59 results on '"Naomi, Ariel"'
Search Results
2. Disruption of the NlpD lipoprotein of the plague pathogen Yersinia pestis affects iron acquisition and the activity of the twin-arginine translocation system.
- Author
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Avital Tidhar, Yinon Levy, Ayelet Zauberman, Yaron Vagima, David Gur, Moshe Aftalion, Ofir Israeli, Theodor Chitlaru, Naomi Ariel, Yehuda Flashner, Anat Zvi, and Emanuelle Mamroud
- Subjects
Arctic medicine. Tropical medicine ,RC955-962 ,Public aspects of medicine ,RA1-1270 - Abstract
We have previously shown that the cell morphogenesis NlpD lipoprotein is essential for virulence of the plague bacteria, Yersinia pestis. To elucidate the role of NlpD in Y. pestis pathogenicity, we conducted a whole-genome comparative transcriptome analysis of the wild-type Y. pestis strain and an nlpD mutant under conditions mimicking early stages of infection. The analysis suggested that NlpD is involved in three phenomena: (i) Envelope stability/integrity evidenced by compensatory up-regulation of the Cpx and Psp membrane stress-response systems in the mutant; (ii) iron acquisition, supported by modulation of iron metabolism genes and by limited growth in iron-deprived medium; (iii) activity of the twin-arginine (Tat) system, which translocates folded proteins across the cytoplasmic membrane. Virulence studies of Y. pestis strains mutated in individual Tat components clearly indicated that the Tat system is central in Y. pestis pathogenicity and substantiated the assumption that NlpD essentiality in iron utilization involves the activity of the Tat system. This study reveals a new role for NlpD in Tat system activity and iron assimilation suggesting a modality by which this lipoprotein is involved in Y. pestis pathogenesis.
- Published
- 2019
- Full Text
- View/download PDF
3. Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates
- Author
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Tal Noy-Porat, Ronit Rosenfeld, Naomi Ariel, Eyal Epstein, Ron Alcalay, Anat Zvi, Chanoch Kronman, Arie Ordentlich, and Ohad Mazor
- Subjects
ricin ,antibody ,neutralization ,affinity ,immunization ,non-human primates ,Medicine - Abstract
Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit) was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (koff < 1 × 10−7 s−1) that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication.
- Published
- 2016
- Full Text
- View/download PDF
4. Disruption of the NlpD lipoprotein of the plague pathogen Yersinia pestis affects iron acquisition and the activity of the twin-arginine translocation system
- Author
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Yaron Vagima, Moshe Aftalion, Anat Zvi, David Gur, Ofir Israeli, Ayelet Zauberman, Yinon Levy, Naomi Ariel, Emanuelle Mamroud, Theodor Chitlaru, Avital Tidhar, and Yehuda Flashner
- Subjects
0301 basic medicine ,Bacterial Diseases ,Mutant ,RC955-962 ,Pathology and Laboratory Medicine ,Biochemistry ,Transcriptome ,Gene Knockout Techniques ,Mice ,0302 clinical medicine ,Fluorescence Microscopy ,Arctic medicine. Tropical medicine ,Gene expression ,Medicine and Health Sciences ,Pathogen ,Twin-Arginine-Translocation System ,Microscopy ,Light Microscopy ,Animal Models ,Yersinia ,Bacterial Pathogens ,Mutant Strains ,Phenotypes ,Infectious Diseases ,Experimental Organism Systems ,Medical Microbiology ,Female ,Public aspects of medicine ,RA1-1270 ,Pathogens ,Research Article ,Yersinia Pestis ,Virulence Factors ,Lipoproteins ,Iron ,030231 tropical medicine ,Virulence ,Mouse Models ,Biology ,Research and Analysis Methods ,Microbiology ,Iron assimilation ,03 medical and health sciences ,Model Organisms ,Bacterial Proteins ,Genetics ,Animals ,Microbial Pathogens ,Bacteria ,Cell morphogenesis ,Gene Expression Profiling ,Public Health, Environmental and Occupational Health ,Organisms ,Biology and Life Sciences ,Proteins ,Biological Transport ,Gene Expression Regulation, Bacterial ,biology.organism_classification ,Plagues ,030104 developmental biology ,Yersinia pestis ,Mutation ,Animal Studies - Abstract
We have previously shown that the cell morphogenesis NlpD lipoprotein is essential for virulence of the plague bacteria, Yersinia pestis. To elucidate the role of NlpD in Y. pestis pathogenicity, we conducted a whole-genome comparative transcriptome analysis of the wild-type Y. pestis strain and an nlpD mutant under conditions mimicking early stages of infection. The analysis suggested that NlpD is involved in three phenomena: (i) Envelope stability/integrity evidenced by compensatory up-regulation of the Cpx and Psp membrane stress-response systems in the mutant; (ii) iron acquisition, supported by modulation of iron metabolism genes and by limited growth in iron-deprived medium; (iii) activity of the twin-arginine (Tat) system, which translocates folded proteins across the cytoplasmic membrane. Virulence studies of Y. pestis strains mutated in individual Tat components clearly indicated that the Tat system is central in Y. pestis pathogenicity and substantiated the assumption that NlpD essentiality in iron utilization involves the activity of the Tat system. This study reveals a new role for NlpD in Tat system activity and iron assimilation suggesting a modality by which this lipoprotein is involved in Y. pestis pathogenesis., Author summary We have previously shown that the NlpD lipoprotein, which is involved in the regulation of cell morphogenesis, is essential for virulence of the plague bacteria, Yersinia pestis. To uncover the role of NlpD in Y. pestis pathogenicity, we conducted a whole-genome comparative transcriptome analysis as well as phenotypic and virulence evaluation analyses of the nlpD and related mutants. The study reveals a new role for the Y. pestis NlpD lipoprotein in iron assimilation and Tat system activity.
- Published
- 2019
5. A Rapid Molecular Test for Determining Yersinia pestis Susceptibility to Ciprofloxacin by the Quantification of Differentially Expressed Marker Genes
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Ida Steinberger-Levy, Sharon Maoz, David Gur, Ofir Israeli, Naomi Ariel, Anat Zvi, Raphael Ber, Moshe Aftalion, Adi Beth-Din, and Ohad Shifman
- Subjects
0301 basic medicine ,Microbiology (medical) ,medicine.drug_class ,030106 microbiology ,Antibiotics ,lcsh:QR1-502 ,Virulence ,minimal inhibitory concentration (MIC) ,Biology ,Microbiology ,lcsh:Microbiology ,antibiotics ,03 medical and health sciences ,Minimum inhibitory concentration ,minimal inhibitory concentrations (MIC) ,In vivo ,ciprofloxacin ,medicine ,Original Research ,molecular testing ,rapid AST ,qRT-PCR ,RNA expression ,biology.organism_classification ,Ciprofloxacin ,030104 developmental biology ,Real-time polymerase chain reaction ,Yersinia pestis ,Y. pestis ,Bacteria ,medicine.drug - Abstract
Standard antimicrobial susceptibility tests used to determine bacterial susceptibility to antibiotics are growth dependent and time consuming. The long incubation time required for standard tests may render susceptibility results irrelevant, particularly for patients infected with lethal bacteria that are slow growing on agar but progress rapidly in vivo, such as Yersinia pestis. Here, we present an alternative approach for the rapid determination of antimicrobial susceptibility, based on the quantification of the changes in the expression levels of specific marker genes following exposure to growth-inhibiting concentrations of the antibiotic, using Y. pestis and ciprofloxacin as a model. The marker genes were identified by transcriptomic DNA microarray analysis of the virulent Y. pestis Kimberley53 strain after exposure to specific concentrations of ciprofloxacin for various time periods. We identified several marker genes that were induced following exposure to growth-inhibitory concentrations of ciprofloxacin, and we confirmed the marker expression profiles at additional ciprofloxacin concentrations using quantitative RT-PCR. Eleven candidate marker transcripts were identified, of which four mRNA markers were selected for a rapid quantitative RT-PCR susceptibility test that correctly determined the Minimal Inhibitory Concentration (MIC) values and the categories of susceptibility of several Y. pestis strains and isolates harboring various ciprofloxacin MIC values. The novel molecular susceptibility test requires just 2 h of antibiotic exposure in a 7-h overall test time, in contrast to the 24 h of antibiotic exposure required for a standard microdilution test.
- Published
- 2016
6. Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates
- Author
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Naomi Ariel, Ronit Rosenfeld, Anat Zvi, Eyal Epstein, Arie Ordentlich, Ohad Mazor, Ron Alcalay, Chanoch Kronman, and Tal Noy-Porat
- Subjects
0301 basic medicine ,endocrine system ,Health, Toxicology and Mutagenesis ,medicine.medical_treatment ,lcsh:Medicine ,Ricin ,Toxicology ,medicine.disease_cause ,immunization ,Neutralization ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Epitopes ,ricin ,antibody ,neutralization ,affinity ,non-human primates ,medicine ,Animals ,Humans ,Glycosides ,Antidote ,Mice, Inbred ICR ,030102 biochemistry & molecular biology ,biology ,Toxin ,Ricinus ,lcsh:R ,biology.organism_classification ,Virology ,Antibodies, Neutralizing ,Macaca mulatta ,In vitro ,Triterpenes ,carbohydrates (lipids) ,enzymes and coenzymes (carbohydrates) ,030104 developmental biology ,Immunization ,chemistry ,Immunology ,Vaccines, Subunit ,biology.protein ,Female ,Antibody ,HeLa Cells - Abstract
Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit) was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (k(off )1 × 10(-7) s(-1)) that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication.
- Published
- 2016
7. The solute-binding component of a putative Mn(II) ABC transporter (MntA) is a novelBacillus anthracisvirulence determinant
- Author
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Haim Grosfeld, Naomi Ariel, Shay Weiss, Theodor Chitlaru, Haim Levy, Orit Gat, Zeev Altboum, Itai Mendelson, Sara Cohen, and Avigdor Shafferman
- Subjects
Plasmid ,Attenuated vaccine ,biology ,In vivo ,Mutant ,Virulence ,ATP-binding cassette transporter ,biology.organism_classification ,Molecular Biology ,Microbiology ,Bacteria ,Bacillus anthracis - Abstract
Here we describe the characterization of a lipoprotein previously proposed as a potential Bacillus anthracis virulence determinant and vaccine candidate. This protein, designated MntA, is the solute-binding component of a manganese ion ATP-binding cassette transporter. Coupled proteomic-serological screen of a fully virulent wild-type B. anthracis Vollum strain, confirmed that MntA is expressed both in vitro and during infection. Expression of MntA is shown to be independent of the virulence plasmids pXO1 and pXO2. An mntA deletion, generated by allelic replacement, results in complete loss of MntA expression and its phenotypic analysis revealed: (i) impaired growth in rich media, alleviated by manganese supplementation; (ii) increased sensitivity to oxidative stress; and (iii) delayed release from cultured macrophages. The DeltamntA mutant expresses the anthrax-associated classical virulence factors, lethal toxin and capsule, in vitro as well as in vivo, and yet the mutation resulted in severe attenuation; a 10(4)-fold drop in LD(50) in a guinea pig model. MntA expressed in trans allowed to restore, almost completely, the virulence of the DeltamntA B. anthracis strain. We propose that MntA is a novel B. anthracis virulence determinant essential for the development of anthrax disease, and that B. anthracisDeltamntA strains have the potential to serve as platform for future live attenuated vaccines.
- Published
- 2005
8. Identification of strain specific markers inBacillus anthracisby random amplification of polymorphic DNA
- Author
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Zeev Altboum, Naomi Ariel, Morly Fisher, Haim Levy, and David Kobiler
- Subjects
DNA, Bacterial ,Genetic Markers ,Genotype ,Bacillus ,Microbiology ,law.invention ,chemistry.chemical_compound ,Species Specificity ,law ,Genetics ,Typing ,Cloning, Molecular ,Molecular Biology ,Phylogeny ,Polymerase chain reaction ,Polymorphism, Genetic ,Base Sequence ,biology ,Strain (biology) ,biology.organism_classification ,Random Amplified Polymorphic DNA Technique ,RAPD ,Bacillus anthracis ,chemistry ,Genetic marker ,Databases, Nucleic Acid ,DNA - Abstract
Classification and differentiation of Bacillus anthracis isolates by genetic markers play an important role in anthrax research. We used a PCR based method – Random Amplification of Polymorphic DNA (RAPD) – to identify genetic markers in B. anthracis strains. Twenty-five differential genetic markers were identified which divided the strains into five different groups. Three selected RAPD-markers were cloned and sequenced. The five RAPD-derived genotypes could be defined by integration of these three markers. This system offers a simple non-expensive method to classify B. anthracis strains in laboratories involved in the research of this bacterium.
- Published
- 2005
9. Identification of chromosomally encoded membranal polypeptides ofBacillus anthracis by a proteomic analysis: Prevalence of proteins containingS-layer homology domains
- Author
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Menahem Lion, Eytan Elhanany, Avigdor Shafferman, Anat Zvi, Naomi Ariel, Theodor Chitlaru, and Baruch Velan
- Subjects
Proteome ,Protein subunit ,Blotting, Western ,Guinea Pigs ,Molecular Sequence Data ,Virulence ,Biology ,Proteomics ,Biochemistry ,Chromosomes ,Microbiology ,Open Reading Frames ,Bacterial Proteins ,Animals ,Urea ,Electrophoresis, Gel, Two-Dimensional ,Trypsin ,Amino Acid Sequence ,Molecular Biology ,Anthrax vaccines ,Immunogenicity ,Cell Membrane ,DNA ,biology.organism_classification ,Protein Structure, Tertiary ,Bacillus anthracis ,Open reading frame ,Databases as Topic ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Electrophoresis, Polyacrylamide Gel ,Rabbits ,Peptides ,S-layer ,Plasmids - Abstract
Bacillus anthracis is the causative agent of anthrax disease. Improvement of existing anthrax vaccines, which are currently based on the administration of Protective Antigen (the highly immunogenic nontoxic subunit of the bacterial toxin) may entail other bacterial immunogenic elements, part of which are predicted to reside on the surface of bacterial cells. In the present study, membranal proteins extracted from a stationary-phase culture of a nonvirulent B. anthracis strain, devoid of the native virulence plasmids pXO1 and pXO2, were separated by two-dimensional electrophoresis (2-DE) and a characteristic protein map was defined. The proteomic analysis allowed matrix-assisted laser desorption/ionization-time of flight mass spectrometry-assisted identification of 86 protein spots which represent the product of 30 individual open reading frames (ORF). Among these, a prevalent class of proteins was the S-layer proteins (which were found to represent more than 75% of the B. anthracis membranal fraction) and proteins containing S-layer homology (SLH)-membranal localization domains. Five novel SLH proteins, previously inferred only from bioinformatic ORF analysis (draft genome sequence), were identified and one was shown to be a highly abundant membranal protein. Western blots of the 2-DE gels were probed with sera from convalescent rabbits and guinea pigs infected with virulent B. anthracis (Vollum strain). This analysis revealed that B. anthracis immune animals exhibit antibodies against at least 14 distinct membranal proteins present in the 2-DE map, establishing that these proteins are expressed in vivo and are able to elicit an immune response. The identification of the protein components of the B. anthracis membranal fraction, as well as the establishment of their potential immunogenicity, underscore the strength of the proteomic approach for identifying molecules which may serve for further analysis of immune and protective abilities.
- Published
- 2004
10. Genome-Based Bioinformatic Selection of Chromosomal Bacillus anthracis Putative Vaccine Candidates Coupled with Proteomic Identification of Surface-Associated Antigens
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Theodor Chitlaru, Avigdor Shafferman, Eytan Elhanany, Naomi Ariel, Anat Zvi, S. Cohen, A. M. Friedlander, K. S. Makarova, and Baruch Velan
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Proteome ,Immunology ,Molecular Genomics ,Anthrax Vaccines ,Proteomics ,Microbiology ,Genome ,Open Reading Frames ,Bacterial Proteins ,Animals ,Humans ,ORFS ,Adhesins, Bacterial ,Gene ,Cellular localization ,Genetics ,Antigens, Bacterial ,Anthrax vaccines ,Virulence ,biology ,Computational Biology ,biology.organism_classification ,Enzymes ,Bacillus anthracis ,Infectious Diseases ,Genes, Bacterial ,Antigens, Surface ,Parasitology ,Genome, Bacterial - Abstract
Bacillus anthracis (Ames strain) chromosome-derived open reading frames (ORFs), predicted to code for surface exposed or virulence related proteins, were selected as B. anthracis -specific vaccine candidates by a multistep computational screen of the entire draft chromosome sequence (February 2001 version, 460 contigs, The Institute for Genomic Research, Rockville, Md.). The selection procedure combined preliminary annotation (sequence similarity searches and domain assignments), prediction of cellular localization, taxonomical and functional screen and additional filtering criteria (size, number of paralogs). The reductive strategy, combined with manual curation, resulted in selection of 240 candidate ORFs encoding proteins with putative known function, as well as 280 proteins of unknown function. Proteomic analysis of two-dimensional gels of a B. anthracis membrane fraction, verified the expression of some gene products. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analyses allowed identification of 38 spots cross-reacting with sera from B. anthracis immunized animals. These spots were found to represent eight in vivo immunogens, comprising of EA1, Sap, and 6 proteins whose expression and immunogenicity was not reported before. Five of these 8 immunogens were preselected by the bioinformatic analysis (EA1, Sap, 2 novel SLH proteins and peroxiredoxin/AhpC), as vaccine candidates. This study demonstrates that a combination of the bioinformatic and proteomic strategies may be useful in promoting the development of next generation anthrax vaccine.
- Published
- 2003
11. Search for Potential Vaccine Candidate Open Reading Frames in the Bacillus anthracis Virulence Plasmid pXO1: In Silico and In Vitro Screening
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Y. Inbar, Avigdor Shafferman, Orit Gat, Naomi Ariel, Baruch Velan, Haim Grosfeld, S. Cohen, and Anat Zvi
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DNA, Bacterial ,Transcription, Genetic ,Guinea Pigs ,Immunology ,Virulence ,Molecular Genomics ,Anthrax Vaccines ,Bacillus subtilis ,Microbiology ,Open Reading Frames ,Plasmid ,Bacterial Proteins ,Animals ,Cloning, Molecular ,ORFS ,Genetics ,Comparative genomics ,Antigens, Bacterial ,biology ,Immune Sera ,Computational Biology ,biology.organism_classification ,Bacillus anthracis ,Infectious Diseases ,Protein Biosynthesis ,Bacillus halodurans ,Parasitology ,Rabbits ,Functional genomics ,Plasmids - Abstract
A genomic analysis of the Bacillus anthracis virulence plasmid pXO1, aimed at identifying potential vaccine candidates and virulence-related genes, was carried out. The 143 previously defined open reading frames (ORFs) (R. T. Okinaka, K. Cloud, O. Hampton, A. R. Hoffmaster, K. K. Hill, P. Keim, T. M. Koehler, G. Lamke, S. Kumano, J. Mahillon, D. Manter, Y. Martinez, D. Ricke, R. Svensson, and P. J. Jackson, J. Bacteriol. 181: 6509-6515, 1999) were subjected to extensive sequence similarity searches (with the nonredundant and unfinished microbial genome databases), as well as motif, cellular location, and domain analyses. A comparative genomics analysis was conducted with the related genomes of Bacillus subtilis , Bacillus halodurans , and Bacillus cereus and the pBtoxis plasmid of Bacillus thuringiensis var. israeliensis. As a result, the percentage of ORFs with clues about their functions increased from ∼30% (as previously reported) to more than 60%. The bioinformatics analysis permitted identification of novel genes with putative relevance for pathogenesis and virulence. Based on our analyses, 11 putative proteins were chosen as targets for functional genomics studies. A rapid and efficient functional screening method was developed, in which PCR-amplified full-length linear DNA products of the selected ORFs were transcribed and directly translated in vitro and their immunogenicities were assessed on the basis of their reactivities with hyperimmune anti- B. anthracis antisera. Of the 11 ORFs selected for analysis, 9 were successfully expressed as full-length polypeptides, and 3 of these were found to be antigenic and to have immunogenic potential. The latter ORFs are currently being evaluated to determine their vaccine potential.
- Published
- 2002
12. Structures of recombinant native and E202Q mutant human acetylcholinesterase complexed with the snake-venom toxin fasciculin-II
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Michal Harel, Joel L. Sussman, Naomi Ariel, Baruch Velan, Gitay Kryger, Kurt Giles, Chanoch Kronman, Arie Lazar, Lilly Toker, Dov Barak, Avigdor Shafferman, and Israel Silman
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Protein Conformation ,Stereochemistry ,Molecular Sequence Data ,Mutant ,Venom ,Biology ,Crystallography, X-Ray ,law.invention ,chemistry.chemical_compound ,Structural Biology ,law ,Catalytic triad ,Animals ,Humans ,Molecular replacement ,Amino Acid Sequence ,Elapidae ,Elapid Venoms ,chemistry.chemical_classification ,Sequence Homology, Amino Acid ,General Medicine ,Acetylcholinesterase ,Recombinant Proteins ,Enzyme ,Biochemistry ,chemistry ,Mutagenesis, Site-Directed ,Recombinant DNA ,Cholinesterase Inhibitors ,Torpedo - Abstract
Structures of recombinant wild-type human acetylcholinesterase and of its E202Q mutant as complexes with fasciculin-II, a 'three-finger' polypeptide toxin purified from the venom of the eastern green mamba (Dendroaspis angusticeps), are reported. The structure of the complex of the wild-type enzyme was solved to 2.8 A resolution by molecular replacement starting from the structure of the complex of Torpedo californica acetylcholinesterase with fasciculin-II and verified by starting from a similar complex with mouse acetylcholinesterase. The overall structure is surprisingly similar to that of the T. californica enzyme with fasciculin-II and, as expected, to that of the mouse acetylcholinesterase complex. The structure of the E202Q mutant complex was refined starting from the corresponding wild-type human acetylcholinesterase structure, using the 2.7 A resolution data set collected. Comparison of the two structures shows that removal of the charged group from the protein core and its substitution by a neutral isosteric moiety does not disrupt the functional architecture of the active centre. One of the elements of this architecture is thought to be a hydrogen-bond network including residues Glu202, Glu450, Tyr133 and two bridging molecules of water, which is conserved in other vertebrate acetylcholinesterases as well as in the human enzyme. The present findings are consistent with the notion that the main role of this network is the proper positioning of the Glu202 carboxylate relative to the catalytic triad, thus defining its functional role in the interaction of acetylcholinesterase with substrates and inhibitors.
- Published
- 2000
13. Functional Characteristics of the Oxyanion Hole in Human Acetylcholinesterase
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Arie Ordentlich, Baruch Velan, Dov Barak, Naomi Ariel, Yoffi Segall, Chanoch Kronman, and Avigdor Shafferman
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Steric effects ,Stereochemistry ,Organophosphonates ,Biochemistry ,Michaelis–Menten kinetics ,Phosphates ,Substrate Specificity ,Active center ,Organophosphorus Compounds ,Transition state analog ,medicine ,Humans ,Enzyme Inhibitors ,Phosphorylation ,Molecular Biology ,chemistry.chemical_classification ,Binding Sites ,Molecular Structure ,Paraoxon ,Hydrolysis ,Cell Biology ,Recombinant Proteins ,Turnover number ,Kinetics ,Enzyme ,chemistry ,Acetylcholinesterase ,Mutagenesis, Site-Directed ,Carbamates ,Oxyanion hole ,Pyridostigmine Bromide ,medicine.drug - Abstract
The contribution of the oxyanion hole to the functional architecture and to the hydrolytic efficiency of human acetylcholinesterase (HuAChE) was investigated through single replacements of its elements, residues Gly-121, Gly-122 and the adjacent residue Gly-120, by alanine. All three substitutions resulted in about 100-fold decrease of the bimolecular rate constants for hydrolysis of acetylthiocholine; however, whereas replacements of Gly-120 and Gly-121 affected only the turnover number, mutation of residue Gly-122 had an effect also on the Michaelis constant. The differential behavior of the G121A and G122A enzymes was manifested also toward the transition state analog m-(N,N, N-trimethylammonio)trifluoroacetophenone (TMTFA), organophosphorous inhibitors, carbamates, and toward selected noncovalent active center ligands. Reactivity of both mutants toward TMTFA was 2000-11, 000-fold lower than that of the wild type HuAChE; however, the G121A enzyme exhibited a rapid inhibition pattern, as opposed to the slow binding kinetics shown by the G122A enzyme. For both phosphates (diethyl phosphorofluoridate, diisopropyl phosphorofluoridate, and paraoxon) and phosphonates (sarin and soman), the decrease in inhibitory activity toward the G121A enzyme was very substantial (2000-6700-fold), irrespective of size of the alkoxy substituents on the phosphorus atom. On the other hand, for the G122A HuAChE the relative decline in reactivity toward phosphonates (500-460-fold) differed from that toward the phosphates (12-95-fold). Although formation of Michaelis complexes with substrates does not seem to involve significant interaction with the oxyanion hole, interactions with this motif are a major stabilizing element in accommodation of covalent inhibitors like organophosphates or carbamates. These observations and molecular modeling suggest that replacements of residues Gly-120 or Gly-121 by alanine alter the structure of the oxyanion hole motif, abolishing the H-bonding capacity of residue at position 121. These mutations weaken the interaction between HuAChE and the various ligands by 2.7-5.0 kcal/mol. In contrast, variations in reactivity due to replacement of residue Gly-122 seem to result from steric hindrance at the active center acyl pocket.
- Published
- 1998
14. Aging of phosphylated human acetylcholinesterase: catalytic processes mediated by aromatic and polar residues of the active centre
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Avigdor Shafferman, Naomi Ariel, Baruch Velan, Dov Barak, Dana Stein, and Arie Ordentlich
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Models, Molecular ,Aging ,Protein Conformation ,Stereochemistry ,Mutant ,In Vitro Techniques ,Biochemistry ,Catalysis ,Cell Line ,Serine ,chemistry.chemical_compound ,Enzyme Stability ,Aspartic acid ,Catalytic triad ,Humans ,Moiety ,Molecular Biology ,Alanine ,Binding Sites ,Wild type ,Hydrogen Bonding ,Cell Biology ,Hydrogen-Ion Concentration ,Acetylcholinesterase ,Recombinant Proteins ,Phosphorus Acids ,chemistry ,Mutagenesis, Site-Directed ,Thermodynamics ,Research Article - Abstract
We have examined the effects of 11 substitutions of active centre gorge residues of human acetylcholinesterase (HuAChE) on the rates of phosphonylation by 1,2,2-trimethylpropyl methylphosphonofluoridate (soman) and the aging of the resulting conjugates. The rates of phosphonylation were reduced to as little as one-seventieth, mainly in mutants of the hydrogen-bond network (Glu-202, Glu-450, Tyr-133). These recombinant enzymes as well as the F338A, W86A, W86F and D74N mutant HuAChEs varied in their resistance to aging (15–3300-fold relative to the wild type). The most dramatic resistance to aging was observed for the phosphonyl conjugate of the mutant W86A enzyme (1850–3300-fold relative to the wild type). It is proposed that Trp-86 contributes to the aging process by stabilizing the evolving carbonium ion on the 1,2,2-trimethylpropyl moiety, via charge–π interaction. The rate-enhancing effect of Trp-86 provides a rationale for the unique facility of aging in soman-inhibited cholinesterases, compared with the corresponding conjugates in other serine hydrolases. Replacements of Glu-202 by aspartic acid, glutamine or alanine residues resulted in a similar (1/130–1/300) decrease of the rates of aging. A comparable decrease was also observed for the conjugate of the F338A mutant. These results, and the similar pH dependence of aging rates for the wild-type and E202Q and F338A mutant HuAChEs, indicate that Glu-202 is not involved in proton transfer to the phosphonyl moiety. On the basis of these findings and of molecular modelling we suggest that Glu-202 and Phe-338 contribute to the aging process by stabilizing the imidazolium of the catalytic triad His-447 via charge–charge and charge–π interactions respectively, thereby facilitating an oxonium formation on the phosphonyl moiety.
- Published
- 1996
15. The Architecture of Human Acetylcholinesterase Active Center Probed by Interactions with Selected Organophosphate Inhibitors
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Avigdor Shafferman, Dov Barak, Baruch Velan, Arie Ordentlich, Yoffi Segall, Chanoch Kronman, and Naomi Ariel
- Subjects
Stereochemistry ,Substituent ,Biochemistry ,Active center ,Structure-Activity Relationship ,chemistry.chemical_compound ,Organophosphorus Compounds ,medicine ,Humans ,Reactivity (chemistry) ,Phosphorylation ,Molecular Biology ,Binding Sites ,Paraoxon ,Organophosphate ,Leaving group ,Hydrogen Bonding ,Cell Biology ,Acetylcholinesterase ,Kinetics ,chemistry ,Mutagenesis, Site-Directed ,Alkoxy group ,Cholinesterase Inhibitors ,medicine.drug - Abstract
The role of the functional architecture of human acetylcholinesterase (HuAChE) active center in facilitating reactions with organophosphorus inhibitors was examined by a combination of site-directed mutagenesis and kinetic studies of phosphorylation with organophosphates differing in size of their alkoxy substituents and in the nature of the leaving group. Replacements of residues Phe-295 and Phe-297, constituting the HuAChE acyl pocket, increase up to 80-fold the reactivity of the enzymes toward diisopropyl phosphorofluoridate, diethyl phosphorofluoridate, and p-nitrophenyl diethyl phosphate (paraoxon), indicating the role of this subsite in accommodating the phosphate alkoxy substituent. On the other hand, a decrease of up to 160-fold in reactivity was observed for enzymes carrying replacements of residues Tyr-133, Glu-202, and Glu-450, which are constituents of the hydrogen bond network in the HuAChE active center, which maintains its unique functional architecture. Replacement of residues Trp-86, Tyr-337, and Phe-338 in the alkoxy pocket affected reactivity toward diisopropyl phosphorofluoridate and paraoxon, but to a lesser extent that toward diethyl phosphorofluoridate, indicating that both the alkoxy substituent and the p-nitrophenoxy leaving group interact with this subsite. In all cases the effects on reactivity toward organophosphates, demonstrated in up to 10,000-fold differences in the values of bimolecular rate constants, were mainly a result of altered affinity of the HuAChE mutants, while the apparent first order rate constants of phosphorylation varied within a narrow range. This finding indicates that the main role of the functional architecture of HuAChE active center in phosphorylation is to facilitate the formation of enzyme-inhibitor Michaelis complexes and that this affinity, rather than the nucleophilic activity of the enzyme catalytic machinery, is a major determinant of HuAChE reactivity toward organophosphates.
- Published
- 1996
16. Enzyme Engineering Towards Novel Op-Hydrolases Based on the Human Acetylcholinesterase Template
- Author
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Naomi Ariel, Yoffi Segall, Dov Barak, Avigdor Shafferman, Dana Stein, Chanoch Kronman, Baruch Velan, Arie Ordentlich, and Haim Grosfeld
- Subjects
Inorganic Chemistry ,chemistry.chemical_classification ,Active center ,chemistry.chemical_compound ,Enzyme ,chemistry ,Biochemistry ,Organic Chemistry ,Protein engineering ,Acetylcholinesterase - Abstract
Protein engineering technologies were used to generate various derivatives of human acetylcholinesterase (HuAChE). These enzymes were reacted with various organophosphates (OP) and reactivators in order to identify key element in the active center determining the efficiency of phosphylation, reactivation and of the aging process. Based on the results obtained we believe that novel biocatalysts with efficient OP-scavenging and possibly even OP-hydrolase activity may be generated, in the near future.
- Published
- 1996
17. Contribution of Aromatic Moieties of Tyrosine 133 and of the Anionic Subsite Tryptophan 86 to Catalytic Efficiency and Allosteric Modulation of Acetylcholinesterase
- Author
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Baruch Velan, Yoffi Segall, Chanoch Kronman, Naomi Ariel, Dov Barak, Arie Ordentlich, and Avigdor Shafferman
- Subjects
Models, Molecular ,Conformational change ,Protein Conformation ,Stereochemistry ,Allosteric regulation ,In Vitro Techniques ,Ligands ,Biochemistry ,Catalysis ,Active center ,Structure-Activity Relationship ,chemistry.chemical_compound ,Allosteric Regulation ,Humans ,Phosphorylation ,Molecular Biology ,chemistry.chemical_classification ,Binding Sites ,biology ,Chemistry ,Hydrogen bond ,Tryptophan ,Active site ,Cell Biology ,Acetylcholinesterase ,Kinetics ,Enzyme ,Mutagenesis, Site-Directed ,biology.protein ,Tyrosine - Abstract
Substitution of Trp-86, in the active center of human acetylcholinesterase (HuAChE), by aliphatic but not by aromatic residues resulted in a several thousandfold decrease in reactivity toward charged substrate and inhibitors but only a severalfold decrease for noncharged substrate and inhibitors. The W86A and W86E HuAChE enzymes exhibit at least a 100-fold increase in the Michaelis-Menten constant or 100-10,000-fold increase in inhibition constants toward various charged inhibitors, as compared to W86F HuAChE or the wild type enzyme. On the other hand, replacement of Glu-202, the only acidic residue proximal to the catalytic site, by glutamine resulted in a nonselective decrease in reactivity toward charged and noncharged substrates or inhibitors. Thus, the quaternary nitrogen groups of substrates and other active center ligands, are stabilized by cation-aromatic interaction with Trp-86 rather than by ionic interactions, while noncharged ligands appear to bind to distinct site(s) in HuAChE. Analysis of the Y133F and Y133A HuAChE mutated enzymes suggests that the highly conserved Tyr-133 plays a dual role in the active center: (a) its hydroxyl appears to maintain the functional orientation of Glu-202 by hydrogen bonding and (b) its aromatic moiety maintains the functional orientation of the anionic subsite Trp-86. In the absence of aromatic interactions between Tyr-133 and Trp-86, the tryptophan acquires a conformation that obstructs the active site leading, in the Y133A enzyme, to several hundredfold decrease in rates of catalysis, phosphorylation, or in affinity to reversible active site inhibitors. It is proposed that allosteric modulation of acetylcholinesterase activity, induced by binding to the peripheral anionic sites, proceeds through such conformational change of Trp-86 from a functional anionic subsite state to one that restricts access of substrates to the active center.
- Published
- 1995
18. Electrostatic attraction by surface charge does not contribute to the catalytic efficiency of acetylcholinesterase
- Author
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Baruch Velan, Tamar Bino, Roman Osman, Arie Ordentlich, C. Kronman, Raphael Ber, Dov Barak, Naomi Ariel, and Avigdor Shafferman
- Subjects
Models, Molecular ,Biology ,Catalysis ,General Biochemistry, Genetics and Molecular Biology ,law.invention ,Active center ,chemistry.chemical_compound ,law ,Electric field ,Computer Graphics ,Humans ,Surface charge ,Amino Acids ,Molecular Biology ,Binding Sites ,Molecular Structure ,General Immunology and Microbiology ,General Neuroscience ,Osmolar Concentration ,Substrate (chemistry) ,Charge density ,Acetylcholinesterase ,Kinetics ,Mutagenesis, Insertional ,Crystallography ,chemistry ,Biochemistry ,Vicinal ,Torpedo ,Research Article - Abstract
Acetylcholinesterases (AChEs) are characterized by a high net negative charge and by an uneven surface charge distribution, giving rise to a negative electrostatic potential extending over most of the molecular surface. To evaluate the contribution of these electrostatic properties to the catalytic efficiency, 20 single- and multiple-site mutants of human AChE were generated by replacing up to seven acidic residues, vicinal to the rim of the active-center gorge (Glu84, Glu285, Glu292, Asp349, Glu358, Glu389 and Asp390), by neutral amino acids. Progressive simulated replacement of these charged residues results in a gradual decrease of the negative electrostatic potential which is essentially eliminated by neutralizing six or seven charges. In marked contrast to the shrinking of the electrostatic potential, the corresponding mutations had no significant effect on the apparent bimolecular rate constants of hydrolysis for charged and non-charged substrates, or on the Ki value for a charged active center inhibitor. Moreover, the kcat values for all 20 mutants are essentially identical to that of the wild type enzyme, and the apparent bimolecular rate constants show a moderate dependence on the ionic strength, which is invariant for all the enzymes examined. These findings suggest that the surface electrostatic properties of AChE do not contribute to the catalytic rate, that this rate is probably not diffusion-controlled and that long-range electrostatic interactions play no role in stabilization of the transition states of the catalytic process.
- Published
- 1994
19. Direct observation and elucidation of the structures of aged and nonaged phosphorylated cholinesterases by phosphorus-31 NMR spectroscopy
- Author
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Yoffi Segall, D. Waysbort, Yacov Ashani, J. Grunwald, Naomi Ariel, Dov Barak, and Bhupendra P. Doctor
- Subjects
Cholinesterase Reactivators ,Protein Denaturation ,Isoflurophate ,Magnetic Resonance Spectroscopy ,Alkylation ,Stereochemistry ,Soman ,Biochemistry ,Adduct ,chemistry.chemical_compound ,Organophosphorus Compounds ,Nucleophile ,Chymotrypsin ,Moiety ,Phosphorylation ,Binding Sites ,Molecular Structure ,biology ,Leaving group ,Active site ,Stereoisomerism ,Acetylcholinesterase ,chemistry ,Butyrylcholinesterase ,biology.protein ,Cholinesterase Inhibitors - Abstract
31P NMR spectroscopy of butyrylcholinesterase (BChE), acetylcholinesterase (AChE), and chymotrypsin (Cht) inhibited by pinacolyl methylphosphonofluoridate (soman), methylphosphonodifluoridate (MPDF), and diisopropyl phosphorofluoridate (DFP) allowed direct observation of the OP-linked moiety of aged (nonreactivatable) and nonaged organophosphorus (OP)-ChE conjugates. The 31P NMR chemical shifts of OP-ChE conjugates clearly demonstrated insertion of a P-O- bond into the active site of aged OP-ChE adducts. The OP moiety of nonaged OP-ChEs was shown to be uncharged. The OP-bound pinacolyl moiety of soman-inhibited and aged AChE was detached completely, whereas only partial dealkylation of the pinacolyl group was observed for soman-inhibited BChEs. This suggests that the latter enzyme reacted with the less active stereoisomer(s) of soman. In the case of soman-inhibited Cht, no dealkylation could be experimentally detected for any of the four stereoisomers of OP-Cht adducts. Results are consistent with the contention that the phenomenon of enzyme-catalyzed dealkylation of OP adducts of serine hydrolases strongly depends on the orientation of both the catalytic His and the carboxyl side chain of either Glu or Asp positioned next to the catalytic Ser. The denatured protein of aged OP-ChE or OP-Cht is a convenient leaving group in nucleophilic displacements of tetrahedral OP compounds despite the presence of a P-O- bond. This indicates that the unusual resistance to reactivation of the aged enzyme cannot be ascribed to simple electrostatic repulsion of an approaching nucleophile. The broadening of the 31P NMR signal of native OP-ChEs relative to that of OP-Cht is in agreement with the crystal structure of AChE, showing that the active site region of ChEs in solution resides in a deep, narrow gorge.
- Published
- 1993
20. A Rapid Method for Bacillus anthracis Genotyping
- Author
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David Kobiler, Shay Weiss, Anat Zvi, Haim Levy, Naomi Ariel, Morly Fisher, and Zeev Altboum
- Subjects
Genetics ,biology ,Genetic marker ,In silico ,Genotype ,Nucleic acid sequence ,biology.organism_classification ,Genotyping ,Genome ,Bacillus anthracis ,RAPD - Abstract
Classification and differentiation of Bacillus anthracis isolates by genetic markers play an important role in the study of anthrax epidemiology. We have applied a PCR based method – Random Amplification of Polymorphic DNA (RAPD) to identify twenty-five B. anthracis genetic markers. These markers allowed for classification of the studied strains into five different groups. Three selected RAPD markers were cloned and sequenced. Typical integration of the three markers allowed for specific definition of the five RAPD derived genotypes. To test the universal power of these markers to discriminate between diverse B. anthracis strains, the nucleotide sequence of each marker was searched against all available B. anthracis genome sequences (both finished and unfinished). The three markers system could differentiate between strains belonging to the genetic groups Aβ, A1a, A1b, A4 and B1 (as defined by Keim et al., 2000; Maho et al., 2006) and gave rise to a unique combination for group A3, but couldn’t distinguish between the sub groups A3a and A3b. In addition, this system could not distinguish between groups B2 and C (identical three marker combination). In an attempt to improve the resolution of this system we introduced a fourth marker. In silico analysis revealed that the resulting four markers system could now differentiate between group B2 and C, adding a second genotype to groups A1a, A3a and A4. This four marker system could potentially provide an accurate, simple, and inexpensive agarose-based system for classification B. anthracis strains in laboratories involved in research of this bacterium.
- Published
- 2010
21. Genetic Detection of Vesicle Forming Pathogens by Arrayed Primer Extension (APEX)
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Dana Stein, Anat Zvi, Ohad Shifman, Arie Ordentlich, Shlomo Lustig, Ofir Israeli, Adi Beth-Din, Nir Paran, Naomi Ariel, and Einat Ben-Arie
- Subjects
biology ,animal diseases ,viruses ,Vesicle ,virus diseases ,Anatomy ,biology.organism_classification ,medicine.disease ,complex mixtures ,Virology ,Primer extension ,medicine ,Smallpox ,Variola virus ,Orthopoxvirus ,Smallpox disease - Abstract
The Variola virus is the causative agent of smallpox disease. Accurate identification of Variola and differential diagnosis between Variola and other vesicle forming (smallpox-like) pathogens are both technically challenging and of great importance. We compiled a list of vesicle forming pathogens that are prone to be misdiagnosed as smallpox. Some of the pathogens (members of the Orthopoxvirus genus) are also genetically highly similar to Variola.
- Published
- 2010
22. Data Mining, Bioinformatic and Immunoinformatic Analyses of Francisella tularensis Schu S4 Genome in Search for Novel Vaccine Candidates
- Author
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Anat Zvi, Naomi Ariel, and Avigdor Shafferman
- Subjects
Whole genome sequencing ,biology ,In silico ,Virulence ,Identification (biology) ,Human pathogen ,Data mining ,biology.organism_classification ,computer.software_genre ,Genome ,computer ,Francisella tularensis ,Tularemia vaccine - Abstract
The pathogenesis mechanism and the identity of major virulence factors of the highly infectious human pathogen F. tularensis tularensis, are still poorly characterized. The restricted efficacy of the sole available vaccine (the LVS attenuated strain) and residual toxicity has motivated extensive R&D efforts directed toward the identification of alternative tularemia vaccine formulations based on attenuated mutants, subunit vaccines and T-cell epitope-based vaccines. Identification of vaccine candidates by bioinformatic approaches is mostly driven by the availability of genome sequence data for numerous human-virulent as well as avirulent strains. In an attempt to select for F. tularensis Schu S4 potent antigens, we have developed a strategy based on genome-scale in silico analyses and data mining of published global experimental studies. The compiled information was divided into distinct biologically relevant categories. Protocols for qualitative and quantitative scoring for each of the categories, were developed. Together with implementation of a biological rationale, these served for ranking and prioritization of the putative antigens, providing a basis for subsequent selection of the top-ranking candidates to be evaluated experimentally.
- Published
- 2010
23. Reverse Vaccinology in Bacillus anthracis
- Author
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Haim Grosfeld, Ronit Aloni-Grinstein, Avigdor Shafferman, Anat Zvi, Sara Cohen, Orit Gat, Izhak Inbar, Galia Zaide, Naomi Ariel, and Theodor Chitlaru
- Subjects
Immunogen ,Antigen ,biology ,Reverse vaccinology ,Virulence ,ATP-binding cassette transporter ,biology.organism_classification ,Functional genomics ,Bacillus anthracis ,Microbiology ,DNA vaccination - Abstract
In search for antigens which may form the basis for improved subunit or live attenuated B. anthracis vaccines, extensive genomic, proteomic and serologic analyses coupled with functional screens for surface exposed and/or secreted proteins, were carried out. The screens resulted in selection of over 50 promising novel in-vivo expressed immunogens, classified as S-Layer Homology (SLH) proteins, repeat proteins, hydrolytic enzymes and ABC transporters. DNA vaccination experiments established that most of these novel antigens are indeed able to elicit a strong humoral response. Yet, unlike the major B. anthracis immunogen Protective Antigen (PA), none of the selected immunogens could provide protection against a subsequent virulent B. anthracis strain challenge. When over-expressed in an attenuated non-toxinogenic and non-encapsulated B. anthracis platform strain, at least three of the novel antigens did confer partial protection against a lethal B. anthracis challenge.
- Published
- 2010
24. Mutagenesis of human acetylcholinesterase. Identification of residues involved in catalytic activity and in polypeptide folding
- Author
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Avigdor Shafferman, Naomi Ariel, Yehuda Flashner, Yehoshua Gozes, Arie Ordentlich, Haim Grosfeld, C. Kronman, M Leitner, Dov Barak, and Sara Cohen
- Subjects
Models, Molecular ,Protein Conformation ,Stereochemistry ,Restriction Mapping ,Transfection ,Biochemistry ,Cell Line ,chemistry.chemical_compound ,Catalytic triad ,Hydrolase ,Humans ,Amino Acid Sequence ,Codon ,Molecular Biology ,Butyrylcholinesterase ,Alanine ,chemistry.chemical_classification ,Binding Sites ,Base Sequence ,Mutagenesis ,Wild type ,Antibodies, Monoclonal ,Cell Biology ,Acetylcholinesterase ,Recombinant Proteins ,Amino acid ,Kinetics ,chemistry ,Mutagenesis, Site-Directed - Abstract
Evidence for the involvement of Ser-203, His-447, and Glu-334 in the catalytic triad of human acetylcholinesterase was provided by substitution of these amino acids by alanine residues. Of 20 amino acid positions mutated so far in human acetylcholinesterase (AChE), these three were unique in abolishing detectable enzymatic activity (less than 0.0003 of wild type), yet allowing proper production, folding, and secretion. This is the first biochemical evidence for the involvement of a glutamate in a hydrolase triad (Schrag, J.D., Li, Y., Wu, M., and Cygler, M. (1991) Nature 351, 761-764), supporting the x-ray crystal structure data of the Torpedo californica acetylcholinesterase (Sussman, J.L., Harel, M., Frolow, F., Oefner, C., Goldman, A., Toker, L. and Silman, I. (1991) Science 253, 872-879). Attempts to convert the AChE triad into a Cys-His-Glu or Ser-His-Asp configuration by site-directed mutagenesis did not yield effective AChE activity. Another type of substitution, that of Asp-74 by Gly or Asn, generated an active enzyme with increased resistance to succinylcholine and dibucaine; thus mimicking in an AChE molecule the phenotype of the atypical butyrylcholinesterase natural variant (D70G mutation). Mutations of other carboxylic residues Glu-84, Asp-95, Asp-333, and Asp-349, all conserved among cholinesterases, did not result in detectable alteration in the recombinant AChE, although polypeptide productivity of the D95N mutant was considerably lower. In contrast, complete absence of secreted human AChE polypeptide was observed when Asp-175 or Asp-404 were substituted by Asn. These two aspartates are conserved in the entire cholinesterase/thyroglobulin family and appear to play a role in generating and/or maintaining the folded state of the polypeptide. The x-ray structure of the Torpedo acetylcholinesterase supports this assumption by revealing the participation of these residues in salt bridges between neighboring secondary structure elements.
- Published
- 1992
25. Whole genome identification of Mycobacterium tuberculosis vaccine candidates by comprehensive data mining and bioinformatic analyses
- Author
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Avigdor Shafferman, John Fulkerson, Anat Zvi, Naomi Ariel, and Jerald C. Sadoff
- Subjects
lcsh:Internal medicine ,education.field_of_study ,Tuberculosis ,lcsh:QH426-470 ,In silico ,Population ,Genomics ,Computational biology ,Biology ,biology.organism_classification ,medicine.disease ,Genome ,Virology ,Mycobacterium tuberculosis ,lcsh:Genetics ,Genetics ,medicine ,Genetics(clinical) ,DNA microarray ,lcsh:RC31-1245 ,education ,Gene ,Genetics (clinical) ,Research Article - Abstract
Background Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), infects ~8 million annually culminating in ~2 million deaths. Moreover, about one third of the population is latently infected, 10% of which develop disease during lifetime. Current approved prophylactic TB vaccines (BCG and derivatives thereof) are of variable efficiency in adult protection against pulmonary TB (0%–80%), and directed essentially against early phase infection. Methods A genome-scale dataset was constructed by analyzing published data of: (1) global gene expression studies under conditions which simulate intra-macrophage stress, dormancy, persistence and/or reactivation; (2) cellular and humoral immunity, and vaccine potential. This information was compiled along with revised annotation/bioinformatic characterization of selected gene products and in silico mapping of T-cell epitopes. Protocols for scoring, ranking and prioritization of the antigens were developed and applied. Results Cross-matching of literature and in silico-derived data, in conjunction with the prioritization scheme and biological rationale, allowed for selection of 189 putative vaccine candidates from the entire genome. Within the 189 set, the relative distribution of antigens in 3 functional categories differs significantly from their distribution in the whole genome, with reduction in the Conserved hypothetical category (due to improved annotation) and enrichment in Lipid and in Virulence categories. Other prominent representatives in the 189 set are the PE/PPE proteins; iron sequestration, nitroreductases and proteases, all within the Intermediary metabolism and respiration category; ESX secretion systems, resuscitation promoting factors and lipoproteins, all within the Cell wall category. Application of a ranking scheme based on qualitative and quantitative scores, resulted in a list of 45 best-scoring antigens, of which: 74% belong to the dormancy/reactivation/resuscitation classes; 30% belong to the Cell wall category; 13% are classical vaccine candidates; 9% are categorized Conserved hypotheticals, all potentially very potent T-cell antigens. Conclusion The comprehensive literature and in silico-based analyses allowed for the selection of a repertoire of 189 vaccine candidates, out of the whole-genome 3989 ORF products. This repertoire, which was ranked to generate a list of 45 top-hits antigens, is a platform for selection of genes covering all stages of M. tuberculosis infection, to be incorporated in rBCG or subunit-based vaccines.
- Published
- 2007
26. Search for Bacillus anthracis potential vaccine candidates by a functional genomic-serologic screen
- Author
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Itzhak Inbar, Orit Gat, Anat Zvi, Sara Cohen, Naomi Ariel, Haim Grosfeld, Dana Stein, Avigdor Shafferman, Yehoshua Broder, Zeev Altboum, Theodor Chitlaru, and Galia Zaide
- Subjects
Immunology ,Guinea Pigs ,Genomics ,Molecular Genomics ,Anthrax Vaccines ,Biology ,Microbiology ,DNA vaccination ,Anthrax ,Mice ,Open Reading Frames ,Plasmid ,Vaccines, DNA ,Animals ,ORFS ,Genetics ,Antigens, Bacterial ,Mice, Inbred ICR ,Anthrax vaccines ,Immune Sera ,Computational Biology ,Chromosomes, Bacterial ,biology.organism_classification ,Virology ,Bacillus anthracis ,Bacterial adhesin ,Open reading frame ,Infectious Diseases ,Parasitology ,Rabbits ,Genome, Bacterial - Abstract
Bacillus anthracis proteins that possess antigenic properties and are able to evoke an immune response were identified by a reductive genomic-serologic screen of a set of in silico-preselected open reading frames (ORFs). The screen included in vitro expression of the selected ORFs by coupled transcription and translation of linear PCR-generated DNA fragments, followed by immunoprecipitation with antisera from B. anthracis- infected animals. Of the 197 selected ORFs, 161 were chromosomal and 36 were on plasmids pXO1 and pXO2, and 138 of the 197 ORFs had putative functional annotations (known ORFs) and 59 had no assigned functions (unknown ORFs). A total of 129 of the known ORFs (93%) could be expressed, whereas only 38 (64%) of the unknown ORFs were successfully expressed. All 167 expressed polypeptides were subjected to immunoprecipitation with the anti- B. anthracis antisera, which revealed 52 seroreactive immunogens, only 1 of which was encoded by an unknown ORF. The high percentage of seroreactive ORFs among the functionally annotated ORFs (37%; 51/129) attests to the predictive value of the bioinformatic strategy used for vaccine candidate selection. Furthermore, the experimental findings suggest that surface-anchored proteins and adhesins or transporters, such as cell wall hydrolases, proteins involved in iron acquisition, and amino acid and oligopeptide transporters, have great potential to be immunogenic. Most of the seroreactive ORFs that were tested as DNA vaccines indeed appeared to induce a humoral response in mice. We list more than 30 novel B. anthracis immunoreactive virulence-related proteins which could be useful in diagnosis, pathogenesis studies, and future anthrax vaccine development.
- Published
- 2006
27. Differential Proteomic Analysis of the Bacillus anthracis Secretome: Distinct Plasmid and Chromosome CO2-Dependent Cross Talk Mechanisms Modulate Extracellular Proteolytic Activities†
- Author
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Orit Gat, Theodor Chitlaru, Avigdor Shafferman, Naomi Ariel, and Yael Gozlan
- Subjects
Proteomics ,Genomics and Proteomics ,Anthrax toxin ,Molecular Sequence Data ,Virulence ,Biology ,Protein Sorting Signals ,Microbiology ,Plasmid ,Bacterial Proteins ,Secretion ,Electrophoresis, Gel, Two-Dimensional ,Amino Acid Sequence ,Molecular Biology ,Regulator gene ,Genetics ,Carbon Dioxide ,Chromosomes, Bacterial ,biology.organism_classification ,Bacillus anthracis ,Culture Media ,Kinetics ,Regulon ,Membrane protein ,Sulfatases ,Peptide Hydrolases ,Plasmids - Abstract
Bacillus anthracis is a gram-positive spore-forming bacterium that is the etiological agent of anthrax, a lethal disease sporadically affecting humans and animals, in particular herbivores. In its most severe manifestation, B. anthracis infection is initiated by inhalation of spores, which are taken up by alveolar macrophages and germinate into fast-dividing vegetative cells which secrete toxins and virulence factors during growth (81, 99). If untreated by prompt antibiotic administration, the bacteria invade the bloodstream, resulting in massive bacteremia and consequently generalized systemic failure and death. B. anthracis is considered to represent a potential biothreat agent, owing to the severity of the anthrax disease, the ease of respiratory contamination, and the perpetual environmental stability of the infective spores. The recent deliberate dissemination of B. anthracis (15) accelerated the efforts to identify new B. anthracis virulence-related determinants for the design of novel diagnostic, preventive, and/or therapeutic strategies. Fully virulent B. anthracis strains harbor two native plasmids, pXO1 and pXO2, which encode critical pathogenicity factors. The absence of either one of the two plasmids results in a pronounced attenuation of B. anthracis virulence. The pXO2 plasmid encodes proteins involved in the biosynthesis of the poly-d-glutamic acid capsule, which may inhibit phagocytosis of bacteria during infection; pXO1 encodes the three toxin components protective antigen (PA), lethal factor (LF) (a zinc-dependent metalloprotease which proteolytically inactivates protein kinase kinases 1 and 2), and edema factor (EF) (a calmodulin-dependent adenylate cyclase), which form two binary toxins, lethal toxin and edema toxin. PA, the common component of both toxins, is not toxic by itself, yet it plays the central role of binding a specific receptor on the host cells and translocating LF and EF into the cytosol of infected cells, where they exert their detrimental activities. Anthrax is acknowledged as a toxinogenic disease, owing to the lethality of pure toxin preparations (77); on the other hand, additional B. anthracis secreted proteins are most probably involved in the onset and course of the disease and in survival of the bacteria in the host. The regulatory circuits governing the virulence of B. anthracis are still to be fully deciphered, yet certain observations suggest that the virulence of the bacteria entails cross talk mechanisms which link expression of plasmid-encoded and chromosomally encoded genes. The regulatory AtxA protein, encoded by pXO1, is essential for expression of the toxin and capsule synthesis genes in vivo (a situation which can be mimicked by growing the bacteria in minimal medium under high bicarbonate-CO2 conditions) (75). Two additional regulatory proteins, AcpA and AcpB, encoded by pXO2, were suggested to act downstream of AtxA and to affect capsule synthesis (35, 36). AtxA was found also to influence expression of chromosomal genes, either directly or via AcpA and AcpB. In addition, the protein AbrB, which is a chromosomally encoded transition state regulator, was suggested to negatively control the activity of the toxin gene promoters (123, 131) via AtxA. Secreted proteins include factors involved in pathogenicity, in particular in gram-positive bacteria (79). Such proteins may serve as possible targets for diagnostic purposes and/or therapeutic intervention. Bacteria of the Bacillus cereus phylogenetic group (B. cereus and Bacillus thuringiensis), to which B. anthracis belongs, secrete a diversity of factors that are essential for virulence, including toxins, hemolysins, proteases, and lecithinases. Notably, in these bacteria the secretion of certain virulence factors is regulated by a pleiotropic regulator, PlcR (2, 82, 87, 110), which is inactive in B. anthracis (2). It has been suggested that the evolutionary inactivation of the PlcR regulon in B. anthracis was due to incompatibility with the AtxA-controlled regulon and reflects the fact that the PlcR target genes are not essential for anthrax pathogenicity (96). Several studies have postulated that secreted proteases, other than those belonging to the silenced PlcR regulon, are responsible for some clinical manifestations of anthrax (1, 9, 117, 140). Such proteases could damage host tissues, interfere with immune effectors of the host, and/or provide nutrients for bacterial survival (103). Some chromosomally encoded B. anthracis extracellular proteases were suggested to be controlled by Cot43, a novel regulatory gene encoded by pXO1 (9). The availability of the B. anthracis genomic DNA sequence (109, 121) paved the way for high-throughput genomic, transcriptomic, and proteomic analyses of B. anthracis (6, 7, 8, 17, 25, 41, 50, 64, 78, 85, 121, 144) in an effort to elucidate pathogenicity mechanisms by identification of novel virulence factors or in search for specific therapeutic and/or diagnostic targets. Indeed, bioinformatic surveys of the B. anthracis genome (7, 8, 121) suggested that proteins other than PA, LF, and EF, may participate in anthrax pathogenesis. Furthermore, many B. anthracis open reading frames (ORFs) encode potentially secreted or membrane-bound proteins exhibiting homology to known virulence factors from other bacteria (7, 8). A preliminary proteomic study carried out in our laboratory examined membrane proteins prepared from a nonvirulent B. anthracis strain and led to the recognition of a number of immunodominant exposed proteins (8, 25). Here, we document an extended proteomic study, focusing on B. anthracis secreted proteins, which expands the data set of expressed B. anthracis proteins from both virulent and nonvirulent strains (6, 25, 41, 50, 64, 78, 85, 144). Based on identification of more than 400 two-dimensional electrophoresis (2-DE)-separated protein spots, we report the expression of 64 proteins which represent the most abundant B. anthracis secreted proteins, many of which resemble factors involved in the virulence of other pathogens. Comparison of the relative abundances of proteins in pXO1- and pXO2-containing and plasmid-cured strains reveals about 30 ORFs which are either preferentially expressed or repressed in the virulent B. anthracis strain under conditions which are considered to simulate those encountered within the mammalian host. The pattern of expression of these specific proteins demonstrates that B. anthracis possesses distinct regulatory pathways which involve plasmid- or chromosome-encoded CO2-inducible responsive factors.
- Published
- 2006
28. Identification of Genes Involved in Yersinia pestis Virulence by Signature-tagged Mutagenesis
- Author
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Yehuda Flashner, Emanuelle Mamroud, Anat Zvi, Sara Cohen, Naomi Ariel, Avital Tidhar, David Gur, Moshe Aftalion, Baruch Velan, Avigdor Shafferman, and Raphael Ber
- Subjects
Genetics ,Signature-tagged mutagenesis ,Yersinia pestis ,biology ,Mutagenesis (molecular biology technique) ,Yersinia pseudotuberculosis ,Virulence ,Identification (biology) ,biology.organism_classification ,Yersinia enterocolitica ,Gene - Published
- 2006
29. The solute-binding component of a putative Mn(II) ABC transporter (MntA) is a novel Bacillus anthracis virulence determinant
- Author
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Orit, Gat, Itai, Mendelson, Theodor, Chitlaru, Naomi, Ariel, Zeev, Altboum, Haim, Levy, Shay, Weiss, Haim, Grosfeld, Sara, Cohen, and Avigdor, Shafferman
- Subjects
Manganese ,Proteome ,Iron ,Lipoproteins ,Macrophages ,Guinea Pigs ,Molecular Sequence Data ,Bacterial Proteins ,Bacillus anthracis ,Animals ,Humans ,ATP-Binding Cassette Transporters ,Female ,Amino Acid Sequence ,Sequence Alignment ,Cells, Cultured ,Plasmids - Abstract
Here we describe the characterization of a lipoprotein previously proposed as a potential Bacillus anthracis virulence determinant and vaccine candidate. This protein, designated MntA, is the solute-binding component of a manganese ion ATP-binding cassette transporter. Coupled proteomic-serological screen of a fully virulent wild-type B. anthracis Vollum strain, confirmed that MntA is expressed both in vitro and during infection. Expression of MntA is shown to be independent of the virulence plasmids pXO1 and pXO2. An mntA deletion, generated by allelic replacement, results in complete loss of MntA expression and its phenotypic analysis revealed: (i) impaired growth in rich media, alleviated by manganese supplementation; (ii) increased sensitivity to oxidative stress; and (iii) delayed release from cultured macrophages. The DeltamntA mutant expresses the anthrax-associated classical virulence factors, lethal toxin and capsule, in vitro as well as in vivo, and yet the mutation resulted in severe attenuation; a 10(4)-fold drop in LD(50) in a guinea pig model. MntA expressed in trans allowed to restore, almost completely, the virulence of the DeltamntA B. anthracis strain. We propose that MntA is a novel B. anthracis virulence determinant essential for the development of anthrax disease, and that B. anthracisDeltamntA strains have the potential to serve as platform for future live attenuated vaccines.
- Published
- 2005
30. Attempts to engineer an enzyme mimic of butyrylcholinesterase by substitution of the six divergent aromatic amino acids in the active center of acetylcholinesterase
- Author
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Baruch Velan, Dov Barak, Arie Ordentlich, Dana Kaplan, Avigdor Shafferman, Naomi Ariel, and Chanoch Kronman
- Subjects
Active center ,chemistry.chemical_compound ,Chemistry ,Stereochemistry ,Substitution (logic) ,Aromatic amino acids ,Enzyme mimic ,Organic chemistry ,Acetylcholinesterase ,Butyrylcholinesterase - Published
- 2004
31. Surprising findings from the functional analysis of human acetylcholinesterase adducts of Alzheimer’s disease drugs
- Author
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Baruch Velan, Naomi Ariel, Avigdor Shafferman, Arie Ordentlich, Dana Kaplan, Dov Barak, and Chanoch Kronman
- Subjects
chemistry.chemical_compound ,Chemistry ,Disease ,Pharmacology ,Neuroscience ,Acetylcholinesterase ,Functional analysis (psychology) - Published
- 2004
32. Generation of Yersinia pestis attenuated strains by signature-tagged mutagenesis in search of novel vaccine candidates
- Author
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T. Avital Tidhar, Baruch Velan, Yehuda Flashner, Emanuelle Mamroud, Avigdor Shafferman, Shirley Lazar, Raphael Ber, Tamar Bino, Naomi Ariel, David Gur, Anat Zvi, Sara Cohen, and Moshe Aftalion
- Subjects
Plague Vaccine ,Signature-tagged mutagenesis ,Attenuated vaccine ,biology ,Yersinia pestis ,Immunology ,Mutant ,Wild type ,Virulence ,Molecular Genomics ,biology.organism_classification ,Vaccines, Attenuated ,Microbiology ,Virology ,Enterobacteriaceae ,Mice ,Infectious Diseases ,Phenotype ,Mutagenesis ,Plague vaccine ,Animals ,Parasitology ,Female - Abstract
In a search for novel attenuated vaccine candidates for use againstYersinia pestis, the causative agent of plague, a signature-tagged mutagenesis strategy was used and optimized for a subcutaneously infected mouse model. A library of tagged mutants of the virulentY. pestisKimberley53 strain was generated. Screening of 300 mutants through two consecutive cycles resulted in selection of 16 mutant strains that were undetectable in spleens 48 h postinfection. Each of these mutants was evaluated in vivo by assays for competition against the wild-type strain and for virulence following inoculation of 100 CFU (equivalent to 100 50% lethal doses [LD50] of the wild type). A wide spectrum of attenuation was obtained, ranging from avirulent mutants exhibiting competition indices of 10−5to 10−7to virulent mutants exhibiting a delay in the mean time to death or mutants indistinguishable from the wild type in the two assays. Characterization of the phenotypes and genotypes of the selected mutants led to identification of virulence-associated genes coding for factors involved in global bacterial physiology (e.g.,purH,purK,dnaE, andgreA) or for hypothetical polypeptides, as well as for the virulence regulator genelcrF. One of the avirulent mutant strains (LD50, >107CFU) was found to be disrupted in thepcmlocus, which is presumably involved in the bacterial response to environmental stress. This Kimberley53pcmmutant was superior to the EV76 live vaccine strain because it induced 10- to 100-fold-higher antibody titers to the protective V and F1 antigens and because it conferred efficacious protective immunity.
- Published
- 2004
33. Identification of genes involved in Yersinia pestis virulence by signature-tagged mutagenesis
- Author
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Yehuda, Flashner, Emanuelle, Mamroud, Avital, Tidhar, Raphael, Ber, Moshe, Aftalion, David, Gur, Anat, Zvi, Naomi, Ariel, Baruch, Velan, Avigdor, Shafferman, and Sara, Cohen
- Subjects
Mice ,Virulence ,Mutagenesis ,Yersinia pestis ,Animals - Published
- 2003
34. Comparison of sequence and structure alignments for protein domains
- Author
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Anna R. Panchenko, Stephen H. Bryant, Aron Marchler-Bauer, and Naomi Ariel
- Subjects
Genetics ,Protein structure database ,Models, Molecular ,Molecular model ,Sequence Homology, Amino Acid ,Conserved Domain Database ,Sequence analysis ,Structural alignment ,Protein domain ,Proteins ,Sequence alignment ,Computational biology ,Biology ,Biochemistry ,Protein Structure, Tertiary ,Structural Biology ,Sequence Analysis, Protein ,Sequence comparison ,Animals ,Databases, Protein ,Molecular Biology ,Sequence Alignment - Abstract
Profile search methods based on protein domain alignments have proven to be useful tools in comparative sequence analysis. Domain alignments used by currently available search methods have been computed by sequence comparison. With the growth of the protein structure database, however, alignments of many domain pairs have also been computed by structure comparison. Here, we examine the extent to which information from these two sources agrees. We measure agreement with respect to identification of homologous regions in each protein, that is, with respect to the location of domain boundaries. We also measure agreement with respect to identification of homologous residue sites by comparing alignments and assessing the accuracy of the molecular models they predict. We find that domain alignments in publicly available collections based on sequence and structure comparison are largely consistent. However, the homologous regions identified by sequence comparison are often shorter than those identified by 3D structure comparison. In addition, when overall sequence similarity is low alignments from sequence comparison produce less accurate molecular models, suggesting that they less accurately identify homologous sites. These observations suggest that structure comparison results might be used to improve the overall accuracy of domain alignment collections and the performance of profile search methods based on them.
- Published
- 2002
35. The aromatic 'trapping' of the catalytic histidine is essential for efficient catalysis in acetylcholinesterase
- Author
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Arie Ordentlich, Avigdor Shafferman, Baruch Velan, Dov Barak, Dana Kaplan, and Naomi Ariel
- Subjects
chemistry.chemical_classification ,Stereochemistry ,Protein Conformation ,Restriction Mapping ,Biochemistry ,Acetylcholinesterase ,Catalysis ,Recombinant Proteins ,Amino acid ,Active center ,Residue (chemistry) ,chemistry.chemical_compound ,Kinetics ,Mutagenesis, Insertional ,Enzyme ,chemistry ,Amino Acid Substitution ,Mutagenesis, Site-Directed ,Humans ,Histidine ,Butyrylcholinesterase - Abstract
While substitution of the aromatic residues (Phe295, Phe338), located in the vicinity of the catalytic His447 in human acetylcholinesterase (HuAChE) had little effect on catalytic activity, simultaneous replacement of both residues by aliphatic amino acids resulted in a 680-fold decrease in catalytic activity. Molecular simulations suggested that the activity decline is related to conformational destabilization of His447, similar to that observed for the hexamutant HuAChE which mimics the active center of butyrylcholinesterase. On the basis of model structures of other cholinesterases (ChEs), we predicted that catalytically nonproductive mobility of His447 could be restricted by introduction of aromatic residue in a different location adjacent to this histidine (Val407). Indeed, the F295A/F338A/V407F enzyme is 170-fold more reactive than the corresponding double mutant and only 3-fold less reactive than the wild-type HuAChE. However, analogous substitution of Val407 in the hexamutant HuAChE (generating the heptamutant Y72N/Y124Q/W286A/F295L/F297V/Y337A/V407F) did not enhance catalytic activity. Reactivity of these double, triple, hexa, and hepta mutant HuAChEs was monitored toward covalent ligands such as organophosphates and the transition state analogue TMFTA, which probe, respectively, the facility of the enzymes to accommodate Michaelis complexes and to undergo the acylation process. The findings suggest that in the F295A/F338A mutant the two His447 conformational states, which are essential for the different stages of the catalytic process, seem to be destabilized. On the other hand, in the F295A/F338A/V407F mutant only the state involved in acylation is impaired. Such differential effects on the His447 conformational properties demonstrate the general role of aromatic residues in cholinesterases, and probably in other serine hydrolases, in "trapping" of the catalytic histidine and thereby in optimization of catalytic activity.
- Published
- 2002
36. Structural and Functional Correlates of Human Acetylcholinesterase Mutants for Evaluating Alzheimer’s Disease Treatments
- Author
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Arie Ordentlich, Dana Kaplan, Baruch Velan, Avigdor Shafferman, Naomi Ariel, Dov Barak, and Chanoch Kronman
- Subjects
Rivastigmine ,chemistry.chemical_classification ,Stereochemistry ,Acetylcholinesterase ,chemistry.chemical_compound ,Enzyme ,chemistry ,Tetrahedral carbonyl addition compound ,Covalent bond ,Tacrine ,Catalytic triad ,medicine ,Site-directed mutagenesis ,Neuroscience ,medicine.drug - Abstract
Inhibitors of AChE constitute presently the only successful approach to symptomatic treatment of senile dementia of the Alzheimer’s type, with three such agents (tacrine, E2020 and rivastigmine) in clinical use1. The intensive effort to develop more therapeutically efficatious inhibitors is currently aided by the remarkable progress, made during the last decade, in elucidating the structural and functional properties of the enzyme through x-ray crystallography 2,3 and site directed mutagenesis 4–8. Investigation of the specific roles for most of the residues in this active-center gorge allowed for identification of several functional subsites including the catalytic triad (S203(200)**, H447(440) , E334(327)); the acyl pocket (F295(288) and F297(290)) and the ‘hydrophobic subsite’. The latter accommodates the alcoholic portion of the covalent adduct (tetrahedral intermediate) and may include residues W86 (84) , Y133 (130) , Y337(330) and F338(331), which operate through nonpolar and/or stacking interactions, depending on the substrate. Stabilization of the charged moieties of substrates and other ligands at the active-centre is mediated by cation -π interactions with the residue at position 86 rather than through true ionic interactions2,6,8
- Published
- 2002
37. Exploring the active center of human acetylcholinesterase with stereomers of an organophosphorus inhibitor with two chiral centers
- Author
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Baruch Velan, Arie Ordentlich, H.P. Benschop, Avigdor Shafferman, Yoffi Segall, Naomi Ariel, C. Kronman, L. P. A. De Jong, Dov Barak, and Ruth Barak
- Subjects
Cholinesterase reactivator ,Models, Molecular ,Soman, 96-64-0 ,Amino acid substitution ,Alkylation ,Stereochemistry ,Enzyme structure ,Phenylalanine ,Binding sites ,Soman ,Stereoisomerism ,Kidney ,Biochemistry ,Adduct ,Active center ,Binding site ,Enzyme binding ,Enzyme analysis ,Humans ,Enzyme activity ,Chirality ,Alanine ,Phenylalanine, 63-91-2 ,Chemistry ,Phosphorus, 7723-14-0 ,Diastereomer ,Cholinesterase inhibitors ,Phosphorus ,Hydrogen Bonding ,Alanine, 56-41-7 ,Enzyme substrate ,Human cell ,Dealkylation ,Macromolecular substances ,Acetylcholinesterase ,Mutagenesis, Site-Directed ,Enzyme specificity ,Protein expression ,Stereoselectivity ,Chirality (chemistry) ,Cell line ,Acetylcholinesterase, EC 3.1.1.7 - Abstract
The stereoselectivity of the phosphonylation reaction and the effects of adduct configuration on the aging process were examined for human acetylcholinesterase (HuAChE) and its selected active center mutants, using the four stereomers of 1,2,2-trimethylpropyl methylphosphonofluoridate (soman). The reactivity of wild type HuAChE toward the P(S)-soman diastereomers was 4.0-7.5 x 104-fold higher than that toward the P(R)- diastereomers. Aging of the P(S)C(S)-somanyl-HuAChE conjugate was also > 1.6 x 104-fold faster than that of the corresponding P(R)C(S)-somanyl adduct, as shown by both reactivation and electrospray mass spectrometry (ESI/MS) experiments. On the other hand, both processes exhibited very limited sensitivity to the chirality of the alkoxy group C(α) of either P(S)- or P(R)-diastereomers. These stereoselectivities presumably reflect the relative participation of the enzyme in stabilization of the Michaelis complexes and in dealkylation of the respective covalent conjugates, and therefore could be utilized for further probing of the HuAChE active center functional architecture. Reactivities of HuAChE enzymes carrying replacements at the acyl pocket (F295A, F297A, and F295L/F297V) indicate that stereoselectivity with respect to the soman phosphorus chirality depends on the structure of this binding subsite, but this stereoselectivity cannot be explained only by limitation in the capacity to accommodate the P(R)-diastereomers. In addition, these acyl pocket enzyme mutants display some (5-10-fold) preference for the P(R)C(R)-soman over the P(R)C(S)-stereomer, while reactivity of the hydrophobic pocket mutant enzyme W86F toward the P(R)C(S)- soman resembles that of the wild type HuAChE. Residue substitutions in the H- bond network (E202Q, E450A, Y133F, and Y133A) and the hydrophobic pocket (F338A, W86A, W86F, and Y337A) result in a limited stereoselectivity for the P(S)C(S)- over the P(S)C(R)-stereomer. Aging of the P(S)-somanyl conjugates with all the HuAChE mutant enzymes tested practically lacked stereoselectivity with respect to the C(α) of the alkoxy moiety. Thus, the inherent asymmetry of the active center does not seem to affect the rate- determining step of the dealkylation process, possibly because both the P(S)C(S)- and the P(S)C(R)-somanyl moieties yield the same carbocationic intermediate.
- Published
- 1999
38. The 'aromatic patch' of three proximal residues in the human acetylcholinesterase active centre allows for versatile interaction modes with inhibitors
- Author
-
Naomi Ariel, Avigdor Shafferman, Arie Ordentlich, Tamar Bino, Baruch Velan, and Dov Barak
- Subjects
Models, Molecular ,Stereochemistry ,Protein Conformation ,Physostigmine ,Crystallography, X-Ray ,Ligands ,Biochemistry ,law.invention ,chemistry.chemical_compound ,Structure-Activity Relationship ,Protein structure ,Alkaloids ,law ,medicine ,Structure–activity relationship ,Humans ,Binding site ,Molecular Biology ,Huperzine A ,Binding Sites ,Chemistry ,Hydrogen bond ,Hydrogen Bonding ,Cell Biology ,Acetylcholinesterase ,Kinetics ,Models, Chemical ,Tacrine ,Cholinesterase Inhibitors ,Sesquiterpenes ,Torpedo ,medicine.drug ,Pyridostigmine Bromide ,Research Article - Abstract
The role of the functional architecture of the human acetylcholinesterase (HuAChE) active centre in accommodating the non-covalent inhibitors tacrine and huperzine A, or the carbamates pyridostigmine and physostigmine, was analysed using 16 mutants of residues lining the active-centre gorge. Despite the structural diversity of the ligands, certain common properties of the complexes could be observed: (a) replacement of aromatic residues Tyr133, Tyr337 and especially Trp86, resulted in pronounced changes in stability of all the complexes examined; (b) effects due to replacements of the five other aromatic residues along the active-centre gorge, such as the acyl pocket (Phe295, Phe297) or at the peripheral anionic site (Tyr124, Trp286, Tyr341) were relatively small; (c) effects due to substitution of the carboxylic residues in the gorge (Glu202, Glu450) were moderate. These results and molecular modelling indicate that the aromatic side chains of residues Trp86, Tyr133 and Tyr337 form together a continuous ‘aromatic patch ’ lining the wall of the active-centre gorge, allowing for the accommodation of the different ligands via multiple modes of interaction. Studies with HuAChE mutants carrying replacements at positions 86, 133 and 337 indicate that the orientations of huperzine A and tacrine in the HuAChE complexes in solution are significantly different from those observed in X-ray structures of the corresponding complexes with Torpedo californica AChE (TcAChE). These discrepancies may be explained in terms of structural differences between the complexes of HuAChE and TcAChE or, more likely, by the enhanced flexibility of the AChE active-centre gorge in solution as compared with the crystalline state.
- Published
- 1998
39. Bovine acetylcholinesterase: cloning, expression and characterization
- Author
-
Avigdor Shafferman, Chanoch Kronman, Naomi Ariel, Itai Mendelson, and Baruch Velan
- Subjects
Models, Molecular ,Glycosylation ,Macromolecular Substances ,Protein Conformation ,Molecular Sequence Data ,Sequence alignment ,Biology ,Kidney ,Transfection ,Biochemistry ,law.invention ,Cell Line ,Evolution, Molecular ,chemistry.chemical_compound ,Protein structure ,law ,Animals ,Humans ,Amino Acid Sequence ,Cloning, Molecular ,Molecular Biology ,Peptide sequence ,Gene ,chemistry.chemical_classification ,Sequence Homology, Amino Acid ,Cell Biology ,Fetal Blood ,Molecular biology ,Acetylcholinesterase ,Recombinant Proteins ,Amino acid ,chemistry ,Recombinant DNA ,Cattle ,Dimerization ,Sequence Alignment ,Fetal bovine serum ,Research Article - Abstract
The bovine acetylcholinesterase (BoAChE) gene was cloned from genomic DNA and its structure was determined. Five exons coding for the AChE T-subunit and the alternative H-subunit were identified and their organization suggests high conservation of structure in mammalian AChE genes. The deduced amino acid sequence of the bovine T-subunit is highly similar to the human sequence, showing differences at 34 positions only. However, the cloned BoAChE sequence differs from the published amino acid sequence of AChE isolated from fetal bovine serum (FBS) by: (1) 13 amino acids, 12 of which are conserved between BoAChE and human AChE, and (2) the presence of four rather than five potential N-glycosylation sites. The full coding sequence of the mature BoAChE T-subunit was expressed in human embryonal kidney 293 cells (HEK-293). The catalytic properties of recombinant BoAChE and its reactivity towards various inhibitors were similar to those of the native bovine enzyme. Soluble recombinant BoAChE is composed of monomers, dimers and tetramers, yet in contrast to FBS-AChE, tetramer formation is not efficient. Comparative SDS/PAGE analysis reveals that all four potential N-glycosylation sites identified by DNA sequencing appear to be utilized, and that recombinant BoAChE comigrates with FBS-AChE. A major difference between the recombinant enzyme and the native enzyme was observed when clearance from circulation was examined. The HEK-293-derived enzyme was cleared from the circulation at a much faster rate than FBS-AChE. This difference in behaviour, together with previous studies on the effect of post-translation modification on human AChE clearance [Kronman, Velan, Marcus, Ordentlich, Reuveny and Shafferman (1995) Biochem. J. 311, 959–967] suggests that cell-dependent glycosylation plays a key role in AChE circulatory residence.
- Published
- 1998
40. Does Electrostatic Attraction or Steering by Charged Residues within the Gorge Contribute to the Reactivity of AChE?
- Author
-
C. Kronman, Dov Barak, Dana Stein, Avigdor Shafferman, Naomi Ariel, Dana Berman, Baruch Velan, and Arie Ordentlich
- Subjects
Active center ,Transition state analog ,Echothiophate ,Chemistry ,Ionic strength ,Stereochemistry ,Catalytic triad ,Acetylthiocholine ,medicine ,Substrate (chemistry) ,Reactivity (chemistry) ,medicine.drug - Abstract
Studies with mutant AChEs where up to seven surface negative charges were neutralized demonstrated that electrostatic attraction does not contribute to the catalytic efficiency of the enzyme [1,2]. Yet, electrostatic steering due to the negatively charged residues in the gorge (D74 at its rim and E202 and E450 near its bottom) remained difficult to assess [3, 4]. We therefore extended the studies of these mutants with isosteric pairs of charged and noncharged substrates (ATC- acetylthiocholine; 3, 3-dimethylbutyl thioacetate-TB), charged and noncharged phosphate inhibitors (echothiophate, S-3,3-di-methylbutyl diethylthiophosphate) as well as with the transition state analog m-trimethylammonio trifluoroacetophenone (TFK). Replacements of D74 by negatively charged (D74E) positively charged (D74K) or neutral (D74N, D74G) residues resulted in small and uniform increase in values of Km for ATC except for D74K where the increase was much larger. On the other hand, the bimolecular rates of reactions with echothiophate and TFK were affected much more in cases of D74N and D74K than in those of D74E and E74G. All these mutations have almost no effect on catalytic activity toward the non-charged substrate, yet a small increase of phosphorylation rates by the noncharged OP inhibitor was observed for enzymes with noncharged residues at position 74 but not for D74K or D74E. These findings, together with the observation that ionic strength had small and equivalent effect on reactivities of all these enzymes, indicate that D74 probably does not enhance reactivity by steering charged ligands into the gorge but rather influences indirectly specific interactions of charged ligands with elements of the active center.
- Published
- 1998
41. Compatibility of Structures Inferred from Mutagenesis and from X-Ray Crystallography for Various AChE Complexes
- Author
-
Arie Ordentlich, Baruch Velan, Tamar Bino, Naomi Ariel, Dov Barak, and Avigdor Shafferman
- Subjects
Molecular model ,Stereochemistry ,Ligand ,Biology ,Acetylcholinesterase ,law.invention ,Dissociation constant ,Active center ,Crystallography ,chemistry.chemical_compound ,chemistry ,law ,Tacrine ,medicine ,Torpedo ,Huperzine A ,medicine.drug - Abstract
Interactions of the potential therapeutic agents tacrine, huperzine A and edrophonium, with 15 mutants of human acetylcholinesterase (HuAChE) were investigated. The score of new inhibition and dissociation constants measured, expand considerably the corresponding data found in literature. Despite the structural diversity of the ligands certain common properties of the complexes could be observed: a. replacement of aromatic residues Y133, Y337 and especially W86, resulted in pronounced changes in stability of all the complexes examined, b. effects due to replacements of the five other aromatic residues along the active-center gorge such as the acyl pocket (F295, F297) or at the peripheral anionic site (Y124, W286, Y341) were relatively small, c. effects due to substitution of the carboxylic residues in the gorge (E202, E450) were moderate. These results and the corresponding molecular models indicate that the aromatic side chains of residues W86, Y133 and Y337 form together a continuous “aromatic patch” lining the wall of the active center gorge allowing for the accommodation of the different ligands via various modes of interactions (cation-π, π-π, H-bond, hydrophobic). The HuAChE-bound orientations of edrophonium, huperzine A and tacrine inferred from the mutagenesis studies, were compared to those in the available X-ray structures of the corresponding complexes with Torpedo californica AChE (TcAChE). For edrophonium the two structures are very similar with respect to the ligand and its binding environment. On the other hand, the x-ray orientation for tacrine and huperzine A is inconsistent with mutagenesis results - some mutations do not support expected interactions while others suggest interactions that are not observed in the x-ray structures. These inconsistencies may be explained in terms of: a. structural differences between the complexes of HuAChE and TcAChE, even though the active center regions of the two enzymes, consisting of over 30 residues, differ in only one position [Y337(F330)]; b. the active-center gorge of AChE in solution is more flexible than in the crystalline state allowing for modified binding orientations of the same ligand in the two states.
- Published
- 1998
42. What can be Learned from the use of HuAChE Mutants for Evaluation of Potential Alzheimer’s Drugs
- Author
-
Baruch Velan, Tamar Bino, Naomi Ariel, Arie Ordentlich, Chanoch Kronman, Dino Marcus, Dov Barak, Arie Lazar, M Leitner, and Avigdor Shafferman
- Subjects
Aché ,business.industry ,Central nervous system ,Cognition ,Pharmacology ,medicine.disease ,Acetylcholinesterase ,Choline acetyltransferase ,language.human_language ,chemistry.chemical_compound ,medicine.anatomical_structure ,chemistry ,medicine ,language ,Cholinergic ,Alzheimer's disease ,business ,Acetylcholine ,medicine.drug - Abstract
Senile dementia of the Alzheimer’s type (SDAT) is characterised by loss of cholinergic neuronal markers like the enzymes choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), in selected brain regions (Bierer et al., 1995). These indications of progressive depletion of cholinergic synapses led to the hypothesis that increasing the central nervous system (CNS) levels of acetylcholine (ACh), through inhibition of AChE, will improve cognition in SDAT (Court & Perry, 1991). This approach is theoretically preferable to other means of cholinergic augmentation since it may amplify the natural temporal pattern of ACh release, rather than globally stimulating the cholinergic system. Yet up to the present most of these agents show only mild to moderate ameliorating effects on memory deficits (Schneider & Tariot, 1994; Kan, 1992). To further optimize the therapeutic efficacy of these agents, a better understanding is needed of the structural features determining their interactions with AChE. The recent progress in the elucidation of structure-function characteristics of AChE, is therefore of considerable importance for these efforts.
- Published
- 1998
43. 3D Structure of a Complex of Human Acetylcholinesterase with Fasciculin-II at 2.7 Å Resolution
- Author
-
Joel L. Sussman, Gitay Kryger, Lilly Toker, Baruch Velan, Dov Barak, Kurt Giles, Ariel Lazar, Israel Silman, Avigdor Shafferman, Chanoch Kronman, Naomi Ariel, and Michal Harel
- Subjects
chemistry.chemical_classification ,Ammonium sulfate ,Chemistry ,Toxin ,Stereochemistry ,HEK 293 cells ,Mutant ,medicine.disease_cause ,Acetylcholinesterase ,law.invention ,chemistry.chemical_compound ,Enzyme ,law ,medicine ,Recombinant DNA ,Torpedo - Abstract
A knowledge of the 3D structure of human acetylcholinesterase (AChE) is of importance for the development of anti-Alzheimer drugs, for the general understanding of organophosphate toxicity and for the design of safer and more specific insecticides. A mutant of recombinant human acetylcholinesterase (rhAChE) was constructed in which the C-terminus had been truncated to give rise to a homogeneous monomeric form. The monomeric rhAChE was expressed in HEK 293 cells and purified by affinity chromatog-raphy. The purified enzyme was co-crystallized from ammonium sulfate at pH 7.2 and 19°C as a stoichiometric complex with the mamba venom polypeptide toxin, fasciculin-II (FAS-II). X-ray data were collected at the BNL-NSLS X12-C source from a single cryogenically cooled crystal. The overall completeness of the 30A-2.7A data is 99.0% and the R-merge is 8.1%. The model was refined starting from the mouse AChE-FAS-II structure to Rfree of 29% and R of 21.9% at 2.7A resolution. The overall fold is similar to that of Torpedo californica and mouse complexes with FAS-II. A mutant E202(199)Q was prepared and crystallized in a similar fashion. Although significantly different from the native enzyme in activity, the E202(199)Q is almost identical to the native in its 3D structure.
- Published
- 1998
44. Structural Modifications of the Ω Loop in Human Acetylcholinesterase
- Author
-
Naomi Ariel, Baruch Velan, Dov Barak, Moshe Leitner, Tamar Bino, Arie Ordentlich, and Avigdor Shafferman
- Published
- 1998
45. 3D Structure at 2.7 Å Resolution of Native and E202Q Mutant Human Acetylcholinesterase Complexed with Fasciculin-II
- Author
-
Michal Harel, Israel Silman, Baruch Velan, Joel L. Sussman, Naomi Ariel, Kurt Giles, Gitay Kryger, Ariel Lazar, Dov Barak, Lilly Toker, Avigdor Shafferman, and Chanoch Kronman
- Subjects
chemistry.chemical_compound ,chemistry ,Resolution (mass spectrometry) ,Biochemistry ,Mutant ,medicine ,Acetylcholinesterase ,Fasciculin ,Nerve agent ,medicine.drug - Abstract
Knowledge of the 3D structure of human acetylcholinesterase (AChE) is of importance for drug design, in particular of anti-Alzheimer drugs (1, 2), for development of improved procedures for treatment of intoxication by nerve agents (3), and for the design of safer and more effective insecticides.
- Published
- 1998
46. Contribution of the Active Center Functional Architecture to AChE Reactivity Toward Substrates and Inhibitors
- Author
-
Baruch Velan, Avigdor Shafferman, Chanoch Kronman, Naomi Ariel, Arie Ordentlich, and Dov Barak
- Subjects
chemistry.chemical_classification ,biology ,Stereochemistry ,Active site ,Acetylcholinesterase ,Amino acid ,Active center ,chemistry.chemical_compound ,Enzyme ,chemistry ,Transition state analog ,Catalytic triad ,biology.protein ,Site-directed mutagenesis - Abstract
The role of acetylcholinesterase (AChE) in cholinergic neurotransmission, as well as its significance as the molecular target for toxic and for potentially therapeutic agents led to an ongoing effort aimed to unravel the origin of the AChE outstanding catalytic efficiency and affinity toward specific ligands. Resolution of the three-dimensional structure of AChE (1) brought a new impetus into the field, guiding a comprehensive mapping of the enzyme functional architecture by site directed mutagenesis (2–11) and inspiring generation of new structure - function models (4, 9, 12). The X-ray structure is characterized by a deep and narrow ‘gorge’, which penetrates halfway into the enzyme and contains the catalytic site at about 4A from its base (1). The structure reveals also two remarkable features of the enzyme which could bear upon its reactivity toward substrates and inhibitors. One of these is the uneven overall distribution of negative charge giving rise to a large electrostatic dipole, aligned with the axis of the active site gorge, that could both attract positively charged species to the protein surface as well as guide them down the gorge to the active-center (13). The possible role of electrostatic attraction due to negatively charged surface amino acids, in the reactivity characteristics of HuAChE was investigated by site directed mutagenesis showing that the attraction does not contribute to either the catalytic rate of the enzyme or to its affinity towards the selective active-center inhibitor -edrophonium (14). Here we report certain results regarding the facility of electrostatic guidance due to the acidic amino acids Asp74, Glu202, Glu450, located in the HuAChE active center gorge.
- Published
- 1998
47. Structural modifications of the omega loop in human acetylcholinesterase
- Author
-
Naomi Ariel, Tamar Bino, Avigdor Shafferman, Dov Barak, Baruch Velan, M Leitner, and Arie Ordentlich
- Subjects
Proline ,Stereochemistry ,Protein Conformation ,Phenylalanine ,Allosteric regulation ,Mutant ,Molecular Sequence Data ,Biophysics ,Biochemistry ,Esterase ,Protein Structure, Secondary ,Deletion ,Active center ,Allosteric Regulation ,Structural Biology ,Hydrolase ,Genetics ,Humans ,Amino Acid Sequence ,Cysteine ,Lipase ,Molecular Biology ,chemistry.chemical_classification ,Allosteric effect ,Binding Sites ,biology ,Chemistry ,Ligand ,Tryptophan ,Hydrogen Bonding ,Cell Biology ,Kinetics ,Mutagenesis, Insertional ,Enzyme ,Mutagenesis ,biology.protein ,Acetylcholinesterase ,Ω loop - Abstract
The surface Ω loop (Cys69-Cys96) in human acetylcholinesterase (HuAChE) was recently implicated by molecular simulations in enhancing substrate and ligand accessibility to the active center and in the allosteric modulation of enzymatic activity. In lipases which together with AChEs belong to the esterase/lipase family sharing the α/β hydrolase fold, such Ω loop mobility was also shown to be functionally significant. In order to examine the relation between the loop structure and reactivity characteristics of the enzyme, several mutant HuAChE’s carrying modifications of the loop sequence were generated and kinetically evaluated. These modifications included replacements at the loop ends (Tyr72, Glu95), substitution of residues involved in hydrogen-bond interactions of the loop with the protein core (Tyr77, Glu84) as well as residues participating in such interactions within the loop (Met85, Asn87), replacement of the conserved proline residues (Pro 78, Pro 88) and deletion of 5 residue segment (Pro 78-Gly82) from the loop sequence. Most of the residue replacements, including those of the prolines, had very limited or no effect on enzyme reactivity. These findings suggest that unlike the case of lipase the Ω loop in HuAChE is not involved in in large lid-like motions. In cases where modifications of loop sequence had some effect on reactivity, the effects could be attributed to an altered position of residue Trp86 as implied by the altered catalytic activity, the interactions with quaternary ligands and the effect on the inhibitory activity of the peripheral site ligand propidium.
- Published
- 1996
48. Allosteric modulation of acetylcholinesterase activity by peripheral ligands involves a conformational transition of the anionic subsite
- Author
-
Arie Lazar, Baruch Velan, Arie Ordentlich, Dino Marcus, Avigdor Shafferman, Dov Barak, Chanoch Kronman, Avraham Bromberg, and Naomi Ariel
- Subjects
chemistry.chemical_classification ,Anions ,Conformational change ,Chromatography ,Binding Sites ,Ligand ,Stereochemistry ,Protein Conformation ,Allosteric regulation ,Ligands ,Biochemistry ,Active center ,Enzyme Activation ,Enzyme activator ,Enzyme ,Protein structure ,chemistry ,Allosteric Regulation ,Acetylcholinesterase ,Mutagenesis, Site-Directed ,Humans ,Cysteine ,Binding site ,Propidium - Abstract
Replacement of residues Asp74, Trp286, and Tyr72, which are constituents of the peripheral anionic site (PAS) of human acetylcholinesterase (HuAChE), affected similarly both the binding and the inhibition constants of the PAS-specific ligand propidium, demonstrating that changes in the inhibitory activity are a direct consequence of altered binding to the PAS. In contrast, the active center HuAChE mutants W86A and Y133A show respective 350- and 25-fold increased resistance to inhibition by propidium but no change in binding affinities, demonstrating that the allosteric mechanism of PAS-mediated inhibition involves a conformational change of these Trp86 and Tyr133 residues rather than physical obstruction of substrate access by the inhibitor itself. These findings support the recent proposal that the allosteric mechanism operates via transition between active and nonactive conformations of the anionic subsite Trp86 and that replacement of Tyr133 by alanine may stabilize a nonactive Trp86 conformation that occludes the active center [Ordentlich et al. (1995) J. Biol. Chem. 270, 2082]. In further support of this mechanism and the role of Tyr133, we find that (a) the dissociation constants (Kd) for the noncovalent complexes of the irreversible inhibitors diisopropyl phosphorofluoridate or paraoxon with Y133A HuAChE are increased 20-500-fold, relative to either wild-type enzyme or its Y133F or W86A mutants; and (b) access of substrates such as 3,3-dimethylbutyl thioacetate is restored by removal of Trp86 from the Y133A enzyme (i.e., the W86A/Y133A mutant). We suggest that the conformational transition of Trp86 is coupled to the motions of the cysteine loop (Cys69-Cys96) of HuAChE and is inherent to the dynamics of the native enzyme.
- Published
- 1995
49. Molecular Aspects of Catalysis and of Allosteric Regulation of Aceytlcholinesterases
- Author
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Baruch Velan, C. Kronman, Yoffi Segall, Shaul Reuveny, M Leitner, Naomi Ariel, Dov Barak, S. Cohen, Arie Ordentlich, Arye Lazar, Dino Marcus, A. Bromberg, Tamar Bino, and Avigdor Shafferman
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biology ,Stereochemistry ,Allosteric regulation ,Serine hydrolase ,Acetylcholinesterase ,law.invention ,chemistry.chemical_compound ,chemistry ,Allosteric enzyme ,law ,Catalytic triad ,biology.protein ,Cholinergic synapse ,Oxyanion hole ,Torpedo - Abstract
Acetylcholinesterase is a serine hydrolase whose function at the cholinergic synapse, is the rapid hydrolysis of the neurotransmitter acetylcholine (ACh). The recently resolved 3D structure of Torpedo californica AChE (TcAChE) revealed a deep and narrow ‘gorge’, which penetrates halfway into the enzyme and contains the catalytic site at about 4A from its base (Sussman et al., 1991). The active center interacts with ACh through several subsites including the catalytic triad (Ser203(200), His447(440), Glu334(327): Sussman et al., 1991; Gibney et al., 1990; Shafferman et al., 1992a, b), the oxyanion hole (Gly121(119), Gly 122(120), Ala204(201); Sussman et al., 1991), the acyl pocket (Phe295 (288) and Phe297(290); Vellom et al., 1993; Ordentlich et al., 1993a).
- Published
- 1995
50. Acetylcholinesterase peripheral anionic site degeneracy conferred by amino acid arrays sharing a common core
- Author
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Avigdor Shafferman, C. Kronman, Arie Ordentlich, Naomi Ariel, A. Bromberg, Arye Lazar, Dov Barak, Baruch Velan, and Dino Marcus
- Subjects
Molecular model ,Stereochemistry ,Allosteric regulation ,Genetic Vectors ,Fluorescence spectrometry ,Transfection ,Biochemistry ,Cell Line ,Substrate Specificity ,Active center ,Decamethonium ,medicine ,Humans ,Point Mutation ,Amino Acid Sequence ,Binding site ,Molecular Biology ,chemistry.chemical_classification ,Binding Sites ,biology ,Active site ,Cell Biology ,Embryo, Mammalian ,Recombinant Proteins ,Amino acid ,Kinetics ,Mutagenesis, Insertional ,chemistry ,biology.protein ,Acetylcholinesterase ,Cholinesterase Inhibitors ,medicine.drug - Abstract
Several of the residues constituting the peripheral anionic site (PAS) in human acetylcholinesterase (HuAChE) were identified by a combination of kinetic studies with 19 single and multiple HuAChE mutants, fluorescence binding studies with the Trp-286 mutant, and by molecular modeling. Mutants were analyzed with three structurally distinct positively charged PAS ligands, propidium, decamethonium, and di(p-allyl-N-dimethylaminophenyl)pentane-3-one (BW284C51), as well as with selective active center inhibitors, hexamethonium and edrophonium. Single mutations of residues Tyr-72, Tyr-124, Glu-285, Trp-286, and Tyr-341 resulted in up to 10-fold increase in inhibition constants for PAS ligands, whereas for multiple mutants up to 400-fold increase was observed. The 6th PAS element residue Asp-74 is unique in its ability to affect conformation of both the active site and the PAS (Shafferman, A., Velan, B., Ordentlich, A., Kronman, C., Grosfeld, H., Leitner, M., Flashner, Y., Cohen, S., Barak, D., and Ariel, N. (1992) EMBO J. 11, 3561-3568) as demonstrated by the several hundred-fold increase in Ki for D74N inhibition by the bisquaternary ligands decamethonium and BW284C51. Based on these studies, singular molecular models for the various HuAChE inhibitor complexes were defined. Yet, for the decamethonium complex two distinct conformations were generated, accommodating the quaternary ammonium group by interactions with either Trp-286 or with Tyr-341. We propose that the PAS consists of a number of binding sites, close to the entrance of the active site gorge, sharing residues Asp-74 and Trp-286 as a common core. Binding of ligands to these residues may be the key to the allosteric modulation of HuAChE catalytic activity. This functional degeneracy is a result of the ability of the Trp-286 indole moiety to interact either via stacking, aromatic-aromatic, or via pi-cation attractions and the involvement of the carboxylate of Asp-74 in charge-charge or H-bond interactions.
- Published
- 1994
Catalog
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