47 results on '"Whisstock, James C"'
Search Results
2. Structure of the metastatic factor P-Rex1 reveals a two-layered autoinhibitory mechanism
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Chang, Yong-Gang, Lupton, Christopher J., Bayly-Jones, Charles, Keen, Alastair C., D’Andrea, Laura, Lucato, Christina M., Steele, Joel R., Venugopal, Hari, Schittenhelm, Ralf B., Whisstock, James C., Halls, Michelle L., and Ellisdon, Andrew M.
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P-Rex (PI(3,4,5)P3-dependent Rac exchanger) guanine nucleotide exchange factors potently activate Rho GTPases. P-Rex guanine nucleotide exchange factors are autoinhibited, synergistically activated by Gβγ and PI(3,4,5)P3binding and dysregulated in cancer. Here, we use X-ray crystallography, cryogenic electron microscopy and crosslinking mass spectrometry to determine the structural basis of human P-Rex1 autoinhibition. P-Rex1 has a bipartite structure of N- and C-terminal modules connected by a C-terminal four-helix bundle that binds the N-terminal Pleckstrin homology (PH) domain. In the N-terminal module, the Dbl homology (DH) domain catalytic surface is occluded by the compact arrangement of the DH-PH-DEP1 domains. Structural analysis reveals a remarkable conformational transition to release autoinhibition, requiring a 126° opening of the DH domain hinge helix. The off-axis position of Gβγ and PI(3,4,5)P3binding sites further suggests a counter-rotation of the P-Rex1 halves by 90° facilitates PH domain uncoupling from the four-helix bundle, releasing the autoinhibited DH domain to drive Rho GTPase signaling.
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- 2022
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3. A mechanism for hereditary angioedema caused by a lysine 311–to–glutamic acid substitution in plasminogen
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Dickeson, S. Kent, Kumar, Sunil, Sun, Mao-Fu, Mohammed, Bassem M., Phillips, Dennis R., Whisstock, James C., Quek, Adam J., Feener, Edward P., Law, Ruby H.P., and Gailani, David
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Patients with hereditary angioedema (HAE) experience episodes of bradykinin (BK)-induced swelling of skin and mucosal membranes. The most common cause is reduced plasma activity of C1 inhibitor, the main regulator of the proteases plasma kallikrein (PKa) and factor XIIa (FXIIa). Recently, patients with HAE were described with a Lys311to glutamic acid substitution in plasminogen (Plg), the zymogen of the protease plasmin (Plm). Adding tissue plasminogen activator to plasma containing Plg-Glu311vs plasma containing wild-type Plg (Plg-Lys311) results in greater BK generation. Similar results were obtained in plasma lacking prekallikrein or FXII (the zymogens of PKa and FXIIa) and in normal plasma treated with a PKa inhibitor, indicating Plg-Glu311induces BK generation independently of PKa and FXIIa. Plm-Glu311cleaves high and low molecular weight kininogens (HK and LK, respectively), releasing BK more efficiently than Plm-Lys311. Based on the plasma concentrations of HK and LK, the latter may be the source of most of the BK generated by Plm-Glu311. The lysine analog ε-aminocaproic acid blocks Plm-catalyzed BK generation. The Glu311substitution introduces a lysine-binding site into the Plg kringle 3 domain, perhaps altering binding to kininogens. Plg residue 311 is glutamic acid in most mammals. Glu311in patients with HAE, therefore, represents reversion to the ancestral condition. Substantial BK generation occurs during Plm-Glu311cleavage of human HK, but not mouse HK. Furthermore, mouse Plm, which has Glu311, did not liberate BK from human kininogens more rapidly than human Plg-Lys311. This indicates Glu311is pathogenic in the context of human Plm when human kininogens are the substrates.
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- 2022
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4. COOH-terminal clustering of autoantibody and T-cell determinants on the structure of GAD65 provide insights into the molecular basis of autoreactivity
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Fenalti, Gustavo, Hampe, Christiane S., Arafat, Yasir, Law, Ruby H.P., Banga, J. Paul, Mackay, Ian R., Whisstock, James C., Buckle, Ashley M., and Rowley, Merrill J.
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Islands of Langerhans -- Properties -- Research ,Crystals -- Structure ,Type 1 diabetes -- Research ,Pancreatic beta cells -- Properties -- Research ,Carboxylic acids -- Properties -- Influence -- Research ,Islet cell stimulating antibodies -- Properties -- Influence -- Research ,Glutamate decarboxylase -- Properties -- Influence -- Structure -- Research ,Health ,Structure ,Influence ,Observations ,Research ,Properties - Abstract
OBJECTIVE--To gain structural insights into the autoantigenic properties of GAD65 in type 1 diabetes, we analyzed experimental epitope mapping data in the context of the recently determined crystal structures of GAD65 and GAD67, to allow 'molecular positioning' of epitope sites for B- and T-cell reactivity. RESEARCH DESIGN AND METHODS--Data were assembled from analysis of reported effects of mutagenesis of GAD65 on its reactivity with a panel of 11 human monoclonal antibodies (mAbs), supplemented by use of recombinant Fab to cross-inhibit reactivity with GAD65 by radioimmunoprecipitation of the same mAbs. RESULTS--The COOH-terminal region on GAD65 was the major autoantigenic site. B-cell epitopes were distributed within two separate clusters around different faces of the COOH-terminal domain. Inclusion of epitope sites in the pyridoxal phosphate- and N[H.sub.2]-terminal domains was attributed to the juxtaposition of all three domains in the crystal structure. Epitope preferences of different mAbs to GAD65 aligned with different clinical expressions of type 1 diabetes. Epitopes for four of five known reactive T-cell sequences restricted by HLA DRB1*0401 were aligned to solvent-exposed regions of the GAD65 structure and colocalized within the two B-cell epitope clusters. The continuous COOH-terminal epitope region of GAD65 was structurally highly flexible and therefore differed markedly from the equivalent region of GAD67. CONCLUSIONS--Structural features could explain the differing antigenicity, and perhaps immunogenicity, of GAD65 versus GAD67. The proximity of B- and T-cell epitopes within the GAD65 structure suggests that antigen-antibody complexes may influence antigen processing by accessory cells and thereby T-cell reactivity., Type 1 diabetes is characterized by autoimmune reactivity to islet cell antigens that include the 65-kDa isoform of GAD (GAD65). Of the two isoforms of GAD, GAD67 and GAD65, only [...]
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- 2008
5. Functional insights from the distribution and role of homopeptide repeat-containing proteins
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Faux, Noel G., Bottomley, Stephen P., Lesk, Arthur M., Irving, James A., de la Banda, Maria Garcia, Morrison, John R., and Whisstock, James C.
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Proteins -- Natural history ,CAD-CAM systems -- Computer programs ,CAD-CAM systems -- Usage ,Genetic research ,CAD/CAM software ,CAD software ,Health - Abstract
A Web-accessible database of repeat-containing proteins (RCPs) is presented. The distribution and evolution of RCPs that contain homopeptide repeats tracts are considered, and the existence of functional patterns investigated.
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- 2005
6. The cryo-EM structure of the human neurofibromin dimer reveals the molecular basis for neurofibromatosis type 1
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Lupton, Christopher J., Bayly-Jones, Charles, D’Andrea, Laura, Huang, Cheng, Schittenhelm, Ralf B., Venugopal, Hari, Whisstock, James C., Halls, Michelle L., and Ellisdon, Andrew M.
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Neurofibromin (NF1) mutations cause neurofibromatosis type 1 and drive numerous cancers, including breast and brain tumors. NF1 inhibits cellular proliferation through its guanosine triphosphatase-activating protein (GAP) activity against rat sarcoma (RAS). In the present study, cryo-electron microscope studies reveal that the human ~640-kDa NF1 homodimer features a gigantic 30 × 10 nm array of α-helices that form a core lemniscate-shaped scaffold. Three-dimensional variability analysis captured the catalytic GAP-related domain and lipid-binding SEC-PH domains positioned against the core scaffold in a closed, autoinhibited conformation. We postulate that interaction with the plasma membrane may release the closed conformation to promote RAS inactivation. Our structural data further allow us to map the location of disease-associated NF1 variants and provide a long-sought-after structural explanation for the extreme susceptibility of the molecule to loss-of-function mutations. Collectively these findings present potential new routes for therapeutic modulation of the RAS pathway.
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- 2021
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7. Proteoform-Resolved Profiling of Plasminogen Activation Reveals Novel Abundant Phosphorylation Site and Primary N-Terminal Cleavage Site
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Cramer, Dario A.T., Yin, Victor, Caval, Tomislav, Franc, Vojtech, Yu, Dingyi, Wu, Guojie, Lloyd, Gordon, Langendorf, Christopher, Whisstock, James C., Law, Ruby H.P., and Heck, Albert J.R.
- Abstract
Plasminogen (Plg), the zymogen of plasmin (Plm), is a glycoprotein involved in fibrinolysis and a wide variety of other physiological processes. Plg dysregulation has been implicated in a range of diseases. Classically, human Plg is categorized into two types, supposedly having different functional features, based on the presence (type I) or absence (type II) of a single N-linked glycan. Using high-resolution native mass spectrometry, we uncovered that the proteoform profiles of human Plg (and Plm) are substantially more extensive than this simple binary classification. In samples derived from human plasma, we identified up to 14 distinct proteoforms of Plg, including a novel highly stoichiometric phosphorylation site at Ser339. To elucidate the potential functional effects of these post-translational modifications, we performed proteoform-resolved kinetic analyses of the Plg-to-Plm conversion using several canonical activators. This conversion is thought to involve at least two independent cleavage events: one to remove the N-terminal peptide and another to release the active catalytic site. Our analyses reveal that these processes are not independent but are instead tightly regulated and occur in a step-wise manner. Notably, N-terminal cleavage at the canonical site (Lys77) does not occur directly from intact Plg. Instead, an activation intermediate corresponding to cleavage at Arg68 is initially produced, which only then is further processed to the canonical Lys77 product. Based on our results, we propose a refined categorization for human Plg proteoforms. In addition, we reveal that the proteoform profile of human Plg is more extensive than that of rat Plg, which lacks, for instance, the here-described phosphorylation at Ser339.
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- 2024
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8. RCPdb: An evolutionary classification and codon usage database for repeat-containing proteins
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Faux, Noel G., Huttley, Gavin A., Mahmood, Khalid, Webb, Geoffrey I., de la Banda, Maria Garcia, and Whisstock, James C.
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Genetic code -- Analysis ,DNA binding proteins -- Research ,Health - Abstract
A large scale genomic analysis of the variation of genes encoding repeat containing proteins (RCPs) in 13 species was performed. A bias for small subset of codons being reiterated within homopeptides was seen along with single base pair transversions and homopeptides that are conserved across different species lying within regions strong under purifying selection.
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- 2007
9. Tranexamic acid is an active site inhibitor of urokinase plasminogen activator
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Wu, Guojie, Mazzitelli, Blake A., Quek, Adam J., Veldman, Matthew J., Conroy, Paul J., Caradoc-Davies, Tom T., Ooms, Lisa M., Tuck, Kellie L., Schoenecker, Jonathan G., Whisstock, James C., and Law, Ruby H. P.
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- 2019
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10. Highly Potent and Selective Plasmin Inhibitors Based on the Sunflower Trypsin Inhibitor-1 Scaffold Attenuate Fibrinolysis in Plasma
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Swedberg, Joakim E., Wu, Guojie, Mahatmanto, Tunjung, Durek, Thomas, Caradoc-Davies, Tom T., Whisstock, James C., Law, Ruby H. P., and Craik, David J.
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Antifibrinolytic drugs provide important pharmacological interventions to reduce morbidity and mortality from excessive bleeding during surgery and after trauma. Current drugs used for inhibiting the dissolution of fibrin, the main structural component of blood clots, are associated with adverse events due to lack of potency, high doses, and nonselective inhibition mechanisms. These drawbacks warrant the development of a new generation of highly potent and selective fibrinolysis inhibitors. Here, we use the 14-amino acid backbone-cyclic sunflower trypsin inhibitor-1 scaffold to design a highly potent (Ki= 0.05 nM) inhibitor of the primary serine protease in fibrinolysis, plasmin. This compound displays a million-fold selectivity over other serine proteases in blood, inhibits fibrinolysis in plasma more effectively than the gold-standard therapeutic inhibitor aprotinin, and is a promising candidate for development of highly specific fibrinolysis inhibitors with reduced side effects.
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- 2018
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11. Torso-Like Is a Component of the Hemolymph and Regulates the Insulin Signaling Pathway in Drosophila
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Henstridge, Michelle A, Aulsebrook, Lucinda, Koyama, Takashi, Johnson, Travis K, Whisstock, James C, Tiganis, Tony, Mirth, Christen K, and Warr, Coral G
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In Drosophila, key developmental transitions are governed by the steroid hormone ecdysone. A number of neuropeptide-activated signaling pathways control ecdysone production in response to environmental signals, including the insulin signaling pathway, which regulates ecdysone production in response to nutrition. Here, we find that the Membrane Attack Complex/Perforin-like protein Torso-like, best characterized for its role in activating the Torso receptor tyrosine kinase in early embryo patterning, also regulates the insulin signaling pathway in Drosophila. We previously reported that the small body size and developmental delay phenotypes of torso-likenull mutants resemble those observed when insulin signaling is reduced. Here we report that, in addition to growth defects, torso-likemutants also display metabolic and nutritional plasticity phenotypes characteristic of mutants with impaired insulin signaling. We further find that in the absence of torso-like, the expression of insulin-like peptides is increased, as is their accumulation in insulin-producing cells. Finally, we show that Torso-like is a component of the hemolymph and that it is required in the prothoracic gland to control developmental timing and body size. Taken together, our data suggest that the secretion of Torso-like from the prothoracic gland influences the activity of insulin signaling throughout the body in Drosophila.
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- 2018
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12. X-ray crystal structure of plasmin with tranexamic acid–derived active site inhibitors
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Law, Ruby H.P., Wu, Guojie, Leung, Eleanor W.W., Hidaka, Koushi, Quek, Adam J., Caradoc-Davies, Tom T., Jeevarajah, Devadharshini, Conroy, Paul J., Kirby, Nigel M., Norton, Raymond S., Tsuda, Yuko, and Whisstock, James C.
- Abstract
The zymogen protease plasminogen and its active form plasmin perform key roles in blood clot dissolution, tissue remodeling, cell migration, and bacterial pathogenesis. Dysregulation of the plasminogen/plasmin system results in life-threatening hemorrhagic disorders or thrombotic vascular occlusion. Accordingly, inhibitors of this system are clinically important. Currently, tranexamic acid (TXA), a molecule that prevents plasminogen activation through blocking recruitment to target substrates, is the most widely used inhibitor for the plasminogen/plasmin system in therapeutics. However, TXA lacks efficacy on the active form of plasmin. Thus, there is a need to develop specific inhibitors that target the protease active site. Here we report the crystal structures of plasmin in complex with the novel YO (trans-4-aminomethylcyclohexanecarbonyl-l-tyrosine-n-octylamide) class of small molecule inhibitors. We found that these inhibitors form key interactions with the S1 and S3′ subsites of the catalytic cleft. Here, the TXA moiety of the YO compounds inserts into the primary (S1) specificity pocket, suggesting that TXA itself may function as a weak plasmin inhibitor, a hypothesis supported by subsequent biochemical and biophysical analyses. Mutational studies reveal that F587 of the S′ subsite plays a key role in mediating the inhibitor interaction. Taken together, these data provide a foundation for the future development of small molecule inhibitors to specifically regulate plasmin function in a range of diseases and disorders.
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- 2017
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13. Real-time visualization of perforin nanopore assembly
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Leung, Carl, Hodel, Adrian W., Brennan, Amelia J., Lukoyanova, Natalya, Tran, Sharon, House, Colin M., Kondos, Stephanie C., Whisstock, James C., Dunstone, Michelle A., Trapani, Joseph A., Voskoboinik, Ilia, Saibil, Helen R., and Hoogenboom, Bart W.
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Perforin is a key protein of the vertebrate immune system. Secreted by cytotoxic lymphocytes as soluble monomers, perforin can self-assemble into oligomeric pores of 10–20 nm inner diameter in the membranes of virus-infected and cancerous cells. These large pores facilitate the entry of pro-apoptotic granzymes, thereby rapidly killing the target cell. To elucidate the pathways of perforin pore assembly, we carried out real-time atomic force microscopy and electron microscopy studies. Our experiments reveal that the pore assembly proceeds via a membrane-bound prepore intermediate state, typically consisting of up to approximately eight loosely but irreversibly assembled monomeric subunits. These short oligomers convert to more closely packed membrane nanopore assemblies, which can subsequently recruit additional prepore oligomers to grow the pore size.
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- 2017
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14. Cell Traversal Activity Is Important for Plasmodium falciparumLiver Infection in Humanized Mice
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Yang, Annie S.P., O’Neill, Matthew T., Jennison, Charlie, Lopaticki, Sash, Allison, Cody C., Armistead, Jennifer S., Erickson, Sara M., Rogers, Kelly L., Ellisdon, Andrew M., Whisstock, James C., Tweedell, Rebecca E., Dinglasan, Rhoel R., Douglas, Donna N., Kneteman, Norman M., and Boddey, Justin A.
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Malaria sporozoites are deposited into the skin by mosquitoes and infect hepatocytes. The molecular basis of how Plasmodium falciparumsporozoites migrate through host cells is poorly understood, and direct evidence of its importance in vivo is lacking. Here, we generated traversal-deficient sporozoites by genetic disruption of sporozoite microneme protein essential for cell traversal (PfSPECT) or perforin-like protein 1 (PfPLP1). Loss of either gene did not affect P. falciparumgrowth in erythrocytes, in contrast with a previous report that PfPLP1 is essential for merozoite egress. However, although traversal-deficient sporozoites could invade hepatocytes in vitro, they could not establish normal liver infection in humanized mice. This is in contrast with NF54 sporozoites, which infected the humanized mice and developed into exoerythrocytic forms. This study demonstrates that SPECT and perforin-like protein 1 (PLP1) are critical for transcellular migration by P. falciparumsporozoites and demonstrates the importance of cell traversal for liver infection by this human pathogen.
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- 2017
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15. Development of the Cellular Immune System of DrosophilaRequires the Membrane Attack Complex/Perforin-Like Protein Torso-Like
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Forbes-Beadle, Lauren, Crossman, Tova, Johnson, Travis K, Burke, Richard, Warr, Coral G, and Whisstock, James C
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Pore-forming members of the membrane attack complex/perforin-like (MACPF) protein superfamily perform well-characterized roles as mammalian immune effectors. For example, complement component 9 and perforin function to directly form pores in the membrane of Gram-negative pathogens or virally infected/transformed cells, respectively. In contrast, the only known MACPF protein in Drosophila melanogaster, Torso-like, plays crucial roles during development in embryo patterning and larval growth. Here, we report that in addition to these functions, Torso-like plays an important role in Drosophilaimmunity. However, in contrast to a hypothesized effector function in, for example, elimination of Gram-negative pathogens, we find that torso-likenull mutants instead show increased susceptibility to certain Gram-positive pathogens such as Staphylococcus aureusand Enterococcus faecalis. We further show that this deficit is due to a severely reduced number of circulating immune cells and, as a consequence, an impaired ability to phagocytose bacterial particles. Together these data suggest that Torso-like plays an important role in controlling the development of the Drosophilacellular immune system.
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- 2016
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16. Capturing embryonic development from metamorphosis: how did the terminal patterning signalling pathway of Drosophilaevolve?
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Duncan, Elizabeth J, Johnson, Travis K, Whisstock, James C, Warr, Coral G, and Dearden, Peter K
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•The Torso activation module in insects is ancestrally involved in regulation of growth and pupation.•The Torso activation module has been co-opted into controlling terminal patterning in Drosophilaand Tribolium.•Torso-likeis highly conserved, but has a largely unknown function in embryogenesis and in the prothoracic gland.
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- 2014
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17. Perforin forms transient pores on the target cell plasma membrane to facilitate rapid access of granzymes during killer cell attack
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Lopez, Jamie A., Susanto, Olivia, Jenkins, Misty R., Lukoyanova, Natalya, Sutton, Vivien R., Law, Ruby H.P., Johnston, Angus, Bird, Catherina H., Bird, Phillip I., Whisstock, James C., Trapani, Joseph A., Saibil, Helen R., and Voskoboinik, Ilia
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Cytotoxic lymphocytes serve a key role in immune homeostasis by eliminating virus-infected and transformed target cells through the perforin-dependent delivery of proapoptotic granzymes. However, the mechanism of granzyme entry into cells remains unresolved. Using biochemical approaches combined with time-lapse microscopy of human primary cytotoxic lymphocytes engaging their respective targets, we defined the time course of perforin pore formation in the context of the physiological immune synapse. We show that, on recognition of targets, calcium influx into the lymphocyte led to perforin exocytosis and target cell permeabilization in as little as 30 seconds. Within the synaptic cleft, target cell permeabilization by perforin resulted in the rapid diffusion of extracellular milieu–derived granzymes. Repair of these pores was initiated within 20 seconds and was completed within 80 seconds, thus limiting granzyme diffusion. Remarkably, even such a short time frame was sufficient for the delivery of lethal amounts of granzymes into the target cell. Rapid initiation of apoptosis was evident from caspase-dependent target cell rounding within 2 minutes of perforin permeabilization. This study defines the final sequence of events controlling cytotoxic lymphocyte immune defense, in which perforin pores assemble on the target cell plasma membrane, ensuring efficient delivery of lethal granzymes.
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- 2013
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18. The X-ray Crystal Structure of Full-Length Human Plasminogen
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Law, Ruby H.P., Caradoc-Davies, Tom, Cowieson, Nathan, Horvath, Anita J., Quek, Adam J., Encarnacao, Joanna Amarante, Steer, David, Cowan, Angus, Zhang, Qingwei, Lu, Bernadine G.C., Pike, Robert N., Smith, A. Ian, Coughlin, Paul B., and Whisstock, James C.
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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.
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- 2012
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19. X-ray crystal structure of the fibrinolysis inhibitor α2-antiplasmin
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Law, Ruby H. P., Sofian, Trifina, Kan, Wan-Ting, Horvath, Anita J., Hitchen, Corinne R., Langendorf, Christopher G., Buckle, Ashley M., Whisstock, James C., and Coughlin, Paul B.
- Abstract
The serpin α2-antiplasmin (SERPINF2) is the principal inhibitor of plasmin and inhibits fibrinolysis. Accordingly, α2-antiplasmin deficiency in humans results in uncontrolled fibrinolysis and a bleeding disorder. α2-antiplasmin is an unusual serpin, in that it contains extensive N- and C-terminal sequences flanking the serpin domain. The N-terminal sequence is crosslinked to fibrin by factor XIIIa, whereas the C-terminal region mediates the initial interaction with plasmin. To understand how this may happen, we have determined the 2.65Å X-ray crystal structure of an N-terminal truncated murine α2-antiplasmin. The structure reveals that part of the C-terminal sequence is tightly associated with the body of the serpin. This would be anticipated to position the flexible plasmin-binding portion of the C-terminus in close proximity to the serpin Reactive Center Loop where it may act as a template to accelerate serpin/protease interactions.
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- 2008
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20. The Type Iα Inositol Polyphosphate 4-Phosphatase Generates and Terminates Phosphoinositide 3-Kinase Signals on Endosomes and the Plasma Membrane
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Ivetac, Ivan, Munday, Adam D., Kisseleva, Marina V., Zhang, Xiang-Ming, Luff, Susan, Tiganis, Tony, Whisstock, James C., Rowe, Tony, Majerus, Phillip W., and Mitchell, Christina A.
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Endosomal trafficking is regulated by the recruitment of effector proteins to phosphatidylinositol 3-phosphate [PtdIns(3)P] on early endosomes. At the plasma membrane, phosphatidylinositol-(3,4)-bisphosphate [PtdIns(3,4)P2] binds the pleckstrin homology (PH) domain-containing proteins Akt and TAPP1. Type Iα inositol polyphosphate 4-phosphatase (4-phosphatase) dephosphorylates PtdIns(3,4)P2, forming PtdIns(3)P, but its subcellular localization is unknown. We report here in quiescent cells, the 4-phosphatase colocalized with early and recycling endosomes. On growth factor stimulation, 4-phosphatase endosomal localization persisted, but in addition the 4-phosphatase localized at the plasma membrane. Overexpression of the 4-phosphatase in serum-stimulated cells increased cellular PtdIns(3)P levels and prevented wortmannin-induced endosomal dilatation. Furthermore, mouse embryonic fibroblasts from homozygous Weeblemice, which have a mutation in the type I 4-phosphatase, exhibited dilated early endosomes. 4-Phosphatase translocation to the plasma membrane upon growth factor stimulation inhibited the recruitment of the TAPP1 PH domain. The 4-phosphatase contains C2 domains, which bound PtdIns(3,4)P2, and C2-domain-deletion mutants lost PtdIns(3,4)P24-phosphatase activity, did not localize to endosomes or inhibit TAPP1 PH domain membrane recruitment. The 4-phosphatase therefore both generates and terminates phosphoinositide 3-kinase signals at distinct subcellular locations.
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- 2005
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21. Cloning and expression of the major secreted cathepsin B-like protein from juvenile Fasciola hepatica and analysis of immunogenicity following liver fluke infection.
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Law, Ruby H P, Smooker, Peter M, Irving, James A, Piedrafita, David, Ponting, Rebecca, Kennedy, Nicholas J, Whisstock, James C, Pike, Robert N, and Spithill, Terry W
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The functions of the cathepsin B-like proteases in liver flukes are unknown and analysis has been hindered by a lack of protein for study, since the protein is produced in small amounts by juvenile flukes. To circumvent this, we isolated and characterized a cDNA encoding the major secreted cathepsin B from Fasciola hepatica. The predicted preproprotein is 339 amino acids in length, with the mature protease predicted to be 254 amino acids long, and shows significant similarity to parasite and mammalian cathepsin B. Only one of the two conserved histidine residues required for cathepsin B exopeptidase activity is predicted to be present. Recombinant preproprotein was produced in yeast, and it was shown that the recombinant proprotein can undergo a degree of self-processing in vitro to the mature form, which is active against gelatin and synthetic peptide substrates. The recombinant protein is antigenic in vaccinated rats, and antibodies to the protein are detected early after infection of rats and sheep with F. hepatica. The kinetics of the response to cathepsin B and cathepsin L after infection of sheep and rats confirm the temporal expression of these proteins during the life cycle of the parasite.
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- 2003
22. Cloning and Expression of the Major SecretedCathepsin B-Like Protein from Juvenile Fasciola hepaticaandAnalysis of Immunogenicity following Liver FlukeInfection
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Law, Ruby H. P., Smooker, Peter M., Irving, James A., Piedrafita, David, Ponting, Rebecca, Kennedy, Nicholas J., Whisstock, James C., Pike, Robert N., and Spithill, Terry W.
- Abstract
ABSTRACTThe functions of the cathepsin B-like proteases in liver flukes are unknown and analysis has been hindered by a lack of protein for study, since the protein is produced in small amounts by juvenile flukes. To circumvent this, we isolated and characterized a cDNA encoding the major secreted cathepsin B from Fasciola hepatica. The predicted preproprotein is 339 amino acids in length, with the mature protease predicted to be 254 amino acids long, and shows significant similarity to parasite and mammalian cathepsin B. Only one of the two conserved histidine residues required for cathepsin B exopeptidase activity is predicted to be present. Recombinant preproprotein was produced in yeast, and it was shown that the recombinant proprotein can undergo a degree of self-processing in vitro to the mature form, which is active against gelatin and synthetic peptide substrates. The recombinant protein is antigenic in vaccinated rats, and antibodies to the protein are detected early after infection of rats and sheep with F. hepatica. The kinetics of the response to cathepsin B and cathepsin L after infection of sheep and rats confirm the temporal expression of these proteins during the life cycle of the parasite.
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- 2003
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23. Molecular Determinants of the Mechanism Underlying Acceleration of the Interaction between Antithrombin and Factor Xa by Heparin Pentasaccharide*
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Quinsey, Noelene S., Whisstock, James C., Le Bonniec, Bernard, Louvain, Virginie, Bottomley, Stephen P., and Pike, Robert N.
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The control of coagulation enzymes by antithrombin is vital for maintenance of normal hemostasis. Antithrombin requires the co-factor, heparin, to efficiently inhibit target proteinases. A specific pentasaccharide sequence (H5) in high affinity heparin induces a conformational change in antithrombin that is particularly important for factor Xa (fXa) inhibition. Thus, synthetic H5 accelerates the interaction between antithrombin and fXa 100-fold as compared with only 2-fold versusthrombin. We built molecular models and identified residues unique to the active site of fXa that we predicted were important for interacting with the reactive center loop of H5-activated antithrombin. To test our predictions, we generated the mutants E37A, E37Q, E39A, E39Q, Q61A, S173A, and F174A in human fXa and examined the rate of association of these mutants with antithrombin in the presence and absence of H5. fXaQ61Ainteracts with antithrombin alone with a nearly normal kass; however, we observe only a 4-fold increase in kassin the presence of H5. The x-ray crystal structure of fXa reveals that Gln61forms part of the S1′ and S3′ pocket, suggesting that the P′ region of the reactive center loop of antithrombin is crucial for mediating the acceleration in the rate of inhibition of fXa by H5-activated antithrombin.
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- 2002
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24. Inhibitory Activity of a Heterochromatin-associated Serpin (MENT) against Papain-like Cysteine Proteinases Affects Chromatin Structure and Blocks Cell Proliferation∗
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Irving, James A., Shushanov, Sain S., Pike, Robert N., Popova, Evgenya Y., Brömme, Dieter, Coetzer, Theresa H.T., Bottomley, Stephen P., Boulynko, Iaroslava A., Grigoryev, Sergei A., and Whisstock, James C.
- Abstract
MENT (Myeloid andErythroid Nuclear Termination stage-specific protein) is a developmentally regulated chromosomal serpin that condenses chromatin in terminally differentiated avian blood cells. We show that MENT is an effective inhibitor of the papain-like cysteine proteinases cathepsins L and V. In addition, ectopic expression of MENT in mammalian cells is apparently sufficient to inhibit a nuclear papain-like cysteine proteinase and prevent degradation of the retinoblastoma protein, a major regulator of cell proliferation. MENT also accumulates in the nucleus, causes a strong block in proliferation, and promotes condensation of chromatin. Variants of MENT with mutations or deletions within the M-loop, which contains a nuclear localization signal and an AT-hook motif, reveal that this region mediates nuclear transport and morphological changes associated with chromatin condensation. Non-inhibitory mutants of MENT were constructed to determine whether its inhibitory activity has a role in blocking proliferation. These mutations changed the mode of association with chromatin and relieved the block in proliferation, without preventing transport to the nucleus. We conclude that the repressive effect of MENT on chromatin is mediated by its direct interaction with a nuclear protein that has a papain-like cysteine proteinase active site.
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- 2002
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25. Molecular Modeling of the Seven Tandem Leucine-Rich Repeats within the Ligand-Binding Region of Platelet Glycoprotein Ibα
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Whisstock, James C., Shen, Yang, López, José A., Andrews, Robert K., and Berndt, Michael C.
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- 2002
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26. The Serpins Are an Expanding Superfamily of Structurally Similar but Functionally Diverse Proteins
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Silverman, Gary A., Bird, Phillip I., Carrell, Robin W., Church, Frank C., Coughlin, Paul B., Gettins, Peter G.W., Irving, James A, Lomas, David A., Luke, Cliff J., Moyer, Richard W., Pemberton, Philip A., Remold-O'Donnell, Eileen, Salvesen, Guy S., Travis, James, and Whisstock, James C.
- Published
- 2001
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27. The Yeast Inositol Polyphosphate 5-Phosphatase Inp54p Localizes to the Endoplasmic Reticulum via a C-terminal Hydrophobic Anchoring Tail
- Author
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Wiradjaja, Fenny, Ooms, Lisa M., Whisstock, James C., McColl, Brad, Helfenbaum, Leon, Sambrook, Joseph F., Gething, Mary-Jane, and Mitchell, Christina A.
- Abstract
The budding yeast Saccharomyces cerevisiaehas four inositol polyphosphate 5-phosphatase (5-phosphatase) genes, INP51, INP52, INP53,andINP54, all of which hydrolyze phosphatidylinositol (4,5)-bisphosphate. INP54encodes a protein of 44 kDa which consists of a 5-phosphatase domain and a C-terminal leucine-rich tail, but lacks the N-terminal SacI domain and proline-rich region found in the other three yeast 5-phosphatases. We report that Inp54p belongs to the family of tail-anchored proteins and is localized to the endoplasmic reticulum via a C-terminal hydrophobic tail. The hydrophobic tail comprises the last 13 amino acids of the protein and is sufficient to target green fluorescent protein to the endoplasmic reticulum. Protease protection assays demonstrated that the N terminus of Inp54p is oriented toward the cytoplasm of the cell, with the C terminus of the protein also exposed to the cytosol. Null mutation ofINP54resulted in a 2-fold increase in secretion of a reporter protein, compared with wild-type yeast or cells deleted for any of the SacI domain-containing 5-phosphatases. We propose that Inp54p plays a role in regulating secretion, possibly by modulating the levels of phosphatidylinositol (4,5)-bisphosphate on the cytoplasmic surface of the endoplasmic reticulum membrane.
- Published
- 2001
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28. Protein structural alignments and functional genomics
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Irving, James A., Whisstock, James C., and Lesk, Arthur M.
- Abstract
Structural genomics—the systematic solution of structures of the proteins of an organism—will increasingly often produce molecules of unknown function with no close relative of known function. Prediction of protein function from structure has thereby become a challenging problem of computational molecular biology. The strong conservation of active site conformations in homologous proteins suggests a method for identifying them. This depends on the relationship between size and goodness‐of‐fit of aligned substructures in homologous proteins. For all pairs of proteins studied, the root‐mean‐square deviation (RMSD) as a function of the number of residues aligned varies exponentially for large common substructures and linearly for small common substructures. The exponent of the dependence at large common substructures is well correlated with the RMSD of the core as originally calculated by Chothia and Lesk (EMBO J 1986;5:823–826), affording the possibility of reconciling different structural alignment procedures. In the region of small common substructures, reduced aligned subsets define active sites and can be used to suggest the locations of active sites in homologous proteins. Proteins 2001;42:378–382. © 2000 Wiley‐Liss, Inc.
- Published
- 2001
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29. Structure of a Serpin-Enzyme Complex Probed by Cysteine Substitutions and Fluorescence Spectroscopy
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Ludeman, Justin P., Whisstock, James C., Hopkins, Paul C.R., Le Bonniec, Bernard F., and Bottomley, Stephen P.
- Abstract
The x-ray crystal structure of the serpin-proteinase complex is yet to be determined. In this study we have investigated the conformational changes that take place within antitrypsin during complex formation with catalytically inactive (thrombinS195A) and active thrombin. Three variants of antitrypsin Pittsburgh (an effective thrombin inhibitor), each containing a unique cysteine residue (Cys232, CysP3′, and Cys313) were covalently modified with the fluorescence probe N,N′-dimethyl-N-(iodoacetyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine. The presence of the fluorescent label did not affect the structure or inhibitory activity of the serpin. We monitored the changes in the fluorescence emission spectra of each labeled serpin in the native and cleaved state, and in complex with active and inactive thrombin. These data show that the serpin undergoes conformational change upon forming a complex with either active or inactive proteinase. Steady-state fluorescence quenching measurements using potassium iodide were used to further probe the nature and extent of this conformational change. A pronounced conformational change is observed upon locking with an active proteinase; however, our data reveal that docking with the inactive proteinase thrombinS195A is also able to induce a conformational change in the serpin.
- Published
- 2001
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30. Importance of the P4′ Residue in Human Granzyme B Inhibitors and Substrates Revealed by Scanning Mutagenesis of the Proteinase Inhibitor 9 Reactive Center Loop*
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Sun, Jiuru, Whisstock, James C., Harriott, Patrick, Walker, Brian, Novak, Andrea, Thompson, Philip E., Smith, A. Ian, and Bird, Phillip I.
- Abstract
The cytotoxic lymphocyte serine proteinase granzyme B induces apoptosis of abnormal cells by cleaving intracellular proteins at sites similar to those cleaved by caspases. Understanding the substrate specificity of granzyme B will help to identify natural targets and develop better inhibitors or substrates. Here we have used the interaction of human granzyme B with a cognate serpin, proteinase inhibitor 9 (PI-9), to examine its substrate sequence requirements. Cleavage and sequencing experiments demonstrated that Glu340is the P1 residue in the PI-9 RCL, consistent with the preference of granzyme B for acidic P1 residues. Ala-scanning mutagenesis demonstrated that the P4-P4′ region of the PI-9 RCL is important for interaction with granzyme B, and that the P4′ residue (Glu344) is required for efficient serpin-proteinase binding. Peptide substrates based on the P4-P4′ PI-9 RCL sequence and containing either P1 Glu or P1 Asp were cleaved by granzyme B (kcat/Km9.5 × 103and 1.2 × 105s−1m−1, respectively) but were not recognized by caspases. A substrate containing P1 Asp but lacking P4′ Glu was cleaved less efficiently (kcat/Km5.3 × 104s−1m−1). An idealized substrate comprising the previously described optimal P4-P1 sequence (Ile-Glu-Pro-Asp) fused to the PI-9 P1′-P4′ sequence was efficiently cleaved by granzyme B (kcat/Km7.5 × 105s−1m−1) and was also recognized by caspases. This contrasts with the literature value for a tetrapeptide comprising the same P4-P1 sequence (kcat/Km6.7 × 104s−1m−1) and confirms that P′ residues promote efficient interaction of granzyme B with substrates. Finally, molecular modeling predicted that PI-9 Glu344forms a salt bridge with Lys27of granzyme B, and we showed that a K27A mutant of granzyme B binds less efficiently to PI-9 and to substrates containing a P4′ Glu. We conclude that granzyme B requires an extended substrate sequence for specific and efficient binding and propose that an acidic P4′ substrate residue allows discrimination between early (high affinity) and late (lower affinity) targets during the induction of apoptosis.
- Published
- 2001
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31. The Inositol Polyphosphate 5-Phosphatases and the Apurinic/Apyrimidinic Base Excision Repair Endonucleases Share a Common Mechanism for Catalysis*
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Whisstock, James C., Romero, Susana, Gurung, Rajendra, Nandurkar, Harshal, Ooms, Lisa M., Bottomley, Stephen P., and Mitchell, Christina A.
- Abstract
Inositol polyphosphate 5-phosphatases (5-phosphatase) hydrolyze the 5-position phosphate from the inositol ring of phosphatidylinositol-derived signaling molecules; however, the mechanism of catalysis is only partially characterized. These enzymes play critical roles in regulating cell growth, apoptosis, intracellular calcium oscillations, and post-synaptic vesicular trafficking. The UCLA fold recognition server (threader) predicted that the conserved 300-amino acid catalytic domain, common to all 5-phosphatases, adopts the fold of the apurinic/apyrimidinic (AP) base excision repair endonucleases. PSI-BLAST searches of GENPEPT, using the amino acid sequence of AP endonuclease exonuclease III, identified all members of the 5-phosphatase family with highly significant scores. A sequence alignment between exonuclease III and all known 5-phosphatases revealed six highly conserved motifs containing residues that corresponded to the catalytic residues in the AP endonucleases. Mutation of each of these residues to alanine in the mammalian 43-kDa, or yeast Inp52p 5-phosphatase, resulted in complete loss of enzyme activity. We predict the 5-phosphatase enzymes share a similar mechanism of catalysis to the AP endonucleases, consistent with other common functional similarities such as an absolute requirement for magnesium for activity. Based on this analysis, functional roles have been assigned to conserved residues in all 5-phosphatase enzymes.
- Published
- 2000
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32. Conformational changes in serpins: II. the mechanism of activation of antithrombin by heparin†22Edited by J. Thornton†Paper I in this series is .
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Whisstock, James C., Pike, Robert N., Jin, Lei, Skinner, Richard, Pei, Xue Y., Carrell, Robin W., and Lesk, Arthur M.
- Abstract
Antithrombin, uniquely among plasma serpins acting as proteinase inhibitors in the control of the blood coagulation cascade, circulates in a relatively inactive form. Its activation by heparin, and specifically by a pentasaccharide core of heparin, has been shown to involve release of the peptide loop containing the reactive centre from partial insertion in the A sheet of the molecule. Here we compare the structures of the circulating inactive form of antithrombin with the activated structure in complex with heparin pentasaccharide. We show that the rearrangement of the reactive centre loop that occurs upon activation is part of a widespread conformational change involving a realignment of the two major domains of the molecule. We also examine natural mutants that possess high affinity for heparin pentasaccharide, and relate the kinetics of their interaction with heparin pentasaccharide to the structural transitions occuring in the activation process.
- Published
- 2000
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33. Conformational changes in serpins: I. the native and cleaved conformations of α1-antitrypsin11Edited by J. M. Thornton
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Whisstock, James C, Skinner, Richard, Carrell, Robin W, and Lesk, Arthur M
- Abstract
The serpins (SERine Proteinase INhibitors) are a family of proteins with important physiological roles, including but not limited to the inhibition of chymotrypsin-like serine proteinases. The inhibitory mechan- ism involves a large conformational change known as the S→R (stressed→relaxed) transition. The largest structural differences occur in a region around the scissile bond called the reactive centre loop: In the native (S) state, the reactive centre is exposed, and is free to interact with proteinases. In inhibitory serpins, in the cleaved (R) state the reactive centre loop forms an additional strand within the β-sheet. The latent state is an uncleaved state in which the intact reactive centre loop is integrated into the A sheet as in the cleaved form, to give an alternative R state.
- Published
- 2000
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34. Requirement of leucine-rich repeats of glycoprotein (GP) Ibα for shear-dependent and static binding of von Willebrand factor to the platelet membrane GP Ib–IX-V complex
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Shen, Yang, Romo, Gabriel M., Dong, Jing-fei, Schade, Alicia, McIntire, Larry V., Kenny, Dermot, Whisstock, James C., Berndt, Michael C., López, José A., and Andrews, Robert K.
- Abstract
The platelet glycoprotein (GP) Ib–IX-V complex mediates adhesion to von Willebrand factor (vWf) in (patho)physiologic thrombus formation. The vWf-binding site on GP Ib–IX-V is within the N-terminal 282 residues of GP Ib, which consist of an N-terminal flanking sequence (His-1–Ile-35), 7 leucine-rich repeats (Leu-36–Ala-200), a C-terminal flank (Phe-201–Gly-268), and a sulfated tyrosine sequence (Asp-269–Glu-282). We have used mammalian cell expression of canine–human chimeras of GP Ib, corresponding to precise structural boundaries, to demonstrate the first specific requirement for individual leucine-rich repeats for binding of vWf either induced by a modulator, ristocetin, or under hydrodynamic flow. Implicit in this approach was that the GP Ib chimeras retained a functional conformation, a supposition confirmed by analyzing restoration of function to reversed human–canine chimeras and demonstrating that all chimeras bound vWf activated by botrocetin, a modulator that is indiscriminate between species. Leucine-rich repeats 2, 3, and 4 of GP Ib were identified as being critical for vWf adhesion to GP Ib–IX-V.
- Published
- 2000
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35. Structure and function of the von Willebrand factor A1 domain: analysis with monoclonal antibodies reveals distinct binding sites involved in recognition of the platelet membrane glycoprotein Ib-IX-V complex and ristocetin-dependent activation
- Author
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De Luca, Mariagrazia, Facey, David A., Favaloro, Emmanuel J., Hertzberg, Mark S., Whisstock, James C., McNally, Tracy, Andrews, Robert K., and Berndt, Michael C.
- Abstract
Binding of the adhesive glycoprotein, von Willebrand factor (vWf), to the platelet membrane glycoprotein (GP) Ib-IX-V complex initiates platelet adhesion and aggregation at high shear stress in hemostasis and thrombosis. In this study, the GP Ib-IX-V binding site within the vWf A1 domain was analyzed using a panel of murine monoclonal antibodies raised against a 39/34-kd vWf fragment (Leu-480/Val-481–Gly-718) encompassing the A1 domain. One antibody, 6G1, strongly inhibited ristocetin-dependent vWf binding to platelets, but had no effect on botrocetin- or jaracetin-dependent binding, or asialo-vWf–dependent platelet aggregation. The 6G1 epitope was mapped to Glu-700–Asp-709, confirming the importance of this region for modulation of vWf by ristocetin. Like ristocetin, 6G1 activated the vWf A1 domain, because it enhanced binding of the 39/34-kd fragment to platelets. In contrast, 5D2 and CR1 completely inhibited asialo-vWf–induced platelet aggregation and ristocetin-induced vWf binding to GP Ib-IX-V. However, only 5D2 blocked botrocetin- and jaracetin-induced vWf binding to platelets and binding of vWf to botrocetin- and jaracetin-coated beads. Epitopes for 5D2 and CR1 were conformationally dependent, but not congruent. Other antibodies mapped to epitopes within the A1 domain (CR2 and CR15, Leu-494–Leu-512; CR2, Phe-536–Ala-554; CR3, Arg-578–Glu-596; CR11 and CR15, Ala-564–Ser-582) were not functional, identifying regions of the vWf A1 domain not directly involved in vWf-GP Ib-IX-V interaction. The combined results provide evidence that the proline-rich sequence Glu-700–Asp-709 constitutes a regulatory site for ristocetin, and that ristocetin and botrocetin induce, at least in part, separate receptor-recognition sites on vWf. (Blood. 2000;95:164-172)
- Published
- 2000
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36. Structural analysis of the PTEN:P-Rex2 signaling complex reveals how cancer-associated mutations coordinate to hyperactivate Rac1
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D’Andrea, Laura, Lucato, Christina M., Marquez, Elsa A., Chang, Yong-Gang, Civciristov, Srgjan, Mastos, Chantel, Lupton, Christopher J., Huang, Cheng, Elmlund, Hans, Schittenhelm, Ralf B., Mitchell, Christina A., Whisstock, James C., Halls, Michelle L., and Ellisdon, Andrew M.
- Published
- 2021
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37. α1-Antitrypsin Polymerisation Can Occur by both Loop A and C Sheet Mechanisms
- Author
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Bottomley, Stephen P., Hopkins, Paul C.R., and Whisstock, James C.
- Abstract
A number of disease states are attributable to α1-antitrypsin polymerisation within the endoplasmic reticulum of hepatocytes and subsequent plasma deficiency. Two distinct mechanisms describing the process of α1-antitrypsin polymerisation have been proposed, the loop A-sheet and C-sheet mechanisms. We report fluorescence studies using α1-antitrypsin covalently modified with pyrene maleimide. These results in conjunction with detailed molecular modelling studies, show that α1-antitrypsin is capable of undergoing both loop A-sheet and loop C-sheet mechanisms of polymerisation, depending upon thein vitrobuffer conditions.
- Published
- 1998
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38. Serpins in the Caenorhabditis elegansgenome
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Whisstock, James C., Irving, James A., Bottomley, Stephen P., Pike, Robert N., and Lesk, Arthur M.
- Abstract
Data mining in genome sequences can identify distant homologues of known protein families, and is most powerful if solved structures are available to reveal the three‐dimensional implications of very dissimilar sequences. Here we describe putative serpin sequences identified with very high statistical significance in the Caenorhabditis elegansgenome. When mapped onto vertebrate serpins such as α1‐antitrypsin, they suggest novel structural features. Some appear complete, some show extensive deletions, and others appear to contain only the C‐terminal part of the known serpin fold, probably in partnership with N‐terminal regions that have conformations unlike those of known serpins. The observation of such striking sequence similarity, in proteins that must have significantly different overall structures, substantially extends the structural characteristics of the serpin family of proteins. Proteins 1999;36:31–41. © 1999 Wiley‐Liss, Inc.
- Published
- 1999
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39. Serpins in the <TOGGLE>Caenorhabditis elegans</TOGGLE> genome
- Author
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Whisstock, James C., Irving, James A., Bottomley, Stephen P., Pike, Robert N., and Lesk, Arthur M.
- Abstract
Data mining in genome sequences can identify distant homologues of known protein families, and is most powerful if solved structures are available to reveal the three-dimensional implications of very dissimilar sequences. Here we describe putative serpin sequences identified with very high statistical significance in the Caenorhabditis elegans genome. When mapped onto vertebrate serpins such as α
1 -antitrypsin, they suggest novel structural features. Some appear complete, some show extensive deletions, and others appear to contain only the C-terminal part of the known serpin fold, probably in partnership with N-terminal regions that have conformations unlike those of known serpins. The observation of such striking sequence similarity, in proteins that must have significantly different overall structures, substantially extends the structural characteristics of the serpin family of proteins. Proteins 1999;36:3141. © 1999 Wiley-Liss, Inc.- Published
- 1999
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40. Expression and Purification of Recombinant Human Granzyme B from Pichia pastoris
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Sun, Jiuru, Bird, Catherina H., Buzza, Marguerite S., McKee, Katherine E., Whisstock, James C., and Bird, Phillip I.
- Abstract
Granzyme B is a cytotoxic lymphocyte granule serine proteinase that is pivotal in the induction of target cell apoptosis. Here we describe the expression of recombinant human granzyme B in Pichia pastorisas a chimeric zymogen comprising the α-factor signal sequence, a prodomain including an enterokinase cleavage site, and the mature granzyme B sequence followed by a hexahistidine tag. Inactive zymogen is purified from the medium by immobilized cobalt chelate affinity chromatography and then activated by enterokinase (final yield is approximately 1 mg per liter). The recombinant enzyme resembles native granzyme B in size and glycosylation, hydrolyzes the substrate Boc-Ala-Ala-Asp-thiobenzyl ester with equivalent efficiency (Km82 μM; kcat12 s−1), processes procaspase-3 to subunit form, and is inhibited by the cognate serpin PI-9. It efficiently induces DNA degradation and apoptosis of human cells. The availability of recombinant human granzyme B will facilitate further investigation of its structure and role in immune effector cells.
- Published
- 1999
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41. The 2.6 Å structure of antithrombin indicates a conformational change at the heparin binding site11Edited by R. Huber
- Author
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Skinner, Richard, Abrahams, Jan-Pieter, Whisstock, James C, Lesk, Arthur M, Carrell, Robin W, and Wardell, Mark R
- Abstract
The crystal structure of a dimeric form of intact antithrombin has been solved to 2.6 Å, representing the highest-resolution structure of an active, inhibitory serpin to date. The crystals were grown under microgravity conditions on Space Shuttle mission STS-67. The overall confidence in the structure, determined earlier from lower resolution data, is increased and new insights into the structure-function relationship are gained. Clear and continuous electron density is present for the reactive centre loop region P12 to P14 inserting into the top of the A-β-sheet. Areas of the extended amino terminus, unique to antithrombin and important in the binding of the glycosaminoglycan heparin, can now be traced further than in the earlier structures. As in the earlier studies, the crystals contain one active and one latent molecule per asymmetric unit. Better definition of the electron density surrounding the D-helix and of the residues implicated in the binding of the heparin pentasaccharide (Arg47, Lys114, Lys125, Arg129) provides an insight into the change of affinity of binding that accompanies the change in conformation. In particular, the observed hydrogen bonding of these residues to the body of the molecule in the latent form explains the mechanism for the release of newly formed antithrombin-protease complexes into the circulation for catabolic removal.
- Published
- 1997
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42. α1-Antitrypsin Mmalton (Phe52-deleted) Forms Loop-Sheet Polymers in Vivo.
- Author
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Lomas, David A., Elliott, Peter R., Sidhar, Sanjiv K., Foreman, Richard C., Finch, John T., Cox, Diane W., Whisstock, James C., and Carrell, Robin W.
- Abstract
The Z (Glu342→ Lys) and Siiyama (Ser53→ Phe) deficiency variants of α1-antitrypsin result in the retention of protein in the endoplasmic reticulum of the hepatocyte by loop-sheet polymerization in which the reactive center loop of one molecule is inserted into a β-pleated sheet of a second. We show here that antitrypsin Mmalton (Phe52-deleted), which is associated with the same liver inclusions, is also retained at an endoglycosidase H-sensitive stage of processing in the Xenopusoocyte and spontaneously forms polymers in vivo. These polymers, obtained from the plasma of an Mmalton/QO (null) bolton heterozygote, were much shorter than other antitrypsin polymers and contained a reactive center loop-cleaved species. Monomeric mutant antitrypsin was also isolated from the plasma. The monomeric component had a normal unfolding transition on transverse urea gradient gel electrophoresis and formed polymers in vitromore readily than M, but less readily than Z, antitrypsin. The A β-sheet accommodated a reactive center loop peptide much less readily than Z antitrypsin, which in turn was less receptive than native M antitrypsin. The nonreceptive conformation of the A sheet in antitrypsin Mmalton had little effect on kinetic parameters, the formation of SDS-stable complexes, the S to R transition, and the formation of the latent conformation.
- Published
- 1995
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43. Assembling the machinery of coagulation
- Author
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Whisstock, James C.
- Published
- 2013
- Full Text
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44. Structural and Functional Analysis of the Pore-Forming Toxin NetB from Clostridium perfringens
- Author
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Yan, Xu-Xia, Porter, Corrine J., Hardy, Simon P., Steer, David, Smith, A. Ian, Quinsey, Noelene S., Hughes, Victoria, Cheung, Jackie K., Keyburn, Anthony L., Kaldhusdal, Magne, Moore, Robert J., Bannam, Trudi L., Whisstock, James C., and Rood, Julian I.
- Abstract
ABSTRACTClostridium perfringensis an anaerobic bacterium that causes numerous important human and animal diseases, primarily as a result of its ability to produce many different protein toxins. In chickens, C. perfringenscauses necrotic enteritis, a disease of economic importance to the worldwide poultry industry. The secreted pore-forming toxin NetB is a key virulence factor in the pathogenesis of avian necrotic enteritis and is similar to alpha-hemolysin, a β-barrel pore-forming toxin from Staphylococcus aureus. To address the molecular mechanisms underlying NetB-mediated tissue damage, we determined the crystal structure of the monomeric form of NetB to 1.8 Å. Structural comparisons with other members of the alpha-hemolysin family revealed significant differences in the conformation of the membrane binding domain. These data suggested that NetB may recognize different membrane receptors or use a different mechanism for membrane-protein interactions. Consistent with this idea, electrophysiological experiments with planar lipid bilayers revealed that NetB formed pores with much larger single-channel conductance than alpha-hemolysin. Channel conductance varied with phospholipid net charge. Furthermore, NetB differed in its ion selectivity, preferring cations over anions. Using hemolysis as a screen, we carried out a random-mutagenesis study that identified several residues that are critical for NetB-induced cell lysis. Mapping of these residues onto the crystal structure revealed that they were clustered in regions predicted to be required for oligomerization or membrane binding. Together these data provide an insight into the mechanism of NetB-mediated pore formation and will contribute to our understanding of the mode of action of this important toxin.IMPORTANCENecrotic enteritis is an economically important disease of the worldwide poultry industry and is mediated by Clostridium perfringensstrains that produce NetB, a β-pore-forming toxin. We carried out structural and functional studies of NetB to provide a mechanistic insight into its mode of action and to assist in the development of a necrotic enteritis vaccine. We determined the structure of the monomeric form of NetB to 1.8 Å, used both site-directed and random mutagenesis to identify key residues that are required for its biological activity, and analyzed pore formation by NetB and its substitution-containing derivatives in planar lipid bilayers.
- Published
- 2013
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45. A Novel DNM2 Mutation Displaying Embryonic Lethality and Impaired Transferrin Uptake Identified in a Mouse ENU Mutagenesis Screen for Genes Perturbing Erythropoiesis
- Author
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Brown, Fiona C, Collett, Michael, Robinson, Phillip J, Whisstock, James C, Hilton, Douglas J., Kile, Benjamin T., Jane, Stephen M, and Curtis, David J.
- Abstract
No relevant conflicts of interest to declare.
- Published
- 2012
- Full Text
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46. Structural biology: Torqueing about pores.
- Author
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Whisstock JC and Dunstone MA
- Subjects
- Aeromonas chemistry, Bacterial Toxins chemistry, Ion Channels chemistry, Protein Precursors chemistry
- Abstract
Cryo-EM, crystallography, biochemical experiments and computational approaches have been used to study different intermediate states of the Aeromonas hydrophila toxin aerolysin en route to pore formation. These results reveal that an unexpected and marked rotation of the core aerolysin machinery is required to unleash the membrane-spanning regions.
- Published
- 2013
- Full Text
- View/download PDF
47. Structural biology: Serpins' mystery solved.
- Author
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Whisstock JC and Bottomley SP
- Subjects
- Amyloid chemistry, Amyloid metabolism, Animals, Biopolymers chemistry, Biopolymers metabolism, Crystallography, X-Ray, Dimerization, Humans, Models, Molecular, Protein Conformation, Protein Folding, Antithrombin III chemistry, Antithrombin III metabolism
- Published
- 2008
- Full Text
- View/download PDF
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