28 results on '"David L. Burk"'
Search Results
2. Supplementary Tables and Figures from Functionally Null RAD51D Missense Mutation Associates Strongly with Ovarian Carcinoma
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William D. Foulkes, Alexandre Orthwein, Patricia N. Tonin, Christopher J. Lord, Mohammad R. Akbari, Steven A. Narod, Reiner Siebert, Susanne Bens, Jacek Majewski, Anne-Marie Mes-Masson, Jacques L. Michaud, Guy A. Rouleau, Diane Provencher, Albert M. Berghuis, Francois Rousseau, George Zogopoulos, Alexandre Dionne-Laporte, Fadi F. Hamdan, Sylvie Giroux, Olga Aleynikova, Rabea Wagener, Nancy Hamel, Valerie Hastings, Eva Tomiak, Damien Grapton, David L. Burk, Suzanna L. Arcand, Somayyeh Fahiminiya, Javad Nadaf, Jessica Frankum, Massimo Di Iorio, and Barbara Rivera
- Abstract
This file includes supplementary Figure S1, Supplementary Figure S2 and Supplementary Figure S3. It also includes Supplementary Table S1 - RAD51D c.620C
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- 2023
3. Supplementary Table 3 from Functionally Null RAD51D Missense Mutation Associates Strongly with Ovarian Carcinoma
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William D. Foulkes, Alexandre Orthwein, Patricia N. Tonin, Christopher J. Lord, Mohammad R. Akbari, Steven A. Narod, Reiner Siebert, Susanne Bens, Jacek Majewski, Anne-Marie Mes-Masson, Jacques L. Michaud, Guy A. Rouleau, Diane Provencher, Albert M. Berghuis, Francois Rousseau, George Zogopoulos, Alexandre Dionne-Laporte, Fadi F. Hamdan, Sylvie Giroux, Olga Aleynikova, Rabea Wagener, Nancy Hamel, Valerie Hastings, Eva Tomiak, Damien Grapton, David L. Burk, Suzanna L. Arcand, Somayyeh Fahiminiya, Javad Nadaf, Jessica Frankum, Massimo Di Iorio, and Barbara Rivera
- Abstract
'Supplementary Table3 -Presence of allelic imbalanced alleles in RAD51D and TP53 somatic mutations in RAD51D c.620C>T;p.S207L HGSC carriers.'
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- 2023
4. Data from Functionally Null RAD51D Missense Mutation Associates Strongly with Ovarian Carcinoma
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William D. Foulkes, Alexandre Orthwein, Patricia N. Tonin, Christopher J. Lord, Mohammad R. Akbari, Steven A. Narod, Reiner Siebert, Susanne Bens, Jacek Majewski, Anne-Marie Mes-Masson, Jacques L. Michaud, Guy A. Rouleau, Diane Provencher, Albert M. Berghuis, Francois Rousseau, George Zogopoulos, Alexandre Dionne-Laporte, Fadi F. Hamdan, Sylvie Giroux, Olga Aleynikova, Rabea Wagener, Nancy Hamel, Valerie Hastings, Eva Tomiak, Damien Grapton, David L. Burk, Suzanna L. Arcand, Somayyeh Fahiminiya, Javad Nadaf, Jessica Frankum, Massimo Di Iorio, and Barbara Rivera
- Abstract
RAD51D is a key player in DNA repair by homologous recombination (HR), and RAD51D truncating variant carriers have an increased risk for ovarian cancer. However, the contribution of nontruncating RAD51D variants to cancer predisposition remains uncertain. Using deep sequencing and case–control genotyping studies, we show that in French Canadians, the missense RAD51D variant c.620C>T;p.S207L is highly prevalent and is associated with a significantly increased risk for ovarian high-grade serous carcinoma (HGSC; 3.8% cases vs. 0.2% controls). The frequency of the p.S207L variant did not significantly differ from that of controls in breast, endometrial, pancreas, or colorectal adenocarcinomas. Functionally, we show that this mutation impairs HR by disrupting the RAD51D–XRCC2 interaction and confers PARP inhibitor sensitivity. These results highlight the importance of a functional RAD51D–XRCC2 interaction to promote HR and prevent the development of HGSC. This study identifies c.620C>T;p.S207L as the first bona fide pathogenic RAD51D missense cancer susceptibility allele and supports the use of targeted PARP-inhibitor therapies in ovarian cancer patients carrying deleterious missense RAD51D variants. Cancer Res; 77(16); 4517–29. ©2017 AACR.
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- 2023
5. Structural analysis of a novel cyclohexylamine oxidase from Brevibacterium oxydans IH-35A.
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I Ahmad Mirza, David L Burk, Bing Xiong, Hiroaki Iwaki, Yoshie Hasegawa, Stephan Grosse, Peter C K Lau, and Albert M Berghuis
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Medicine ,Science - Abstract
Cyclohexylamine oxidase (CHAO) is a flavoprotein first described in Brevibacterium oxydans strain IH-35A that carries out the initial step of the degradation of the industrial chemical cyclohexylamine to cyclohexanone. We have cloned and expressed in Escherichia coli the CHAO-encoding gene (chaA) from B. oxydans, purified CHAO and determined the structures of both the holoenzyme form of the enzyme and a product complex with cyclohexanone. CHAO is a 50 kDa monomer with a PHBH fold topology. It belongs to the flavin monooxygenase family of enzymes and exhibits high substrate specificity for alicyclic amines and sec-alkylamines. The overall structure is similar to that of other members of the flavin monooxygenase family, but lacks either of the C- or N-terminal extensions observed in these enzymes. Active site features of the flavin monooxygenase family are conserved in CHAO, including the characteristic aromatic cage. Differences in the orientations of residues of the CHAO aromatic cage result in a substrate-binding site that is more open than those of its structural relatives. Since CHAO has a buried hydrophobic active site with no obvious route for substrates and products, a random acceleration molecular dynamics simulation has been used to identify a potential egress route. The path identified includes an intermediate cavity and requires transient conformation changes in a shielding loop and a residue at the border of the substrate-binding cavity. These results provide a foundation for further studies with CHAO aimed at identifying features determining substrate specificity and for developing the biocatalytic potential of this enzyme.
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- 2013
- Full Text
- View/download PDF
6. Functionally Null RAD51D Missense Mutation Associates Strongly with Ovarian Carcinoma
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Jacek Majewski, Rabea Wagener, Jacques L. Michaud, Guy A. Rouleau, Patricia N. Tonin, Mohammad R. Akbari, William D. Foulkes, Diane Provencher, Christopher J. Lord, Somayyeh Fahiminiya, Nancy Hamel, Barbara Rivera, Damien Grapton, David L. Burk, Susanne Bens, George Zogopoulos, Massimo Di Iorio, Alexandre Dionne-Laporte, Jessica Frankum, Anne-Marie Mes-Masson, Sylvie Giroux, Steven A. Narod, Suzanna L. Arcand, Olga Aleynikova, Valerie Hastings, Javad Nadaf, François Rousseau, Eva Tomiak, Alexandre Orthwein, Fadi F. Hamdan, Albert M. Berghuis, and Reiner Siebert
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0301 basic medicine ,Cancer Research ,education.field_of_study ,Serous carcinoma ,Population ,Cancer ,Biology ,medicine.disease ,Bioinformatics ,03 medical and health sciences ,030104 developmental biology ,Germline mutation ,Oncology ,Ovarian carcinoma ,PARP inhibitor ,medicine ,Cancer research ,Missense mutation ,Ovarian cancer ,education - Abstract
RAD51D is a key player in DNA repair by homologous recombination (HR), and RAD51D truncating variant carriers have an increased risk for ovarian cancer. However, the contribution of nontruncating RAD51D variants to cancer predisposition remains uncertain. Using deep sequencing and case–control genotyping studies, we show that in French Canadians, the missense RAD51D variant c.620C>T;p.S207L is highly prevalent and is associated with a significantly increased risk for ovarian high-grade serous carcinoma (HGSC; 3.8% cases vs. 0.2% controls). The frequency of the p.S207L variant did not significantly differ from that of controls in breast, endometrial, pancreas, or colorectal adenocarcinomas. Functionally, we show that this mutation impairs HR by disrupting the RAD51D–XRCC2 interaction and confers PARP inhibitor sensitivity. These results highlight the importance of a functional RAD51D–XRCC2 interaction to promote HR and prevent the development of HGSC. This study identifies c.620C>T;p.S207L as the first bona fide pathogenic RAD51D missense cancer susceptibility allele and supports the use of targeted PARP-inhibitor therapies in ovarian cancer patients carrying deleterious missense RAD51D variants. Cancer Res; 77(16); 4517–29. ©2017 AACR.
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- 2017
7. DrugViz: a Cytoscape plugin for visualizing and analyzing small molecule drugs in biological networks.
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Bing Xiong, Ke Liu, Jie Wu, David L. Burk, Hualiang Jiang, and Jingkang Shen
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- 2008
- Full Text
- View/download PDF
8. BSSF: a fingerprint based ultrafast binding site similarity search and function analysis server.
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Bing Xiong, Jie Wu, David L. Burk, Mengzhu Xue, Hualiang Jiang, and Jingkang Shen
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- 2010
- Full Text
- View/download PDF
9. Functionally Null
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Barbara, Rivera, Massimo, Di Iorio, Jessica, Frankum, Javad, Nadaf, Somayyeh, Fahiminiya, Suzanna L, Arcand, David L, Burk, Damien, Grapton, Eva, Tomiak, Valerie, Hastings, Nancy, Hamel, Rabea, Wagener, Olga, Aleynikova, Sylvie, Giroux, Fadi F, Hamdan, Alexandre, Dionne-Laporte, George, Zogopoulos, Francois, Rousseau, Albert M, Berghuis, Diane, Provencher, Guy A, Rouleau, Jacques L, Michaud, Anne-Marie, Mes-Masson, Jacek, Majewski, Susanne, Bens, Reiner, Siebert, Steven A, Narod, Mohammad R, Akbari, Christopher J, Lord, Patricia N, Tonin, Alexandre, Orthwein, and William D, Foulkes
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Adult ,DNA-Binding Proteins ,Ovarian Neoplasms ,Genotype ,Case-Control Studies ,Mutation ,Mutation, Missense ,Humans ,Female ,Middle Aged ,Polymorphism, Single Nucleotide ,Aged ,Pedigree - Abstract
RAD51D is a key player in DNA repair by homologous recombination (HR), and
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- 2017
10. Functional characterization of the human dendritic cell immunodeficiency associated with the IRF8K108E mutation
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Karina Butler, Jean-Laurent Casanova, Philippe Gros, David Langlais, David L. Burk, Francois Lefebvre, Albert M. Berghuis, Timothy Ronan Leahy, Muzz Haniffa, Sophie Hambleton, Venetia Bigley, Guillaume Bourque, and Sandra Salem
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Myeloid ,Immunology ,Mutation, Missense ,Biology ,Biochemistry ,Lymphohistiocytosis, Hemophagocytic ,Antigen ,T-Lymphocyte Subsets ,medicine ,Humans ,Immunodeficiency ,Immunobiology ,Clonal Anergy ,Antigen Presentation ,Protein Stability ,Homozygote ,Immunologic Deficiency Syndromes ,Infant ,Dendritic Cells ,Cell Biology ,Hematology ,Dendritic cell ,medicine.disease ,Transplantation ,HEK293 Cells ,medicine.anatomical_structure ,Amino Acid Substitution ,Cord blood ,Interferon Regulatory Factors ,Cancer research ,RNA ,Female ,Mutant Proteins ,Cord Blood Stem Cell Transplantation ,IRF8 ,Protein Processing, Post-Translational ,CD81 - Abstract
We have previously reported on a unique patient in whom homozygosity for a mutation at IRF8 (IRF8(K108E)) causes a severe immunodeficiency. Laboratory evaluation revealed a highly unusual myeloid compartment, remarkable for the complete absence of CD141 and CD161 monocytes, absence of CD11c1 conventional dendritic cells (DCs) and CD11c1/CD1231 plasmacytoid DCs, and striking granulocytic hyperplasia. The patient initially presented with severe disseminated mycobacterial and mucocutaneous fungal infections and was ultimately cured by cord blood transplant. Sequencing RNA from the IRF8(K108E) patient's primary blood cells prior to transplant shows not only depletion of IRF8-bound and IRF8-regulated transcriptional targets, in keeping with the distorted composition of the myeloid compartment, but also a paucity of transcripts associated with activated CD41 and CD81 T lymphocytes. This suggests that T cells reared in the absence of a functional antigen-presenting compartment in IRF8(K108E) are anergic. Biochemical characterization of the IRF8(K108E) mutant in vitro shows that loss of the positively charged side chain at K108 causes loss of nuclear localization and loss of transcriptional activity, which is concomitant with decreased protein stability, increased ubiquitination, increased small ubiquitin-like modification, and enhanced proteasomal degradation. These findings provide functional insight into the molecular basis of immunodeficiency associated with loss of IRF8.
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- 2014
11. Structural Basis for Kinase-Mediated Macrolide Antibiotic Resistance
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Amy Y. Yan, David L. Burk, Albert M. Berghuis, Desiree H. Fong, and Jonathan Blanchet
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0301 basic medicine ,GTP' ,Stereochemistry ,030106 microbiology ,Biology ,Ribosome ,Substrate Specificity ,Phosphotransferase ,03 medical and health sciences ,Bacterial Proteins ,Structural Biology ,Drug Resistance, Bacterial ,Transferase ,Molecular Biology ,50S ,chemistry.chemical_classification ,Binding Sites ,Bacteria ,Aminoglycoside ,3. Good health ,Phosphotransferases (Alcohol Group Acceptor) ,030104 developmental biology ,Enzyme ,Biochemistry ,chemistry ,Guanosine Triphosphate ,Macrolides ,Phosphotransferases ,Protein Binding - Abstract
The macrolides are a class of antibiotic, characterized by a large macrocyclic lactone ring that can be inactivated by macrolide phosphotransferase enzymes. We present structures for MPH(2')-I and MPH(2')-II in the apo state, and in complex with GTP analogs and six different macrolides. These represent the first structures from the two main classes of macrolide phosphotransferases. The structures show that the enzymes are related to the aminoglycoside phosphotransferases, but are distinguished from them by the presence of a large interdomain linker that contributes to an expanded antibiotic binding pocket. This pocket is largely hydrophobic, with a negatively charged patch located at a conserved aspartate residue, rationalizing the broad-spectrum resistance conferred by the enzymes. Complementary mutation studies provide insights into factors governing substrate specificity. A comparison with macrolides bound to their natural target, the 50S ribosome, suggests avenues for next-generation antibiotic development.
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- 2016
12. Abstract 756: Whole exome sequencing identifies frequent mutations of PTPRB and KDR in secondary angiosarcoma
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Tenzin Gayden, Brendan C. Dickson, Hamid Nikbakht, Pierre-Olivier Fiset, Nicolas De Jay, Javad Nadaf, David L. Burk, Albert Berghuis, Rebecca Gladdy, Jay Wunder, Robert Turcotte, Jacek Majewski, and Nada Jabado
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Cancer Research ,Oncology - Abstract
Angiosarcoma is an uncommon malignant vascular tumor that can occur anywhere on the body, with a predilection for the skin or soft tissues of the scalp and face. By whole exome sequencing, we identified recurrent somatic mutations in PTPRB, which encodes an endothelial-specific phosphatase that negatively regulates angiogenesis, in three secondary angiosarcoma tumors (3/10). In addition, four tumors harbored missense variants in the KDR gene, including three germline substitutions. Neither the PTPRB nor the KDR alterations were found in the 5 primary tumors studied. Copy number analysis derived from the exome and methylation data revealed frequent MYC amplification in the majority of secondary angiosarcoma tumors (9/10). Altogether, our data provide a comprehensive overview of genetic and epigenetic landscapes of angiosarcoma, and demonstrate that aberrant angiogenesis associated with PTPRB and KDR mutations may contribute to the pathogenesis of angiosarcoma. Citation Format: Tenzin Gayden, Brendan C. Dickson, Hamid Nikbakht, Pierre-Olivier Fiset, Nicolas De Jay, Javad Nadaf, David L. Burk, Albert Berghuis, Rebecca Gladdy, Jay Wunder, Robert Turcotte, Jacek Majewski, Nada Jabado. Whole exome sequencing identifies frequent mutations of PTPRB and KDR in secondary angiosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 756.
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- 2019
13. A Recurrent PDGFRB Mutation Causes Familial Infantile Myofibromatosis
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Yanfang Guan, Van-Hung Nguyen, Brendan Lee, Ming Qi, James T. Lu, Donna Russo, Richard A. Gibbs, David Orchard, David Malkin, Karen W. Eldin, Steffen Albrecht, Jiancheng Guo, Kym M. Boycott, Albert M. Berghuis, Sherif Emil, Tenzin Gayden, Wendy K. Chung, Philippe M. Campeau, Jeremy Schwartzentruber, Brenda Moroz, Yee Him Cheung, Nada Jabado, Megan R. Vanstone, Charles A. LeDuc, and David L. Burk
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Male ,Models, Molecular ,Heterozygote ,Mutation, Missense ,Infantile myofibromatosis ,PDGFRB ,Biology ,medicine.disease_cause ,Myofibromatosis ,Germline ,Receptor, Platelet-Derived Growth Factor beta ,03 medical and health sciences ,0302 clinical medicine ,Germline mutation ,Report ,Genetics ,medicine ,Humans ,Missense mutation ,Genetics(clinical) ,Amino Acid Sequence ,Receptor, Notch3 ,Genetic Association Studies ,Germ-Line Mutation ,Genetics (clinical) ,Genes, Dominant ,030304 developmental biology ,0303 health sciences ,Mutation ,Receptors, Notch ,Heterozygote advantage ,Sequence Analysis, DNA ,medicine.disease ,Pedigree ,Protein Structure, Tertiary ,3. Good health ,030220 oncology & carcinogenesis ,Cancer research ,Female - Abstract
Infantile myofibromatosis (IM) is the most common benign fibrous tumor of soft tissues affecting young children. By using whole-exome sequencing, RNA sequencing, and targeted sequencing, we investigated germline and tumor DNA in individuals from four distinct families with the familial form of IM and in five simplex IM cases with no previous family history of this disease. We identified a germline mutation c.1681C>T (p.Arg561Cys) in platelet-derived growth factor receptor β (PDGFRB) in all 11 affected individuals with familial IM, although none of the five individuals with nonfamilial IM had mutations in this gene. We further identified a second heterozygous mutation in PDGFRB in two myofibromas from one of the affected familial cases, indicative of a potential second hit in this gene in the tumor. PDGFR-β promotes growth of mesenchymal cells, including blood vessels and smooth muscles, which are affected in IM. Our findings indicate p.Arg561Cys substitution in PDGFR-β as a cause of the dominant form of this disease. They provide a rationale for further investigations of this specific mutation and gene to assess the benefits of targeted therapies against PDGFR-β in aggressive life-threatening familial forms of the disease.
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- 2013
14. Germline and somatic FGFR1 abnormalities in dysembryoplastic neuroepithelial tumors
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Paul J. Lockhart, Thomas Niederstadt, Ronald Sträter, Jacek Majewski, Werner Paulus, Duncan MacGregor, Jian Carrot-Zhang, Ulrich Schüller, Jason Karamchandani, Maria Thom, Camelia M. Monoranu, William D. Foulkes, Nada Jabado, Steffen Albrecht, Krzysztof Zakrzewski, Magdalena Zakrzewska, Michele Zeinieh, Martin Hasselblatt, Gerhard Kurlemann, Albert M. Berghuis, Richard J. Leventer, Benjamin Ellezam, Roméo Sébastien Blanc, Zuzanna Michalak, Talia Boshari, Damien Faury, Susanne Bens, Pawel P. Liberski, Reiner Siebert, Somayyeh Fahiminiya, Sanjay M. Sisodiya, Javad Nadaf, Eric Bareke, David L. Burk, Milou Ohm, David T.W. Jones, Celia M. T. Greenwood, Tenzin Gayden, Barbara Rivera, and Kornelius Kerl
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0301 basic medicine ,Adult ,Male ,Proto-Oncogene Proteins B-raf ,Pathology ,medicine.medical_specialty ,Adolescent ,DNA Copy Number Variations ,MAP Kinase Signaling System ,Brain tumor ,Germline ,Article ,Pathology and Forensic Medicine ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,symbols.namesake ,Young Adult ,0302 clinical medicine ,Gene duplication ,medicine ,Humans ,Copy-number variation ,Receptor, Fibroblast Growth Factor, Type 1 ,Exome sequencing ,DNET ,Sanger sequencing ,business.industry ,Brain Neoplasms ,Dysembryoplastic Neuroepithelial Tumor ,Glioma ,medicine.disease ,3. Good health ,stomatognathic diseases ,030104 developmental biology ,HEK293 Cells ,Mutation ,symbols ,Female ,Neurology (clinical) ,business ,030217 neurology & neurosurgery - Abstract
Dysembryoplastic neuroepithelial tumor (DNET) is a benign brain tumor associated with intractable drug-resistant epilepsy. In order to identify underlying genetic alterations and molecular mechanisms, we examined three family members affected by multinodular DNETs as well as 100 sporadic tumors from 96 patients, which had been referred to us as DNETs. We performed whole-exome sequencing on 46 tumors and targeted sequencing for hotspot FGFR1 mutations and BRAF p.V600E was used on the remaining samples. FISH, copy number variation assays and Sanger sequencing were used to validate the findings. By whole-exome sequencing of the familial cases, we identified a novel germline FGFR1 mutation, p.R661P. Somatic activating FGFR1 mutations (p.N546K or p.K656E) were observed in the tumor samples and further evidence for functional relevance was obtained by in silico modeling. The FGFR1 p.K656E mutation was confirmed to be in cis with the germline p.R661P variant. In 43 sporadic cases, in which the diagnosis of DNET could be confirmed on central blinded neuropathology review, FGFR1 alterations were also frequent and mainly comprised intragenic tyrosine kinase FGFR1 duplication and multiple mutants in cis (25/43; 58.1 %) while BRAF p.V600E alterations were absent (0/43). In contrast, in 53 cases, in which the diagnosis of DNET was not confirmed, FGFR1 alterations were less common (10/53; 19 %; p
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- 2016
15. IRF8Mutations and Human Dendritic-Cell Immunodeficiency
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Frederic Geissmann, Xiao-Fei Kong, Rainer Doffinger, Anny Fortin, Sandra Salem, Damien Chaussabel, Dewton Moraes-Vasconcelos, Chris M. Bacon, Venetia Bigley, Anete Sevciovic Grumach, Jacqueline Feinberg, Katia Abarca, Matthew Collin, Karina Butler, Lourdes Ceron-Gutierrez, Florent Ginhoux, Laurent Abel, Muzlifah Haniffa, Marjorie Hubeau, Jacinta Bustamante, Joana Azevedo, Dinakantha S. Kumararatne, Timothy J. Zumwalt, Albert M. Berghuis, David L. Burk, Jean-Laurent Casanova, Alberto José da Silva Duarte, Stéphanie Boisson-Dupuis, Geetha Menon, Eduardo Talesnik, Sophie Hambleton, Andrew J. Cant, Laia Alsina, David McDonald, Céline Trouillet, Philippe Gros, Peter Carey, and Saleh Al-Muhsen
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Severe combined immunodeficiency ,business.industry ,Monocyte ,General Medicine ,Dendritic cell ,medicine.disease ,Virology ,Transplantation ,medicine.anatomical_structure ,Immunity ,Immunology ,medicine ,IRF8 ,Antigen-presenting cell ,business ,Immunodeficiency - Abstract
BACKGROUND The genetic analysis of human primary immunodeficiencies has defined the contribution of specific cell populations and molecular pathways in the host defense against infection. Disseminated infection caused by bacille Calmette-Guerin (BCG) vaccines is an early manifestation of primary immunodeficiencies, such as severe combined immunodeficiency. In many affected persons, the cause of disseminated BCG disease is unexplained. METHODS We evaluated an infant presenting with features of severe immunodeficiency, including early-onset disseminated BCG disease, who required hematopoietic stem-cell transplantation. We also studied two otherwise healthy subjects with a history of disseminated but curable BCG disease in childhood. We characterized the monocyte and dendritic-cell compartments in these three subjects and sequenced candidate genes in which mutations could plausibly confer susceptibility to BCG disease. RESULTS We detected two distinct disease-causing mutations affecting interferon regulatory factor 8 (IRF8). Both K108E and T80A mutations impair IRF8 transcriptional activity by disrupting the interaction between IRF8 and DNA. The K108E variant was associated with an autosomal recessive severe immunodeficiency with a complete lack of circulating monocytes and dendritic cells. The T80A variant was associated with an autosomal dominant, milder immunodeficiency and a selective depletion of CD11c+CD1c+ circulating dendritic cells. CONCLUSIONS These findings define a class of human primary immunodeficiencies that affect the differentiation of mononuclear phagocytes. They also show that human IRF8 is critical for the development of monocytes and dendritic cells and for antimycobacterial immunity. (Funded by the Medical Research Council and others.).
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- 2011
16. Structural Studies of the Final Enzyme in the α-Aminoadipate Pathway-Saccharopine Dehydrogenase from Saccharomyces cerevisiae
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Albert M. Berghuis, David L. Burk, J. Hwang, Laura Marrone, Valerie J. Goodfellow, E. Kwok, and Gary I. Dmitrienko
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Models, Molecular ,Conformational change ,Protein Conformation ,Stereochemistry ,Molecular Sequence Data ,Saccharomyces cerevisiae ,Crystallography, X-Ray ,Catalysis ,chemistry.chemical_compound ,Structural Biology ,Oxidoreductase ,Amino Acid Sequence ,Molecular Biology ,Ternary complex ,chemistry.chemical_classification ,Binding Sites ,Sequence Homology, Amino Acid ,biology ,Lysine ,Saccharopine dehydrogenase ,Alpha-aminoadipate pathway ,NAD ,biology.organism_classification ,Alanine Dehydrogenase ,Biochemistry ,chemistry ,Saccharopine ,Saccharopine Dehydrogenases ,NAD+ kinase - Abstract
The 1.64 A structure of the apoenzyme form of saccharopine dehydrogenase (SDH) from Saccharomyces cerevisiae shows the enzyme to be composed of two domains with similar dinucleotide binding folds with a deep cleft at the interface. The structure reveals homology to alanine dehydrogense, despite low primary sequence similarity. A model of the ternary complex of SDH, NAD, and saccharopine identifies residues Lys77 and Glu122 as potentially important for substrate binding and/or catalysis, consistent with a proton shuttle mechanism. Furthermore, the model suggests that a conformational change is required for catalysis and that residues Lys99 and Asp281 may be instrumental in mediating this change. Analysis of the crystal structure in the context of other homologous enzymes from pathogenic fungi and human sources sheds light into the suitability of SDH as a target for antimicrobial drug development.
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- 2007
17. Inhibition of Outer Membrane Proteases of the Omptin Family by Aprotinin
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John R. Brannon, David L. Burk, Jenny-Lee Thomassin, Samantha Gruenheid, Albert M. Berghuis, Hervé Le Moual, Andrea Portt, and Jean-Mathieu Leclerc
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Models, Molecular ,Proteases ,Serine Proteinase Inhibitors ,Protein Conformation ,medicine.medical_treatment ,Omptin ,Immunology ,Antimicrobial peptides ,Biology ,Microbiology ,Gene Expression Regulation, Enzymologic ,Aprotinin ,Species Specificity ,Cathelicidins ,Catalytic Domain ,medicine ,Fluorescence Resonance Energy Transfer ,Amino Acid Sequence ,Serine protease ,Protease ,Escherichia coli Proteins ,Gene Expression Regulation, Bacterial ,OmpT ,Molecular Pathogenesis ,Infectious Diseases ,Biochemistry ,biology.protein ,Citrobacter rodentium ,Parasitology ,Serine Proteases ,Bacterial outer membrane ,medicine.drug ,Antimicrobial Cationic Peptides ,Bacterial Outer Membrane Proteins ,Peptide Hydrolases - Abstract
Bacterial proteases are important virulence factors that inactivate host defense proteins and contribute to tissue destruction and bacterial dissemination. Outer membrane proteases of the omptin family, exemplified by Escherichia coli OmpT, are found in some Gram-negative bacteria. Omptins cleave a variety of substrates at the host-pathogen interface, including plasminogen and antimicrobial peptides. Multiple omptin substrates relevant to infection have been identified; nonetheless, an effective omptin inhibitor remains to be found. Here, we purified native CroP, the OmpT ortholog in the murine pathogen Citrobacter rodentium . Purified CroP was found to readily cleave both a synthetic fluorescence resonance energy transfer substrate and the murine cathelicidin-related antimicrobial peptide. In contrast, CroP was found to poorly activate plasminogen into active plasmin. Although classical protease inhibitors were ineffective against CroP activity, we found that the serine protease inhibitor aprotinin displays inhibitory potency in the micromolar range. Aprotinin was shown to act as a competitive inhibitor of CroP activity and to interfere with the cleavage of the murine cathelicidin-related antimicrobial peptide. Importantly, aprotinin was able to inhibit not only CroP but also Yersinia pestis Pla and, to a lesser extent, E. coli OmpT. We propose a structural model of the aprotinin-omptin complex in which Lys 15 of aprotinin forms salt bridges with conserved negatively charged residues of the omptin active site.
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- 2015
18. X-ray structure of the AAC(6')-Ii antibiotic resistance enzyme at 1.8 A resolution; examination of oligomeric arrangements in GNAT superfamily members
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Leanne E. Wybenga-Groot, Navleen Ghuman, David L. Burk, and Albert M. Berghuis
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Models, Molecular ,Enterococcus faecium ,Biology ,Crystallography, X-Ray ,Biochemistry ,Article ,Evolution, Molecular ,Protein structure ,Acetyl Coenzyme A ,Acetyltransferases ,Drug Resistance, Bacterial ,medicine ,Transferase ,Gnat ,Structural motif ,Molecular Biology ,Genetics ,Binding Sites ,Aminoglycoside ,Kanamycin ,Enzyme structure ,Anti-Bacterial Agents ,Protein Structure, Tertiary ,Aminoglycosides ,Multigene Family ,Dimerization ,Protein Binding ,medicine.drug - Abstract
The rise of antibiotic resistance as a public health concern has led to increased interest in studying the ways in which bacteria avoid the effects of antibiotics. Enzymatic inactivation by several families of enzymes has been observed to be the predominant mechanism of resistance to aminoglycoside antibiotics such as kanamycin and gentamicin. Despite the importance of acetyltransferases in bacterial resistance to aminoglycoside antibiotics, relatively little is known about their structure and mechanism. Here we report the three-dimensional atomic structure of the aminoglycoside acetyltransferase AAC(6′)-Ii in complex with coenzyme A (CoA). This structure unambiguously identifies the physiologically relevant AAC(6′)-Ii dimer species, and reveals that the enzyme structure is similar in the AcCoA and CoA bound forms. AAC(6′)-Ii is a member of the GCN5-related N-acetyltransferase (GNAT) superfamily of acetyltransferases, a diverse group of enzymes that possess a conserved structural motif, despite low sequence homology. AAC(6′)-Ii is also a member of a subset of enzymes in the GNAT superfamily that form multimeric complexes. The dimer arrangements within the multimeric GNAT superfamily members are compared, revealing that AAC(6′)-Ii forms a dimer assembly that is different from that observed in the other multimeric GNAT superfamily members. This different assembly may provide insight into the evolutionary processes governing dimer formation.
- Published
- 2003
19. Abstract 2479: A functionally null RAD51D missense mutation is strongly associated with ovarian carcinoma
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Barbara Rivera, Somayyeh Fahiminiya, François Rousseau, Mohammad Esmaeil Akbari, Damien Grapton, Alexandre Orthwein, Massimo Di Iorio, Olga Aleynikova, Valerie Hastings, Anne-Marie Mes-Masson, Guy A. Rouleau, Jacques L. Michaud, Sylvie Giroux, Jessica Frankum, Patricia N. Tonin, George Zogopoulos, William D. Foulkes, Suzanna L. Arcand, Diane Provencher, Christopher J. Lord, David L. Burk, Alexandre Dionne-Laporte, Nancy Hamel, Susanne Bens, Jacek Majewski, Rabea Wagener, Javad Nadaf, Eva Tomiak, Fadi F. Hamdan, Albert M. Berghuis, Reiner Siebert, and Steven A. Narod
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Oncology ,Cancer Research ,medicine.medical_specialty ,Internal medicine ,Ovarian carcinoma ,Mutation (genetic algorithm) ,medicine ,Biology - Abstract
Background and goal: RAD51D is a key player in DNA repair by homologous recombination (HR) and carriers of truncating RAD51D mutations have an increased risk for ovarian cancer (OC). However, the contribution of non-truncating RAD51D variants to cancer predisposition remains uncertain. We sought to fully characterize the previously described missense RAD51D variant c.620C>T;p.S207L in order to elucidate its role in OC. Methods: A clinical panel screening was used to identify the RAD51D variant c.620C>T;p.S207L in two French Canadian (FC) kindred affected with familial High Grade Serous Cancer (HGSC) of the ovary or endometrium. High resolution melting, TaqMan genotyping and Sanger sequencing were used to genotype the p.S207L variant in a series of unselected cases of HGSC of the ovary and endometrium, breast, pancreas and colorectal cancer and healthy controls, all of a FC origin. Whole exome sequencing (WES) was performed to study the genetic signature characterizing RAD51D associated tumors. RAD51 foci formation and CRISPR-Cas9-stimulated and HR-mediated gene targeting assays were used to assess HR activity of RAD51D-S207L mutated CHO cells. HR activity in RAD51D-S207L mutated human cells was tested by a DR-GFP assay. The effect of RAD51D p.S207L on RAD51D-XRCC2 interactions was analyzed by co-immunoprecipitation and quantified in-vivo in a single cell colocalization assay. Sensitivity to PARP inhibitors (PARPi) was evaluated in a cell survival assay. Results: Using deep sequencing and case-control genotyping studies, we showed that the missense RAD51D variant c.620C>T;p.S207L is over-represented in the French Canadian population affected by HGSC of the ovary (3.8% cases vs 0.002% controls; p < 0.0001).The frequency of the p.S207L variant did not differ from that of controls in breast, endometrial, pancreas and colorectal adenocarcinomas. A common haplotype shared by all the carriers suggested a founder origin for c.620C>T;p.S207L mutation. WES analysis of RAD51D tumor profiles revealed the presence of signature 3 which is known to be associated with HR defects. Functionally, we show that this mutation impairs HR by disrupting the RAD51D-XRCC2 interaction and confers PARP-inhibitor sensitivity to CHO cells. Conclusions: This work identifies RAD51D p.S207L as the first bona fide pathogenic missense susceptibility allele for HGSC of the ovary and supports the use of targeted PARPi therapies in OC patients carrying missense RAD51D mutations. Citation Format: Barbara Rivera, Massimo R. Di Iorio, Jessica Frankum, Javad Nadaf, Somayyeh Fahiminiya, Suzanna L. Arcand, David Burk, Damien Grapton, Eva Tomiak, Valerie Hastings, Nancy Hamel, Rabea Wagener, Olga Aleynikova, Sylvie Giroux, Fadi F. Hamdan, Alexandre Orthwein, George Zogopoulos, Francois Rousseau, Albert Berghuis, Diane M. Provencher, Guy A. Rouleau, Jacques L. Michaud, Anne-Marie Mes-Masson, Jacek Majewski, Susanne Bens, Reiner Siebert, Steven Narod, Mohammad Akbari, Chris J. Lord, Patricia N. Tonin, Alexandre Dionne-Laporte, William D. Foulkes. A functionally null RAD51D missense mutation is strongly associated with ovarian carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2479. doi:10.1158/1538-7445.AM2017-2479
- Published
- 2017
20. Structural analysis of a novel cyclohexylamine oxidase from Brevibacterium oxydans IH-35A
- Author
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Yoshie Hasegawa, I. Ahmad Mirza, Bing Xiong, Hiroaki Iwaki, Albert M. Berghuis, Stephan Grosse, David L. Burk, and Peter C. K. Lau
- Subjects
molecular cloning ,cyclohexylamine oxidase ,Cyclohexylamine oxidase ,Applied Microbiology ,lcsh:Medicine ,enzyme purification ,Molecular Dynamics ,01 natural sciences ,Biochemistry ,Substrate Specificity ,chemistry.chemical_compound ,Computational Chemistry ,Macromolecular Structure Analysis ,Brevibacterium ,lcsh:Science ,Biochemistry Simulations ,oxidoreductase ,chemistry.chemical_classification ,0303 health sciences ,Oxidoreductases Acting on CH-NH Group Donors ,conformational transition ,Multidisciplinary ,biology ,aliphatic amine ,structure analysis ,Enzyme structure ,unclassified drug ,Enzymes ,Chemistry ,chaA gene ,bacterial gene ,cyclohexanone ,Research Article ,Biotechnology ,crystal structure ,Protein Structure ,Flavoprotein ,Flavin group ,Cyclohexylamine ,010402 general chemistry ,Microbiology ,03 medical and health sciences ,Bacterial Proteins ,Oxidoreductase ,Escherichia coli ,enzyme specificity ,Biology ,hydrophobicity ,030304 developmental biology ,Microbial Metabolism ,flavine adenine nucleotide ,binding site ,lcsh:R ,Active site ,Proteins ,Computational Biology ,nucleotide sequence ,unspecific monooxygenase ,biology.organism_classification ,0104 chemical sciences ,brevibacterium oxydans ,chemistry ,Microbacterium oxydans ,Enzyme Structure ,gene expression ,biology.protein ,Biocatalysis ,lcsh:Q ,alicyclic amine - Abstract
Cyclohexylamine oxidase (CHAO) is a flavoprotein first described in Brevibacterium oxydans strain IH-35A that carries out the initial step of the degradation of the industrial chemical cyclohexylamine to cyclohexanone. We have cloned and expressed in Escherichia coli the CHAO-encoding gene (chaA) from B. oxydans, purified CHAO and determined the structures of both the holoenzyme form of the enzyme and a product complex with cyclohexanone. CHAO is a 50 kDa monomer with a PHBH fold topology. It belongs to the flavin monooxygenase family of enzymes and exhibits high substrate specificity for alicyclic amines and sec-alkylamines. The overall structure is similar to that of other members of the flavin monooxygenase family, but lacks either of the C- or N-terminal extensions observed in these enzymes. Active site features of the flavin monooxygenase family are conserved in CHAO, including the characteristic aromatic cage. Differences in the orientations of residues of the CHAO aromatic cage result in a substrate-binding site that is more open than those of its structural relatives. Since CHAO has a buried hydrophobic active site with no obvious route for substrates and products, a random acceleration molecular dynamics simulation has been used to identify a potential egress route. The path identified includes an intermediate cavity and requires transient conformation changes in a shielding loop and a residue at the border of the substrate-binding cavity. These results provide a foundation for further studies with CHAO aimed at identifying features determining substrate specificity and for developing the biocatalytic potential of this enzyme. © 2013 Mirza et al.
- Published
- 2012
21. Abstract LB-019: FGFR1 abnormalities in seizure-associated familial and sporadic dysembryoplastic neuroepithelial tumors
- Author
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Werner Paulus, Jian Zhang, Thomas Niederstadt, Jason Karamchandani, Paul J. Lockhart, Krzysztof Zakrzewski, Magdalena Zakrzewska, Gerhard Kurlemann, Roméo Sébastien Blanc, Maria Thom, Albert M. Berghuis, Steffen Albrecht, Somayyeh Fahiminiya, Damien Faury, Zuzanna Michalak, Jacek Majewski, Ronald Sträter, Michele Zeinieh, Reiner Siebert, Camelia M. Monoranu, Talia Boshari, Milou Ohm, Sanjay M. Sisodiya, Eric Bareke, Nada Jabado, Javad Nadaf, Duncan McGregor, David T.W. Jones, Pawel P. Liberski, Celia M. T. Greenwood, Susanne Bens, Benjamin Ellezam, Tenzin Gayden, David L. Burk, Barbara Rivera, Kornelius Kerl, William D. Foulkes, Martin Hasselblatt, Richard J. Leventer, and Ulrich Schueller
- Subjects
Sanger sequencing ,Cancer Research ,Pathology ,medicine.medical_specialty ,Fibroblast growth factor receptor 1 ,Neuropathology ,Biology ,medicine.disease ,Germline ,Epilepsy ,symbols.namesake ,Germline mutation ,Oncology ,Gene duplication ,medicine ,symbols ,Copy-number variation - Abstract
Background: Dysembryoplastic neuroepithelial tumors (DNETs) are benign developmental brain tumors associated with intractable, drug-resistant epilepsy. Distinguishing this entity from other low-grade ganglioneuronal tumors is challenging for neuro-pathologists. We set out to identify the genetic causes of DNETs and to clarify the molecular mechanisms underlying this condition. Experimental procedures: We collected a family with three individuals with seizures and multinodular DNETs together with 100 sporadic tumors from 96 persons referred to us as DNETs. Whole-exome sequencing was performed on 46 tumours and targeted sequencing for hotspot FGFR1 mutations and BRAFp.V600E was used on the remaining samples. Blind neuropathology review and molecular characterization were performed. FISH, Copy Number Variation assays and Sanger sequencing were used to validate the findings. Supporting evidence for functional defects was obtained by in silico modelling of novel FGFR1p.R661P variant. Functional impact of this and other FGFR1 mutations were assessed using Flow Cytometry and β-galactosidase staining in HEK293 cells. Results: We identified a novel germline FGFR1 mutation, p.R661P, in a father and his two children with DNETs. Somatic activating FGFR1 mutations (p.N546K or p.K656E) were observed in cis in the tumors with the germline mutation. Pathology review distinguished DNETs (WHO grade I) (45%) from DNET-like tumors (55%). FGFR1 alterations, mainly intragenic tyrosine kinase FGFR1 duplication and multiple mutants in cis, characterized DNETs (25/43;58.1%) whereas FGFR1 mutations (10/53;19%) (p < 0.0001) and hotspot BRAFp.V600E (12/53;22.6%) (p < 0.001) were identified in DNET-like tumors. Phospho-ERK overexpression in FGFR1p.R661P and p.N546K mutant cells support enhanced MAPK/ERK activation in this condition. Conclusions: This study identifies constitutional and somatic FGFR1 alterations and hotspot BRAFV600E as key events in DNETs and DNET-like tumors respectively. The final common effect of these alterations appears to be a balanced level of signalling that results in benign rather than malignant tumors. The integrated pathology and molecular characterization performed here reveals the key role of the MAP-Kinase pathway in these epileptogenic low-grade glioneuronal tumors, pointing the way towards existing targeted therapies. Citation Format: Barbara Rivera, Tenzin Gayden, Jian Zhang, Javad Nadaf, Talia Boshari, Damien Faury, Michele Zeinieh, Romeo Blanc, David Burk, Somayyeh Fahiminiya, Eric Bareke, Ulrich Schueller, Camelia M. Monoranu, Ronald Sträter, Kornelius Kerl, Thomas Niederstadt, Gerhard Kurlemann, Benjamin Ellezam, Zuzanna Michalak, Maria Thom, Paul Lockhart, Richard Leventer, Milou Ohm, Duncan McGregor, David Jones, Jason Karamchandani, Celia Greenwood, Albert Berghuis, Susanne Bens, Reiner Siebert, Magdalena Zakrzewska, Pawel Liberski, Krzysztof Zakrzewski, Sanjay Sisodiya, Werner Paulus, Steffen Albrecht, Martin Hasselblatt, Nada Jabado, William D. Foulkes, Jacek Majewski. FGFR1 abnormalities in seizure-associated familial and sporadic dysembryoplastic neuroepithelial tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-019.
- Published
- 2016
22. Preparation and characterization of bacterial protein complexes for structural analysis
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Albert M. Berghuis, Irena Ekiel, Guennadi Kozlov, Zongchao Jia, Allan Matte, Jean-François Trempe, Miroslaw Cygler, Kalle Gehring, Mark A. Currie, and David L. Burk
- Subjects
Bacterial protein ,Chemistry ,Biophysics ,Nuclear magnetic resonance spectroscopy ,Computational biology ,Function (biology) ,Characterization (materials science) ,Macromolecule - Abstract
Bacteria mediate a large variety of biological processes using protein complexes. These complexes range from simple binary heterodimeric enzymes to more complex multi-subunit complexes that can be described as macromolecular machines. A key to understanding how these complexes function is obtaining structural information using methods that include electron microscopy, small-angle X-ray scattering, NMR spectroscopy, and X-ray crystallography. Here we describe a variety of approaches to the expression, purification, and biophysical characterization of bacterial protein complexes as a prerequisite to structural analysis. We also give several examples of the kinds of information these different biophysical approaches can provide and various experimental approaches to obtaining structure information for a given system. Further, we describe several examples of protein complexes where we have obtained structural data that have led to new biological insights.
- Published
- 2011
23. BSSF: a fingerprint based ultrafast binding site similarity search and function analysis server
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Jingkang Shen, Hualiang Jiang, David L. Burk, Bing Xiong, Jie Wu, and Mengzhu Xue
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Computer science ,Nearest neighbor search ,Protein Data Bank (RCSB PDB) ,Genomics ,lcsh:Computer applications to medicine. Medical informatics ,computer.software_genre ,Biochemistry ,Protein Structure, Secondary ,DNA sequencing ,Structural genomics ,Similarity (network science) ,Structural Biology ,Binding site ,lcsh:QH301-705.5 ,Molecular Biology ,Gene ,Structure (mathematical logic) ,Binding Sites ,Methodology Article ,Applied Mathematics ,Fingerprint (computing) ,Computational Biology ,Proteins ,Enzyme Commission number ,Computer Science Applications ,lcsh:Biology (General) ,lcsh:R858-859.7 ,Data mining ,DNA microarray ,computer ,Software - Abstract
Background Genome sequencing and post-genomics projects such as structural genomics are extending the frontier of the study of sequence-structure-function relationship of genes and their products. Although many sequence/structure-based methods have been devised with the aim of deciphering this delicate relationship, there still remain large gaps in this fundamental problem, which continuously drives researchers to develop novel methods to extract relevant information from sequences and structures and to infer the functions of newly identified genes by genomics technology. Results Here we present an ultrafast method, named BSSF(Binding Site Similarity & Function), which enables researchers to conduct similarity searches in a comprehensive three-dimensional binding site database extracted from PDB structures. This method utilizes a fingerprint representation of the binding site and a validated statistical Z-score function scheme to judge the similarity between the query and database items, even if their similarities are only constrained in a sub-pocket. This fingerprint based similarity measurement was also validated on a known binding site dataset by comparing with geometric hashing, which is a standard 3D similarity method. The comparison clearly demonstrated the utility of this ultrafast method. After conducting the database searching, the hit list is further analyzed to provide basic statistical information about the occurrences of Gene Ontology terms and Enzyme Commission numbers, which may benefit researchers by helping them to design further experiments to study the query proteins. Conclusions This ultrafast web-based system will not only help researchers interested in drug design and structural genomics to identify similar binding sites, but also assist them by providing further analysis of hit list from database searching.
- Published
- 2010
24. The type IA topoisomerase catalytic cycle: A normal mode analysis and molecular dynamics simulation
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Xiaomin Luo, David L. Burk, Bing Xiong, Albert M. Berghuis, Jingkang Shen, Hong Liu, and Jianhua Shen
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DNA, Bacterial ,Models, Molecular ,Time Factors ,DNA, Single-Stranded ,Biology ,Crystallography, X-Ray ,Biochemistry ,Models, Biological ,Catalysis ,chemistry.chemical_compound ,Structural Biology ,Escherichia coli ,Topoisomerase III ,Protein–DNA interaction ,Computer Simulation ,Protein Structure, Quaternary ,Molecular Biology ,Genetics ,Binding Sites ,Topoisomerase ,DNA replication ,Temperature ,Protein Structure, Tertiary ,DNA binding site ,chemistry ,Catalytic cycle ,DNA Topoisomerases, Type I ,Biophysics ,biology.protein ,DNA supercoil ,Apoproteins ,DNA ,Protein Binding - Abstract
Type IA topoisomerases alter the topological state of DNA to relax the supercoils introduced during the DNA replication and transcription process, giving them critical roles in many cellular functions. To manipulate the DNA, type IA topoisomerases first cleave one DNA strand and form a covalent linkage between a catalytic tyrosine residue and the 5'-phosphoryl of the DNA. This is followed by a movement of domain III of the topoisomerase to accommodate the second DNA strand in the center channel of the topoisomerase. Domain III is then closed for religation of the cleaved DNA and subsequently reopened to release the passing strand. Although numerous biophysical and biochemical studies have examined this catalytic cycle, fundamental questions remain such as how domain III opens and closes during this process. We have used computational simulation methods, namely normal mode analysis and molecular dynamics, to investigate the catalytic cycle of Escherichia coli topoisomerase III as a representative of the type IA topoisomerases. It was found that domain II is intrinsically flexible and may empower the enzyme to perform its function by triggering domain III opening and closing. A molecular dynamics simulation and MM-PBSA analysis shows that topoisomerase III alone cannot overcome the large energy barrier of the conformational transition. A detailed examination of the DNA binding sites suggests that the processing DNA cooperates with the topoisomerase to accomplish this dramatic conformational change. These findings will guide future mutagenesis studies of type IA topoisomerases aimed at dissecting the driving forces and conformations in the catalytic cycle.
- Published
- 2008
25. Roles of the equatorial tyrosyl iron ligand of protocatechuate 3,4-dioxygenase in catalysis
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John D. Lipscomb, David L. Burk, C. Kent Brown, Douglas H. Ohlendorf, Matthew W. Vetting, and Michael P. Valley
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Denticity ,Stereochemistry ,Iron ,Parabens ,Protocatechuate-3,4-Dioxygenase ,Crystallography, X-Ray ,Iron Chelating Agents ,Biochemistry ,Ferric Compounds ,Catalysis ,Bacterial Proteins ,Dioxygenase ,Oxidoreductase ,Hydroxides ,Histidine ,Lewis acids and bases ,Enzyme Inhibitors ,chemistry.chemical_classification ,Binding Sites ,biology ,Ligand ,Pseudomonas putida ,Active site ,Protein Structure, Tertiary ,Oxygen ,Catalytic cycle ,chemistry ,Amino Acid Substitution ,biology.protein ,Tyrosine ,Protein Binding - Abstract
The active site Fe(III) of protocatechuate 3,4-dioxygenase (3,4-PCD) from Pseudomonas putida is ligated axially by Tyr447 and His462 and equatorially by Tyr408, His460, and OH - . Tyr447 and OH - are displaced as protocatechuate (3,4-dihydroxybenzoate, PCA) chelates the iron and appear to serve as in situ bases to promote this process. The role(s) of Tyr408 is (are) explored here using mutant enzymes that exhibit less than 0.1% wild-type activity. The X-ray crystal structures of the mutants and their PCA complexes show that the new shorter residues in the 408 position cannot ligate the iron and instead interact with the iron through solvents. Moreover, PCA binds as a monodentate rather than a bidentate ligand, and Tyr447 fails to dissociate. Although the new residues at position 408 do not directly bind to the iron, large changes in the spectroscopic and catalytic properties are noted among the mutant enzymes. Resonance Raman features show that the Fe-O bond of the monodentate 4-hydroxybenzoate (4HB) inhibitor complex is significantly stronger in the mutants than in wild-type 3,4-PCD. Transient kinetic studies show that PCA and 4HB bind to 3,4-PCD in a fast, reversible step followed by a step in which coordination to the metal occurs; the latter process is at least 50-fold slower in the mutant enzymes. It is proposed that, in wild-type 3,4-PCD, the Lewis base strength of Tyr408 lowers the Lewis acidity of the iron to foster the rapid exchange of anionic ligands during the catalytic cycle. Accordingly, the increase in Lewis acidity of the iron caused by substitution of this residue by solvent tends to make the iron substitution inert. Tyr447 cannot be released to allow formation of the usual dianionic PCA chelate complex with the active site iron, and the rate of electrophilic attack by O 2 becomes rate limiting overall. The structures of the PCA complexes of these mutant enzymes show that hydrogen-bonding interactions between the new solvent ligand and the new second-sphere residue in position 408 allow this residue to significantly influence the spectroscopic and kinetic properties of the enzymes. Protocatechuate 3,4-dioxygenase (3,4-PCD) 1 (E.C. 1.13.11.3) catalyzes the ring cleavage of protocatechuate (PCA, 3,4- dihydroxybenzoate) with the incorporation of both atoms of molecular oxygen to form ‚-carboxy-cis,cis-muconate (1, 2).
- Published
- 2005
26. Structures of aminoglycoside acetyltransferase AAC(6')-Ii in a novel crystal form: structural and normal-mode analyses
- Author
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Caroline Breitbach, Albert M. Berghuis, Bing Xiong, and David L. Burk
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chemistry.chemical_classification ,biology ,Stereochemistry ,Protein Conformation ,Dimer ,Coenzyme A ,Aminoglycoside ,Acetyltransferases ,General Medicine ,Protomer ,Crystallography, X-Ray ,Cofactor ,chemistry.chemical_compound ,Enzyme ,chemistry ,Structural Biology ,biology.protein ,Escherichia coli ,Transferase ,Cloning, Molecular ,Crystallization ,Pliability - Abstract
The aminoglycoside-modifying enzyme aminoglycoside 6'-N-acetyltransferase type Ii [AAC(6')-Ii] has been crystallized with its cofactor coenzyme A in space group C222(1), with unit-cell parameters a = 71.5, b = 127.4, c = 76.9 A and one physiologically relevant dimer species per asymmetric unit. The space group previously observed for this complex was P2(1)2(1)2(1), with two dimers per asymmetric unit. By comparing the six available protomer structures of the AAC(6')-Ii-CoA complex, it has been possible to identify regions of plasticity within the protein. Normal-mode analysis of this complex suggests that this plasticity is not an artefact of crystal-packing forces, but that the region of the protomer that displays multiple conformations is intrinsically flexible. It is conjectured that the flexibility is relevant for the cooperative activity observed for the enzyme.
- Published
- 2005
27. Protein kinase inhibitors and antibiotic resistance
- Author
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David L. Burk and Albert M. Berghuis
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Pharmacology ,chemistry.chemical_classification ,Binding Sites ,biology ,Kinase ,Aminoglycoside ,Drug Resistance ,Intracellular Signaling Peptides and Proteins ,Active site ,Microbiology ,Anti-Bacterial Agents ,Structure-Activity Relationship ,Enzyme ,Antibiotic resistance ,Aminoglycosides ,chemistry ,Biochemistry ,biology.protein ,Phosphorylation ,Pharmacology (medical) ,Binding site ,Protein kinase A ,Carrier Proteins - Abstract
While antibiotics revolutionized the treatment of infectious disease in the 20th century, bacterial resistance now threatens to render many of them ineffective. Aminoglycosides are a class of clinically important antibiotics used in the treatment of infections caused by Gram-positive and -negative organisms. They are bactericidal, targeting the bacterial ribosome, where they bind to the A-site and disrupt protein synthesis. Clinical resistance to these drugs occurs mainly via enzymatic inactivation by aminoglycoside-modifying enzymes that phosphorylate, adenylate, or acetylate the aminoglycoside. Those that phosphorylate (i.e., aminoglycoside kinases) have been shown to be structurally related to eukaryotic protein kinases. This was surprising, given the low degree of sequence similarity between the groups of enzymes. The nucleotide-binding site, specifically, is very similar in structure, suggesting that the two classes of enzymes share a common mechanism of phosphoryl transfer. Three strategies can be envisaged for combating aminoglycoside kinase-mediated bacterial resistance. The first involves compounds that target the antibiotic binding region. Secondly, protein kinase inhibitors have been identified that disable aminoglycoside-modifying enzymes by targeting the ATP-binding site. Lastly, compounds are being developed that exploit the bridged nature of the active site, incorporating nucleotide and substrate motifs. A strategy using bifunctional aminoglycoside dimers has also been pursued, yielding molecules that bind to the target site on the bacterial ribosome, while serving as poor substrates for modifying enzymes. This work holds out the promise that effective inhibitors of aminoglycoside-modifying enzymes may eventually restore the usefulness of aminoglycoside antibiotics.
- Published
- 2002
28. DrugViz: a Cytoscape plugin for visualizing and analyzing small molecule drugs in biological networks
- Author
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Keqin Liu, Jingkang Shen, Hualiang Jiang, David L. Burk, Bing Xiong, and Jie Wu
- Subjects
Statistics and Probability ,Databases, Factual ,Proteome ,Computer science ,Systems biology ,computer.software_genre ,Biochemistry ,Interactome ,User-Computer Interface ,chemistry.chemical_compound ,Metabolomics ,Computer Graphics ,Chemogenomics ,Computer Simulation ,Databases, Protein ,Cluster analysis ,Molecular Biology ,Fingerprint (computing) ,Small molecule ,Computer Science Applications ,Visualization ,Computational Mathematics ,Pharmaceutical Preparations ,Computational Theory and Mathematics ,chemistry ,Drug Design ,Data mining ,computer ,Software ,Biological network - Abstract
Summary: DrugViz is a Cytoscape plugin that is designed to visualize and analyze small molecules within the framework of the interactome. DrugViz can import drug–target network information in an extended SIF file format to Cytoscape and display the two-dimensional (2D) structures of small molecule nodes in a unified visualization environment. It also can identify small molecule nodes by means of three different 2D structure searching methods, namely isomorphism, substructure and fingerprint-based similarity searches. After selections, users can furthermore conduct a two-side clustering analysis on drugs and targets, which allows for a detailed analysis of the active compounds in the network, and elucidate relationships between these drugs and targets. DrugViz represents a new tool for the analysis of data from chemogenomics, metabolomics and systems biology. Availability: DrugViz and data set used in Application are freely available for download at http://202.127.30.184:8080/software.html Contact: jkshen@mail.shcnc.ac.cn
- Published
- 2008
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