them. 3C273 exhibited a prominent flare in γ-rays in September 2009 which was followed by a strong flare in the 7 mm VLBI core and emergence of a new feature in the parsec scale jet. We have used time delay between flares in different wavebands together with kinematic analysis to determine that the γ-ray emission zone in 3C273 is located 3.6-5.3 pc upstream from the apparent 7 mm core. We have also analyzed frequency dependent core position to measure a deprojected distance between 7 mm core and the true base of the jet: 1-6 pc for 3C273 and 1-3 pc for 3C279, depending on observing epoch. For 3C279 light curve analysis did not give a robust γ-radio delay because there were too many overlapping flares in this source during considered period.

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

    Fyfe, Cameron D.; Grinter, Rhys; Josts, Inokentijs; Mosbahi, Khedidja [University of Glasgow, Glasgow G12 8QQ, Scotland (United Kingdom); Roszak, Aleksander W. [University of Glasgow, Glasgow G12 8QQ, Scotland (United Kingdom); University of Glasgow, Glasgow G12 8QQ, Scotland (United Kingdom); Cogdell, Richard J.; Wall, Daniel M.; Burchmore, Richard J. S.; Byron, Olwyn; Walker, Daniel, E-mail: daniel.walker@glasgow.ac.uk [University of Glasgow, Glasgow G12 8QQ, Scotland (United Kingdom)

    2015-06-30

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

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

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

  6. Isolation of a thiol-dependent serine protease in peanut and investigation of its role in the complement and the allergic reaction.

    Javaux, Cédric; Stordeur, Patrick; Azarkan, Mohamed; Mascart, Françoise; Baeyens-Volant, Danielle

    2016-07-01

    A serine protease activity was detected in aqueous peanuts seeds extracts, partially purified and characterized as a thiol-dependent serine protease. The potential role of this proteolytic activity on allergic reaction to peanuts was prospected through complement activation studies in human plasma and serum, and MDCK cells to investigate a possible occludin degradation in tight junctions. The peanut protease activity induced the production of anaphylatoxins C3a and C5a, and of the terminal membrane attack complex SC5b-9 whatever the complement activation pathway. The protease activity was also involved in the partial digestion of occludin within tight junctions, with for result, an increase of the epithelial permeability to antigen absorption. PMID:27280846

  7. Series of HIV-1 protease nanomolar inhibitors; binding to WT and mutant protease

    Skálová, Tereza; Dohnálek, Jan; Dušková, Jarmila; Petroková, Hana; Vondráčková, Eva; Hašek, Jindřich

    Florence : International Union of Crystallography, 2005. C243. [Congress of the International Union of Crystallography /20./. 23.8.2005-31.8.2005, Florence] R&D Projects: GA AV ČR(CZ) KJB4050312 Keywords : HIV retroviral proteases * HIV drug design * macromolecular crystal structure Subject RIV: EB - Genetics ; Molecular Biology

  8. Reversible Unfolding of Rhomboid Intramembrane Proteases.

    Panigrahi, Rashmi; Arutyunova, Elena; Panwar, Pankaj; Gimpl, Katharina; Keller, Sandro; Lemieux, M Joanne

    2016-03-29

    Denaturant-induced unfolding of helical membrane proteins provides insights into their mechanism of folding and domain organization, which take place in the chemically heterogeneous, anisotropic environment of a lipid membrane. Rhomboid proteases are intramembrane proteases that play key roles in various diseases. Crystal structures have revealed a compact helical bundle with a buried active site, which requires conformational changes for the cleavage of transmembrane substrates. A dimeric form of the rhomboid protease has been shown to be important for activity. In this study, we examine the mechanism of refolding for two distinct rhomboids to gain insight into their secondary structure-activity relationships. Although helicity is largely abolished in the unfolded states of both proteins, unfolding is completely reversible for HiGlpG but only partially reversible for PsAarA. Refolding of both proteins results in reassociation of the dimer, with a 90% regain of catalytic activity for HiGlpG but only a 70% regain for PsAarA. For both proteins, a broad, gradual transition from the native, folded state to the denatured, partly unfolded state was revealed with the aid of circular dichroism spectroscopy as a function of denaturant concentration, thus arguing against a classical two-state model as found for many globular soluble proteins. Thermal denaturation has irreversible destabilizing effects on both proteins, yet reveals important functional details regarding substrate accessibility to the buried active site. This concerted biophysical and functional analysis demonstrates that HiGlpG, with a simple six-transmembrane-segment organization, is more robust than PsAarA, which has seven predicted transmembrane segments, thus rendering HiGlpG amenable to in vitro studies of membrane-protein folding. PMID:27028647

  9. Substrate profiling of tobacco etch virus protease using a novel fluorescence-assisted whole-cell assay.

    George Kostallas

    Full Text Available Site-specific proteolysis of proteins plays an important role in many cellular functions and is often key to the virulence of infectious organisms. Efficient methods for characterization of proteases and their substrates will therefore help us understand these fundamental processes and thereby hopefully point towards new therapeutic strategies. Here, a novel whole-cell in vivo method was used to investigate the substrate preference of the sequence specific tobacco etch virus protease (TEVp. The assay, which utilizes protease-mediated intracellular rescue of genetically encoded short-lived fluorescent substrate reporters to enhance the fluorescence of the entire cell, allowed subtle differences in the processing efficiency of closely related substrate peptides to be detected. Quantitative screening of large combinatorial substrate libraries, through flow cytometry analysis and cell sorting, enabled identification of optimal substrates for TEVp. The peptide, ENLYFQG, identical to the protease's natural substrate peptide, emerged as a strong consensus cleavage sequence, and position P3 (tyrosine, Y and P1 (glutamine, Q within the substrate peptide were confirmed as being the most important specificity determinants. In position P1', glycine (G, serine (S, cysteine (C, alanine (A and arginine (R were among the most prevalent residues observed, all known to generate functional TEVp substrates and largely in line with other published studies stating that there is a strong preference for short aliphatic residues in this position. Interestingly, given the complex hydrogen-bonding network that the P6 glutamate (E is engaged in within the substrate-enzyme complex, an unexpectedly relaxed residue preference was revealed for this position, which has not been reported earlier. Thus, in the light of our results, we believe that our assay, besides enabling protease substrate profiling, also may serve as a highly competitive platform for directed evolution of

  10. EBV Nuclear Antigen 3C Mediates Regulation of E2F6 to Inhibit E2F1 Transcription and Promote Cell Proliferation.

    Pei, Yonggang; Banerjee, Shuvomoy; Sun, Zhiguo; Jha, Hem Chandra; Saha, Abhik; Robertson, Erle S

    2016-08-01

    Epstein-Barr virus (EBV) is considered a ubiquitous herpesvirus with the ability to cause latent infection in humans worldwide. EBV-association is evidently linked to different types of human malignancies, mainly of epithelial and lymphoid origin. Of interest is the EBV nuclear antigen 3C (EBNA3C) which is critical for EBV-mediated immortalization. Recently, EBNA3C was shown to bind the E2F1 transcription regulator. The E2F transcription factors have crucial roles in various cellular functions, including cell cycle, DNA replication, DNA repair, cell mitosis, and cell fate. Specifically, E2F6, one of the unique E2F family members, is known to be a pRb-independent transcription repressor of E2F-target genes. In our current study, we explore the role of EBNA3C in regulating E2F6 activities. We observed that EBNA3C plays an important role in inducing E2F6 expression in LCLs. Our study also shows that EBNA3C physically interacts with E2F6 at its amino and carboxy terminal domains and they form a protein complex in human cells. In addition, EBNA3C stabilizes the E2F6 protein and is co-localized in the nucleus. We also demonstrated that both EBNA3C and E2F6 contribute to reduction in E2F1 transcriptional activity. Moreover, E2F1 forms a protein complex with EBNA3C and E2F6, and EBNA3C competes with E2F1 for E2F6 binding. E2F6 is also recruited by EBNA3C to the E2F1 promoter, which is critical for EBNA3C-mediated cell proliferation. These results demonstrate a critical role for E2F family members in EBV-induced malignancies, and provide new insights for targeting E2F transcription factors in EBV-associated cancers as potential therapeutic intervention strategies. PMID:27548379

  11. Production and cleavage specificity determination of serine proteases mMCP-4, mMCP-5, rMCP-2 and two platypus serine proteases of the chymase locus.

    Sidibeh, Cherno Omar

    2013-01-01

    Serine proteases are a family of enzymes with a wide array of functions across both eukaryotes and prokaryotes. Here we have attempted to produce the serine proteases rat mast cell protease 2 and mouse mast cell protease 5 in a culture of HEK 293 cells; and mouse mast cell protease 4, platypus granzyme B-like protease and platypus hypothetical protease in a baculovirus expression system. Following production we wanted to analyse these serine proteases using a phage display assay and a battery...

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

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

    2016-01-01

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

  13. A review on production of serine alkaline protease by Bacillus spp

    Biswanath Bhunia; Bikram Basak; Apurba Dey

    2012-01-01

    In recent times, protease has gained considerable importance in the world market. Proteases are groups of proteins included in the subclass hydrolases, within the main class enzymes. Serine alkaline proteases (SAP) are one of the most important groups of industrial enzymes. They account for approximately 35% of the total microbial enzyme sales. Serine protease is produced by various types of fermentation techniques using microorganism. Among the proteases, bacterial proteases are more signifi...

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

    Fluhrer, Regina; Steiner, Harald; Haass, Christian

    2009-01-01

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

  15. Probing the Disk-Jet Connection of the Radio Galaxy 3C120 Observed With Suzaku

    Broad line radio galaxies (BLRGs) are a rare type of radio-loud AGN, in which the broad optical permitted emission lines have been detected in addition to the extended jet emission. Here we report on deep (40ksec x 4) observations of the bright BLRG 3C 120 using Suzaku. The observations were spaced a week apart, and sample a range of continuum fluxes. An excellent broadband spectrum was obtained over two decades of frequency (0.6 to 50 keV) within each 40 ksec exposure. We clearly resolved the iron K emission line complex, finding that it consists of a narrow Kα core (σ ≅ 110 eV or an EW of 60 eV), a 6.9 keV line, and an underlying broad iron line. Our confirmation of the broad line contrasts with the XMM-Newton observation in 2003, where the broad line was not required. The most natural interpretation of the broad line is iron K line emission from a face-on accretion disk which is truncated at ∼ 10 rg. Above 10 keV, a relatively weak Compton hump was detected (reflection fraction of R ≅ 0.6), superposed on the primary X-ray continuum of Λ ≅ 1.75. Thanks to the good photon statistics and low background of the Suzaku data, we clearly confirm the spectral evolution of 3C 120, whereby the variability amplitude decreases with increasing energy. More strikingly, we discovered that the variability is caused by a steep power-law component of Λ ≅ 2.7, possibly related to the non-thermal jet emission. We discuss our findings in the context of similarities and differences between radio-loud/quiet objects

  16. Probing the Disk-Jet Connection of the Radio Galaxy 3C120 Observed With Suzaku

    Kataoka, Jun; Reeves, James N.; Iwasawa, Kazushi; Markowitz, Alex G.; Mushotzky, Richard F.; Arimoto, Makoto; Takahashi, Tadayuki; Tsubuku, Yoshihiro; Ushio, Masayoshi; Watanabe, Shin; Gallo, Luigi C.; Madejski, Greg M.; Terashima, Yuichi; Isobe, Naoki; Tashiro, Makoto S.; Kohmura, Takayoshi; /Tokyo Inst. Tech. /NASA, Goddard /Garching, Max Planck

    2007-01-03

    Broad line radio galaxies (BLRGs) are a rare type of radio-loud AGN, in which the broad optical permitted emission lines have been detected in addition to the extended jet emission. Here we report on deep (40ksec x 4) observations of the bright BLRG 3C 120 using Suzaku. The observations were spaced a week apart, and sample a range of continuum fluxes. An excellent broadband spectrum was obtained over two decades of frequency (0.6 to 50 keV) within each 40 ksec exposure. We clearly resolved the iron K emission line complex, finding that it consists of a narrow K{sub {alpha}} core ({sigma} {approx_equal} 110 eV or an EW of 60 eV), a 6.9 keV line, and an underlying broad iron line. Our confirmation of the broad line contrasts with the XMM-Newton observation in 2003, where the broad line was not required. The most natural interpretation of the broad line is iron K line emission from a face-on accretion disk which is truncated at {approx} 10 r{sub g}. Above 10 keV, a relatively weak Compton hump was detected (reflection fraction of R {approx_equal} 0.6), superposed on the primary X-ray continuum of {Lambda} {approx_equal} 1.75. Thanks to the good photon statistics and low background of the Suzaku data, we clearly confirm the spectral evolution of 3C 120, whereby the variability amplitude decreases with increasing energy. More strikingly, we discovered that the variability is caused by a steep power-law component of {Lambda} {approx_equal} 2.7, possibly related to the non-thermal jet emission. We discuss our findings in the context of similarities and differences between radio-loud/quiet objects.

  17. The High Energy view of the Broad Line Radio Galaxy 3C 111

    Ballo, L; Reeves, J N; Sambruna, R M; Tombesi, F

    2011-01-01

    We present the analysis of Suzaku and XMM-Newton observations of the broad-line radio galaxy (BLRG) 3C 111. Its high energy emission shows variability, a harder continuum with respect to the radio quiet AGN population, and weak reflection features. Suzaku found the source in a minimum flux level; a comparison with the XMM-Newton data implies an increase of a factor of 2.5 in the 0.5-10 keV flux, in the 6 months separating the two observations. The iron K complex is detected in both datasets, with rather low equivalent width(s). The intensity of the iron K complex does not respond to the change in continuum flux. An ultra-fast, high-ionization outflowing gas is clearly detected in the XIS data; the absorber is most likely unstable. Indeed, during the XMM-Newton observation, which was 6 months after, the absorber was not detected. No clear roll-over in the hard X-ray emission is detected, probably due to the emergence of the jet as a dominant component in the hard X-ray band, as suggested by the detection above...

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

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

    2009-05-19

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

  19. Cloning and analysis of a Trichinella pseudospiralis muscle larva secreted serine protease gene.

    Cwiklinski, Krystyna; Meskill, Diana; Robinson, Mark W; Pozio, Eduardo; Appleton, Judith A; Connolly, Bernadette

    2009-02-23

    Nematode parasites of the genus Trichinella are intracellular and distinct life cycle stages invade intestinal epithelial and skeletal muscle cells. Within the genus, Trichinella spiralis and Trichinella pseudospiralis exhibit species-specific differences with respect to host-parasite complex formation and host immune modulation. Parasite excretory-secretory (ES) proteins play important roles at the host-parasite interface and are thought to underpin these differences in biology. Serine proteases are among the most abundant group of T. spiralis ES proteins and multiple isoforms of the muscle larvae-specific TspSP-1 serine protease have been identified. Recently, a similar protein (TppSP-1) in T. pseudospiralis muscle larvae was identified. Here we report the cloning and characterisation of the full-length transcript of TppSP-1 and present comparative data between TspSP-1 and TppSP-1. PMID:19054614

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

    Murray, Andrew S.; Varela, Fausto A.

    2016-01-01

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

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

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

    2015-01-01

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

  2. Expression and characterization of Coprothermobacter proteolyticus alkaline serine protease

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

  3. Heterologous expression of Hordeum vulgare cysteine protease in yeast

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

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

  4. Effect of proteases on the β-thromboglobulin radioimmunoassay

    Rat peritoneal mast cells and mast cell granules were evaluated by radioimmunoassay for the presence of β-thromboglobulin and platelet factor 4. The initial assays indicated that a β-thromboglobulin cross reacting material was released from mast cells by compound 48/80 in a similar dose-dependent manner as histamine release. The material was also found to be associated with purified granules. However, the use of protease inhibitors in the buffers completely abolished the positive assays. Further evaluation of the effects of various proteases on the β-thromboglobulin assay indicated that elastase would also generate a false positive assay which could then be neutralized by the use of α1-antitrypsin as a protease inhibitor. There was no protease effect on the platelet factor 4 radioimmunoassay which always showed no detectable amounts with mast cells, granules or proteases. These results clearly indicate the artifactual positive assays which can arise when using certain radioimmunoassay tests in the presence of cell proteases. The use of protease inhibitors is a necessary control when applying a radioimmunoassay to a system with potentially active proteases. 24 references, 2 figures, 4 tables

  5. A NOVEL APPROACH TO REGULATE NITROGEN MINERALIZATION USING PROTEASE INHIBITORS

    Mineralization of organic N sources by extracellular proteases affects both the availability of inorganic N to plants and losses of N to the environment. We hypothesized that (i) application of purified protease inhibitors would slow down soil N mineralization, and (ii) elevated concentrations of pr...

  6. Isolation and characterization of proteases from Bacteroides melaninogenicus.

    Fujimura, S.; Nakamura, T.(International Center for Elementary Particle Physics and Department of Physics, The University of Tokyo, Tokyo, Japan)

    1981-01-01

    We isolated two types of intracellular proteases from a strain of Bacteroides melaninogenicus. These enzymes were extracted from cells by ultrasonic treatment and were partially purified. These two enzymes (proteases I and II) differed in molecular weight, heat stability, sensitivity to reducing agents, Km value, and optimum pH for activity.

  7. A Recombinant Adenovirus Expressing P12A and 3C Protein of the Type O Foot-and-Mouth Disease Virus Stimulates Systemic and Mucosal Immune Responses in Mice

    Yinli Xie; Peng Gao; Zhiyong Li

    2016-01-01

    Foot-and-mouth disease (FMD) is a highly contagious livestock disease of cloven-hoofed animals which causes severe economic losses. The replication-deficient, human adenovirus-vectored FMD vaccine has been proven effective against FMD. However, the role of T-cell-mediated antiviral responses and the mucosae-mediated antiviral responses induced by the adenovirus-vectored FMD vaccine was rarely examined. Here, the capsid protein precursor P1-2A and viral protease 3C of the type O FMDV were expr...

  8. A Recombinant Adenovirus Expressing P12A and 3C Protein of the Type O Foot-and-Mouth Disease Virus Stimulates Systemic and Mucosal Immune Responses in Mice.

    Xie, Yinli; Gao, Peng; Li, Zhiyong

    2016-01-01

    Foot-and-mouth disease (FMD) is a highly contagious livestock disease of cloven-hoofed animals which causes severe economic losses. The replication-deficient, human adenovirus-vectored FMD vaccine has been proven effective against FMD. However, the role of T-cell-mediated antiviral responses and the mucosae-mediated antiviral responses induced by the adenovirus-vectored FMD vaccine was rarely examined. Here, the capsid protein precursor P1-2A and viral protease 3C of the type O FMDV were expressed in replicative-deficient human adenovirus type 5 vector. BALB/c mice immunized intramuscularly and intraperitoneally with recombinant adenovirus rAdv-P12A3C elicited higher FMDV-specific IgG antibodies, IFN-γ, and IL-4 cytokines than those in mice immunized with inactivated FMDV vaccine. Moreover, BALB/c mice immunized with recombinant adenovirus rAdv-P12A3C by oral and intraocular-nasal immunization induced high FMDV-specific IgA antibodies. These results show that the recombinant adenovirus rAdv-P12A3C could resist FMDV comprehensively. This study highlights the potential of rAdv-P12A3C to serve as a type O FMDV vaccine. PMID:27478836

  9. A Recombinant Adenovirus Expressing P12A and 3C Protein of the Type O Foot-and-Mouth Disease Virus Stimulates Systemic and Mucosal Immune Responses in Mice

    Gao, Peng

    2016-01-01

    Foot-and-mouth disease (FMD) is a highly contagious livestock disease of cloven-hoofed animals which causes severe economic losses. The replication-deficient, human adenovirus-vectored FMD vaccine has been proven effective against FMD. However, the role of T-cell-mediated antiviral responses and the mucosae-mediated antiviral responses induced by the adenovirus-vectored FMD vaccine was rarely examined. Here, the capsid protein precursor P1-2A and viral protease 3C of the type O FMDV were expressed in replicative-deficient human adenovirus type 5 vector. BALB/c mice immunized intramuscularly and intraperitoneally with recombinant adenovirus rAdv-P12A3C elicited higher FMDV-specific IgG antibodies, IFN-γ, and IL-4 cytokines than those in mice immunized with inactivated FMDV vaccine. Moreover, BALB/c mice immunized with recombinant adenovirus rAdv-P12A3C by oral and intraocular-nasal immunization induced high FMDV-specific IgA antibodies. These results show that the recombinant adenovirus rAdv-P12A3C could resist FMDV comprehensively. This study highlights the potential of rAdv-P12A3C to serve as a type O FMDV vaccine. PMID:27478836

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

    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 (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. PMID:26999188

  11. CVD growth and properties of boron phosphide on 3C-SiC

    Padavala, Balabalaji; Frye, C. D.; Wang, Xuejing; Raghothamachar, Balaji; Edgar, J. H.

    2016-09-01

    Improving the crystalline quality of boron phosphide (BP) is essential for realizing its full potential in semiconductor device applications. In this study, 3C-SiC was tested as a substrate for BP epitaxy. BP films were grown on 3C-SiC(100)/Si, 3C-SiC(111)/Si, and 3C-SiC(111)/4H-SiC(0001) substrates in a horizontal chemical vapor deposition (CVD) system. Films were produced with good crystalline orientation and morphological features in the temperature range of 1000-1200 °C using a PH3+B2H6+H2 mixture. Rotational twinning was absent in the BP due to the crystal symmetry-matching with 3C-SiC. Confocal 3D Raman imaging of BP films revealed primarily uniform peak shift and peak widths across the scanned area, except at defects on the surface. Synchrotron white beam X-ray topography showed the epitaxial relationship between BP and 3C-SiC was (100) BP||(100) 3C-SiC and (111) BP||(111) 3C-SiC. Scanning electron microscopy, Raman spectroscopy and X-ray diffraction analysis indicated residual tensile strain in the films and improved crystalline quality at temperatures below 1200 °C. These results indicated that BP properties could be further enhanced by employing high quality bulk 3C-SiC or 3C-SiC epilayers on 4H-SiC substrates.

  12. Cathepsin Protease Inhibition Reduces Endometriosis Lesion Establishment.

    Porter, Kristi M; Wieser, Friedrich A; Wilder, Catera L; Sidell, Neil; Platt, Manu O

    2016-05-01

    Endometriosis is a gynecologic disease characterized by the ectopic presence of endometrial tissue on organs within the peritoneal cavity, causing debilitating abdominal pain and infertility. Current treatments alleviate moderate pain symptoms associated with the disorder but exhibit limited ability to prevent new or recurring lesion establishment and growth. Retrograde menstruation has been implicated for introducing endometrial tissue into the peritoneal cavity, but molecular mechanisms underlying attachment and invasion are not fully understood. We hypothesize that cysteine cathepsins, a group of powerful extracellular matrix proteases, facilitate endometrial tissue invasion and endometriosis lesion establishment in the peritoneal wall and inhibiting this activity would decrease endometriosis lesion implantation. To test this, we used an immunocompetent endometriosis mouse model and found that endometriotic lesions exhibited a greater than 5-fold increase in active cathepsins compared to tissue from peritoneal wall or eutopic endometrium, with cathepsins L and K specifically implicated. Human endometriosis lesions also exhibited greater cathepsin activity than adjacent peritoneum tissue, supporting the mouse results. Finally, we tested the hypothesis that inhibiting cathepsin activity could block endometriosis lesion attachment and implantation in vivo. Intraperitoneal injection of the broad cysteine cathepsin inhibitor, E-64, significantly reduced the number of attached endometriosis lesions in our murine model compared to vehicle-treated controls demonstrating that cathepsin proteases contribute to endometriosis lesion establishment, and their inhibition may provide a novel, nonhormonal therapy for endometriosis. PMID:26482207

  13. Purification and Characterization of An Alkaline Protease from Acetes chinensis

    XU Jiachao; LIU Xin; LI Zhaojie; XU Jie; XUE Changhu; GAO Xin

    2005-01-01

    An alkaline protease from Acetes chinensis was purified and characterized in this study. The steps of purification include ammonium sulfate precipitation, ion-exchange chromatography with Q-sepharose Fast Flow, gel filtration chromatography with S300 and the second ion-exchange chromatography with Q-sepharose Fast Flow. The protease was isolated and purified, which was present and active on protein substrates (azocasein and casein). The specific protease activity was 17.15folds and the recovery was 4.67. The molecular weight of the protease was estimated at 23.2 kD by SDS-PAGE. With azocasein as the susbstrate, the optimal temperature was 55 ℃ and the optimal pH value was 5.5. Ion Ca2+ could enhance the proteolytic activity of the protease, while Cu2+ , EDTA and PMSF could inhibit its activity.

  14. Alkaline Protease Production by a Strain of Marine Yeasts

    WANG Ping; CHI Zhenming; MA Chunling

    2006-01-01

    Yeast strain 10 with high yield of protease was isolated from sediments of saltern near Qingdao, China.The protease had the highest activity at pH 9.0 and 45 ℃.The optimal medium for the maximum alkaline protease production of strain 10 was 2.5 g soluble starch and 2.0 g NaNO3 in 100 mL seawater with initial pH6.0.The optimal cultivation conditions for the maximum protease production were temperature 24.5 ℃, aeration rate 8.0 L min -1 and agitation speed 150 r min-1.Under the optimal conditions, 623.1 Umg-1 protein of alkaline protease was reached in the culture within 30 h of fermentation.

  15. Comparative Study of Dermatophytic Fungi for Extra Cellular Proteases Efficacy

    Sanchita Chaturvedi

    2013-07-01

    Full Text Available Fungi are known to produce proteases of different kind. The dermatphytic fungal strains were isolated from human skin tissues for extra cellular proteases efficacy. The present study deals with purification, estimation and comparison of extracellular proteases from five fungal species. (Fusarium sp., Curvularia sp. , Fumigatus Sp. , Aspergillus Sp. and Mucor Sp.. All the five fungal strains showed good amount of extra cellular protease activity in terms of unit total protein content. By testing all the crude extracts for enzyme activity, Fusarium sp. was found to show the highest activity whereas Mucor sp. showed the lowest. The study supports the notion that fungi can be a good source of extracellular enzymes especially proteases.

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

    Polacek, Charlotta; Belsham, Graham; McInerney, Gerald

    2014-01-01

    have found that foot-and-mouth disease virus (FMDV) triggers SG formation early during infection in IBRS-2 cells. These SGs contain G3BP and TIA-1, but not dsRNA. However, the presence of the FMDV-induced SGs is transient due to the cleavage of G3BP by the viral L-protease (Lpro), which results...... in subsequent SG dispersal. Cells infected with an Lpro-deficient mutant FMDV are not subjected to G3BP cleavage and the SGs formed upon infection with this mutant maintain throughout the infection. In vitro studies using different variants of the Lpro show different G3BP cleavage efficiencies, suggesting...... a superior function of the full length Lpro for this substrate. Furthermore, the Lpro-directed G3BP cleavage is not dependent on virus replication, as investigated by transfecting FMDV RNAs lacking a functional 3D-polymerase. Finally, FMDV RNAs that contain Lpro, but lack the FMDV 3C-protease, also induce...

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

    Sowdhamini R

    2006-08-01

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

  18. The family of Deg/HtrA proteases in plants

    Schuhmann Holger

    2012-04-01

    Full Text Available Abstract Background The Deg/HtrA family of ATP-independent serine endopeptidases is present in nearly all organisms from bacteria to human and vascular plants. In recent years, multiple deg/htrA protease genes were identified in various plant genomes. During genome annotations most proteases were named according to the order of discovery, hence the same names were sometimes given to different types of Deg/HtrA enzymes in different plant species. This can easily lead to false inference of individual protease functions based solely on a shared name. Therefore, the existing names and classification of these proteolytic enzymes does not meet our current needs and a phylogeny-based standardized nomenclature is required. Results Using phylogenetic and domain arrangement analysis, we improved the nomenclature of the Deg/HtrA protease family, standardized protease names based on their well-established nomenclature in Arabidopsis thaliana, and clarified the evolutionary relationship between orthologous enzymes from various photosynthetic organisms across several divergent systematic groups, including dicots, a monocot, a moss and a green alga. Furthermore, we identified a “core set” of eight proteases shared by all organisms examined here that might provide all the proteolytic potential of Deg/HtrA proteases necessary for a hypothetical plant cell. Conclusions In our proposed nomenclature, the evolutionarily closest orthologs have the same protease name, simplifying scientific communication when comparing different plant species and allowing for more reliable inference of protease functions. Further, we proposed that the high number of Deg/HtrA proteases in plants is mainly due to gene duplications unique to the respective organism.

  19. Phase-Variable Expression of an Operon Encoding Extracellular Alkaline Protease, a Serine Protease Homolog, and Lipase in Pseudomonas brassicacearum

    Chabeaud, Philippe; de Groot, Arjan; Bitter, Wilbert; Tommassen, Jan; Heulin, Thierry; Achouak, Wafa

    2001-01-01

    The rhizobacterium Pseudomonas brassicacearum forms phenotypic variants which do not show extracellular protease and lipase activity. The operon encoding these enzymes, a serine protease homolog, and a type I secretion machinery was characterized. Transcriptional lacZ gene fusions revealed that the expression of the operon is under the control of phase variation.

  20. The exponential law for superluminal expansion of quasar 3C273

    The radioastronomical technique of VLBI achieves an angular resolution better than 1 marcs, equivalent to a physical resolution of the order of light-years. In even the most distant galaxies such objects can be easily detected. In four extreme cases, three quasars (3C273, 3C279 and 3C345) and one seyfert galaxy (3C120) have been seen to expand with an apparent speed more than twice the speed of light. The evidence for such 'superluminal' expansion has been based on rather limited data, and the interpretation has been uncertain; but the recent development of the VLBI technique of hybrid mapping allows us to map the radio brightness distribution to show the expansion directly and unambiguously. This article uses the data of the quasar 3C273 presented earlier and presents a formula for the observation of superluminal expansion velocity of quasar 3C273. (Auth.)

  1. X-Ray Photoelectron Spectrum Analysis of Yb3C60 Compound

    CAO Xue-Wei; SHAO Yue; WANG Yu-Fang; LAN Guo-Xiang

    2001-01-01

    The nominal composition of the Yb3 C60 compound is characterized by means of x-ray photoelectron spectroscopy.Evidence of the divalent state for the Yb cation in the as-grown crystalline Yb3C60 is obtained. After exposure to air, the Yb3C60 compound transforms to an amorphous phase and Yb2O3 compound, while the valence state of the Yb cations changes from divalent to trivalent.

  2. Effectiveness of Ritonavir-Boosted Protease Inhibitor Monotherapy in Clinical Practice Even with Previous Virological Failures to Protease Inhibitor-Based Regimens

    López-Cortés, Luis F.; Castaño, Manuel A.; López-Ruz, Miguel A.; Rios-Villegas, María J.; Hernández-Quero, José; Merino, Dolores; Jiménez-Aguilar, Patricia; Marquez-Solero, Manuel; Terrón-Pernía, Alberto; Tellez-Pérez, Francisco; Viciana, Pompeyo; Orihuela-Cañadas, Francisco; Palacios-Baena, Zaira; Vinuesa-Garcia, David; Fajardo-Pico, Jose M.; Romero-Palacios, Alberto; Ojeda-Burgos, Guillermo; Pasquau-Liaño, Juan

    2016-01-01

    Background and Objective Significant controversy still exists about ritonavir-boosted protease inhibitor monotherapy (mtPI/rtv) as a simplification strategy that is used up to now to treat patients that have not experienced previous virological failure (VF) while on protease inhibitor (PI) -based regimens. We have evaluated the effectiveness of two mtPI/rtv regimens in an actual clinical practice setting, including patients that had experienced previous VF with PI-based regimens. Methods This retrospective study analyzed 1060 HIV-infected patients with undetectable viremia that were switched to lopinavir/ritonavir or darunavir/ritonavir monotherapy. In cases in which the patient had previously experienced VF while on a PI-based regimen, the lack of major HIV protease resistance mutations to lopinavir or darunavir, respectively, was mandatory. The primary endpoint of this study was the percentage of participants with virological suppression after 96 weeks according to intention-to-treat analysis (non-complete/missing = failure). Results A total of 1060 patients were analyzed, including 205 with previous VF while on PI-based regimens, 90 of whom were on complex therapies due to extensive resistance. The rates of treatment effectiveness (intention-to-treat analysis) and virological efficacy (on-treatment analysis) at week 96 were 79.3% (CI95, 76.8−81.8) and 91.5% (CI95, 89.6–93.4), respectively. No relationships were found between VF and earlier VF while on PI-based regimens, the presence of major or minor protease resistance mutations, the previous time on viral suppression, CD4+ T-cell nadir, and HCV-coinfection. Genotypic resistance tests were available in 49 out of the 74 patients with VFs and only four patients presented new major protease resistance mutations. Conclusion Switching to mtPI/rtv achieves sustained virological control in most patients, even in those with previous VF on PI-based regimens as long as no major resistance mutations are present for

  3. Molecular Mechanisms of Viral and Host Cell Substrate Recognition by Hepatitis C Virus NS3/4A Protease

    Romano, Keith P.; Laine, Jennifer M.; Deveau, Laura M.; Cao, Hong; Massi, Francesca; Schiffer, Celia A. (UMASS, MED)

    2011-08-16

    Hepatitis C NS3/4A protease is a prime therapeutic target that is responsible for cleaving the viral polyprotein at junctions 3-4A, 4A4B, 4B5A, and 5A5B and two host cell adaptor proteins of the innate immune response, TRIF and MAVS. In this study, NS3/4A crystal structures of both host cell cleavage sites were determined and compared to the crystal structures of viral substrates. Two distinct protease conformations were observed and correlated with substrate specificity: (i) 3-4A, 4A4B, 5A5B, and MAVS, which are processed more efficiently by the protease, form extensive electrostatic networks when in complex with the protease, and (ii) TRIF and 4B5A, which contain polyproline motifs in their full-length sequences, do not form electrostatic networks in their crystal complexes. These findings provide mechanistic insights into NS3/4A substrate recognition, which may assist in a more rational approach to inhibitor design in the face of the rapid acquisition of resistance.

  4. 3C-SiC/ZnS heterostructured nanospheres with high photocatalytic activity and enhancement mechanism

    Zhang, J. [Key Laboratory of Modern Acoustics, MOE, Institute of Acoustics and Collaborative Innovation Center of Advanced Microstructures, National Laboratory of Solid State Microstructures, Nanjing University, Nanjing 210093 (China); Department of Physics, Huaiyin Institute of Technology, Huaian 223003 (China); Wu, X. L., E-mail: hkxlwu@nju.edu.cn, E-mail: paul.chu@cityu.edu.hk [Key Laboratory of Modern Acoustics, MOE, Institute of Acoustics and Collaborative Innovation Center of Advanced Microstructures, National Laboratory of Solid State Microstructures, Nanjing University, Nanjing 210093 (China); Department of Physics, NingBo University, NingBo 315001 (China); Liu, L. Z.; Yang, L.; Gan, Z. X. [Key Laboratory of Modern Acoustics, MOE, Institute of Acoustics and Collaborative Innovation Center of Advanced Microstructures, National Laboratory of Solid State Microstructures, Nanjing University, Nanjing 210093 (China); Chu, Paul K., E-mail: hkxlwu@nju.edu.cn, E-mail: paul.chu@cityu.edu.hk [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong (China)

    2015-03-15

    3C-SiC/n-type ZnS heterostructured nanospheres synthesized hydrothermally deliver enhanced photocatalytic performance under visible light excitation. The heterostructured catalysts consisting of 3C-SiC and ZnS nanocrystals with a mean size being less than 5 nm exhibit extended light absorption to the visible range. The proper band structure of the 3C-SiC and ZnS nanocrystals and intrinsic electric field induced by the heterojunction promote separation of photoexcited electrons and holes in the ZnS and 3C-SiC nanocrystals resulting in the increased photocatalytic efficiency. The associated mechanism is studied and proposed.

  5. 3C-SiC/ZnS heterostructured nanospheres with high photocatalytic activity and enhancement mechanism

    3C-SiC/n-type ZnS heterostructured nanospheres synthesized hydrothermally deliver enhanced photocatalytic performance under visible light excitation. The heterostructured catalysts consisting of 3C-SiC and ZnS nanocrystals with a mean size being less than 5 nm exhibit extended light absorption to the visible range. The proper band structure of the 3C-SiC and ZnS nanocrystals and intrinsic electric field induced by the heterojunction promote separation of photoexcited electrons and holes in the ZnS and 3C-SiC nanocrystals resulting in the increased photocatalytic efficiency. The associated mechanism is studied and proposed

  6. Simultaneous uncoupled expression and purification of the Dengue virus NS3 protease and NS2B co-factor domain.

    Shannon, A E; Chappell, K J; Stoermer, M J; Chow, S Y; Kok, W M; Fairlie, D P; Young, P R

    2016-03-01

    Dengue Virus (DENV) infection is responsible for the world's most significant insect-borne viral disease. Despite an increasing global impact, there are neither prophylactic nor therapeutic options available for the effective treatment of DENV infection. An attractive target for antiviral drugs is the virally encoded trypsin-like serine protease (NS3pro) and its associated cofactor (NS2B). The NS2B-NS3pro complex is responsible for cleaving the viral polyprotein into separate functional viral proteins, and is therefore essential for replication. Recombinant expression of an active NS2B-NS3 protease has primarily been based on constructs linking the C-terminus of the approximately 40 amino acid hydrophilic cofactor domain of NS2B to the N-terminus of NS3pro via a flexible glycine linker. The resulting complex can be expressed in high yield, is soluble and catalytically active and has been used for most in vitro screening, inhibitor, and X-ray crystallographic studies over the last 15 years. Despite extensive analysis, no inhibitor drug candidates have been identified yet. Moreover, the effect of the artificial linker introduced between the protease and its cofactor is unknown. Two alternate methods for bacterial expression of non-covalently linked, catalytically active, NS2B-NS3pro complex are described here along with a comparison of the kinetics of substrate proteolysis and binding affinities of substrate-based aldehyde inhibitors. Both expression methods produced high yields of soluble protein with improved substrate proteolysis kinetics and inhibitor binding compared to their glycine-linked equivalent. The non-covalent association between NS2B and NS3pro is predicted to be more relevant for examining inhibitors that target cofactor-protease interactions rather than the protease active site. Furthermore, these approaches offer alternative strategies for the high yield co-expression of other protein assemblies. PMID:26647367

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

    Catherine S. Adamson

    2012-01-01

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

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

    Ratia, Kiira; Pegan, Scott; Takayama, Jun; Sleeman, Katrina; Coughlin, Melissa; Baliji, Surendranath; Chaudhuri, Rima; Fu, Wentao; Prabhakar, Bellur S.; Johnson, Michael E.; Baker, Susan C.; Ghosh, Arun K.; Mesecar, Andrew D. (Loyola); (Purdue); (UIC)

    2008-10-27

    We report the discovery and optimization of a potent inhibitor against the papain-like protease (PLpro) from the coronavirus that causes severe acute respiratory syndrome (SARS-CoV). This unique protease is not only responsible for processing the viral polyprotein into its functional units but is also capable of cleaving ubiquitin and ISG15 conjugates and plays a significant role in helping SARS-CoV evade the human immune system. We screened a structurally diverse library of 50,080 compounds for inhibitors of PLpro and discovered a noncovalent lead inhibitor with an IC{sub 50} value of 20 {mu}M, which was improved to 600 nM via synthetic optimization. The resulting compound, GRL0617, inhibited SARS-CoV viral replication in Vero E6 cells with an EC{sub 50} of 15 {mu}M and had no associated cytotoxicity. The X-ray structure of PLpro in complex with GRL0617 indicates that the compound has a unique mode of inhibition whereby it binds within the S4-S3 subsites of the enzyme and induces a loop closure that shuts down catalysis at the active site. These findings provide proof-of-principle that PLpro is a viable target for development of antivirals directed against SARS-CoV, and that potent noncovalent cysteine protease inhibitors can be developed with specificity directed toward pathogenic deubiquitinating enzymes without inhibiting host DUBs.

  9. Discovery of Nanomolar Dengue and West Nile Virus Protease Inhibitors Containing a 4-Benzyloxyphenylglycine Residue.

    Behnam, Mira A M; Graf, Dominik; Bartenschlager, Ralf; Zlotos, Darius P; Klein, Christian D

    2015-12-10

    The dengue virus (DENV) and West Nile Virus (WNV) NS2B-NS3 proteases are attractive targets for the development of dual-acting therapeutics against these arboviral pathogens. We present the synthesis and extensive biological evaluation of inhibitors that contain benzyl ethers of 4-hydroxyphenylglycine as non-natural peptidic building blocks synthesized via a copper-complex intermediate. A three-step optimization strategy, beginning with fragment growth of the C-terminal 4-hydroxyphenylglycine to the benzyloxy ether, followed by C- and N-terminal optimization, and finally fragment merging generated compounds with in vitro affinities in the low nanomolar range. The most promising derivative reached Ki values of 12 nM at the DENV-2 and 39 nM at the WNV proteases. Several of the newly discovered protease inhibitors yielded a significant reduction of dengue and West Nile virus titers in cell-based assays of virus replication, with an EC50 value of 3.4 μM at DENV-2 and 15.5 μM at WNV for the most active analogue. PMID:26562070

  10. Cysteine Protease Inhibitors as Chemotherapy: Lessons from a Parasite Target

    Selzer, Paul M.; Pingel, Sabine; Hsieh, Ivy; Ugele, Bernhard; Chan, Victor J.; Engel, Juan C.; Bogyo, Matthew; Russell, David G.; Sakanari, Judy A.; McKerrow, James H.

    1999-09-01

    Papain family cysteine proteases are key factors in the pathogenesis of cancer invasion, arthritis, osteoporosis, and microbial infections. Targeting this enzyme family is therefore one strategy in the development of new chemotherapy for a number of diseases. Little is known, however, about the efficacy, selectivity, and safety of cysteine protease inhibitors in cell culture or in vivo. We now report that specific cysteine protease inhibitors kill Leishmania parasites in vitro, at concentrations that do not overtly affect mammalian host cells. Inhibition of Leishmania cysteine protease activity was accompanied by defects in the parasite's lysosome/endosome compartment resembling those seen in lysosomal storage diseases. Colocalization of anti-protease antibodies with biotinylated surface proteins and accumulation of undigested debris and protease in the flagellar pocket of treated parasites were consistent with a pathway of protease trafficking from flagellar pocket to the lysosome/endosome compartment. The inhibitors were sufficiently absorbed and stable in vivo to ameliorate the pathology associated with a mouse model of Leishmania infection.

  11. Cold-adapted proteases as an emerging class of therapeutics.

    Fornbacke, Marcus; Clarsund, Mats

    2013-06-01

    Proteases have been used in medicine for several decades and are an established and well tolerated class of therapeutic agent. These proteases were sourced from mammals or bacteria that exist or have adapted to moderate temperatures (mesophilic organisms); however, proteases derived from organisms from cold environments-cold-adapted or psychrophilic proteases-generally have high specific activity, low substrate affinity, and high catalytic rates at low and moderate temperatures. Made possible by greater flexibility, psychrophilic enzymes interact with and transform the substrate at lower energy costs. Cold-adapted proteases have been used in a wide range of applications, including industrial functions, textiles, cleaning/hygiene products, molecular biology, environmental bioremediations, consumer food products, cosmetics, and pharmaceutical production. In addition to these applications, they have also shown promise as therapeutic modalities for cosmeceutical applications (by reducing glabellar [frown] lines) and a number of disease conditions, including bacterial infections (by disrupting biofilms to prevent bacterial infection), topical wound management (when used as a debridement agent to remove necrotic tissue and fibrin clots), oral/dental health management (by removing plaque and preventing periodontal disease), and in viral infections (by reducing the infectivity of viruses, such as human rhinovirus 16 and herpes simplex virus). Psychrophilic proteases with greater activity and stability (than the original organism-derived variant) have been developed; this coupled with available manufacturing recombinant production techniques suggests that cold-adapted proteases have a promising future as a distinct therapeutic class with diverse clinical applications. PMID:25135820

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

    Koh-Stenta X

    2015-12-01

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

  13. Fluorometric CCHFV OTU protease assay with potent inhibitors.

    Kocabas, Fatih; Aslan, Galip S

    2015-10-01

    Crimean-Congo hemorrhagic fever virus (CCHFV) is a deadly virus that has been listed in the Category C as a potential bioterror agent. There are no specific therapies against CCHFV, which urges identification of potential therapeutic targets and development of CCHFV therapies. CCHFV OTU protease takes an important role in viral invasion through antagonizing NF-κB signaling. Inhibition of CCHFV OTU protease by small molecules warrants an exciting potential as antiviral therapeutics. Here we report the expression and purification of a C-His-tagged recombinant CCHFV OTU protease in E. coli BL21 (DE3) host strain. Activity of the refolded purified recombinant viral OTU protease has been validated with a UB-AMC fluorescent assay. In addition, we show a dose-dependent inhibition of the viral OTU protease by two small molecules. This study provides a reliable approach for recombinant expression and purification of CCHFV OTU protease, and demonstrates validation of OTU protease activity and its inhibition based on a UB-AMC florescent assay. PMID:26156848

  14. Exploring a new serine protease from Cucumis sativus L.

    Nafeesa, Zohara; Shivalingu, B R; Vivek, H K; Priya, B S; Swamy, S Nanjunda

    2015-03-01

    Coagulation is an important physiological process in hemostasis which is activated by sequential action of proteases. This study aims to understand the involvement of aqueous fruit extract of Cucumis sativus L. (AqFEC) European burp less variety in blood coagulation cascade. AqFEC hydrolyzed casein in a dose-dependent manner. The presence of protease activity was further confirmed by casein zymography which revealed the possible presence of two high molecular weight protease(s). The proteolytic activity was inhibited only by phenyl methyl sulphonyl fluoride suggesting the presence of serine protease(s). In a dose-dependent manner, AqFEC also hydrolysed Aα and Bβ subunits of fibrinogen, whereas it failed to degrade the γ subunit of fibrinogen even at a concentration as high as 100 μg and incubation time up to 4 h. AqFEC reduced the clotting time of citrated plasma by 87.65%. The protease and fibrinogenolytic activity of AqFEC suggests its possible role in stopping the bleeding and ensuing wound healing process. PMID:25577345

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

    E. V. Маtseliukh

    2015-04-01

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

  16. Laundry detergent compatibility of the alkaline protease from Bacillus cereus.

    Banik, Rathindra Mohan; Prakash, Monika

    2004-01-01

    The endogenous protease activity in various commercially available laundry detergents of international companies was studied. The maximum protease activity was found at 50 degrees C in pH range 10.5-11.0 in all the tested laundry detergents. The endogenous protease activity in the tested detergents retained up to 70% on incubation at 40 degrees C for 1 h, whereas less than 30% activity was only found on incubation at 50 degrees C for 1 h. The alkaline protease from an alkalophilic strain of Bacillus cereus was studied for its compatibility in commercial detergents. The cell free fermented broth from shake flask culture of the organism showed maximum activity at pH 10.5 and 50 degrees C. The protease from B. cereus showed much higher residual activity (more than 80%) on incubation with laundry detergents at 50 degrees C for 1 h or longer. The protease enzyme from B. cereus was found to be superior over the endogenous proteases present in the tested commercial laundry detergents in comparison to the enzyme stability during the washing at higher temperature, e.g., 40-50 degrees C. PMID:15293947

  17. A comparison of BCF-12 organic scintillators and Al2O3:C crystals for real-time medical dosimetry

    Beierholm, Anders Ravnsborg; Andersen, Claus Erik; Lindvold, Lars;

    2008-01-01

    Radioluminescence (RL) from aluminium oxide (Al2O3:C) crystals and organic scintillators such as the blue-emitting BCF-12 can be used for precise real-time dose rate measurements during radiation therapy of cancer patients. Attaching the dosimeters to thin light-guiding fiber cables enables in vivo...... can be circumvented for pulsed beams due to the long life-time of the main luminescence center. In contrast, chromatic removal seems to be the most effective method for organic scintillators, but is found to yield some experimental complexities. In this paper, we report on dose rate measurements using...

  18. Effect of psychosocial stress on FKBP5 and NR3C1 gene expression in healthy young men

    Nina Höhne; Hildegard Pfister; Tanja Brückl; Petra Zimmermann; Manfred Uhr; Florian Holsboer; Marcus Ising

    2012-01-01

    Stress diseases such as affective disorders are often characterized by a disturbed regulation of the hypothalamus-pituitary-adrenocortical (HPA) axis. This dysregulation can be explained by an impaired function of the receptors involved in the HPA-axis regulation, for example, the glucocorticoid receptors (GR). The regulation process of the HPA axis and the GR function are influenced by several genes, for instance by NR3C1 coding for the GR and also by FKBP5, a co-chaperone in the GR-complex....

  19. The Central Engine Structure of 3C120: Evidence for a Retrograde Black Hole or a Refilling Accretion Disk

    Cowperthwaite, Philip S.; Reynolds, Christopher S.

    2012-01-01

    The broad-line radio galaxy 3C120 is a powerful source of both X-ray and radio emission including superluminal jet outflows. We report on our reanalysis of 160 ks of Suzaku data taken in 2006, previously examined by Kataoka et al. (2007). Spectral fits to the XIS and HXD/PIN data over a range of 0.7-45 keV reveal a well-defined iron K line complex with a narrow Ka core and relativistically broadened features consistent with emission from the inner regions of the accretion disk. Furthermore, t...

  20. An outburst scenario for the X-ray spectral variability in 3C 111

    Tombesi, F; Reynolds, C S; Garcia, J; Lohfink, A

    2013-01-01

    We present a combined Suzaku and Swift BAT broad-band E=0.6-200keV spectral analysis of three 3C 111 observations obtained in 2010. The data are well described with an absorbed power-law continuum and a weak (R~0.2) cold reflection component from distant material. We constrain the continuum cutoff at E_c~150-200keV, which is in accordance with X-ray Comptonization corona models and supports claims that the jet emission is only dominant at much higher energies. Fe XXVI Ly\\alpha emission and absorption lines are also present in the first and second observations, respectively. The modelling and interpretation of the emission line is complex and we explore three possibilities. If originating from ionized disc reflection, this should be emitted at r_in> 50r_g or, in the lamp-post configuration, the illuminating source should be at a height of h> 30r_g over the black hole. Alternatively, the line could be modeled with a hot collisionally ionized plasma with temperature kT = 22.0^{+6.1}_{-3.2} keV or a photo-ionized...