202 results on '"Robert N. Pike"'
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2. The X-ray Crystal Structure of Full-Length Human Plasminogen
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Ruby H.P. Law, Tom Caradoc-Davies, Nathan Cowieson, Anita J. Horvath, Adam J. Quek, Joanna Amarante Encarnacao, David Steer, Angus Cowan, Qingwei Zhang, Bernadine G.C. Lu, Robert N. Pike, A. Ian Smith, Paul B. Coughlin, and James C. Whisstock more...
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Biology (General) ,QH301-705.5 - Abstract
Plasminogen is the proenzyme precursor of the primary fibrinolytic protease plasmin. Circulating plasminogen, which comprises a Pan-apple (PAp) domain, five kringle domains (KR1-5), and a serine protease (SP) domain, adopts a closed, activation-resistant conformation. The kringle domains mediate interactions with fibrin clots and cell-surface receptors. These interactions trigger plasminogen to adopt an open form that can be cleaved and converted to plasmin by tissue-type and urokinase-type plasminogen activators. Here, the structure of closed plasminogen reveals that the PAp and SP domains, together with chloride ions, maintain the closed conformation through interactions with the kringle array. Differences in glycosylation alter the position of KR3, although in all structures the loop cleaved by plasminogen activators is inaccessible. The ligand-binding site of KR1 is exposed and likely governs proenzyme recruitment to targets. Furthermore, analysis of our structure suggests that KR5 peeling away from the PAp domain may initiate plasminogen conformational change. more...
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- 2012
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3. Twenty years of bioinformatics research for protease-specific substrate and cleavage site prediction: a comprehensive revisit and benchmarking of existing methods.
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Fuyi Li, Yanan Wang 0003, Chen Li 0021, Tatiana T. Marquez-Lago, André Leier, Neil D. Rawlings, Gholamreza Haffari, Jerico Nico De Leon Revote, Tatsuya Akutsu, Kuo-Chen Chou, Anthony W. Purcell, Robert N. Pike, Geoffrey I. Webb, Alexander Ian Smith, Trevor Lithgow, Roger J. Daly, James C. Whisstock, and Jiangning Song more...
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- 2019
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4. PROSPERous: high-throughput prediction of substrate cleavage sites for 90 proteases with improved accuracy.
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Jiangning Song, Fuyi Li, André Leier, Tatiana T. Marquez-Lago, Tatsuya Akutsu, Gholamreza Haffari, Kuo-Chen Chou, Geoffrey I. Webb, and Robert N. Pike
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- 2018
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5. Effect of a protease‐activated receptor‐2 antagonist ( <scp>GB88</scp> ) on inflammation‐related loss of alveolar bone in periodontal disease
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Nidhish Francis, Reza Sanaei, Babatunde A. Ayodele, Neil M. O'Brien‐Simpson, David P. Fairlie, Lakshmi C. Wijeyewickrema, Robert N. Pike, Eleanor Jean Mackie, and Charles Neil Pagel
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Periodontics - Published
- 2023
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6. PoPS: A Computational Tool for Modeling and Predicting Protease Specificity.
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Sarah E. Boyd, Maria J. García de la Banda, Robert N. Pike, James C. Whisstock, and George B. Rudy
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- 2004
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7. Bioinformatic Approaches for Predicting substrates of Proteases.
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Jiangning Song, Hao Tan, Sarah E. Boyd, Hong-Bin Shen, Khalid Mahmood 0001, Geoffrey I. Webb, Tatsuya Akutsu, James C. Whisstock, and Robert N. Pike
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- 2011
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8. Pops: a Computational Tool for Modeling and Predicting Protease Specificity.
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Sarah E. Boyd, Robert N. Pike, George B. Rudy, James C. Whisstock, and Maria J. García de la Banda
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- 2005
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9. Determination of the crystal structure and substrate specificity of ananain
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Tang Yongqing, Jing Pan, Tracey J. Brown, Robert N. Pike, Pascal G. Wilmann, Tracey Mynott, Lakshmi C. Wijeyewickrema, and Michael L. West
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Models, Molecular ,0301 basic medicine ,Binding Sites ,030102 biochemistry & molecular biology ,Bromelain (pharmacology) ,Plant Extracts ,Chemistry ,Stereochemistry ,Substrate (chemistry) ,General Medicine ,Tripeptide ,Ananas ,Bromelains ,Biochemistry ,Cysteine protease ,Substrate Specificity ,Cysteine Endopeptidases ,Kinetics ,03 medical and health sciences ,Residue (chemistry) ,030104 developmental biology ,Plant protein ,Stem bromelain ,Enzyme kinetics ,Plant Proteins - Abstract
Ananain (EC 3.4.22.31) accounts for less than 10% of the total enzyme in the crude pineapple stem extract known as bromelain, yet yields the majority of the proteolytic activity of bromelain. Despite a high degree of sequence identity between ananain and stem bromelain, the most abundant bromelain cysteine protease, ananain displays distinct chemical properties, substrate preference and inhibitory profile compared to stem bromelain. A tripeptidyl substrate library (REPLi) was used to further characterize the substrate specificity of ananain and identified an optimal substrate for cleavage by ananain. The optimal tripeptide, PLQ, yielded a high kcat/Km value of 1.7 x 106 M−1s−1, with cleavage confirmed to occur after the Gln residue. Crystal structures of unbound ananain and an inhibitory complex of ananain and E−64, solved at 1.73 and 1.98 A, respectively, revealed a geometrically flat and open S1 subsite for ananain. This subsite accommodates diverse P1 substrate residues, while a narrow and deep hydrophobic pocket-like S2 subsite would accommodate a non-polar P2 residue, such as the preferred Leu residue observed in the specificity studies. A further illustration of the atomic interactions between E−64 and ananain explains the high inhibitory efficiency of E−64 toward ananain. These data reveal the first in depth structural and functional data for ananain and provide a basis for further study of the natural properties of the enzyme. more...
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- 2019
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10. Twenty years of bioinformatics research for protease-specific substrate and cleavage site prediction: a comprehensive revisit and benchmarking of existing methods
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Roger J. Daly, Jerico Revote, Tatiana T. Marquez-Lago, Geoffrey I. Webb, Jiangning Song, James C. Whisstock, A. Ian Smith, Gholamreza Haffari, André Leier, Yanan Wang, Kuo-Chen Chou, Robert N. Pike, Chen Li, Neil D. Rawlings, Fuyi Li, Tatsuya Akutsu, Anthony W. Purcell, and Trevor Lithgow more...
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Computer science ,0206 medical engineering ,Review Article ,02 engineering and technology ,Bioinformatics ,Cleavage (embryo) ,Substrate Specificity ,Machine Learning ,03 medical and health sciences ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,business.industry ,Research ,Deep learning ,Computational Biology ,Robustness (evolution) ,Usability ,Benchmarking ,Ensemble learning ,Scalability ,Artificial intelligence ,business ,Algorithms ,020602 bioinformatics ,Peptide Hydrolases ,Information Systems ,Test data - Abstract
The roles of proteolytic cleavage have been intensively investigated and discussed during the past two decades. This irreversible chemical process has been frequently reported to influence a number of crucial biological processes (BPs), such as cell cycle, protein regulation and inflammation. A number of advanced studies have been published aiming at deciphering the mechanisms of proteolytic cleavage. Given its significance and the large number of functionally enriched substrates targeted by specific proteases, many computational approaches have been established for accurate prediction of protease-specific substrates and their cleavage sites. Consequently, there is an urgent need to systematically assess the state-of-the-art computational approaches for protease-specific cleavage site prediction to further advance the existing methodologies and to improve the prediction performance. With this goal in mind, in this article, we carefully evaluated a total of 19 computational methods (including 8 scoring function-based methods and 11 machine learning-based methods) in terms of their underlying algorithm, calculated features, performance evaluation and software usability. Then, extensive independent tests were performed to assess the robustness and scalability of the reviewed methods using our carefully prepared independent test data sets with 3641 cleavage sites (specific to 10 proteases). The comparative experimental results demonstrate that PROSPERous is the most accurate generic method for predicting eight protease-specific cleavage sites, while GPS-CCD and LabCaS outperformed other predictors for calpain-specific cleavage sites. Based on our review, we then outlined some potential ways to improve the prediction performance and ease the computational burden by applying ensemble learning, deep learning, positive unlabeled learning and parallel and distributed computing techniques. We anticipate that our study will serve as a practical and useful guide for interested readers to further advance next-generation bioinformatics tools for protease-specific cleavage site prediction. more...
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- 2018
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11. A T cell-specific knockout reveals an important role for protease-activated receptor 2 in lymphocyte development
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Robert N. Pike, Eleanor J. Mackie, Charles N. Pagel, Babatunde A. Ayodele, Nidhish Francis, and Alison L. Every
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0301 basic medicine ,T-Lymphocytes ,Lymphocyte ,T cell ,Cellular differentiation ,Apoptosis ,Thymus Gland ,Biology ,Lymphocyte Activation ,Biochemistry ,Mice ,03 medical and health sciences ,Interleukin 21 ,medicine ,Animals ,Receptor, PAR-2 ,Cytotoxic T cell ,Mice, Knockout ,Hyperplasia ,ZAP70 ,CD28 ,Cell Biology ,Molecular biology ,030104 developmental biology ,medicine.anatomical_structure ,Immunology ,Spleen ,CD8 - Abstract
Activation of protease-activated receptor-2 (PAR2) expressed by T cells has been linked to the bone loss associated with periodontitis. We generated PAR2 conditional-null mice and crossed these with mice expressing Cre recombinase under control of the Lck proximal promoter, to produce T cell-specific PAR2-null mice in order to further study the cellular mechanism involved in periodontitis. Here we report that efficient deletion of PAR2 in thymocytes isolated from T cell-specific PAR2-null mice resulted in thymic and splenic hypoplasia and a reduction in the cells of the cortex and a loss of distinction between the cortex and the medulla of the thymus. FACS analysis confirmed significant reductions in CD4 and CD8 double negative (DN3 and DN4) sub-populations, as well as double positive and single positive T cells, in T cell-specific PAR2-null mice compared to Cre expressing PAR2 wild-type mice. The proportion of annexin V positive and propidium iodide negative cells was increased in CD4 and CD8 double negative, double positive and single positive T cells from T cell-specific PAR2-null mice. No change in the proportion of Ki67 positive cells was observed in sections of thymus from T cell-specific PAR2-null mice, suggesting that the depletion of T cell sub-populations in T cell-specific PAR2-null mice resulted from increased apoptosis rather than reduced proliferation. Together, these results demonstrate that PAR2 plays an important and previously unrecognised anti-apoptotic role in T cell development and suggest that the PAR2 conditional-null mouse will be an important resource for determining tissue and cell specific effects of PAR2. more...
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- 2017
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12. Recruitment of Human C1 Esterase Inhibitor Controls Complement Activation on Blood Stage Plasmodium falciparum Merozoites
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Alisee Huglo, Alan F. Cowman, Wai-Hong Tham, Lakshmi C. Wijeyewickrema, Alexander T. Kennedy, Clara S. Lin, and Robert N. Pike
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0301 basic medicine ,biology ,CD46 ,Immunology ,Complement receptor ,Virology ,Cell biology ,Complement system ,C1-inhibitor ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Factor H ,biology.protein ,Immunology and Allergy ,Merozoite surface protein ,MASP2 ,030215 immunology ,Complement C1s - Abstract
The complement system is a front-line defense system that opsonizes and lyses invading pathogens. To survive, microbes exposed to serum must evade the complement response. To achieve this, many pathogens recruit soluble human complement regulators to their surfaces and hijack their regulatory function for protection from complement activation. C1 esterase inhibitor (C1-INH) is a soluble regulator of complement activation that negatively regulates the classical and lectin pathways of complement to protect human tissue from aberrant activation. In this article, we show that Plasmodium falciparum merozoites, the invasive form of blood stage malaria parasites, actively recruit C1-INH to their surfaces when exposed to human serum. We identified PfMSP3.1, a member of the merozoite surface protein 3 family of merozoite surface proteins, as the direct interaction partner. When bound to the merozoite surface, C1-INH retains its ability to complex with and inhibit C1s, MASP1, and MASP2, the activating proteases of the complement cascade. P. falciparum merozoites that lack PfMSP3.1 showed a marked reduction in C1-INH recruitment and increased C3b deposition on their surfaces. However, these ΔPfMSP3.1 merozoites exhibit enhanced invasion of RBCs in the presence of active complement. This study characterizes an immune-evasion strategy used by malaria parasites and highlights the complex relationship between merozoites and the complement system. more...
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- 2017
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13. Mapping the binding site of C1-inhibitor for polyanion cofactors
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James C. Whisstock, Jing Pan, Lilian Hor, Robert N. Pike, and Lakshmi C. Wijeyewickrema
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0301 basic medicine ,Polymers ,Immunology ,Serpin ,Cofactor ,C1-inhibitor ,03 medical and health sciences ,Classical complement pathway ,0302 clinical medicine ,Polyphosphates ,medicine ,Binding site ,Anion binding ,Molecular Biology ,Cofactor binding ,Binding Sites ,biology ,Complement C1s ,Chemistry ,Heparin ,Polyelectrolytes ,Recombinant Proteins ,030104 developmental biology ,Mechanism of action ,Mutation ,Biophysics ,biology.protein ,Mutagenesis, Site-Directed ,medicine.symptom ,Complement C1 Inhibitor Protein ,030215 immunology ,Protein Binding - Abstract
The serpin, C1-inhibitor (also known as SERPING1), plays a vital anti-inflammatory role in the body by controlling pro-inflammatory pathways such as complement and coagulation. The inhibitor's action is enhanced in the presence of polyanionic cofactors, such as heparin and polyphosphate, by increasing the rate of association with key enzymes such as C1s of the classical pathway of complement. The cofactor binding site of the serpin has never been mapped. Here we show that residues Lys284, Lys285 and Arg287 of C1-inhibitor play key roles in binding heparin and delivering the rate enhancement seen in the presence of polyanions and thus most likely represent the key cofactor binding residues for the serpin. We also show that simultaneous binding of the anion binding site of C1s by the polyanion is required to deliver the rate enhancement. Finally, we have shown that it is unlikely that the two positively charged zones of C1-inhibitor and C1s interact in the encounter complex between molecules as ablation of the charged zones did not in itself deliver a rate enhancement as might have been expected if the zones interacted. These insights provide crucial information as to the mechanism of action of this key serpin in the presence and absence of cofactor molecules. more...
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- 2019
14. Polyphosphate is a novel cofactor for regulation of complement by a serpin, C1 inhibitor
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Piyushkumar R. Kapopara, Hugh Kim, Toshikazu Shiba, Robert N. Pike, Richard J. Travers, Renee C. Duncan, Stephanie A. Smith, Victor Lei, Lakshmi C. Wijeyewickrema, James H. Morrissey, Edward M. Conway, Lilian Hor, and Emilie Lameignere more...
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0301 basic medicine ,Proteases ,Complement component 2 ,Immunology ,Cell Biology ,Hematology ,Biology ,Serpin ,Biochemistry ,Thrombosis and Hemostasis ,C1-inhibitor ,Complement system ,03 medical and health sciences ,Classical complement pathway ,Polyps ,030104 developmental biology ,biology.protein ,Humans ,Platelet activation ,Complement C1s - Abstract
The complement system plays a key role in innate immunity, inflammation, and coagulation. The system is delicately balanced by negative regulatory mechanisms that modulate the host response to pathogen invasion and injury. The serpin, C1-esterase inhibitor (C1-INH), is the only known plasma inhibitor of C1s, the initiating serine protease of the classical pathway of complement. Like other serpin-protease partners, C1-INH interaction with C1s is accelerated by polyanions such as heparin. Polyphosphate (polyP) is a naturally occurring polyanion with effects on coagulation and complement. We recently found that polyP binds to C1-INH, prompting us to consider whether polyP acts as a cofactor for C1-INH interactions with its target proteases. We show that polyP dampens C1s-mediated activation of the classical pathway in a polymer length- and concentration-dependent manner by accelerating C1-INH neutralization of C1s cleavage of C4 and C2. PolyP significantly increases the rate of interaction between C1s and C1-INH, to an extent comparable to heparin, with an exosite on the serine protease domain of the enzyme playing a major role in this interaction. In a serum-based cell culture system, polyP significantly suppressed C4d deposition on endothelial cells, generated via the classical and lectin pathways. Moreover, polyP and C1-INH colocalize in activated platelets, suggesting that their interactions are physiologically relevant. In summary, like heparin, polyP is a naturally occurring cofactor for the C1s:C1-INH interaction and thus an important regulator of complement activation. The findings may provide novel insights into mechanisms underlying inflammatory diseases and the development of new therapies. more...
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- 2016
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15. Protein unfolding is essential for cleavage within the α-helix of a model protein substrate by the serine protease, thrombin
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Martin J. Scanlon, Stephen J. Headey, Stephen P. Bottomley, Amy L. Robertson, Robert N. Pike, Lakshmi C. Wijeyewickrema, and Natasha M. Ng
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Models, Molecular ,0301 basic medicine ,Proteases ,Magnetic Resonance Spectroscopy ,medicine.medical_treatment ,Molecular Sequence Data ,Biochemistry ,Protein Structure, Secondary ,Substrate Specificity ,alpha-2-Macroglobulin ,03 medical and health sciences ,Thrombin ,Bacterial Proteins ,medicine ,Native state ,Humans ,Amino Acid Sequence ,Protein secondary structure ,Protein Unfolding ,Serine protease ,Protease ,Sequence Homology, Amino Acid ,030102 biochemistry & molecular biology ,biology ,Active site ,General Medicine ,Kinetics ,030104 developmental biology ,Mutation ,Proteolysis ,biology.protein ,Electrophoresis, Polyacrylamide Gel ,Serine Proteases ,medicine.drug - Abstract
Proteolysis has a critical role in transmitting information within a biological system and therefore an important element of biology is to determine the subset of proteins amenable to proteolysis. Until recently, it has been thought that proteases cleave native protein substrates only within solvent exposed loops, but recent evidence indicates that cleavage sites located within α-helices can also be cleaved by proteases, despite the conformation of this secondary structure being generally incompatible with binding into an active site of a protease. In this study, we address the mechanism by which a serine endopeptidase, thrombin, recognizes and cleaves a target sequence located within an α-helix. Thrombin was able to cleave a model substrate, protein G, within its α-helix when a suitable cleavage sequence for the enzyme was introduced into this region. However, structural data for the complex revealed that thrombin was not perturbing the structure of the α-helix, thus it was not destabilizing the helix in order to allow it to fit within its active site. This indicated that thrombin was only cleaving within the α-helix when it was in an unfolded state. In support of this, the introduction of destabilizing mutations within the protein increased the efficiency of cleavage by the enzyme. Our data suggest that a folded α-helix cannot be proteolytically cleaved by thrombin, but the species targeted are the unfolded conformations of the native state ensemble. more...
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- 2016
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16. PROSPERous: high-throughput prediction of substrate cleavage sites for 90 proteases with improved accuracy
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Gholamreza Haffari, Fuyi Li, Kuo-Chen Chou, André Leier, Jiangning Song, Robert N. Pike, Tatiana T. Marquez-Lago, Geoffrey I. Webb, and Tatsuya Akutsu
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0301 basic medicine ,Statistics and Probability ,Proteases ,Sequence analysis ,Computer science ,Proteolysis ,In silico ,0206 medical engineering ,02 engineering and technology ,Computational biology ,Cleavage (embryo) ,Biochemistry ,Substrate Specificity ,03 medical and health sciences ,Protein methods ,Sequence Analysis, Protein ,medicine ,Computer Simulation ,Molecular Biology ,Peptide sequence ,Caspase ,chemistry.chemical_classification ,medicine.diagnostic_test ,biology ,Substrate (chemistry) ,Computational Biology ,Applications Notes ,Computer Science Applications ,Data Accuracy ,Computational Mathematics ,030104 developmental biology ,Peptide backbone ,Enzyme ,Computational Theory and Mathematics ,chemistry ,Peptide Hydrolases ,biology.protein ,020602 bioinformatics ,Software - Abstract
Summary Proteases are enzymes that specifically cleave the peptide backbone of their target proteins. As an important type of irreversible post-translational modification, protein cleavage underlies many key physiological processes. When dysregulated, proteases’ actions are associated with numerous diseases. Many proteases are highly specific, cleaving only those target substrates that present certain particular amino acid sequence patterns. Therefore, tools that successfully identify potential target substrates for proteases may also identify previously unknown, physiologically relevant cleavage sites, thus providing insights into biological processes and guiding hypothesis-driven experiments aimed at verifying protease–substrate interaction. In this work, we present PROSPERous, a tool for rapid in silico prediction of protease-specific cleavage sites in substrate sequences. Our tool is based on logistic regression models and uses different scoring functions and their pairwise combinations to subsequently predict potential cleavage sites. PROSPERous represents a state-of-the-art tool that enables fast, accurate and high-throughput prediction of substrate cleavage sites for 90 proteases. Availability and implementation http://prosperous.erc.monash.edu/ Supplementary information Supplementary data are available at Bioinformatics online. more...
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- 2017
17. Molecular basis for the folding of β-helical autotransporter passenger domains
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Jing Zhang, Robert N. Pike, Lakshmi C. Wijeyewickrema, Mark A. Schembri, Denisse L. Leyton, Alvin W. Lo, Matthew D. Johnson, Ian R. Henderson, Gerard H. M. Huysmans, and Xiaojun Yuan
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0301 basic medicine ,Models, Molecular ,Protein Conformation, alpha-Helical ,Protein Folding ,Type V Secretion Systems ,Science ,030106 microbiology ,Bacterial Toxins ,Genetic Vectors ,Beta sheet ,General Physics and Astronomy ,Gene Expression ,Context (language use) ,Computational biology ,Crystallography, X-Ray ,General Biochemistry, Genetics and Molecular Biology ,Article ,Domain (software engineering) ,Substrate Specificity ,03 medical and health sciences ,Enterotoxins ,Protein structure ,Escherichia coli ,Protein Interaction Domains and Motifs ,Amino Acid Sequence ,Cloning, Molecular ,lcsh:Science ,Physics ,Multidisciplinary ,Binding Sites ,Sequence Homology, Amino Acid ,Escherichia coli Proteins ,Serine Endopeptidases ,General Chemistry ,Recombinant Proteins ,Folding (chemistry) ,Kinetics ,Protein Transport ,030104 developmental biology ,Thermodynamics ,Protein folding ,lcsh:Q ,Protein Conformation, beta-Strand ,Bacterial outer membrane ,Sequence Alignment ,Autotransporters ,Protein Binding - Abstract
Bacterial autotransporters comprise a C-terminal β-barrel domain, which must be correctly folded and inserted into the outer membrane to facilitate translocation of the N-terminal passenger domain to the cell exterior. Once at the surface, the passenger domains of most autotransporters are folded into an elongated β-helix. In a cellular context, key molecules catalyze the assembly of the autotransporter β-barrel domain. However, how the passenger domain folds into its functional form is poorly understood. Here we use mutational analysis on the autotransporter Pet to show that the β-hairpin structure of the fifth extracellular loop of the β-barrel domain has a crucial role for passenger domain folding into a β-helix. Bioinformatics and structural analyses, and mutagenesis of a homologous autotransporter, suggest that this function is conserved among autotransporter proteins with β-helical passenger domains. We propose that the autotransporter β-barrel domain is a folding vector that nucleates folding of the passenger domain., Autotransporter passenger domains are presented on or released from the bacterial surface upon translocation through an outer membrane β-barrel anchor. Here the authors study the two E. coli autotransporters Pet and EspP and propose that the β-barrel anchor acts as a vector to nucleate the folding of the passenger domain. more...
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- 2017
18. The Structural Basis for Complement Inhibition by Gigastasin, a Protease Inhibitor from the Giant Amazon Leech
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Emilie Lameignere, Sheareen Tan, Edward M. Conway, Anna M. Blom, James C. Whisstock, Lilian Hor, Robert N. Pike, Xuyu Liu, Frida C. Mohlin, Lakshmi C. Wijeyewickrema, Siew Siew Pang, and Richard J. Payne more...
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0301 basic medicine ,Serine Proteinase Inhibitors ,Immunology ,Biology ,03 medical and health sciences ,Classical complement pathway ,Complement C1 ,Catalytic Domain ,Leeches ,medicine ,Immunology and Allergy ,Animals ,Humans ,Complement Pathway, Classical ,Complement Activation ,Cells, Cultured ,Mannan-binding lectin ,Complement component 2 ,Complement Pathway, Mannose-Binding Lectin ,Protease inhibitor (biology) ,Recombinant Proteins ,Complement system ,030104 developmental biology ,Complement Inactivating Agents ,Biochemistry ,Lectin pathway ,Factor H ,Mannose-Binding Protein-Associated Serine Proteases ,Endothelium, Vascular ,Complement membrane attack complex ,Peptides ,medicine.drug - Abstract
Complement is crucial to the immune response, but dysregulation of the system causes inflammatory disease. Complement is activated by three pathways: classical, lectin, and alternative. The classical and lectin pathways are initiated by the C1r/C1s (classical) and MASP-1/MASP-2 (lectin) proteases. Given the role of complement in disease, there is a requirement for inhibitors to control the initiating proteases. In this article, we show that a novel inhibitor, gigastasin, from the giant Amazon leech, potently inhibits C1s and MASP-2, whereas it is also a good inhibitor of MASP-1. Gigastasin is a poor inhibitor of C1r. The inhibitor blocks the active sites of C1s and MASP-2, as well as the anion-binding exosites of the enzymes via sulfotyrosine residues. Complement deposition assays revealed that gigastasin is an effective inhibitor of complement activation in vivo, especially for activation via the lectin pathway. These data suggest that the cumulative effects of inhibiting both MASP-2 and MASP-1 have a greater effect on the lectin pathway than the more potent inhibition of only C1s of the classical pathway. more...
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- 2017
19. A molecular basis for the association of the HLA-DRB1 locus, citrullination, and rheumatoid arthritis
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Lakshmi C. Wijeyewickrema, Hendrik J. Nel, Jamie Rossjohn, Khai Lee Loh, S.W. Scally, Jurgen van Heemst, Hugh H. Reid, James McCluskey, Ranjeny Thomas, Sidonia B G Eckle, Anthony W. Purcell, Nadine L. Dudek, Nicole L. La Gruta, René E. M. Toes, Jan Petersen, Robert N. Pike, and Soi Cheng Law more...
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musculoskeletal diseases ,CD4-Positive T-Lymphocytes ,Models, Molecular ,T cell ,Immunology ,HLA-DR beta-Chains ,Molecular Sequence Data ,Vimentin ,Mice, Transgenic ,Human leukocyte antigen ,Major histocompatibility complex ,Autoantigens ,Epitope ,Article ,Arthritis, Rheumatoid ,chemistry.chemical_compound ,Epitopes ,Mice ,immune system diseases ,Citrulline ,medicine ,HLA-DR4 Antigen ,Immunology and Allergy ,Animals ,Humans ,Aggrecans ,Amino Acid Sequence ,skin and connective tissue diseases ,HLA-DRB1 ,Genetic Association Studies ,Antigen Presentation ,Polymorphism, Genetic ,biology ,Citrullination ,Molecular biology ,medicine.anatomical_structure ,chemistry ,biology.protein ,HLA-DRB1 Chains - Abstract
A comprehensive structural portrait of the association between citrullination, the HLA-DRB1 locus, and T cell autoreactivity in rheumatoid arthritis., Rheumatoid arthritis (RA) is strongly associated with the human leukocyte antigen (HLA)-DRB1 locus that possesses the shared susceptibility epitope (SE) and the citrullination of self-antigens. We show how citrullinated aggrecan and vimentin epitopes bind to HLA-DRB1*04:01/04. Citrulline was accommodated within the electropositive P4 pocket of HLA-DRB1*04:01/04, whereas the electronegative P4 pocket of the RA-resistant HLA-DRB1*04:02 allomorph interacted with arginine or citrulline-containing epitopes. Peptide elution studies revealed P4 arginine–containing peptides from HLA-DRB1*04:02, but not from HLA-DRB1*04:01/04. Citrullination altered protease susceptibility of vimentin, thereby generating self-epitopes that are presented to T cells in HLA-DRB1*04:01+ individuals. Using HLA-II tetramers, we observed citrullinated vimentin- and aggrecan-specific CD4+ T cells in the peripheral blood of HLA-DRB1*04:01+ RA-affected and healthy individuals. In RA patients, autoreactive T cell numbers correlated with disease activity and were deficient in regulatory T cells relative to healthy individuals. These findings reshape our understanding of the association between citrullination, the HLA-DRB1 locus, and T cell autoreactivity in RA. more...
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- 2013
20. Molecular Mechanisms Underlying the Actions of the Complement System
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Robert N. Pike and Lakshmi C. Wijeyewickrema
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Complement component 2 ,Biochemistry ,biology ,Factor H ,Lectin pathway ,biology.protein ,Complement receptor ,Complement membrane attack complex ,CFHR5 ,Complement system ,Complement control protein - Abstract
Complement is crucial to the immune system. It is initiated by the classical, lectin, and alternative pathways. Each pathway involves initial binding to a foreign surface, triggering the activation of proteases. These cleave and combine with further molecules of the system to effect amplification and assembly of structures such as the membrane-attack complex, which kills the targeted pathogen or altered cell. Targeted cells are also tagged for removal by immune system cells attracted to the site of complement activation by peptides released. The system is highly regulated, but is also involved in many inflammatory diseases. more...
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- 2016
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21. Identification of a Catalytic Exosite for Complement Component C4 on the Serine Protease Domain of C1s
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James A. Huntington, Robert N. Pike, Lakshmi C. Wijeyewickrema, Anna M. Blom, Theresa H.T. Coetzer, Richard J. Payne, Renee C. Duncan, Frida C. Mohlin, and Deni Taleski
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medicine.medical_treatment ,Molecular Sequence Data ,Immunology ,Complement factor I ,Biology ,Classical complement pathway ,Catalytic Domain ,medicine ,Humans ,Immunology and Allergy ,Amino Acid Sequence ,Complement Pathway, Classical ,Complement Activation ,Serine protease ,Protease ,Complement C1s ,Complement component 2 ,Complement component 7 ,Complement C4 ,Peptide Fragments ,Complement system ,Biochemistry ,biology.protein ,Binding Sites, Antibody ,Serine Proteases ,MASP1 - Abstract
The classical pathway of complement is crucial to the immune system, but it also contributes to inflammatory diseases when dysregulated. Binding of the C1 complex to ligands activates the pathway by inducing autoactivation of associated C1r, after which C1r activates C1s. C1s cleaves complement component C4 and then C2 to cause full activation of the system. The interaction between C1s and C4 involves active site and exosite-mediated events, but the molecular details are unknown. In this study, we identified four positively charged amino acids on the serine protease domain that appear to form a catalytic exosite that is required for efficient cleavage of C4. These residues are coincidentally involved in coordinating a sulfate ion in the crystal structure of the protease. Together with other evidence, this pointed to the involvement of sulfate ions in the interaction with the C4 substrate, and we showed that the protease interacts with a peptide from C4 containing three sulfotyrosine residues. We present a molecular model for the interaction between C1s and C4 that provides support for the above data and poses questions for future research into this aspect of complement activation. more...
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- 2012
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22. Analysis of Fasciola cathepsin L5 by S2 subsite substitutions and determination of the P1–P4 specificity reveals an unusual preference
- Author
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Youngchool Choe, Luke J. Norbury, Terence W. Spithill, Andrew Hung, Robert N. Pike, Charles S. Craik, Simone A. Beckham, Peter M. Smooker, and John V. Fecondo
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Proteases ,E-64 ,Biology ,Biochemistry ,Protein Structure, Secondary ,Substrate Specificity ,Serine ,Cathepsin L ,chemistry.chemical_compound ,Cathepsin O ,parasitic diseases ,Animals ,Cathepsin ,chemistry.chemical_classification ,Fasciola ,General Medicine ,Fasciola hepatica ,biology.organism_classification ,Cathepsins ,Molecular biology ,Amino acid ,chemistry ,Mutagenesis, Site-Directed ,biology.protein ,Protein Binding - Abstract
Fasciola parasites (liver flukes) express numerous cathepsin L proteases that are believed to be involved in important functions related to host invasion and parasite survival. These proteases are evolutionarily divided into clades that are proposed to reflect their substrate specificity, most noticeably through the S 2 subsite. Single amino acid substitutions to residues lining this site, including amino acid residue 69 (aa69; mature cathepsin L5 numbering) can have profound influences on subsite architecture and influence enzyme specificity. Variations at aa69 among known Fasciola cathepsin L proteases include leucine, tyrosine, tryptophan, phenylalanine and glycine. Other amino acids (cysteine, serine) might have been expected at this site due to codon usage as cathepsin L isoenzymes evolved, but C69 and S69 have not been observed. The introduction of L69C and L69S substitutions into FhCatL5 resulted in low overall activity indicating their expression provides no functional advantage, thus explaining the absence of such variants in Fasciola . An FhCatL5 L69F variant showed an increase in the ability to cleave substrates with P 2 proline, indicating F69 variants expressed by the fluke would likely have this ability. An FhCatL2 Y69L variant showed a decreased acceptance of P 2 proline, further highlighting the importance of Y69 for FhCatL2 P 2 proline acceptance. Finally, the P 1 –P 4 specificity of Fasciola cathepsin L5 was determined and, unexpectedly, aspartic acid was shown to be well accepted at P 2, which is unique amongst Fasciola cathepsins examined to date. more...
- Published
- 2012
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23. The X-ray Crystal Structure of Full-Length Human Plasminogen
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Qingwei Zhang, Bernadine G.C. Lu, Nathan Cowieson, Angus Cowan, Tom T. Caradoc-Davies, Robert N. Pike, Adam J. Quek, Joanna Amarante Encarnacao, Anita J Horvath, Ruby H. P. Law, James C. Whisstock, A. Ian Smith, David Steer, and Paul Bernard Coughlin more...
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Models, Molecular ,Conformational change ,Glycosylation ,Plasmin ,medicine.medical_treatment ,Crystallography, X-Ray ,Protein Structure, Secondary ,General Biochemistry, Genetics and Molecular Biology ,Kringle domain ,chemistry.chemical_compound ,Protein structure ,Kringles ,medicine ,Humans ,lcsh:QH301-705.5 ,Urokinase ,Serine protease ,Protease ,biology ,Plasminogen ,Enzyme Activation ,lcsh:Biology (General) ,chemistry ,Biochemistry ,Mutation ,Biophysics ,biology.protein ,Protein Binding ,medicine.drug - Abstract
SummaryPlasminogen is the proenzyme precursor of the primary fibrinolytic protease plasmin. Circulating plasminogen, which comprises a Pan-apple (PAp) domain, five kringle domains (KR1-5), and a serine protease (SP) domain, adopts a closed, activation-resistant conformation. The kringle domains mediate interactions with fibrin clots and cell-surface receptors. These interactions trigger plasminogen to adopt an open form that can be cleaved and converted to plasmin by tissue-type and urokinase-type plasminogen activators. Here, the structure of closed plasminogen reveals that the PAp and SP domains, together with chloride ions, maintain the closed conformation through interactions with the kringle array. Differences in glycosylation alter the position of KR3, although in all structures the loop cleaved by plasminogen activators is inaccessible. The ligand-binding site of KR1 is exposed and likely governs proenzyme recruitment to targets. Furthermore, analysis of our structure suggests that KR5 peeling away from the PAp domain may initiate plasminogen conformational change. more...
- Published
- 2012
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24. S1 Pocket of a Bacterially Derived Subtilisin-like Protease Underpins Effective Tissue Destruction
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Wilson Wong, Shane Reeve, Robert N. Pike, Julian I. Rood, Corrine Joy Porter, Cyril F. Reboul, Lakshmi C. Wijeyewickrema, David Steer, James C. Whisstock, A. Ian Smith, and Ruth M. Kennan
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Models, Molecular ,endocrine system ,Proteases ,animal structures ,Phenylalanine ,Virulence ,Dichelobacter nodosus ,Crystallography, X-Ray ,Models, Biological ,Biochemistry ,Bacterial Proteins ,Leucine ,Hydrolase ,Animals ,Humans ,Amino Acids ,Foot Rot ,Molecular Biology ,chemistry.chemical_classification ,Serine protease ,Sheep ,biology ,fungi ,Serine Endopeptidases ,Subtilisin ,Congo Red ,Cell Biology ,biology.organism_classification ,Endopeptidase ,Fibronectins ,Protein Structure, Tertiary ,enzymes and coenzymes (carbohydrates) ,Kinetics ,Enzyme ,chemistry ,biological sciences ,Protein Structure and Folding ,biology.protein ,Crystallization - Abstract
The ovine footrot pathogen, Dichelobacter nodosus, secretes three subtilisin-like proteases that play an important role in the pathogenesis of footrot through their ability to mediate tissue destruction. Virulent and benign strains of D. nodosus secrete the basic proteases BprV and BprB, respectively, with the catalytic domain of these enzymes having 96% sequence identity. At present, it is not known how sequence variation between these two putative virulence factors influences their respective biological activity. We have determined the high resolution crystal structures of BprV and BprB. These data reveal that that the S1 pocket of BprV is more hydrophobic but smaller than that of BprB. We show that BprV is more effective than BprB in degrading extracellular matrix components of the host tissue. Mutation of two residues around the S1 pocket of BprB to the equivalent residues in BprV dramatically enhanced its proteolytic activity against elastin substrates. Application of a novel approach for profiling substrate specificity, the Rapid Endopeptidase Profiling Library (REPLi) method, revealed that both enzymes prefer cleaving after hydrophobic residues (and in particular P1 leucine) but that BprV has more restricted primary substrate specificity than BprB. Furthermore, for P1 Leu-containing substrates we found that BprV is a significantly more efficient enzyme than BprB. Collectively, these data illuminate how subtle changes in D. nodosus proteases may significantly influence tissue destruction as part of the ovine footrot pathogenesis process. more...
- Published
- 2011
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25. BIOINFORMATIC APPROACHES FOR PREDICTING SUBSTRATES OF PROTEASES
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Hao Tan, Robert N. Pike, James C. Whisstock, Geoffrey I. Webb, Hong-Bin Shen, Tatsuya Akutsu, Khalid Mahmood, Sarah Elizabeth Boyd, and Jiangning Song
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Proteases ,medicine.medical_treatment ,Molecular Sequence Data ,Context (language use) ,Biology ,Biochemistry ,Protein Structure, Secondary ,Substrate Specificity ,Protein structure ,Artificial Intelligence ,Catalytic Domain ,medicine ,Animals ,Humans ,Amino Acid Sequence ,Databases, Protein ,Molecular Biology ,Protease ,Computational Biology ,Proteins ,Substrate (biology) ,Computer Science Applications ,Solvents ,Identification (biology) ,Target protein ,Algorithms ,Function (biology) ,Peptide Hydrolases - Abstract
Proteases have central roles in "life and death" processes due to their important ability to catalytically hydrolyze protein substrates, usually altering the function and/or activity of the target in the process. Knowledge of the substrate specificity of a protease should, in theory, dramatically improve the ability to predict target protein substrates. However, experimental identification and characterization of protease substrates is often difficult and time-consuming. Thus solving the "substrate identification" problem is fundamental to both understanding protease biology and the development of therapeutics that target specific protease-regulated pathways. In this context, bioinformatic prediction of protease substrates may provide useful and experimentally testable information about novel potential cleavage sites in candidate substrates. In this article, we provide an overview of recent advances in developing bioinformatic approaches for predicting protease substrate cleavage sites and identifying novel putative substrates. We discuss the advantages and drawbacks of the current methods and detail how more accurate models can be built by deriving multiple sequence and structural features of substrates. We also provide some suggestions about how future studies might further improve the accuracy of protease substrate specificity prediction. more...
- Published
- 2011
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26. Mapping the cofactor binding site for polyanions of the serpin, C1-inhibitor
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Robert N. Pike, Lilian Hor, Lakshmi C. Wijeyewickrema, Raj Pannu, and Jing Pan
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Cofactor binding ,Biochemistry ,biology ,Chemistry ,Immunology ,biology.protein ,Serpin ,Molecular Biology ,C1-inhibitor - Published
- 2018
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27. Cathepsin B proteases of flukes: the key to facilitating parasite control?
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Terence W Spithill, Peter M. Smooker, Robert N. Pike, and Rama Jayaraj
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Cathepsin ,Proteases ,Fasciola ,biology ,Virulence Factors ,Vaccination ,Trematode Infections ,biology.organism_classification ,Virology ,Cathepsin B ,Infectious Diseases ,RNA interference ,parasitic diseases ,Animals ,Humans ,Parasite hosting ,Fasciola hepatica ,Parasitology ,Trematoda ,Peptide Hydrolases - Abstract
Cysteine proteases are important virulence factors for parasites. This review will focus on the cathepsin B proteases of trematodes (also known as flukes) which are abundant in juvenile and immature flukes. Recent research, primarily in Fasciola, using inhibitors, RNA interference (RNAi) and vaccination studies indicates that cathepsin Bs play a key role in the biology of trematodes. As these proteases are largely expressed by infective parasite stages, their inactivation by chemotherapy or vaccination will greatly reduce the damage wrought by flukes as they invade host tissues. This validates cathepsin Bs as key strategic targets for fluke control. more...
- Published
- 2010
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28. Keratinocyte-specific ablation of protease-activated receptor 2 prevents gingival inflammation and bone loss in a mouse model of periodontal disease
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Eleanor J. Mackie, Neil M O'Brien-Simpson, Charles N. Pagel, Robert N. Pike, Nidhish Francis, Walter Birchmeier, and Babatunde A. Ayodele
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Keratinocytes ,0301 basic medicine ,Pathology ,medicine.medical_specialty ,Proteases ,Immunology ,Alveolar Bone Loss ,Inflammation ,Microbiology ,03 medical and health sciences ,Gingivitis ,Virology ,Bacteroidaceae Infections ,medicine ,Animals ,Receptor, PAR-2 ,Porphyromonas gingivalis ,Periodontal Diseases ,Protease-activated receptor 2 ,Dental alveolus ,Periodontitis ,CD11b Antigen ,030102 biochemistry & molecular biology ,biology ,Interleukin-6 ,biology.organism_classification ,medicine.disease ,Chronic periodontitis ,Mice, Mutant Strains ,Disease Models, Animal ,030104 developmental biology ,Gene Expression Regulation ,medicine.symptom - Abstract
Chronic periodontitis is characterised by gingival inflammation and alveolar bone loss. A major aetiological agent is Porphyromonas gingivalis, which secretes proteases that activate protease-activated receptor 2 (PAR2 ). PAR2 expressed on oral keratinocytes is activated by proteases released by P. gingivalis, inducing secretion of interleukin 6 (IL-6), and global knockout of PAR2 prevents bone loss and inflammation in a periodontal disease model in mice. To test the hypothesis that PAR2 expressed on gingival keratinocytes is required for periodontal disease pathology, keratinocyte-specific PAR2 -null mice were generated using K14-Cre targeted deletion of the PAR2 gene (F2rl1). These mice were subjected to a model of periodontitis involving placement of a ligature around a tooth, combined with P. gingivalis infection ("Lig + Inf"). The intervention caused a significant 44% decrease in alveolar bone volume (assessed by microcomputed tomography) in wildtype (K14-Cre:F2rl1wt/wt ), but not littermate keratinocyte-specific PAR2 -null (K14-Cre:F2rl1fl/fl ) mice. Keratinocyte-specific ablation of PAR2 prevented the significant Lig + Inf-induced increase (2.8-fold) in the number of osteoclasts in alveolar bone and the significant up-regulation (2.4-4-fold) of the inflammatory markers IL-6, IL-1β, interferon-γ, myeloperoxidase, and CD11b in gingival tissue. These data suggest that PAR2 expressed on oral epithelial cells is a critical regulator of periodontitis-induced bone loss and will help in designing novel therapies with which to treat the disease. more...
- Published
- 2018
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29. Synthesis of 'Difficult' Fluorescence Quenched Substrates of Granzyme C
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Richard A. Hughes, Phillip I. Bird, Simon J. Mountford, Matthew Mangan, Robert N. Pike, Dion Kaiserman, Susan E. Northfield, Kade D. Roberts, and Philip E. Thompson
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chemistry.chemical_classification ,Pseudoproline ,Stereochemistry ,Substrate (chemistry) ,Bioengineering ,Sequence (biology) ,Peptide ,Cleavage (embryo) ,Biochemistry ,Fluorescence ,Combinatorial chemistry ,Analytical Chemistry ,Amino acid ,chemistry.chemical_compound ,chemistry ,Drug Discovery ,PEG ratio ,Molecular Medicine - Abstract
The synthesis of fluorescence quenched peptide substrates of granzyme C is presented. These peptides which incorporate some unusual amino acids and have “difficult sequence” elements, in some cases could not be prepared by standard Fmoc-based SPPS. Application of three different contemporary strategies, namely the use of pseudoproline dipeptides, PEG-based solid supports and the application of microwave heating were able to provide for successful synthesis of our desired substrate peptides. more...
- Published
- 2010
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30. Fluorinated β²- and β³-Amino Acids: Synthesis and Inhibition of α-Chymotrypsin
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Robert N. Pike, Victoria Peddie, Peter J. Duggan, Andrew D. Abell, Karen Maree Bromfield, and Markus Pietsch
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chemistry.chemical_classification ,Chymotrypsin ,biology ,Stereochemistry ,Chemistry ,Organic Chemistry ,biology.protein ,Catalysis ,Amino acid - Abstract
Victoria Peddie, Markus Pietsch, Karen M. Bromfield, Robert N. Pike, Peter J. Duggan, Andrew D. Abell
- Published
- 2010
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31. Vector-based RNA interference of cathepsin B1 in Schistosoma mansoni
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Bernd H. Kalinna, Paul J. Brindley, Robert N. Pike, Elissaveta B Tchoubrieva, and Poh Chee Ong
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Pharmacology ,biology ,Genetic Vectors ,Proteolytic enzymes ,Schistosoma mansoni ,Cell Biology ,biology.organism_classification ,Virology ,Schistosomiasis mansoni ,Virus ,Cathepsin B ,Viral vector ,Cellular and Molecular Neuroscience ,RNA silencing ,Retrovirus ,Parasitic Sensitivity Tests ,RNA interference ,parasitic diseases ,Animals ,Molecular Medicine ,RNA Interference ,Molecular Biology - Abstract
In helminth parasites, proteolytic enzymes have been implicated in facilitating host invasion, moulting, feeding, and evasion of the host immune response. These key functions render them potential targets for anti-parasite chemotherapy and immunotherapy. Schistosomes feed on host blood and the digested haemoglobin is their major source of amino acids. Haemoglobin digestion is essential for parasite development, growth, and reproduction. We recently reported the use of pseudotyped Moloney murine leukaemia virus to accomplish transformation of Schistosoma mansoni. Here, we report the design of a viral vector expressing a dsRNA hairpin to silence expression of the schistosome cathepsin B1 (SmCB1) gene. We observed 80% reduction in transcript level 72 h after virus exposure and complete silencing of enzyme activity in transduced worms. This is the first report using this technology in any helminth parasite. It will facilitate the evaluation of potential drug targets and biochemical pathways for novel interventions in schistosomes. more...
- Published
- 2010
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32. The effects of exosite occupancy on the substrate specificity of thrombin
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Philip E. Thompson, Phillip I. Bird, Antony Yaron Matthews, Dion Kaiserman, Noelene Sheila Quinsey, Lakshmi C. Wijeyewickrema, Natasha May-Yoke Ng, and Robert N. Pike
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Thrombomodulin ,medicine.medical_treatment ,Biophysics ,Peptide ,Ligands ,Biochemistry ,Substrate Specificity ,Thrombin ,Peptide Library ,Catalytic Domain ,medicine ,Humans ,Molecular Biology ,chemistry.chemical_classification ,Protease ,biology ,Heparin ,Chemistry ,Substrate (chemistry) ,Active site ,Hirudins ,Ligand (biochemistry) ,Peptide Fragments ,Enzyme ,biology.protein ,sense organs ,Protein Binding ,medicine.drug - Abstract
Thrombin (EC 3.4.4.13) has two exosites that mediate interactions between the enzyme and its substrates and cofactors. The binding of ligands to the exosites alters the functions of the protease, for example, when the cofactor thrombomodulin binds to both exosites I and II, it converts the enzyme from a procoagulant to an anticoagulant factor. It is unknown whether ligand binding to a thrombin exosite will alter the substrate specificity of the enzyme and thus contribute to the changed substrate repertoire of the enzyme upon engagement with cofactors. We first examined whether binding of ligands to exosites I and II altered the activity of the enzyme against fluorogenic peptide substrates. The efficiency of cleavage of substrates by thrombin did change when thrombomodulin or hirugen was present, indicating that exosite I occupancy changed the active site of the protease. The presence of heparin did not change the activity of the enzyme, indicating that exosite II occupancy had little effect on active site function. Investigation of the effects of exosite I occupancy by hirugen on thrombin specificity using phage display substrate libraries revealed that the ligand only changed the specificity of the enzyme to a small degree. Occupancy of both exosites by thrombomodulin induced greater changes to the specificity of the enzyme, with the prime side showing broader changes in amino acid frequencies. Thus, exosite I ligands do affect the activity and specificity of thrombin, but not greatly enough to explain the altered substrate profile of the enzyme when complexed with thrombomodulin. more...
- Published
- 2009
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33. A major cathepsin B protease from the liver fluke Fasciola hepatica has atypical active site features and a potential role in the digestive tract of newly excysted juvenile parasites
- Author
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Terence W Spithill, Peter M. Smooker, Deanne L.V. Greenwood, Ruby H. P. Law, Carolyn I Phillips, Robert N. Pike, Nirma Samarawickrema, Lakshmi C. Wijeyewickrema, Boris Turk, David Piedrafita, Noelene Sheila Quinsey, Theresa H.T. Coetzer, James A. Irving, Matthew Bogyo, Steven H. L. Verhelst, and Simone A. Beckham more...
- Subjects
Cysteine Proteinase Inhibitors ,Biochemistry ,Article ,Cathepsin B ,Substrate Specificity ,Enzyme activator ,Catalytic Domain ,Animals ,Humans ,Fasciola hepatica ,Parasites ,Cathepsin ,Life Cycle Stages ,Exopeptidase activity ,Sheep ,biology ,Cell Biology ,Exopeptidase ,biology.organism_classification ,Cathepsins ,Cystatins ,Cysteine protease ,Enzyme Activation ,Gastrointestinal Tract ,Kinetics ,Protein Transport ,Structural Homology, Protein ,Molecular Probes ,biology.protein ,Cystatin - Abstract
The newly excysted juvenile (NEJ) stage of the Fasciola hepatica lifecycle occurs just prior to invasion into the wall of the gut of the host, rendering it an important target for drug development. The cathepsin B enzymes from NEJ flukes have recently been demonstrated to be crucial to invasion and migration by the parasite. Here we characterize one of the cathepsin B enzymes (recombinant FhcatB1) from NEJ flukes. FhcatB1 has biochemical properties distinct from mammalian cathepsin B enzymes, with an atypical preference for Ile over Leu or Arg residues at the P(2) substrate position and an inability to act as an exopeptidase. FhcatB1 was active across a broad pH range (optimal activity at pH 5.5-7.0) and resistant to inhibition by cystatin family inhibitors from sheep and humans, suggesting that this enzyme would be able to function in extracellular environments in its mammalian hosts. It appears, however, that the FhcatB1 protease functions largely as a digestive enzyme in the gut of the parasite, due to the localization of a specific, fluorescently labeled inhibitor with an Ile at the P(2) position. Molecular modelling and dynamics were used to predict the basis for the unusual substrate specificity: a P(2) Ile residue positions the substrate optimally for interaction with catalytic residues of the enzyme, and the enzyme lacks an occluding loop His residue crucial for exopeptidase activity. The unique features of the enzyme, particularly with regard to its specificity and likely importance to a vital stage of the parasite's life cycle, make it an excellent target for therapeutic inhibitors or vaccination. more...
- Published
- 2009
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34. Structural Mechanisms of Inactivation in Scabies Mite Serine Protease Paralogues
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James C. Whisstock, David J. Kemp, James A. Irving, Charlene Willis, Sundy N.Y. Yang, Simone L. Reynolds, Katja Fischer, Tanya Ann Bashtannyk-Puhalovich, Robert N. Pike, Ashley M. Buckle, Christopher G. Langendorf, Simone A. Beckham, Ruby H. P. Law, and Sheena McGowan more...
- Subjects
Models, Molecular ,Proteases ,Protein Conformation ,medicine.medical_treatment ,Sarcoptes scabiei ,Crystallography, X-Ray ,Microbiology ,Serine ,Peptide Library ,Structural Biology ,Catalytic Domain ,Catalytic triad ,medicine ,Mite ,Animals ,Molecular Biology ,Phylogeny ,Serine protease ,Protease ,biology ,Serine Endopeptidases ,biology.organism_classification ,Enzyme Activation ,Mutation ,biology.protein ,Antibody - Abstract
The scabies mite (Sarcoptes scabiei) is a parasite responsible for major morbidity in disadvantaged communities and immuno-compromised patients worldwide. In addition to the physical discomfort caused by the disease, scabies infestations facilitate infection by Streptococcal species via skin lesions, resulting in a high prevalence of rheumatic fever/heart disease in affected communities. The scabies mite produces 33 proteins that are closely related to those in the dust mite group 3 allergen and belong to the S1-like protease family (chymotrypsin-like). However, all but one of these molecules contain mutations in the conserved active-site catalytic triad that are predicted to render them catalytically inactive. These molecules are thus termed scabies mite inactivated protease paralogues (SMIPPs). The precise function of SMIPPs is unclear; however, it has been suggested that these proteins might function by binding and protecting target substrates from cleavage by host immune proteases, thus preventing the host from mounting an effective immune challenge. In order to begin to understand the structural basis for SMIPP function, we solved the crystal structures of SMIPP-S-I1 and SMIPP-S-D1 at 1.85 A and 2.0 A resolution, respectively. Both structures adopt the characteristic serine protease fold, albeit with large structural variations over much of the molecule. In both structures, mutations in the catalytic triad together with occlusion of the S1 subsite by a conserved Tyr200 residue is predicted to block substrate ingress. Accordingly, we show that both proteases lack catalytic function. Attempts to restore function (via site-directed mutagenesis of catalytic residues as well as Tyr200) were unsuccessful. Taken together, these data suggest that SMIPPs have lost the ability to bind substrates in a classical "canonical" fashion, and instead have evolved alternative functions in the lifecycle of the scabies mite. more...
- Published
- 2009
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35. Gingipain enzymes fromPorphyromonas gingivalispreferentially bind immobilized extracellular proteins: a mechanism favouring colonization?
- Author
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Rebecca E. Fitzpatrick, Robert N. Pike, Noelene Sheila Quinsey, James Travis, Aneta Sroka, Jan Potempa, and Adrian Dale McAlister
- Subjects
medicine.medical_treatment ,Fimbria ,matrix proteins ,Enzyme-Linked Immunosorbent Assay ,Plasma protein binding ,Article ,Bacterial Adhesion ,Microbiology ,Fimbriae Proteins ,medicine ,Extracellular ,Humans ,Vitronectin ,Adhesins, Bacterial ,Porphyromonas gingivalis ,Extracellular Matrix Proteins ,Protease ,biology ,Fibrinogen ,biochemical phenomena, metabolism, and nutrition ,biology.organism_classification ,Fibronectins ,Gingipain ,Bacterial adhesin ,Cysteine Endopeptidases ,adhesion ,stomatognathic diseases ,Immobilized Proteins ,Biochemistry ,Gingipain Cysteine Endopeptidases ,Periodontics ,gingipains ,Protein Binding - Abstract
Background and Objective: Porphyromonas gingivalis, an anaerobic bacterium associated with adult periodontal disease, employs a number of pathogenic mechanisms, including protease/adhesin complexes (gingipains), fimbriae and hemagglutinins, to maintain attachment within colonized hosts. Here we examined the binding of gingipains and whole, live P. gingivalis cells to immobilized extracellular matrix proteins in the presence of soluble forms of the same proteins, to investigate whether this may constitute a colonization mechanism in the oral environment. Material and Methods: Binding of purified gingipain molecules and whole bacterial cells to immobilized matrix proteins was examined in the presence and absence of soluble competitors using enzyme-linked immunosorbent assays. Results: Purified gingipains or whole, live bacteria preferentially bound immobilized forms of matrix proteins, even in the presence of soluble forms of the same proteins. Fimbriae appeared to be redundant for adhesion to immobilized proteins in the presence of the gingipains, indicating that the protease/adhesins and hemagglutinins may be more important for adhesion under these conditions. Conclusion: The data presented here provide evidence for a model of adhesion for P. gingivalis within the fluid environment of the oral cavity, where preferential binding of matrix-located proteins over soluble forms facilitates colonization of the host. more...
- Published
- 2009
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36. Subsite cooperativity in protease specificity
- Author
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Natasha M. Ng, Robert N. Pike, and Sarah Elizabeth Boyd
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chemistry.chemical_classification ,Proteases ,Protease ,biology ,Cell growth ,medicine.medical_treatment ,Clinical Biochemistry ,Active site ,Cooperativity ,Cleavage (embryo) ,Biochemistry ,Substrate Specificity ,Kinetics ,Enzyme ,chemistry ,Catalytic Domain ,biology.protein ,medicine ,Animals ,Humans ,Substrate specificity ,Molecular Biology ,Peptide Hydrolases - Abstract
Proteases play vital roles in a range of biological processes, such as cell cycle, cell growth and differentiation, apoptosis, haemostasis and signalling. Fundamental to our knowledge of protease action is an understanding of how the active site operates; this has been examined through extensive studies of the substrate specificity of the enzymes. Kinetic and structural analyses have shown that the binding of a particular substrate residue at a protease subsite can have either a positive or negative influence on the binding of particular residues at other subsites. This phenomenon has been termed subsite cooperativity and has been observed in a wide range of proteases, often between non-adjacent subsites. This review aims to highlight studies where subsite cooperativity has been observed, experimental techniques used in the past and potential methods that can be employed to comprehensively examine this phenomenon. Further understanding of how the protease active site recognises and chooses its substrates for cleavage will have a significant impact on the development of pharmaceuticals that target these enzymes. more...
- Published
- 2009
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37. The role of strand 1 of the C β-sheet in the structure and function of α1-antitrypsin
- Author
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Stephen P. Bottomley, Robert N. Pike, Isobel D Lawrenson, Deborah J. Tew, Weiwen Dai, and James C. Whisstock
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Models, Molecular ,Proteases ,animal structures ,Protein Conformation ,Mutant ,Serpin ,Biology ,medicine.disease_cause ,Biochemistry ,Antithrombins ,Protein Structure, Secondary ,Article ,Protein structure ,medicine ,Molecular Biology ,Reactive center ,Serpins ,Mutation ,Circular Dichroism ,Temperature ,Thrombin ,Alanine scanning ,Protease inhibitor (biology) ,Cell biology ,carbohydrates (lipids) ,alpha 1-Antitrypsin ,embryonic structures ,Mutagenesis, Site-Directed ,Electrophoresis, Polyacrylamide Gel ,Protein Binding ,medicine.drug - Abstract
Serpins inhibit cognate serine proteases involved in a number of important processes including blood coagulation and inflammation. Consequently, loss of serpin function or stability results in a number of disease states. Many of the naturally occurring mutations leading to disease are located within strand 1 of the C beta-sheet of the serpin. To ascertain the structural and functional importance of each residue in this strand, which constitutes the so-called distal hinge of the reactive center loop of the serpin, an alanine scanning study was carried out on recombinant alpha(1)-antitrypsin Pittsburgh mutant (P1 = Arg). Mutation of the P10' position had no effect on its inhibitory properties towards thrombin. Mutations to residues P7' and P9' caused these serpins to have an increased tendency to act as substrates rather than inhibitors, while mutations at P6' and P8' positions caused the serpin to behave almost entirely as a substrate. Mutations at the P6' and P8' residues of the C beta-sheet, which are buried in the hydrophobic core in the native structure, caused the serpin to become highly unstable and polymerize much more readily. Thus, P6' and P8' mutants of alpha(1)-antitrypsin had melting temperatures 14 degrees lower than wild-type alpha(1)-antitrypsin. These results indicate the importance of maintaining the anchoring of the distal hinge to both the inhibitory mechanism and stability of serpins, the inhibitory mechanism being particularly sensitive to any perturbations in this region. The results of this study allow more informed analysis of the effects of mutations found at these positions in disease-associated serpin variants. more...
- Published
- 2009
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38. High Molecular Weight Gingipains from Porphyromonas gingivalis Induce Cytokine Responses from Human Macrophage-Like Cells via a Nonproteolytic Mechanism
- Author
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Eleanor J. Mackie, Rebecca E. Fitzpatrick, Jan Potempa, Charles N. Pagel, Andrea Aprico, Robert N. Pike, Lakshmi C. Wijeyewickrema, and David M. Wong
- Subjects
Proteases ,biology ,medicine.medical_treatment ,periodontal disease ,protease ,biology.organism_classification ,Article ,cytokines ,macrophages ,Microbiology ,Bacterial adhesin ,stomatognathic diseases ,Cytokine ,Granulocyte macrophage colony-stimulating factor ,stomatognathic system ,Downregulation and upregulation ,medicine ,Immunology and Allergy ,Macrophage ,gingipains ,Receptor ,Porphyromonas gingivalis ,medicine.drug - Abstract
Periodontal disease is an oral inflammatory disease affecting the supporting structures of teeth. Porphyromonas gingivalis, a major pathogenic agent for the disease, expresses a number of virulence factors, including cysteine proteases called the gingipains. The arginine- and lysine-specific gingipains, HRgpA and Kgp, respectively, are expressed as high molecular weight forms containing both catalytic and adhesin subunits. We examined the expression pattern of cytokines and their receptors in differentiated macrophages following exposure to active and inactive forms of the gingipains, using a cDNA array, quantitative PCR and ELISA analysis. Amongst other pro-inflammatory cytokines, results from the cDNA array suggested that interleukin-1β, granulocyte-macrophage colony stimulatory factor and interferon-γ were upregulated after exposure of the macrophages to the gingipains. Quantitative PCR analysis substantiated these observations and indicated that active or inactive forms of the high molecular weight gingipains were able to upregulate expression of transcripts for these cytokines. The strongly enhanced production of interleukin-1β and granulocyte-macrophage colony stimulatory factor by differentiated macrophages in response to active or inactive forms of the high molecular weight gingipains was confirmed at the protein level by ELISA analysis. The results indicate that the adhesin subunits of the gingipains mediate strong upregulation of the expression of pro-inflammatory cytokines in macrophages. more...
- Published
- 2008
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39. The initiating proteases of the complement system: Controlling the cleavage
- Author
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Renee C. Duncan, Robert N. Pike, and Lakshmi C. Wijeyewickrema
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Proteases ,Complement C1s ,Complement component 2 ,Complement C1r ,General Medicine ,Complement receptor ,Biology ,Biochemistry ,Complement system ,Classical complement pathway ,Mannose-Binding Protein-Associated Serine Proteases ,Lectin pathway ,Alternative complement pathway ,Animals ,Humans ,Complement membrane attack complex ,Complement Activation - Abstract
The complement system is a vital component of the host immune system, but when dysregulated, can also cause disease. The system is activated by three pathways: classical, lectin and alternative. The initiating proteases of the classical and lectin pathways have similar domain structure and employ similar mechanisms of activation. The C1r, C1s and MASP-2 proteases have the most defined roles in the activation of the system. This review focuses on the mechanisms whereby their interaction with substrates and inhibitors is regulated. more...
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- 2008
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40. Structural basis for substrate specificity of Helicobacter pylori M17 aminopeptidase
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Joyanta K. Modak, Robert N. Pike, Marcin Drag, Anna Roujeinikova, Lakshmi C. Wijeyewickrema, and Wioletta Rut
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0301 basic medicine ,chemistry.chemical_classification ,030102 biochemistry & molecular biology ,Helicobacter pylori ,Stereochemistry ,Catabolism ,Substrate (chemistry) ,Peptide ,General Medicine ,Biology ,Biochemistry ,Aminopeptidase ,Aminopeptidases ,Amino acid ,Substrate Specificity ,03 medical and health sciences ,Residue (chemistry) ,030104 developmental biology ,Enzyme ,chemistry ,Leucine ,Hydrolase ,Humans ,Protein Binding - Abstract
The M17 aminopeptidase from the carcinogenic gastric bacterium Helicobacter pylori (HpM17AP) is an important housekeeping enzyme involved in catabolism of endogenous and exogenous peptides. It is implicated in H. pylori defence against the human innate immune response and in the mechanism of metronidazole resistance. Bestatin inhibits HpM17AP and suppresses H. pylori growth. To address the structural basis of catalysis and inhibition of this enzyme, we have established its specificity towards the N-terminal amino acid of peptide substrates and determined the crystal structures of HpM17AP and its complex with bestatin. The position of the D-phenylalanine moiety of the inhibitor with respect to the active-site metal ions, bicarbonate ion and with respect to other M17 aminopeptidases suggested that this residue binds to the S1 subsite of HpM17AP. In contrast to most characterized M17 aminopeptidases, HpM17AP displays preference for L-Arg over L-Leu residues in peptide substrates. Compared to very similar homologues from other bacteria, a distinguishing feature of HpM17AP is a hydrophilic pocket at the end of the S1 subsite that is likely to accommodate the charged head group of the L-Arg residue of the substrate. The pocket is flanked by a sodium ion (not present in M17 aminopeptidases that show preference for L-Leu) and its coordinating water molecules. In addition, the structure suggests that variable loops at the entrance to, and in the middle of, the substrate-binding channel are important determinants of substrate specificity of M17 aminopeptidases. more...
- Published
- 2015
41. The protease cathepsin L regulates Th17 cell differentiation
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Lifei Hou, Eileen Remold-O'Donnell, Richard Swanson, Steven T. Olson, Jessica Cooley, Matthew Bogyo, Robert N. Pike, and Poh Chee Ong
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Male ,Cellular differentiation ,medicine.medical_treatment ,Cathepsin L ,Immunology ,Article ,Mice ,medicine ,Immunology and Allergy ,Animals ,Transcription factor ,Cells, Cultured ,Serpins ,Cathepsin ,Mice, Knockout ,Protease ,biology ,SERPINB1 ,Biological activity ,Cell Differentiation ,Molecular biology ,Protease inhibitor (biology) ,Mice, Inbred C57BL ,Cysteine Endopeptidases ,biology.protein ,Th17 Cells ,Female ,Protein Processing, Post-Translational ,medicine.drug - Abstract
Previously we reported that IL-17(+) T cells, primarily IL-17(+) γδ cells, are increased in mice lacking the protease inhibitor serpinB1 (serpinb1(-/-) mice). Here we show that serpinB1-deficient CD4 cells exhibit a cell-autonomous and selective deficiency in suppressing T helper 17 (Th17) cell differentiation. This suggested an opposing role for one or more protease in promoting Th17 differentiation. We found that several SerpinB1-inhibitable cysteine cathepsins are induced in Th17 cells, most prominently cathepsin L (catL); this was verified by peptidase assays, active site labeling and Western blots. Moreover, Th17 differentiation was suppressed by both broad cathepsin inhibitors and catL selective inhibitors. CatL is present in Th17 cells as single chain (SC)- and two-chain (TC)-forms. Inhibiting asparagine endopeptidase (AEP) blocked conversion of SC-catL to TC-catL and increased generation of serpinb1(-/-) Th17 cells, but not wild-type Th17 cells. These findings suggest that SC-catL is biologically active in promoting Th17 generation and is counter-regulated by serpinB1 and secondarily by AEP. Thus, in addition to regulation by cytokines and transcription factors, differentiation of CD4 cells to Th17 cells is actively regulated by a catL-serpinB1-AEP module. Targeting this protease regulatory module could be an approach to treating Th17 cell-driven autoimmune disorders. more...
- Published
- 2015
42. Investigation of the mechanism of interaction between Mannose-binding lectin-associated serine protease-2 and complement C4
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Paul J. Conroy, Nicole C Drentin, Robert N. Pike, Lakshmi C. Wijeyewickrema, and Menachem J. Gunzburg
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Serine protease ,Time Factors ,Complement component 2 ,Immunology ,Complement C4 ,Biology ,Surface Plasmon Resonance ,Cleavage (embryo) ,Protein Structure, Tertiary ,Serine ,Kinetics ,Immobilized Proteins ,Biochemistry ,Lectin pathway ,Mannose-Binding Protein-Associated Serine Proteases ,Mutation ,biology.protein ,Mutant Proteins ,Molecular Biology ,MASP1 ,Mannan-binding lectin ,Complement control protein ,Protein Binding - Abstract
The interaction between mannose-binding lectin [MBL]-associated serine protease-2 (MASP-2) and its first substrate, C4 is crucial to the lectin pathway of complement, which is vital for innate host immunity, but also involved in a number of inflammatory diseases. Recent data suggests that two areas outside of the active site of MASP-2 (so-called exosites) are crucial for efficient cleavage of C4: one at the junction of the two complement control protein (CCP) domains of the enzyme and the second on the serine protease (SP) domain. Here, we have further investigated the roles of each of these exosites in the binding and cleavage of C4. We have found that both exosites are required for high affinity binding and efficient cleavage of the substrate protein. Within the SP domain exosite, we have shown here that two arginine residues are most important for high affinity binding and efficient cleavage of C4. Finally, we show that the CCP domain exosite appears to play the major role in the initial interaction with C4, whilst the SP domain exosite plays the major role in a secondary conformational change between the two proteins required to form a high affinity complex. This data has provided new insights into the binding and cleavage of C4 by MASP-2, which may be useful in the design of molecules that modulate this important interaction required to activate the lectin pathway of complement. more...
- Published
- 2015
43. Production and processing of a recombinant Fasciola hepatica cathepsin B-like enzyme (FhcatB1) reveals potential processing mechanisms in the parasite
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Noelene Sheila Quinsey, Ruby H-P Law, Simone A. Beckham, Robert N. Pike, James H. McKerrow, Conor R. Caffrey, Terence W Spithill, and Peter M. Smooker
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Models, Molecular ,Proteases ,Time Factors ,Protein Conformation ,Clinical Biochemistry ,Biology ,Crystallography, X-Ray ,Biochemistry ,Cathepsin B ,Pichia pastoris ,law.invention ,Structure-Activity Relationship ,law ,Animals ,Fasciola hepatica ,Molecular Biology ,chemistry.chemical_classification ,Cathepsin ,Binding Sites ,Dose-Response Relationship, Drug ,Heparin ,Dextran Sulfate ,Hydrogen-Ion Concentration ,biology.organism_classification ,Recombinant Proteins ,Endopeptidase ,Protein Structure, Tertiary ,Enzyme Activation ,Enzyme ,chemistry ,Recombinant DNA ,Protein Processing, Post-Translational - Abstract
The liver fluke,Fasciola hepatica, apparently uses a number of cysteine proteases during its life cycle, most likely for feeding, immune evasion and invasion of tissues. A cathepsin B-like enzyme (herein referred to as FhcatB1) appears to be a major enzyme secreted by the invasive, newly excysted juvenile flukes of this parasite. To examine the processing mechanisms for this enzyme, a recombinant form was expressed inPichia pastorisand purified to yield a homogenous pool of the enzyme. The purified enzyme could be autoactivated at low pH via a bi-molecular mechanism, a process that was greatly accelerated by the presence of large, negatively charged molecules such as dextran sulfate. The enzyme could also apparently be processed to the correct size by an asparaginyl endopeptidase via cleavage in an unusual insertion N-terminal to the normal cleavage site used to yield the active form of the enzyme. Thus, there appear to be a number of ways in which this enzyme can be processed to its optimally active form prior to secretion byF. hepatica. more...
- Published
- 2006
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44. Approaches to Selective Peptidic Inhibitors of Factor Xa
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Robert N. Pike, Noelene Sheila Quinsey, Peter J. Duggan, and Karen Maree Bromfield
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Serine Proteinase Inhibitors ,Stereochemistry ,Molecular Sequence Data ,Peptide ,Ligands ,Cleavage (embryo) ,Biochemistry ,Substrate Specificity ,Non-competitive inhibition ,Thrombin ,Catalytic Domain ,Drug Discovery ,medicine ,Humans ,Amino Acid Sequence ,Enzyme kinetics ,Fluorescent Dyes ,Pharmacology ,Serine protease ,chemistry.chemical_classification ,biology ,Chemistry ,Organic Chemistry ,Anticoagulants ,Protease inhibitor (biology) ,Kinetics ,Enzyme ,Drug Design ,Factor Xa ,biology.protein ,Molecular Medicine ,Peptides ,Factor Xa Inhibitors ,medicine.drug - Abstract
Inhibitors of procoagulant enzymes, such as factor Xa (fXa) and thrombin, are important for treating thrombosis. Thrombin has complex pro- and anti-coagulant roles and thus fXa is thought to represent an ideal target. Discrete kcat and Km values for cleavage of a library of fluorescence-quenched substrates by fXa were determined. The results highlighted the low selectivity of fXa at its prime sites, and its poor efficiency compared with thrombin, creating a challenge for the design of fXa-specific peptidic inhibitors. We hypothesized that Km rather than kcat/Km values may be better indicators of inhibitor potential for a peptidic sequence, leading us to design peptide sequences for both fXa and thrombin in three forms: fluorescence-quenched substrates, standard alpha-peptides and peptides containing a beta-homoarginine at the cleavage site. Kinetic and competitive inhibition assays with both fXa and thrombin showed the fluorescence-quenched substrates to be the best inhibitors, while the inhibitory effect of the beta-homoarginine peptides varied for the two proteases. Importantly, fXa was inhibited to a much greater extent by the beta-peptides than the corresponding alpha-peptides, resulting in an increased selectivity for fXa inhibition over thrombin for those peptides containing a beta-amino acid at the cleavage site. more...
- Published
- 2006
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45. X-ray crystal structure of MENT: evidence for functional loop–sheet polymers in chromatin condensation
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Kate Henderson, Poh Chee Ong, James C. Whisstock, A. Ian Smith, Evgenya Y. Popova, Sheena McGowan, Robert N. Pike, Yaroslava A. Bulynko, Wan Ting Kan, Ashley M. Buckle, Jamie Rossjohn, Stephen P. Bottomley, Sergei A. Grigoryev, James A. Irving, and Tanya Ann Bashtannyk-Puhalovich more...
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Models, Molecular ,Protein Conformation ,Cathepsin L ,Serpin ,Biology ,Crystallography, X-Ray ,DNA-binding protein ,Article ,General Biochemistry, Genetics and Molecular Biology ,chemistry.chemical_compound ,Prophase ,Protein structure ,Animals ,Humans ,Nucleosome ,Molecular Biology ,Serpins ,Binding Sites ,General Immunology and Microbiology ,General Neuroscience ,Cathepsins ,Linker DNA ,Molecular biology ,Chromatin ,Nucleosomes ,DNA-Binding Proteins ,Cysteine Endopeptidases ,chemistry ,Mutation ,Biophysics ,DNA - Abstract
Most serpins are associated with protease inhibition, and their ability to form loop-sheet polymers is linked to conformational disease and the human serpinopathies. Here we describe the structural and functional dissection of how a unique serpin, the non-histone architectural protein, MENT (Myeloid and Erythroid Nuclear Termination stage-specific protein), participates in DNA and chromatin condensation. Our data suggest that MENT contains at least two distinct DNA-binding sites, consistent with its simultaneous binding to the two closely juxtaposed linker DNA segments on a nucleosome. Remarkably, our studies suggest that the reactive centre loop, a region of the MENT molecule essential for chromatin bridging in vivo and in vitro, is able to mediate formation of a loop-sheet oligomer. These data provide mechanistic insight into chromatin compaction by a non-histone architectural protein and suggest how the structural plasticity of serpins has adapted to mediate physiological, rather than pathogenic, loop-sheet linkages. more...
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- 2006
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46. The Murine Orthologue of Human Antichymotrypsin
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Stephen P. Bottomley, Noelene Sheila Quinsey, Robert N. Pike, Anita J Horvath, James C. Whisstock, Jamie Rossjohn, Paul Bernard Coughlin, James A. Irving, and Ruby H. P. Law
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biology ,Cell Biology ,Cathepsin G ,Serpin ,Biochemistry ,Molecular biology ,Alpha 1-antichymotrypsin ,Protease inhibitor (biology) ,chemistry.chemical_compound ,Protein structure ,chemistry ,biology.protein ,medicine ,Structural motif ,Molecular Biology ,Peptide sequence ,Reactive center ,medicine.drug - Abstract
Antichymotrypsin (SERPINA3) is a widely expressed member of the serpin superfamily, required for the regulation of leukocyte proteases released during an inflammatory response and with a permissive role in the development of amyloid encephalopathy. Despite its biological significance, there is at present no available structure of this serpin in its native, inhibitory state. We present here the first fully refined structure of a murine antichymotrypsin orthologue to 2.1 A, which we propose as a template for other antichymotrypsin-like serpins. A most unexpected feature of the structure of murine serpina3n is that it reveals the reactive center loop (RCL) to be partially inserted into the A beta-sheet, a structural motif associated with ligand-dependent activation in other serpins. The RCL is, in addition, stabilized by salt bridges, and its plane is oriented at 90 degrees to the RCL of antitrypsin. A biochemical and biophysical analysis of this serpin demonstrates that it is a fast and efficient inhibitor of human leukocyte elastase (ka: 4 +/- 0.9 x 10(6) m(-1) s(-)1) and cathepsin G (ka: 7.9 +/- 0.9 x 10(5) m(-1) s(-)1) giving a spectrum of activity intermediate between that of human antichymotrypsin and human antitrypsin. An evolutionary analysis reveals that residues subject to positive selection and that have contributed to the diversity of sequences in this sub-branch (A3) of the serpin superfamily are essentially restricted to the P4-P6' region of the RCL, the distal hinge, and the loop between strands 4B and 5B. more...
- Published
- 2005
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47. Elucidation of the Substrate Specificity of the C1s Protease of the Classical Complement Pathway
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James C. Whisstock, Phillip I. Bird, Felicity Kate Kerr, Dion Kaiserman, Sarah Elizabeth Boyd, Maria Garcia de la Banda, Antony Yaron Matthews, Noelene Sheila Quinsey, Robert N. Pike, and Grace O'Brien more...
- Subjects
Models, Molecular ,Phage display ,medicine.medical_treatment ,In Vitro Techniques ,Biology ,Biochemistry ,Substrate Specificity ,Classical complement pathway ,Peptide Library ,Catalytic Domain ,medicine ,Humans ,Amino Acid Sequence ,Complement Pathway, Classical ,Peptide library ,Molecular Biology ,Peptide sequence ,chemistry.chemical_classification ,Binding Sites ,Protease ,Base Sequence ,Complement C1s ,Active site ,DNA ,Cell Biology ,Complement system ,Enzyme ,chemistry ,biology.protein ,Oligopeptides - Abstract
The complement system is a central component of host defense but can also contribute to the inflammation seen in pathological conditions. The C1s protease of the first complement component, the C1 complex, initiates the pathway. In this study we have elucidated the full specificity of the enzyme for the first time using a randomized phage display library. It was found that, aside from the crucial P(1) position, the S(3) and S(2) subsites (in that order) played the greatest role in determining specificity. C1s prefers Leu or Val at P(3) and Gly or Ala residues at P(2). Apart from the S(2)' position, which showed specificity for Leu, prime subsites did not greatly affect specificity. It was evident, however, that together they significantly contributed to the efficiency of cleavage of a peptide. A peptide substrate based on the top sequence obtained in the phage display validated these results and produced the best kinetics of any C1s substrate to date. The results allow an understanding of the active site specificity of the C1s protease for the first time and provide a basis for the development of specific inhibitors aimed at controlling inflammation associated with complement activation in adverse pathological situations. more...
- Published
- 2005
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48. Serpins 2005 - fun between the β-sheets
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Robert N. Pike, Paul Bernard Coughlin, James C. Whisstock, Stephen P. Bottomley, and Phillip I. Bird
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Proteases ,animal structures ,Protease ,medicine.diagnostic_test ,biology ,Proteolysis ,medicine.medical_treatment ,Cell Biology ,Computational biology ,Serpin ,biology.organism_classification ,Biochemistry ,Molecular biology ,carbohydrates (lipids) ,Multicellular organism ,Protein structure ,embryonic structures ,medicine ,Eukaryote ,Molecular Biology ,Function (biology) - Abstract
Serpins are the largest family of protease inhibitors and are fundamental for the control of proteolysis in multicellular eukaryotes. Most eukaryote serpins inhibit serine or cysteine proteases, however, noninhibitory members have been identified that perform diverse functions in processes such as hormone delivery and tumour metastasis. More recently inhibitory serpins have been identified in prokaryotes and unicellular eukaryotes, nevertheless, the precise molecular targets of these molecules remains to be identified. The serpin mechanism of protease inhibition is unusual and involves a major conformational rearrangement of the molecule concomitant with a distortion of the target protease. As a result of this requirement, serpins are susceptible to mutations that result in polymerization and conformational diseases such as the human serpinopathies. This review reports on recent major discoveries in the serpin field, based upon presentations made at the 4th International Symposium on Serpin Structure, Function and Biology (Cairns, Australia). more...
- Published
- 2005
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49. Control of the coagulation system by serpins
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Ashley M. Buckle, Robert N. Pike, Bernard Le Bonniec, and Frank C. Church
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Heparin cofactor II ,Serine protease ,Proteases ,Serine Proteinase Inhibitors ,animal structures ,Antithrombin ,Cell Biology ,Serpin ,Biology ,Thrombomodulin ,Biochemistry ,carbohydrates (lipids) ,Thrombin ,medicine ,biology.protein ,Animals ,Humans ,Blood Coagulation ,Molecular Biology ,Serpins ,Glycosaminoglycans ,medicine.drug - Abstract
Members of the serine protease inhibitor (serpin) superfamily play important roles in the inhibition of serine proteases involved in complex systems. This is evident in the regulation of coagulation serine proteases, especially the central enzyme in this system, thrombin. This review focuses on three serpins which are known to be key players in the regulation of thrombin: antithrombin and heparin cofactor II, which inhibit thrombin in its procoagulant role, and protein C inhibitor, which primarily inhibits the thrombin/thrombomodulin complex, where thrombin plays an anticoagulant role. Several structures have been published in the past few years which have given great insight into the mechanism of action of these serpins and have significantly added to a wealth of biochemical and biophysical studies carried out previously. A major feature of these serpins is that they are under the control of glycosaminoglycans, which play a key role in accelerating and localizing their action. While further work is clearly required to understand the mechanism of action of the glycosaminoglycans, the biological mechanisms whereby cognate glycosaminoglycans for each serpin come into contact with the inhibitors also requires much further work in this important field. more...
- Published
- 2005
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50. Studies on the receptors mediating responses of osteoblasts to thrombin
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Shu Jun Song, Eleanor J. Mackie, Charles N. Pagel, and Robert N. Pike
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Peptide binding ,Thrombomodulin ,Biochemistry ,Mice ,Thrombin ,Thrombin receptor ,medicine ,Animals ,Receptor, PAR-1 ,Protease-activated receptor ,Cells, Cultured ,Protease-activated receptor 2 ,Cell Proliferation ,Mice, Knockout ,Osteoblasts ,Chemistry ,Skull ,Cell Differentiation ,Osteoblast ,Cell Biology ,Molecular biology ,medicine.anatomical_structure ,Alkaline phosphatase ,Peptides ,Signal Transduction ,medicine.drug - Abstract
The serine protease thrombin stimulates proliferation in osteoblasts, but decreases alkaline phosphatase (ALP) activity, a marker of osteoblast differentiation. Three thrombin receptors have been identified, protease activated receptor (PAR)-1, PAR-3 and PAR-4; we have previously demonstrated that mouse osteoblasts express PAR-1 and PAR-4. The effect of thrombin on osteoblast proliferation and differentiation was studied to determine which of the thrombin receptors is responsible for the primary effects of thrombin. Primary mouse calvarial osteoblasts from PAR-1-null and wild-type mice, and synthetic peptides that specifically activate PAR-1 (TFFLR-NH2) and PAR-4 (AYPGKF-NH2) were used. Both the PAR-1-activating peptide and thrombin stimulated incorporation of 5-bromo-2′-deoxyuridine (two to four-fold, P < 0.001) and reduced alkaline phosphatase activity (approximately three-fold, P < 0.05) in cells from wild-type mice. The PAR-4-activating peptide, however, had no effect on either alkaline phosphatase activity or proliferation in these cells. Neither thrombin nor PAR-4-activating peptide was able to affect osteoblast proliferation or alkaline phosphatase activity in cells isolated from PAR-1-null mice. The results demonstrate that thrombin stimulates proliferation and inhibits differentiation of osteoblasts through activation of PAR-1. No other thrombin receptor appears to be involved in these effects. more...
- Published
- 2005
- Full Text
- View/download PDF
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