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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

    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

  6. 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.

  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. [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

  7. 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

  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. 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...

  20. 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

  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. 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

  9. 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.

  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; 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.

  13. 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.

  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. 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...

  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. 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.

  12. 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.

  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. 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.

  19. 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)

  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. 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...

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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:...

  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. 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...

  16. 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...

  17. 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...

  18. 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...

  19. 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...

  20. 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...

  1. 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...

  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. 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.

  5. 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

  6. 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.

  7. 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...

  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. 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.

  12. 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...

  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. 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.

  15. 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.

  16. 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...

  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. 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

  2. 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.

  3. 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...

  4. 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...

  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. 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...

  8. 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.

  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. 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...

  4. 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...

  5. 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...

  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

    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.

  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

    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。