A Fluorogenic Peptide Containing the Processing Site of Human SARS Corona Virus S‐Protein: Kinetic Evaluation and NMR Structure Elucidation
Identifieur interne : 000F24 ( Main/Exploration ); précédent : 000F23; suivant : 000F25A Fluorogenic Peptide Containing the Processing Site of Human SARS Corona Virus S‐Protein: Kinetic Evaluation and NMR Structure Elucidation
Auteurs : Ajoy Basak ; Abhijit Mitra [États-Unis] ; Sarmistha Basak ; Carolyn Pasko ; Michel Chrétien ; Pamela Seaton [États-Unis]Source :
- ChemBioChem [ 1439-4227 ] ; 2007-06-18.
Descripteurs français
- KwdFr :
- Acides aminés (analyse), Algorithmes, Chromatographie en phase liquide à haute performance, Cinétique, Colorants fluorescents, Dichroïsme circulaire, Furanes (), Glycoprotéine de spicule des coronavirus, Glycoprotéines membranaires (), Peptides (), Protéines de l'enveloppe virale (), Relation quantitative structure-activité, Spectrométrie de masse, Spectroscopie par résonance magnétique, Virus du SRAS ().
- MESH :
- analyse : Acides aminés.
- Algorithmes, Chromatographie en phase liquide à haute performance, Cinétique, Colorants fluorescents, Dichroïsme circulaire, Furanes, Glycoprotéine de spicule des coronavirus, Glycoprotéines membranaires, Peptides, Protéines de l'enveloppe virale, Relation quantitative structure-activité, Spectrométrie de masse, Spectroscopie par résonance magnétique, Virus du SRAS.
English descriptors
- KwdEn :
- Algorithms, Amino Acids (analysis), Chromatography, High Pressure Liquid, Circular Dichroism, Fluorescent Dyes, Furans (chemistry), Kinetics, Magnetic Resonance Spectroscopy, Mass Spectrometry, Membrane Glycoproteins (chemistry), Peptides (chemistry), Quantitative Structure-Activity Relationship, SARS Virus (chemistry), Spike Glycoprotein, Coronavirus, Viral Envelope Proteins (chemistry).
- MESH :
- chemical , analysis : Amino Acids.
- chemical , chemistry : Furans, Membrane Glycoproteins, Peptides, Viral Envelope Proteins.
- chemistry : SARS Virus.
- Teeft :
- Active enzyme, Algorithms, Amide, Amide protons, Amino, Amino acid analysis, Amino acids, Analytical column, Authentic samples, Basak, Biochem, Bruker biospin, Chembiochem, Chromatography, High Pressure Liquid, Circular Dichroism, Cleavage, Cleavage site, Cleavage sites, Comparative study, Crucial role, Crude digests, Data show, Digestion, Enzyme activity, Experimental section, Fluorescent Dyes, Fluorogenic, Fluorogenic peptide, Furin, Furin cleavage, Furin inhibitors, Further studies, Glutamic acid, Glycoprotein, Gmbh, Hsars coronavirus cleavage, Hsars coronavirus cleavage figure, Human sars corona virus, Identical conditions, Identical levels, Inhibitor, Kcat, Kgaa, Kinetic parameters, Kinetics, Magnetic Resonance Spectroscopy, Mass Spectrometry, Mass spectrometry, Model peptide, Peak intensity, Peptide, Peptide synthesis, Proteolytic maturation, Proton, Quantitative Structure-Activity Relationship, Random structures, Roesy, Roesy correlations, Roesy spectra, Sars, Sars infection, Secondary structure, Seidah, Sheet content, Sheet structure, Soluble, Soluble forms, Soluble pc5a, Spike, Spike Glycoprotein, Coronavirus, Spike protein, Tocsy patterns, Various concentrations, Verlag, Verlag gmbh, Weinheim, Weinheim chembiochem.
Abstract
Human severe acute respiratory syndrome coronavirus (hSARS‐CoV) is the causative agent for SARS infection. Its surface glycoprotein (spike protein) is considered to be one of the prime targets for SARS therapeutics and intervention because its proteolytic maturation by a host protease is crucial for host–virus fusion. Using intramolecularly quenched fluorogenic (IQF) peptides based on hSARS‐CoV spike protein (Abz‐755Glu‐Gln‐Asp‐Arg‐Asn‐Thr‐Arg‐Glu‐Val‐Phe‐Ala‐Gln766‐Tyx‐NH2) and in vitro studies, we show that besides furin, other PCs, like PC5 and PC7, might also be involved in this cleavage event. Through kinetic measurements with recombinant PCs, we observed that the peptide was cleaved efficiently by both furin and PC5, but very poorly by PC7. The cleavage could be blocked by a PC‐inhibitor, α1‐PDX, in a dose‐dependent manner. Circular dichroism spectra indicated that this peptide possesses a high degree of sheet structure. Following cleavage by furin, the sheet content increased, possibly at the expense of turn and random structures. 1H NMR spectra from 2D COSY and ROESY experiments under physiological buffer and pH conditions indicated that this peptide possesses a structure with a turn at its C‐terminal segment, close to the cleavage site. The data suggest that the cleavable peptide bond is located within the most exposed domain; this is supported by the nearby turn structure. Several strong to weak NMR ROESY correlations were detected, and a 3D structure of the spike IQF peptide that contains the crucial cleavage site R761↓E has been proposed.
Cut and fuse. A key event in SARS infection is the proteolytic cleavage of viral spike protein by host proteases. By using intramolecularly quenched fluorogenic (IQF) peptides based on hSARS‐CoV spike protein and in vitro studies, we show that in addition to furin, proprotein convertases PC5 and PC7 cleave the model IQF peptide. Cleavage could be blocked by the PC inhibitor, α1‐PDX, in a dose‐dependent manner. The 3D structure of the model peptide was investigated by circular dichromism and 2D 1H NMR spectroscopy.
Url:
DOI: 10.1002/cbic.200700007
Affiliations:
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Pasko</name><affiliation><wicri:noCountry code="subField">(+1) 613‐761‐4355</wicri:noCountry></affiliation></author><author><name sortKey="Chretien, Michel" sort="Chretien, Michel" uniqKey="Chretien M" first="Michel" last="Chrétien">Michel Chrétien</name><affiliation><wicri:noCountry code="subField">(+1) 613‐761‐4355</wicri:noCountry></affiliation></author><author><name sortKey="Seaton, Pamela" sort="Seaton, Pamela" uniqKey="Seaton P" first="Pamela" last="Seaton">Pamela Seaton</name><affiliation wicri:level="2"><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Chemistry and Biochemistry, University of North Carolina, Wilmington, NC 28403</wicri:regionArea><placeName><region type="state">Caroline du Nord</region></placeName></affiliation></author></analytic><monogr></monogr><series><title level="j" type="main">ChemBioChem</title><title level="j" type="alt">CHEMBIOCHEM</title><idno type="ISSN">1439-4227</idno><idno type="eISSN">1439-7633</idno><imprint><biblScope unit="vol">8</biblScope><biblScope unit="issue">9</biblScope><biblScope unit="page" from="1029">1029</biblScope><biblScope unit="page" to="1037">1037</biblScope><biblScope unit="page-count">9</biblScope><publisher>WILEY‐VCH Verlag</publisher><pubPlace>Weinheim</pubPlace><date type="published" when="2007-06-18">2007-06-18</date></imprint><idno type="ISSN">1439-4227</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">1439-4227</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Algorithms</term><term>Amino Acids (analysis)</term><term>Chromatography, High Pressure Liquid</term><term>Circular Dichroism</term><term>Fluorescent Dyes</term><term>Furans (chemistry)</term><term>Kinetics</term><term>Magnetic Resonance Spectroscopy</term><term>Mass Spectrometry</term><term>Membrane Glycoproteins (chemistry)</term><term>Peptides (chemistry)</term><term>Quantitative Structure-Activity Relationship</term><term>SARS Virus (chemistry)</term><term>Spike Glycoprotein, Coronavirus</term><term>Viral Envelope Proteins (chemistry)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Acides aminés (analyse)</term><term>Algorithmes</term><term>Chromatographie en phase liquide à haute performance</term><term>Cinétique</term><term>Colorants fluorescents</term><term>Dichroïsme circulaire</term><term>Furanes ()</term><term>Glycoprotéine de spicule des coronavirus</term><term>Glycoprotéines membranaires ()</term><term>Peptides ()</term><term>Protéines de l'enveloppe virale ()</term><term>Relation quantitative structure-activité</term><term>Spectrométrie de masse</term><term>Spectroscopie par résonance magnétique</term><term>Virus du SRAS ()</term></keywords><keywords scheme="MESH" type="chemical" qualifier="analysis" xml:lang="en"><term>Amino Acids</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Furans</term><term>Membrane Glycoproteins</term><term>Peptides</term><term>Viral Envelope Proteins</term></keywords><keywords scheme="MESH" qualifier="analyse" xml:lang="fr"><term>Acides aminés</term></keywords><keywords scheme="MESH" qualifier="chemistry" xml:lang="en"><term>SARS Virus</term></keywords><keywords scheme="Teeft" xml:lang="en"><term>Active enzyme</term><term>Algorithms</term><term>Amide</term><term>Amide protons</term><term>Amino</term><term>Amino acid analysis</term><term>Amino acids</term><term>Analytical column</term><term>Authentic samples</term><term>Basak</term><term>Biochem</term><term>Bruker biospin</term><term>Chembiochem</term><term>Chromatography, High Pressure Liquid</term><term>Circular Dichroism</term><term>Cleavage</term><term>Cleavage site</term><term>Cleavage sites</term><term>Comparative study</term><term>Crucial role</term><term>Crude digests</term><term>Data show</term><term>Digestion</term><term>Enzyme activity</term><term>Experimental section</term><term>Fluorescent Dyes</term><term>Fluorogenic</term><term>Fluorogenic peptide</term><term>Furin</term><term>Furin cleavage</term><term>Furin inhibitors</term><term>Further studies</term><term>Glutamic acid</term><term>Glycoprotein</term><term>Gmbh</term><term>Hsars coronavirus cleavage</term><term>Hsars coronavirus cleavage figure</term><term>Human sars corona virus</term><term>Identical conditions</term><term>Identical levels</term><term>Inhibitor</term><term>Kcat</term><term>Kgaa</term><term>Kinetic parameters</term><term>Kinetics</term><term>Magnetic Resonance Spectroscopy</term><term>Mass Spectrometry</term><term>Mass spectrometry</term><term>Model peptide</term><term>Peak intensity</term><term>Peptide</term><term>Peptide synthesis</term><term>Proteolytic maturation</term><term>Proton</term><term>Quantitative Structure-Activity Relationship</term><term>Random structures</term><term>Roesy</term><term>Roesy correlations</term><term>Roesy spectra</term><term>Sars</term><term>Sars infection</term><term>Secondary structure</term><term>Seidah</term><term>Sheet content</term><term>Sheet structure</term><term>Soluble</term><term>Soluble forms</term><term>Soluble pc5a</term><term>Spike</term><term>Spike Glycoprotein, Coronavirus</term><term>Spike protein</term><term>Tocsy patterns</term><term>Various concentrations</term><term>Verlag</term><term>Verlag gmbh</term><term>Weinheim</term><term>Weinheim chembiochem</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Algorithmes</term><term>Chromatographie en phase liquide à haute performance</term><term>Cinétique</term><term>Colorants fluorescents</term><term>Dichroïsme circulaire</term><term>Furanes</term><term>Glycoprotéine de spicule des coronavirus</term><term>Glycoprotéines membranaires</term><term>Peptides</term><term>Protéines de l'enveloppe virale</term><term>Relation quantitative structure-activité</term><term>Spectrométrie de masse</term><term>Spectroscopie par résonance magnétique</term><term>Virus du SRAS</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Human severe acute respiratory syndrome coronavirus (hSARS‐CoV) is the causative agent for SARS infection. Its surface glycoprotein (spike protein) is considered to be one of the prime targets for SARS therapeutics and intervention because its proteolytic maturation by a host protease is crucial for host–virus fusion. Using intramolecularly quenched fluorogenic (IQF) peptides based on hSARS‐CoV spike protein (Abz‐755Glu‐Gln‐Asp‐Arg‐Asn‐Thr‐Arg‐Glu‐Val‐Phe‐Ala‐Gln766‐Tyx‐NH2) and in vitro studies, we show that besides furin, other PCs, like PC5 and PC7, might also be involved in this cleavage event. Through kinetic measurements with recombinant PCs, we observed that the peptide was cleaved efficiently by both furin and PC5, but very poorly by PC7. The cleavage could be blocked by a PC‐inhibitor, α1‐PDX, in a dose‐dependent manner. Circular dichroism spectra indicated that this peptide possesses a high degree of sheet structure. Following cleavage by furin, the sheet content increased, possibly at the expense of turn and random structures. 1H NMR spectra from 2D COSY and ROESY experiments under physiological buffer and pH conditions indicated that this peptide possesses a structure with a turn at its C‐terminal segment, close to the cleavage site. The data suggest that the cleavable peptide bond is located within the most exposed domain; this is supported by the nearby turn structure. Several strong to weak NMR ROESY correlations were detected, and a 3D structure of the spike IQF peptide that contains the crucial cleavage site R761↓E has been proposed.</div><div type="abstract" xml:lang="en">Cut and fuse. A key event in SARS infection is the proteolytic cleavage of viral spike protein by host proteases. By using intramolecularly quenched fluorogenic (IQF) peptides based on hSARS‐CoV spike protein and in vitro studies, we show that in addition to furin, proprotein convertases PC5 and PC7 cleave the model IQF peptide. Cleavage could be blocked by the PC inhibitor, α1‐PDX, in a dose‐dependent manner. The 3D structure of the model peptide was investigated by circular dichromism and 2D 1H NMR spectroscopy.</div></front></TEI><affiliations><list><country><li>États-Unis</li></country><region><li>Caroline du Nord</li><li>État de New York</li></region></list><tree><noCountry><name sortKey="Basak, Ajoy" sort="Basak, Ajoy" uniqKey="Basak A" first="Ajoy" last="Basak">Ajoy Basak</name><name sortKey="Basak, Sarmistha" sort="Basak, Sarmistha" uniqKey="Basak S" first="Sarmistha" last="Basak">Sarmistha Basak</name><name sortKey="Chretien, Michel" sort="Chretien, Michel" uniqKey="Chretien M" first="Michel" last="Chrétien">Michel Chrétien</name><name sortKey="Pasko, Carolyn" sort="Pasko, Carolyn" uniqKey="Pasko C" first="Carolyn" last="Pasko">Carolyn Pasko</name></noCountry><country name="États-Unis"><region name="État de New York"><name sortKey="Mitra, Abhijit" sort="Mitra, Abhijit" uniqKey="Mitra A" first="Abhijit" last="Mitra">Abhijit Mitra</name></region><name sortKey="Seaton, Pamela" sort="Seaton, Pamela" uniqKey="Seaton P" first="Pamela" last="Seaton">Pamela Seaton</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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