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31 results on '"Hewitt, Lorraine"'

1. IL-17–high asthma with features of a psoriasis immunophenotype

Author

Adcock, I.M., Ahmed, H., Auffray, C., Bakke, P., Bansal, A.T., Baribaud, F., Bates, S., Bel, E.H., Bigler, J., Bisgaard, H., Boedigheimer, M.J., Bønnelykke, K., Brandsma, J., Brinkman, P., Bucchioni, E., Burg, D., Bush, A., Caruso, M., Chaiboonchoe, A., Chanez, P., Chung, K.F., Compton, C.H., Corfield, J., D'Amico, A., Dahlen, S.E., De Meulder, B., Djukanovic, R., Erpenbeck, V.J., Erzen, D., Fichtner, K., Fitch, N., Fleming, L.J., Formaggio, E., Fowler, S.J., Frey, U., Gahlemann, M., Geiser, T., Guo, Y., Hashimoto, S., Haughney, J., Hedlin, G., Hekking, P.W., Higenbottam, T., Hohlfeld, J.M., Holweg, C., Horváth, I., Howarth, P., James, A.J., Knowles, R., Knox, A.J., Krug, N., Lefaudeux, D., Loza, M.J., Lutter, R., Manta, A., Masefield, S., Mazein, A., Meiser, A., Middelveld, R.J.M., Miralpeix, M., Montuschi, P., Mores, N., Murray, C.S., Musial, J., Myles, D., Pahus, L., Pandis, I., Pavlidis, S., Powell, P., Praticò, G., Rao, M. Puig N., Riley, J., Roberts, A., Roberts, G., Rowe, A., Sandström, T., Seibold, W., Selby, A., Shaw, D.E., Sigmund, R., Singer, F., Skipp, P.J., Sousa, A.R., Sterk, P.J., Sun, K., Thornton, B., van Aalderen, W.M., van Geest, M., Vestbo, J., Vissing, N.H., Wagener, A.H., Wagers, S.S., Weiszhart, Z., Wheelock, C.E., Wilson, S.J., Aliprantis, Antonios, Allen, David, Alving, Kjell, Badorrek, P., Balgoma, David, Ballereau, S., Barber, Clair, Batuwitage, Manohara Kanangana, Bautmans, A., Bedding, A., Behndig, A.F., Beleta, Jorge, Berglind, A., Berton, A., Bochenek, Grazyna, Braun, Armin, Campagna, D., Carayannopoulos, Leon, Casaulta, C., Chaleckis, Romanas, Dahlén, B., Davison, imothy, De Alba, Jorge, De Lepeleire, Inge, Dekker, Tamara, Delin, Ingrid, Dennison, P., Dijkhuis, Annemiek, Dodson, Paul, Draper, Aleksandra, Dyson, K., Edwards, Jessica, El Hadjam, L., Emma, Rosalia, Ericsson, Magnus, Faulenbach, C., Flood, Breda, Galffy, G., Gallart, Hector, Garissi, D., Gent, J., Gerhardsson de Verdier, M., Gibeon, D., Gomez, Cristina, Gove, Kerry, Gozzard, Neil, Guillmant-Farry, E., Henriksson, E., Hewitt, Lorraine, Hoda, U., Hu, Richard, Hu, Sile, Hu, X., Jeyasingham, E., Johnson, K., Jullian, N., Kamphuis, Juliette, Kennington, Erika J., Kerry, Dyson, Kerry, G., Klüglich, M., Knobel, Hugo, Kolmert, Johan, Konradsen, J.R., Kots, Maxim, Kretsos, Kosmas, Krueger, L., Kuo, Scott, Kupczyk, Maciej, Lambrecht, Bart, Lantz, A.-S., Larminie, Christopher, Larsson, L.X., Latzin, P., Lazarinis, N., Lemonnier, N., Lone-Latif, Saeeda, Lowe, L.A., Manta, Alexander, Marouzet, Lisa, Martin, Jane, Mathon, Caroline, McEvoy, L., Meah, Sally, Menzies-Gow, A., Metcalf, Leanne, Mikus, Maria, Monk, Philip, Naz, Shama, Nething, K., Nicholas, Ben, Nihlén, U., Nilsson, Peter, Niven, R., Nordlund, B., Nsubuga, S., Pacino, Antonio, Palkonen, Susanna, Pellet, J., Pennazza, Giorgio, Petrén, Anne, Pink, Sandy, Pison, C., Postle, Anthony, Rahman-Amin, Malayka, Ravanetti, Lara, Ray, Emma, Reinke, Stacey, Reynolds, Leanne, Riemann, K., Robberechts, Martine, Rocha, J.P., Rossios, C., Russell, Kirsty, Rutgers, Michael, Santini, G., Santoninco, Marco, Saqi, M., Schoelch, Corinna, Schofield, James P.R., Scott, S., Sehgal, N., Sjödin, Marcus, Smids, Barbara, Smith, Caroline, Smith, Jessica, Smith, Katherine M., Söderman, P., Sogbessan, A., Spycher, F., Staykova, Doroteya, Stephan, S., Stokholm, J., Strandberg, K., Sunther, M., Szentkereszty, M., Tamasi, L., Tariq, K., Thörngren, John-Olof, Thorsen, Jonathan, Valente, S., van de Pol, Marianne, van Drunen, C.M., Van Eyll, Jonathan, Versnel, Jenny, Vink, Anton, von Garnier, C., Vyas, A., Wald, Frans, Walker, Samantha, Ward, Jonathan, Wetzel, Kristiane, Wiegman, Coen, Williams, Siân, Yang, Xian, Yeyasingham, Elizabeth, Amgen, W. Yu, Zetterquist, W., Zolkipli, Z., Zwinderman, A.H., Östling, Jörgen, van Geest, Marleen, Jevnikar, Zala, Wilson, Susan, Lutter, Rene, Shaw, Dominick E., Bakke, Per S., Caruso, Massimo, Dahlen, Sven-Erik, Fowler, Stephen J., Horváth, Ildikó, Krug, Norbert, Montuschi, Paolo, Sanak, Marek, Sandström, Thomas, Sun, Kai, Pandis, Ioannis, Auffray, Charles, Sousa, Ana R., Guo, Yike, Adcock, Ian M., Howarth, Peter, Chung, Kian Fan, Bigler, Jeanette, Sterk, Peter J., Skipp, Paul J., Djukanović, Ratko, and Vaarala, Outi

Published
2019
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3. A human mitochondrial poly(A) polymerase mutation reveals the complexities of post-transcriptional mitochondrial gene expression

Author

Wilson, William C., Hornig-Do, Hue-Tran, Bruni, Francesco, Chang, Jeong Ho, Jourdain, Alexis A., Martinou, Jean-Claude, Falkenberg, Maria, Spåhr, Henrik, Larsson, Nils-Göran, Lewis, Richard J., Hewitt, Lorraine, Baslé, Arnaud, Cross, Harold E., Tong, Liang, Lebel, Robert R., Crosby, Andrew H., Chrzanowska-Lightowlers, Zofia M. A., and Lightowlers, Robert N.

Published
2014
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4. Detection of exacerbations in asthma based on electronic diary data: results from the 1-year prospective BIOAIR study

Author

Kupczyk, Maciej, Haque, Shushila, Sterk, Peter J, Niżankowska-Mogilnicka, Ewa, Papi, Alberto, Bel, Elisabeth H, Chanez, Pascal, Dahlén, Barbro, Gaga, Mina, Gjomarkaj, Mark, Howarth, Peter H, Johnston, Sebastian L, Joos, Guy F, Kanniess, Frank, Tzortzaki, Eleni, James, Anna, Middelveld, Roelinde J M, Dahlén, Sven-Erik, Weersink, Els, Papadopoulos, Nikos, Oikonomidou, Erasmia, Zervas, Eleftherios, Contoli, Marco, Pauwels, Romain A., Brusselle, Guy, de Rudder, Isabelle, Schelfhout, Vanessa, Richter, Kai, Gerding, Daisy, Magnussen, Helgo, Siafakas, Nikos M., Samara, Katerina, Plataki, Maria, Papadopouli, Eva, Szczeklik, Andrzej, Ziolkowska-Graca, Bozena, Kania, Aleksander, Gawlewicz-Mroczka, Agnieszka, Duplaga, Mariusz, Figiel, Ewa, Rabe, Klaus F., Hiemstra, Pieter S., Gauw, Stefanie, van Veen, Ilonka, Kips, Johan C., Mallia, Patrick, Campbell, Deborah A, Robinson, Douglas S, Fabbri, Leo M., Romagnoli, Micaela, Vachier, Isabelle, Devautour, Catherine, Meziane, Lahouari, Maurizio Vignola, A., Pace, Elisabetta, Profita, Mirella, Holgate, Stephen T., Wilson, Susan J., Hewitt, Lorraine, Holoway, John, Damm, Katarina, Delin, Ingrid, Eduards, Marianne, Ek, Alexandra, Ekström, Tommy, Gülich, Agneta, Johansson, Lovisa E., Karlsson, Östen, Kumlin, Maria, Martling, Ingrid, and Skedinger, Maria

Published
2013
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6. Crystal structure of NucB, a biofilm-degrading endonuclease

Author

Baslé, Arnaud, Hewitt, Lorraine, Koh, Alan, Lamb, Heather K, Thompson, Paul, Burgess, J Grant, Hall, Michael J, Hawkins, Alastair R, Murray, Heath, and Lewis, Richard J

Subjects
Models, Molecular, Deoxyribonucleases, Bacterial Proteins, Structural Biology, Protein Conformation, Biofilms, Bacillus licheniformis, DNA, Crystallography, X-Ray
Abstract

Bacterial biofilms are a complex architecture of cells that grow on moist interfaces, and are held together by a molecular glue of extracellular proteins, sugars and nucleic acids. Biofilms are particularly problematic in human healthcare as they can coat medical implants and are thus a potential source of disease. The enzymatic dispersal of biofilms is increasingly being developed as a new strategy to treat this problem. Here, we have characterized NucB, a biofilm-dispersing nuclease from a marine strain of Bacillus licheniformis, and present its crystal structure together with the biochemistry and a mutational analysis required to confirm its active site. Taken together, these data support the categorization of NucB into a unique subfamily of the ββα metal-dependent non-specific endonucleases. Understanding the structure and function of NucB will facilitate its future development into an anti-biofilm therapeutic agent.

Published
2017

7. Stratification of asthma phenotypes by airway proteomic signatures

Author

Schofield, James P. R., Burg, Dominic, Nicholas, Ben, Strazzeri, Fabio, Brandsma, Joost, Staykova, Doroteya, Folisi, Caterina, Bansal, Aruna T., Xian, Yang, Guo, Yike, Rowe, Anthony, Corfield, Julie, Wilson, Susan, Ward, Jonathan, Lutter, Rene, Shaw, Dominick E., Bakke, Per S., Caruso, Massimo, Dahlen, Sven-Erik, Fowler, Stephen J., Horvath, Ildiko, Howarth, Peter, Krug, Norbert, Montuschi, Paolo, Sanak, Marek, Sandstrom, Thomas, Sun, Kai, Pandis, Ioannis, Riley, John, Auffray, Charles, De Meulder, Bertrand, Lefaudeux, Diane, Sousa, Ana R., Adcock, Ian M., Chung, Kian Fan, Sterk, Peter J., Skipp, Paul J., Djukanovic, Ratko, Ahmed, H., Allen, D., Badorrek, P., Ballereau, S., Baribaud, F., Batuwitage, M. K., Bedding, A., Behndig, A. F., Berglind, A., Berton, A., Bigler, J., Boedigheimer, M. J., Bonnelykke, K., Brinkman, P., Bush, A., Campagna, D., Casaulta, C., Chaiboonchoe, A., Davison, T., De Meulder, B., Delin, I., Dennison, P., Dodson, P., El Hadjam, L., Erzen, D., Faulenbach, C., Fichtner, K., Fitch, N., Formaggio, E., Gahlemann, M., Galffy, G., Garissi, D., Garret, T., Gent, J., Guillmant-Farry, E., Henriksson, E., Hoda, U., Hohlfeld, J. M., Hu, X., James, A., Johnson, K., Jullian, N., Kerry, G., Klueglich, M., Knowles, R., Konradsen, J. R., Kretsos, K., Krueger, L., Lantz, A. -S, Larminie, C., Latzin, P., Lefaudeux, D., Lemonnier, N., Lowe, L. A., Lutter, R., Manta, A., Mazein, A., McEvoy, L., Menzies-Gow, A., Mores, N., Murray, C. S., Nething, K., Nihlen, U., Niven, R., Nordlund, B., Nsubuga, S., Pellet, J., Pison, C., Pratico, G., Puig Valls, M., Riemann, K., Rocha, J. P., Rossios, C., Santini, G., Saqi, M., Scott, S., Sehgal, N., Selby, A., Soderman, P., Sogbesan, A., Spycher, F., Stephan, S., Stokholm, J., Sunther, M., Szentkereszty, M., Tamasi, L., Tariq, K., Valente, S., van Aalderen, W. M., van Drunen, C. M., Van Eyll, J., Vyas, A., Yu, W., Zetterquist, W., Zolkipli, Z., Zwinderman, A. H., Adriaens, Nora, Aliprantis, Antonios, Alving, Kjell, Bakke, Per, Balgoma, David, Barber, Clair, Baribaud, Frederic, Bates, Stewart, Bautmans, An, Beleta, Jorge, Bochenek, Grazyna, Braun, Armin, Carayannopoulos, Leon, Rocha, Joao Pedro Carvalho da Purificacao, Chaleckis, Romanas, D'Amico, Arnaldo, De Alba, Jorge, De Lepeleire, Inge, Dekker, Tamara, Dijkhuis, Annemiek, Draper, Aleksandra, Edwards, Jessica, Emma, Rosalia, Ericsson, Magnus, Flood, Breda, Gallart, Hector, Gomez, Cristina, Gove, Kerry, Gozzard, Neil, Haughney, John, Hewitt, Lorraine, Hohlfeld, Jens, Holweg, Cecile, Hu, Richard, Hu, Sile, Kamphuis, Juliette, Kennington, Erika J., Kerry, Dyson, Knobel, Hugo, Kolmert, Johan, Kots, Maxim, Kuo, Scott, Kupczyk, Maciej, Lambrecht, Bart, Lone-Latif, Saeeda, Loza, Matthew J., Marouzet, Lisa, Martin, Jane, Masefield, Sarah, Mathon, Caroline, Meah, Sally, Meiser, Andrea, Metcalf, Leanne, Mikus, Maria, Miralpeix, Montse, Monk, Philip, Naz, Shama, Nilsson, Peter, Ostling, Jorgen, Pacino, Antonio, Palkonen, Susanna, Pavlidis, Stelios, Pennazza, Giorgio, Petren, Anne, Pink, Sandy, Postle, Anthony, Powell, Pippa, Rahman-Amin, Malayka, Rao, Navin, Ravanetti, Lara, Ray, Emma, Reinke, Stacey, Reynolds, Leanne, Robberechts, Martine, Roberts, Amanda, Russell, Kirsty, Rutgers, Michael, Santoninco, Marco, Schoelch, Corinna, Sjodin, Marcus, Smids, Barbara, Smith, Caroline, Smith, Jessica, Smith, Katherine M., Thorngren, John-Olof, Thornton, Bob, Thorsen, Jonathan, van de Pol, Marianne, van Geest, Marleen, Versnel, Jenny, Vink, Anton, Wald, Frans, Walker, Samantha, Weiszhart, Zsoka, Wetzel, Kristiane, Wheelock, Craig E., Wiegman, Coen, Williams, Sian, Wilson, Susan J., Woodcock, Ashley, Yang, Xian, Yeyasingham, Elizabeth, Prins, Jan-Bas, Gahlemann, Martina, Visintin, Luigi, Evans, Hazel, Puhl, Martine, Buzermaniene, Lina, Hudson, Val, Bond, Laura, de Boer, Pim, Widdershoven, Guy, Sigmund, Ralf, Supple, David, Hamerlijnck, Dominique, Negus, Jenny, Kamphuis, Julitte, Sergison, Lehanne, Onstein, Susanne, MacNee, William, Bernardini, Renato, Bont, Louis, Wecksell, Per-Ake, Graduate School, AII - Inflammatory diseases, Pulmonology, Ear, Nose and Throat, Epidemiology and Data Science, APH - Methodology, Publica, and Commission of the European Communities

Subjects
0301 basic medicine, Male, Proteomics, Allergy, Proteome, Neutrophils, Respiratory Medicine and Allergy, Transcriptome, 0302 clinical medicine, neutrophils, Forced Expiratory Volume, Immunology and Allergy, CD44, 610 Medicine & health, Lungmedicin och allergi, phenotypes, Middle Aged, medicine.anatomical_structure, Phenotype, 1107 Immunology, Female, eosinophils, medicine.symptom, Life Sciences & Biomedicine, Adult, Settore BIO/14 - FARMACOLOGIA, Immunology, Computational biology, 03 medical and health sciences, Young Adult, proteomics, Eosinophilia, medicine, Humans, U-BIOPRED Study Group, Asthma, Aged, Science & Technology, Microarray analysis techniques, business.industry, Sputum, biomarkers, DEGRADATION, Eosinophil, medicine.disease, Eosinophils, EXACERBATIONS, 030104 developmental biology, 030228 respiratory system, business
Abstract

Background: Stratification by eosinophil and neutrophil counts increases our understanding of asthma and helps target therapy, but there is room for improvement in our accuracy in prediction of treatment responses and a need for better understanding of the underlying mechanisms. Objective: We sought to identify molecular subphenotypes of asthma defined by proteomic signatures for improved stratification. Methods: Unbiased label-free quantitative mass spectrometry and topological data analysis were used to analyze the proteomes of sputum supernatants from 246 participants (206 asthmatic patients) as a novel means of asthma stratification. Microarray analysis of sputum cells provided transcriptomics data additionally to inform on underlying mechanisms. Results: Analysis of the sputum proteome resulted in 10 clusters (ie, proteotypes) based on similarity in proteomic features, representing discrete molecular subphenotypes of asthma. Overlaying granulocyte counts onto the 10 clusters as metadata further defined 3 of these as highly eosinophilic, 3 as highly neutrophilic, and 2 as highly atopic with relatively low granulocytic inflammation. For each of these 3 phenotypes, logistic regression analysis identified candidate protein biomarkers, and matched transcriptomic data pointed to differentially activated underlying mechanisms. Conclusion: This study provides further stratification of asthma currently classified based on quantification of granulocytic inflammation and provided additional insight into their underlying mechanisms, which could become targets for novel therapies. asthma proteomics biomarkers eosinophils neutrophils

Published
2019

8. IL-17–high asthma with features of a psoriasis immunophenotype

Author

Östling, Jörgen, primary, van Geest, Marleen, additional, Schofield, James P.R., additional, Jevnikar, Zala, additional, Wilson, Susan, additional, Ward, Jonathan, additional, Lutter, Rene, additional, Shaw, Dominick E., additional, Bakke, Per S., additional, Caruso, Massimo, additional, Dahlen, Sven-Erik, additional, Fowler, Stephen J., additional, Horváth, Ildikó, additional, Krug, Norbert, additional, Montuschi, Paolo, additional, Sanak, Marek, additional, Sandström, Thomas, additional, Sun, Kai, additional, Pandis, Ioannis, additional, Auffray, Charles, additional, Sousa, Ana R., additional, Guo, Yike, additional, Adcock, Ian M., additional, Howarth, Peter, additional, Chung, Kian Fan, additional, Bigler, Jeanette, additional, Sterk, Peter J., additional, Skipp, Paul J., additional, Djukanović, Ratko, additional, Vaarala, Outi, additional, Adcock, I.M., additional, Ahmed, H., additional, Auffray, C., additional, Bakke, P., additional, Bansal, A.T., additional, Baribaud, F., additional, Bates, S., additional, Bel, E.H., additional, Bigler, J., additional, Bisgaard, H., additional, Boedigheimer, M.J., additional, Bønnelykke, K., additional, Brandsma, J., additional, Brinkman, P., additional, Bucchioni, E., additional, Burg, D., additional, Bush, A., additional, Caruso, M., additional, Chaiboonchoe, A., additional, Chanez, P., additional, Chung, K.F., additional, Compton, C.H., additional, Corfield, J., additional, D'Amico, A., additional, Dahlen, S.E., additional, De Meulder, B., additional, Djukanovic, R., additional, Erpenbeck, V.J., additional, Erzen, D., additional, Fichtner, K., additional, Fitch, N., additional, Fleming, L.J., additional, Formaggio, E., additional, Fowler, S.J., additional, Frey, U., additional, Gahlemann, M., additional, Geiser, T., additional, Guo, Y., additional, Hashimoto, S., additional, Haughney, J., additional, Hedlin, G., additional, Hekking, P.W., additional, Higenbottam, T., additional, Hohlfeld, J.M., additional, Holweg, C., additional, Horváth, I., additional, Howarth, P., additional, James, A.J., additional, Knowles, R., additional, Knox, A.J., additional, Krug, N., additional, Lefaudeux, D., additional, Loza, M.J., additional, Lutter, R., additional, Manta, A., additional, Masefield, S., additional, Mazein, A., additional, Meiser, A., additional, Middelveld, R.J.M., additional, Miralpeix, M., additional, Montuschi, P., additional, Mores, N., additional, Murray, C.S., additional, Musial, J., additional, Myles, D., additional, Pahus, L., additional, Pandis, I., additional, Pavlidis, S., additional, Powell, P., additional, Praticò, G., additional, Rao, M. Puig N., additional, Riley, J., additional, Roberts, A., additional, Roberts, G., additional, Rowe, A., additional, Sandström, T., additional, Seibold, W., additional, Selby, A., additional, Shaw, D.E., additional, Sigmund, R., additional, Singer, F., additional, Skipp, P.J., additional, Sousa, A.R., additional, Sterk, P.J., additional, Sun, K., additional, Thornton, B., additional, van Aalderen, W.M., additional, van Geest, M., additional, Vestbo, J., additional, Vissing, N.H., additional, Wagener, A.H., additional, Wagers, S.S., additional, Weiszhart, Z., additional, Wheelock, C.E., additional, Wilson, S.J., additional, Aliprantis, Antonios, additional, Allen, David, additional, Alving, Kjell, additional, Badorrek, P., additional, Balgoma, David, additional, Ballereau, S., additional, Barber, Clair, additional, Batuwitage, Manohara Kanangana, additional, Bautmans, A., additional, Bedding, A., additional, Behndig, A.F., additional, Beleta, Jorge, additional, Berglind, A., additional, Berton, A., additional, Bochenek, Grazyna, additional, Braun, Armin, additional, Campagna, D., additional, Carayannopoulos, Leon, additional, Casaulta, C., additional, Chaleckis, Romanas, additional, Dahlén, B., additional, Davison, imothy, additional, De Alba, Jorge, additional, De Lepeleire, Inge, additional, Dekker, Tamara, additional, Delin, Ingrid, additional, Dennison, P., additional, Dijkhuis, Annemiek, additional, Dodson, Paul, additional, Draper, Aleksandra, additional, Dyson, K., additional, Edwards, Jessica, additional, El Hadjam, L., additional, Emma, Rosalia, additional, Ericsson, Magnus, additional, Faulenbach, C., additional, Flood, Breda, additional, Galffy, G., additional, Gallart, Hector, additional, Garissi, D., additional, Gent, J., additional, Gerhardsson de Verdier, M., additional, Gibeon, D., additional, Gomez, Cristina, additional, Gove, Kerry, additional, Gozzard, Neil, additional, Guillmant-Farry, E., additional, Henriksson, E., additional, Hewitt, Lorraine, additional, Hoda, U., additional, Hu, Richard, additional, Hu, Sile, additional, Hu, X., additional, Jeyasingham, E., additional, Johnson, K., additional, Jullian, N., additional, Kamphuis, Juliette, additional, Kennington, Erika J., additional, Kerry, Dyson, additional, Kerry, G., additional, Klüglich, M., additional, Knobel, Hugo, additional, Kolmert, Johan, additional, Konradsen, J.R., additional, Kots, Maxim, additional, Kretsos, Kosmas, additional, Krueger, L., additional, Kuo, Scott, additional, Kupczyk, Maciej, additional, Lambrecht, Bart, additional, Lantz, A.-S., additional, Larminie, Christopher, additional, Larsson, L.X., additional, Latzin, P., additional, Lazarinis, N., additional, Lemonnier, N., additional, Lone-Latif, Saeeda, additional, Lowe, L.A., additional, Manta, Alexander, additional, Marouzet, Lisa, additional, Martin, Jane, additional, Mathon, Caroline, additional, McEvoy, L., additional, Meah, Sally, additional, Menzies-Gow, A., additional, Metcalf, Leanne, additional, Mikus, Maria, additional, Monk, Philip, additional, Naz, Shama, additional, Nething, K., additional, Nicholas, Ben, additional, Nihlén, U., additional, Nilsson, Peter, additional, Niven, R., additional, Nordlund, B., additional, Nsubuga, S., additional, Pacino, Antonio, additional, Palkonen, Susanna, additional, Pellet, J., additional, Pennazza, Giorgio, additional, Petrén, Anne, additional, Pink, Sandy, additional, Pison, C., additional, Postle, Anthony, additional, Rahman-Amin, Malayka, additional, Ravanetti, Lara, additional, Ray, Emma, additional, Reinke, Stacey, additional, Reynolds, Leanne, additional, Riemann, K., additional, Robberechts, Martine, additional, Rocha, J.P., additional, Rossios, C., additional, Russell, Kirsty, additional, Rutgers, Michael, additional, Santini, G., additional, Santoninco, Marco, additional, Saqi, M., additional, Schoelch, Corinna, additional, Scott, S., additional, Sehgal, N., additional, Sjödin, Marcus, additional, Smids, Barbara, additional, Smith, Caroline, additional, Smith, Jessica, additional, Smith, Katherine M., additional, Söderman, P., additional, Sogbessan, A., additional, Spycher, F., additional, Staykova, Doroteya, additional, Stephan, S., additional, Stokholm, J., additional, Strandberg, K., additional, Sunther, M., additional, Szentkereszty, M., additional, Tamasi, L., additional, Tariq, K., additional, Thörngren, John-Olof, additional, Thorsen, Jonathan, additional, Valente, S., additional, van de Pol, Marianne, additional, van Drunen, C.M., additional, Van Eyll, Jonathan, additional, Versnel, Jenny, additional, Vink, Anton, additional, von Garnier, C., additional, Vyas, A., additional, Wald, Frans, additional, Walker, Samantha, additional, Wetzel, Kristiane, additional, Wiegman, Coen, additional, Williams, Siân, additional, Yang, Xian, additional, Yeyasingham, Elizabeth, additional, Amgen, W. Yu, additional, Zetterquist, W., additional, Zolkipli, Z., additional, and Zwinderman, A.H., additional

Published
2019
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11. A human mitochondrial poly(A) polymerase mutation reveals the complexities of post-transcriptional mitochondrial gene expression

Author

Wilson, William C., Hornig-Do, Hue-Tran, Bruni, Francesco, Chang, Jeong Ho, Jourdain, Alexis A., Martinou, Jean-Claude, Falkenberg, Maria, Spåhr, Henrik, Larsson, Nils-Göran, Lewis, Richard J., Hewitt, Lorraine, Baslé, Arnaud, Cross, Harold E., Tong, Liang, Lebel, Robert R., Crosby, Andrew H., Chrzanowska-Lightowlers, Zofia M. A., Lightowlers, Robert N., Wilson, William C., Hornig-Do, Hue-Tran, Bruni, Francesco, Chang, Jeong Ho, Jourdain, Alexis A., Martinou, Jean-Claude, Falkenberg, Maria, Spåhr, Henrik, Larsson, Nils-Göran, Lewis, Richard J., Hewitt, Lorraine, Baslé, Arnaud, Cross, Harold E., Tong, Liang, Lebel, Robert R., Crosby, Andrew H., Chrzanowska-Lightowlers, Zofia M. A., and Lightowlers, Robert N.

Abstract

The p.N478D missense mutation in human mitochondrial poly(A) polymerase (mtPAP) has previously been implicated in a form of spastic ataxia with optic atrophy. In this study, we have investigated fibroblast cell lines established from family members. The homozygous mutation resulted in the loss of polyadenylation of all mitochondrial transcripts assessed; however, oligoadenylation was retained. Interestingly, this had differential effects on transcript stability that were dependent on the particular species of transcript. These changes were accompanied by a severe loss of oxidative phosphorylation complexes I and IV, and perturbation of de novo mitochondrial protein synthesis. Decreases in transcript polyadenylation and in respiratory chain complexes were effectively rescued by overexpression of wild-type mtPAP. Both mutated and wild-type mtPAP localized to the mitochondrial RNA-processing granules thereby eliminating mislocalization as a cause of defective polyadenylation. In vitro polyadenylation assays revealed severely compromised activity by the mutated protein, which generated only short oligo(A) extensions on RNA substrates, irrespective of RNA secondary structure. The addition of LRPPRC/SLIRP, a mitochondrial RNA-binding complex, enhanced activity of the wild-type mtPAP resulting in increased overall tail length. The LRPPRC/SLIRP effect although present was less marked with mutated mtPAP, independent of RNA secondary structure. We conclude that (i) the polymerase activity of mtPAP can be modulated by the presence of LRPPRC/SLIRP, (ii) N478D mtPAP mutation decreases polymerase activity and (iii) the alteration in poly(A) length is sufficient to cause dysregulation of post-transcriptional expression and the pathogenic lack of respiratory chain complexes

Published
2017

12. U-BIOPRED clinical adult asthma clusters linked to a subset of sputum omics

Author

Lefaudeux, Diane, primary, De Meulder, Bertrand, additional, Loza, Matthew J., additional, Peffer, Nancy, additional, Rowe, Anthony, additional, Baribaud, Frédéric, additional, Bansal, Aruna T., additional, Lutter, Rene, additional, Sousa, Ana R., additional, Corfield, Julie, additional, Pandis, Ioannis, additional, Bakke, Per S., additional, Caruso, Massimo, additional, Chanez, Pascal, additional, Dahlén, Sven-Erik, additional, Fleming, Louise J., additional, Fowler, Stephen J., additional, Horvath, Ildiko, additional, Krug, Norbert, additional, Montuschi, Paolo, additional, Sanak, Marek, additional, Sandstrom, Thomas, additional, Shaw, Dominic E., additional, Singer, Florian, additional, Sterk, Peter J., additional, Roberts, Graham, additional, Adcock, Ian M., additional, Djukanovic, Ratko, additional, Auffray, Charles, additional, Chung, Kian Fan, additional, Adriaens, Nora, additional, Ahmed, Hassan, additional, Aliprantis, Antonios, additional, Alving, Kjell, additional, Badorek, Philipp, additional, Balgoma, David, additional, Barber, Clair, additional, Bautmans, An, additional, Behndig, Annelie F., additional, Bel, Elisabeth, additional, Beleta, Jorge, additional, Berglind, Ann, additional, Berton, Alix, additional, Bigler, Jeanette, additional, Bisgaard, Hans, additional, Bochenek, Grazyna, additional, Boedigheimer, Michael J., additional, Bøonnelykke, Klaus, additional, Brandsma, Joost, additional, Braun, Armin, additional, Brinkman, Paul, additional, Burg, Dominic, additional, Campagna, Davide, additional, Carayannopoulos, Leon, additional, Carvalho da Purfição Rocha, João P., additional, Chaiboonchoe, Amphun, additional, Chaleckis, Romanas, additional, Coleman, Courtney, additional, Compton, Chris, additional, D'Amico, Arnaldo, additional, Dahlén, Barbro, additional, De Alba, Jorge, additional, de Boer, Pim, additional, De Lepeleire, Inge, additional, Dekker, Tamara, additional, Delin, Ingrid, additional, Dennison, Patrick, additional, Dijkhuis, Annemiek, additional, Draper, Aleksandra, additional, Edwards, Jessica, additional, Emma, Rosalia, additional, Ericsson, Magnus, additional, Erpenbeck, Veit, additional, Erzen, Damijan, additional, Faulenbach, Cornelia, additional, Fichtner, Klaus, additional, Fitch, Neil, additional, Flood, Breda, additional, Frey, Urs, additional, Gahlemann, Martina, additional, Galffy, Gabriella, additional, Gallart, Hector, additional, Garret, Trevor, additional, Geiser, Thomas, additional, Gent, Jilaiha, additional, Gerhardsson de Verdier, Maria, additional, Gibeon, David, additional, Gomez, Cristina, additional, Gove, Kerry, additional, Gozzard, Neil, additional, Guo, Yi-Ke, additional, Hashimoto, Simone, additional, Haughney, John, additional, Hedlin, Gunilla, additional, Hekking, Pieter-Paul, additional, Henriksson, Elisabeth, additional, Hewitt, Lorraine, additional, Higgenbottam, Tim, additional, Hoda, Uruj, additional, Hohlfeld, Jans, additional, Holweg, Cecile, additional, Howarth, Peter, additional, Hu, Richard, additional, Hu, Sile, additional, Hu, Xugang, additional, Hudson, Val, additional, James, Anna J., additional, Kamphuis, Juliette, additional, Kennington, Erika J., additional, Kerry, Dyson, additional, Klüglich, Matthias, additional, Knobel, Hugo, additional, Knowles, Richard, additional, Knox, Alan, additional, Kolmert, Johan, additional, Konradsen, Jon, additional, Kots, Maxim, additional, Krueger, Linn, additional, Kuo, Scott, additional, Kupczyk, Maciej, additional, Lambrecht, Bart, additional, Lantz, Ann-Sofie, additional, Larsson, Lars, additional, Lazarinis, Nikos, additional, Lone-Satif, Saeeda, additional, Marouzet, Lisa, additional, Martin, Jane, additional, Masefield, Sarah, additional, Mathon, Caroline, additional, Matthews, John G., additional, Mazein, Alexander, additional, Meah, Sally, additional, Maiser, Andrea, additional, Menzies-Gow, Andrew, additional, Metcalf, Leanne, additional, Middelveld, Roelinde, additional, Mikus, Maria, additional, Miralpeix, Montse, additional, Monk, Philips, additional, Mores, Nadia, additional, Murray, Clare S., additional, Musial, Jacek, additional, Myles, David, additional, Naz, Shama, additional, Nething, Katja, additional, Nicholas, Ben, additional, Nihlen, Ulf, additional, Nilsson, Peter, additional, Nordlund, Björn, additional, Östling, Jörgen, additional, Pacino, Antonio, additional, Pahus, Laurie, additional, Palkonnen, Susanna, additional, Pavlidis, Stelios, additional, Pennazza, Giorgio, additional, Petrén, Anne, additional, Pink, Sandy, additional, Postle, Anthony, additional, Powel, Pippa, additional, Rahman-Amin, Malayka, additional, Rao, Navin, additional, Ravanetti, Lara, additional, Ray, Emma, additional, Reinke, Stacey, additional, Reynolds, Leanne, additional, Riemann, Kathrin, additional, Riley, John, additional, Robberechts, Martine, additional, Roberts, Amanda, additional, Rossios, Christos, additional, Russell, Kirsty, additional, Rutgers, Michael, additional, Santini, Giuseppe, additional, Sentoninco, Marco, additional, Schoelch, Corinna, additional, Schofield, James P.R., additional, Seibold, Wolfgang, additional, Sigmund, Ralf, additional, Sjödin, Marcus, additional, Skipp, Paul J., additional, Smids, Barbara, additional, Smith, Caroline, additional, Smith, Jessica, additional, Smith, Katherine M., additional, Söderman, Päivi, additional, Sogbesan, Adesimbo, additional, Staykova, Doroteya, additional, Strandberg, Karin, additional, Sun, Kai, additional, Supple, David, additional, Szentkereszty, Marton, additional, Tamasi, Lilla, additional, Tariq, Kamran, additional, Thörngren, John-Olof, additional, Thornton, Bob, additional, Thorsen, Jonathan, additional, Valente, Salvatore, additional, van Aalderenm, Wim, additional, van de Pol, Marianne, additional, van Drunen, Kees, additional, van Geest, Marleen, additional, Versnel, Jenny, additional, Vestbo, Jorgen, additional, Vink, Anton, additional, Vissing, Nadja, additional, von Garnier, Christophe, additional, Wagerner, Arianne, additional, Wagers, Scott, additional, Wald, Frans, additional, Walker, Samantha, additional, Ward, Jonathan, additional, Weiszhart, Zsoka, additional, Wetzel, Kristiane, additional, Wheelock, Craig E., additional, Wiegman, Coen, additional, Williams, Siân, additional, Wilson, Susan J., additional, Woosdcock, Ashley, additional, Yang, Xian, additional, Yeyashingham, Elizabeth, additional, Yu, Wen, additional, Zetterquist, Wilhelm, additional, and Zwinderman, Koos, additional

Published
2017
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13. Control of the Diadenylate Cyclase CdaS in Bacillus subtilis AN AUTOINHIBITORY DOMAIN LIMITS CYCLIC DI-AMP PRODUCTION

Author

Mehne, Felix M. P., Schroeder-Tittmann, Kathrin, Eijlander, Robyn T., Herzberg, Christina, Hewitt, Lorraine, Kaever, Volkhard, Lewis, Richard J., Kuipers, Oscar P., Tittmann, Kai, Stuelke, Joerg, and Molecular Genetics

Subjects
PATHWAY, GENES, IDENTIFICATION, GMP, BACTERIA, REVEALS, CHECKPOINT PROTEIN, BIOSYNTHESIS, SPORULATION, DNA INTEGRITY
Abstract

The Gram-positive bacterium Bacillus subtilis encodes three diadenylate cyclases that synthesize the essential signaling nucleotide cyclic di-AMP. The activities of the vegetative enzymes DisA and CdaA are controlled by protein-protein interactions with their conserved partner proteins. Here, we have analyzed the regulation of the unique sporulation-specific diadenylate cyclase CdaS. Very low expression of CdaS as the single diadenylate cyclase resulted in the appearance of spontaneous suppressor mutations. Several of these mutations in the cdaS gene affected the N-terminal domain of CdaS. The corresponding CdaS mutant proteins exhibited a significantly increased enzymatic activity. The N-terminal domain of CdaS consists of two alpha-helices and is attached to the C-terminal catalytically active diadenylate cyclase (DAC) domain. Deletion of the first or both helices resulted also in strongly increased activity indicating that the N-terminal domain serves to limit the enzyme activity of the DAC domain. The structure of YojJ, a protein highly similar to CdaS, indicates that the protein forms hexamers that are incompatible with enzymatic activity of the DAC domains. In contrast, the mutations and the deletions of the N-terminal domain result in conformational changes that lead to highly increased enzymatic activity. Although the full-length CdaS protein was found to form hexamers, a truncated version with a deletion of the first N-terminal helix formed dimers with high enzyme activity. To assess the role of CdaS in sporulation, we assayed the germination of wild type and cdaS mutant spores. The results indicate that cyclic di-AMP formed by CdaS is required for efficient germination.

Published
2014

14. Structure and function of a spectrin-like regulator of bacterial cytokinesis

Author

Cleverley, Robert M., Barrett, Jeffrey R., Baslé, Arnaud, Bui, Nhat Khai, Hewitt, Lorraine, Solovyova, Alexandra, Xu, Zhi-Qiang, Daniel, Richard A., Dixon, Nicholas E., Harry, Elizabeth J., Oakley, Aaron J., Vollmer, Waldemar, and Lewis, Richard J.

Subjects
cell division, Binding Sites, Applied Microbiology, Spectrin, cytoskeleton, macromolecular substances, Crystallography, X-Ray, Microbiology, Article, Cytoskeletal Proteins, Bacterial Proteins, Genetics, crystallography, Molecular Biology, EzrA, Bacillus subtilis, Cytokinesis
Abstract

Bacterial cell division is facilitated by a molecular machine—the divisome—that assembles at mid-cell in dividing cells. The formation of the cytokinetic Z-ring by the tubulin homologue FtsZ is regulated by several factors, including the divisome component EzrA. Here we describe the structure of the 60-kDa cytoplasmic domain of EzrA, which comprises five linear repeats of an unusual triple helical bundle. The EzrA structure is bent into a semicircle, providing the protein with the potential to interact at both N- and C-termini with adjacent membrane-bound divisome components. We also identify at least two binding sites for FtsZ on EzrA and map regions of EzrA that are responsible for regulating FtsZ assembly. The individual repeats, and their linear organization, are homologous to the spectrin proteins that connect actin filaments to the membrane in eukaryotes, and we thus propose that EzrA is the founding member of the bacterial spectrin family., EzrA regulates the polymerization of FtsZ, a tubulin-like protein and main component of the Z-ring, which drives cell division in bacteria. Here the authors describe the crystal structure of EzrA and demonstrate that it shares structural and functional properties with eukaryotic spectrins.

Published
2014

16. A different slant on DNA

Author

Hewitt, Lorraine

Subjects
DNA -- Research, Molecular structure -- Research
Abstract

One of the most enduring molecules of life is DNA. It replicates, and virtually exact copies of each molecule are passed from one generation of cells to the next. DNA […]

Published
2001

17. Structure and function of a spectrin-like regulator of bacterial cytokinesis

Author

Cleverley, Robert, Barrett, Jeffrey, Basle, Arnaud, Bui, Nhat Khai, Hewitt, Lorraine, Solovyova, Alexandra S, Xu, Zhi-Qiang, Daniel, Richard A, Dixon, Nicholas E, Harry, Elizabeth J, Oakley, Aaron J, Vollmer, Waldemar, Lewis, Richard J, Cleverley, Robert, Barrett, Jeffrey, Basle, Arnaud, Bui, Nhat Khai, Hewitt, Lorraine, Solovyova, Alexandra S, Xu, Zhi-Qiang, Daniel, Richard A, Dixon, Nicholas E, Harry, Elizabeth J, Oakley, Aaron J, Vollmer, Waldemar, and Lewis, Richard J

Abstract

Bacterial cell division is facilitated by a molecular machine-the divisome-that assembles at mid-cell in dividing cells. The formation of the cytokinetic Z-ring by the tubulin homologue FtsZ is regulated by several factors, including the divisome component EzrA. Here we describe the structure of the 60-kDa cytoplasmic domain of EzrA, which comprises five linear repeats of an unusual triple helical bundle. The EzrA structure is bent into a semicircle, providing the protein with the potential to interact at both N- and C-termini with adjacent membrane-bound divisome components. We also identify at least two binding sites for FtsZ on EzrA and map regions of EzrA that are responsible for regulating FtsZ assembly. The individual repeats, and their linear organization, are homologous to the spectrin proteins that connect actin filaments to the membrane in eukaryotes, and we thus propose that EzrA is the founding member of the bacterial spectrin family.

Published
2014

20. Characterisation of a Tip60 Specific Inhibitor, NU9056, in Prostate Cancer

Author

Coffey, Kelly, primary, Blackburn, Timothy J., additional, Cook, Susan, additional, Golding, Bernard T., additional, Griffin, Roger J., additional, Hardcastle, Ian R., additional, Hewitt, Lorraine, additional, Huberman, Kety, additional, McNeill, Hesta V., additional, Newell, David R., additional, Roche, Celine, additional, Ryan-Munden, Claudia A., additional, Watson, Anna, additional, and Robson, Craig N., additional

Published
2012
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24. Screening and Optimizing Protein Production in E. coli.

Author

Walker, John M., Downing, A. Kristina, Hewitt, Lorraine, and McDonnell, James M.

Abstract

Significant improvements in the technologies used for protein production have been driven by impending genome-scale proteomics projects. These initiatives have favored Escherichia coli-based expression systems, which allow rapid cloning and expression of proteins at low cost. The range of commercially available molecular biology kits, vectors, affinity tags, and host cell lines have increased dramatically in recent years. For the structural biology community, where protein production is often a rate-limiting step, these developments have made the process of producing and purifying large amounts of protein for structural studies simpler and faster. The large-scale automated screening approaches for optimizing protein production employed by structural genomics initiatives can be adapted to a more practical targeted approach appropriate for individual structural biology groups. This chapter describes simple, rapid screening methods for testing optimal vector/host combinations using a 96-well format. [ABSTRACT FROM AUTHOR]

Published
2004
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27. Structure of d(TGCGCA)2and a Comparison to Other DNA Hexamers

Author

Harper, Anne, Brannigan, James A., Buck, Martin, Hewitt, Lorraine, Lewis, Richard J., Moore, Madeleine H., and Schneider, Bohdan

Abstract

The X‐ray crystal structure of d(TGCGCA)2has been determined at 120 K to a resolution of 1.3 Å. Hexamer duplexes, in the Z‐DNA conformation, pack in an arrangement similar to the `pure spermine form' [Egli et al.(1991). Biochemistry, 30, 11388–11402] but with significantly different cell dimensions. The phosphate backbone exists in two equally populated discrete conformations at one nucleotide step, around phosphate 11. The structure contains two ordered cobalt hexammine molecules which have roles in stabilization of both the Z‐DNA conformation of the duplex and in crystal packing. A comparison of d(TGCGCA)2with other Z‐DNA hexamer structures available in the Nucleic Acid Database illustrates the elusive nature of crystal packing. A review of the interactions with the metal cations Na+, Mg2+and Co3+reveals a relatively small proportion of phosphate binding and that close contacts between metal ions are common. A prediction of the water structure is compared with the observed pattern in the reported structure.

Published
1998
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28. A human mitochondrial poly(A) polymerase mutation reveals the complexities of post-transcriptional mitochondrial gene expression

Author

Wilson, William C., Hornig-Do, Hue-Tran, Bruni, Francesco, Chang, Jeong Ho, Jourdain, Alexis A., Martinou, Jean-Claude, Falkenberg, Maria, Spåhr, Henrik, Larsson, Nils-Göran, Lewis, Richard J., Hewitt, Lorraine, Baslé, Arnaud, Cross, Harold E., Tong, Liang, Lebel, Robert R., Crosby, Andrew H., Chrzanowska-Lightowlers, Zofia M. A., Lightowlers, Robert N., Wilson, William C., Hornig-Do, Hue-Tran, Bruni, Francesco, Chang, Jeong Ho, Jourdain, Alexis A., Martinou, Jean-Claude, Falkenberg, Maria, Spåhr, Henrik, Larsson, Nils-Göran, Lewis, Richard J., Hewitt, Lorraine, Baslé, Arnaud, Cross, Harold E., Tong, Liang, Lebel, Robert R., Crosby, Andrew H., Chrzanowska-Lightowlers, Zofia M. A., and Lightowlers, Robert N.

Abstract

The p.N478D missense mutation in human mitochondrial poly(A) polymerase (mtPAP) has previously been implicated in a form of spastic ataxia with optic atrophy. In this study, we have investigated fibroblast cell lines established from family members. The homozygous mutation resulted in the loss of polyadenylation of all mitochondrial transcripts assessed; however, oligoadenylation was retained. Interestingly, this had differential effects on transcript stability that were dependent on the particular species of transcript. These changes were accompanied by a severe loss of oxidative phosphorylation complexes I and IV, and perturbation of de novo mitochondrial protein synthesis. Decreases in transcript polyadenylation and in respiratory chain complexes were effectively rescued by overexpression of wild-type mtPAP. Both mutated and wild-type mtPAP localized to the mitochondrial RNA-processing granules thereby eliminating mislocalization as a cause of defective polyadenylation. In vitro polyadenylation assays revealed severely compromised activity by the mutated protein, which generated only short oligo(A) extensions on RNA substrates, irrespective of RNA secondary structure. The addition of LRPPRC/SLIRP, a mitochondrial RNA-binding complex, enhanced activity of the wild-type mtPAP resulting in increased overall tail length. The LRPPRC/SLIRP effect although present was less marked with mutated mtPAP, independent of RNA secondary structure. We conclude that (i) the polymerase activity of mtPAP can be modulated by the presence of LRPPRC/SLIRP, (ii) N478D mtPAP mutation decreases polymerase activity and (iii) the alteration in poly(A) length is sufficient to cause dysregulation of post-transcriptional expression and the pathogenic lack of respiratory chain complexes

30. Control of the diadenylate cyclase CdaS in Bacillus subtilis: an autoinhibitory domain limits cyclic di-AMP production.

Author

Mehne FM, Schröder-Tittmann K, Eijlander RT, Herzberg C, Hewitt L, Kaever V, Lewis RJ, Kuipers OP, Tittmann K, and Stülke J

Subjects
Bacillus subtilis genetics, Protein Structure, Quaternary, Protein Structure, Secondary, Protein Structure, Tertiary, Bacillus subtilis enzymology, Bacterial Proteins chemistry, Bacterial Proteins genetics, Bacterial Proteins metabolism, Dinucleoside Phosphates chemistry, Dinucleoside Phosphates genetics, Dinucleoside Phosphates metabolism, Phosphorus-Oxygen Lyases chemistry, Phosphorus-Oxygen Lyases genetics, Phosphorus-Oxygen Lyases metabolism
Abstract

The Gram-positive bacterium Bacillus subtilis encodes three diadenylate cyclases that synthesize the essential signaling nucleotide cyclic di-AMP. The activities of the vegetative enzymes DisA and CdaA are controlled by protein-protein interactions with their conserved partner proteins. Here, we have analyzed the regulation of the unique sporulation-specific diadenylate cyclase CdaS. Very low expression of CdaS as the single diadenylate cyclase resulted in the appearance of spontaneous suppressor mutations. Several of these mutations in the cdaS gene affected the N-terminal domain of CdaS. The corresponding CdaS mutant proteins exhibited a significantly increased enzymatic activity. The N-terminal domain of CdaS consists of two α-helices and is attached to the C-terminal catalytically active diadenylate cyclase (DAC) domain. Deletion of the first or both helices resulted also in strongly increased activity indicating that the N-terminal domain serves to limit the enzyme activity of the DAC domain. The structure of YojJ, a protein highly similar to CdaS, indicates that the protein forms hexamers that are incompatible with enzymatic activity of the DAC domains. In contrast, the mutations and the deletions of the N-terminal domain result in conformational changes that lead to highly increased enzymatic activity. Although the full-length CdaS protein was found to form hexamers, a truncated version with a deletion of the first N-terminal helix formed dimers with high enzyme activity. To assess the role of CdaS in sporulation, we assayed the germination of wild type and cdaS mutant spores. The results indicate that cyclic di-AMP formed by CdaS is required for efficient germination., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Published
2014
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31. Screening and optimizing protein production in E. coli.

Author

Hewitt L and McDonnell JM

Subjects
Cloning, Molecular, DNA analysis, DNA, Complementary genetics, Electrophoresis, Polyacrylamide Gel methods, Gels, Gene Expression Regulation, Genetic Vectors genetics, Nuclear Magnetic Resonance, Biomolecular methods, Polymerase Chain Reaction methods, Proteins genetics, Proteins metabolism, Recombinant Proteins biosynthesis, Recombinant Proteins genetics, Recombinant Proteins isolation & purification, Escherichia coli metabolism, Protein Biosynthesis
Abstract

Significant improvements in the technologies used for protein production have been driven by impending genome-scale proteomics projects. These initiatives have favored Escherichia coli-based expression systems, which allow rapid cloning and expression of proteins at low cost. The range of commercially available molecular biology kits, vectors, affinity tags, and host cell lines have increased dramatically in recent years. For the structural biology community, where protein production is often a rate-limiting step, these developments have made the process of producing and purifying large amounts of protein for structural studies simpler and faster. The large-scale automated screening approaches for optimizing protein production employed by structural genomics initiatives can be adapted to a more practical targeted approach appropriate for individual structural biology groups. This chapter describes simple, rapid screening methods for testing optimal vector/host combinations using a 96-well format.

Published
2004
Full Text
View/download PDF

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