Catalytic function and substrate specificity of the papain-like protease domain of nsp3 from the Middle East respiratory syndrome coronavirus.
Identifieur interne : 001695 ( Main/Exploration ); précédent : 001694; suivant : 001696Catalytic function and substrate specificity of the papain-like protease domain of nsp3 from the Middle East respiratory syndrome coronavirus.
Auteurs : Yahira M. Báez-Santos [États-Unis] ; Anna M. Mielech [États-Unis] ; Xufang Deng [États-Unis] ; Susan Baker [États-Unis] ; Andrew D. Mesecar [États-Unis]Source :
- Journal of virology [ 1098-5514 ] ; 2014.
Descripteurs français
- KwdFr :
- Cinétique, Clonage moléculaire, Coronavirus du syndrome respiratoire du Moyen-Orient (enzymologie), Coronavirus du syndrome respiratoire du Moyen-Orient (génétique), Expression des gènes, Humains, Lignée cellulaire, Peptide hydrolases (), Peptide hydrolases (génétique), Peptide hydrolases (métabolisme), Protéines recombinantes (), Protéines recombinantes (génétique), Protéines recombinantes (isolement et purification), Protéines recombinantes (métabolisme), Protéines virales non structurales (), Protéines virales non structurales (génétique), Protéines virales non structurales (métabolisme), RNA replicase (), RNA replicase (métabolisme), Spécificité du substrat, Structure quaternaire des protéines.
- MESH :
- enzymologie : Coronavirus du syndrome respiratoire du Moyen-Orient.
- génétique : Coronavirus du syndrome respiratoire du Moyen-Orient, Peptide hydrolases, Protéines recombinantes, Protéines virales non structurales.
- isolement et purification : Protéines recombinantes.
- métabolisme : Peptide hydrolases, Protéines recombinantes, Protéines virales non structurales, RNA replicase.
- Cinétique, Clonage moléculaire, Expression des gènes, Humains, Lignée cellulaire, Peptide hydrolases, Protéines recombinantes, Protéines virales non structurales, RNA replicase, Spécificité du substrat, Structure quaternaire des protéines.
English descriptors
- KwdEn :
- Cell Line, Cloning, Molecular, Gene Expression, Humans, Kinetics, Middle East Respiratory Syndrome Coronavirus (enzymology), Middle East Respiratory Syndrome Coronavirus (genetics), Peptide Hydrolases (chemistry), Peptide Hydrolases (genetics), Peptide Hydrolases (metabolism), Protein Structure, Quaternary, RNA Replicase (chemistry), RNA Replicase (metabolism), Recombinant Proteins (chemistry), Recombinant Proteins (genetics), Recombinant Proteins (isolation & purification), Recombinant Proteins (metabolism), Substrate Specificity, Viral Nonstructural Proteins (chemistry), Viral Nonstructural Proteins (genetics), Viral Nonstructural Proteins (metabolism).
- MESH :
- chemical , chemistry : Peptide Hydrolases, RNA Replicase, Recombinant Proteins, Viral Nonstructural Proteins.
- enzymology : Middle East Respiratory Syndrome Coronavirus.
- genetics : Middle East Respiratory Syndrome Coronavirus, Peptide Hydrolases, Recombinant Proteins, Viral Nonstructural Proteins.
- chemical , isolation & purification : Recombinant Proteins.
- chemical , metabolism : Peptide Hydrolases, RNA Replicase, Recombinant Proteins, Viral Nonstructural Proteins.
- Cell Line, Cloning, Molecular, Gene Expression, Humans, Kinetics, Protein Structure, Quaternary, Substrate Specificity.
Abstract
The papain-like protease (PLpro) domain from the deadly Middle East respiratory syndrome coronavirus (MERS-CoV) was overexpressed and purified. MERS-CoV PLpro constructs with and without the putative ubiquitin-like (UBL) domain at the N terminus were found to possess protease, deubiquitinating, deISGylating, and interferon antagonism activities in transfected HEK293T cells. The quaternary structure and substrate preferences of MERS-CoV PLpro were determined and compared to those of severe acute respiratory syndrome coronavirus (SARS-CoV) PLpro, revealing prominent differences between these closely related enzymes. Steady-state kinetic analyses of purified MERS-CoV and SARS-CoV PLpros uncovered significant differences in their rates of hydrolysis of 5-aminomethyl coumarin (AMC) from C-terminally labeled peptide, ubiquitin, and ISG15 substrates, as well as in their rates of isopeptide bond cleavage of K48- and K63-linked polyubiquitin chains. MERS-CoV PLpro was found to have 8-fold and 3,500-fold higher catalytic efficiencies for hydrolysis of ISG15-AMC than for hydrolysis of the Ub-AMC and Z-RLRGG-AMC substrates, respectively. A similar trend was observed for SARS-CoV PLpro, although it was much more efficient than MERS-CoV PLpro toward ISG15-AMC and peptide-AMC substrates. MERS-CoV PLpro was found to process K48- and K63-linked polyubiquitin chains at similar rates and with similar debranching patterns, producing monoubiquitin species. However, SARS-CoV PLpro much preferred K48-linked polyubiquitin chains to K63-linked chains, and it rapidly produced di-ubiquitin molecules from K48-linked chains. Finally, potent inhibitors of SARS-CoV PLpro were found to have no effect on MERS-CoV PLpro. A homology model of the MERS-CoV PLpro structure was generated and compared to the X-ray structure of SARS-CoV PLpro to provide plausible explanations for differences in substrate and inhibitor recognition.
DOI: 10.1128/JVI.01294-14
PubMed: 25142582
Affiliations:
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Syndrome Coronavirus (enzymology)</term><term>Middle East Respiratory Syndrome Coronavirus (genetics)</term><term>Peptide Hydrolases (chemistry)</term><term>Peptide Hydrolases (genetics)</term><term>Peptide Hydrolases (metabolism)</term><term>Protein Structure, Quaternary</term><term>RNA Replicase (chemistry)</term><term>RNA Replicase (metabolism)</term><term>Recombinant Proteins (chemistry)</term><term>Recombinant Proteins (genetics)</term><term>Recombinant Proteins (isolation & purification)</term><term>Recombinant Proteins (metabolism)</term><term>Substrate Specificity</term><term>Viral Nonstructural Proteins (chemistry)</term><term>Viral Nonstructural Proteins (genetics)</term><term>Viral Nonstructural Proteins (metabolism)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Cinétique</term><term>Clonage moléculaire</term><term>Coronavirus du syndrome respiratoire du Moyen-Orient (enzymologie)</term><term>Coronavirus du syndrome respiratoire du Moyen-Orient (génétique)</term><term>Expression des gènes</term><term>Humains</term><term>Lignée cellulaire</term><term>Peptide hydrolases ()</term><term>Peptide hydrolases (génétique)</term><term>Peptide hydrolases (métabolisme)</term><term>Protéines recombinantes ()</term><term>Protéines recombinantes (génétique)</term><term>Protéines recombinantes (isolement et purification)</term><term>Protéines recombinantes (métabolisme)</term><term>Protéines virales non structurales ()</term><term>Protéines virales non structurales (génétique)</term><term>Protéines virales non structurales (métabolisme)</term><term>RNA replicase ()</term><term>RNA replicase (métabolisme)</term><term>Spécificité du substrat</term><term>Structure quaternaire des protéines</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Peptide Hydrolases</term><term>RNA Replicase</term><term>Recombinant Proteins</term><term>Viral Nonstructural Proteins</term></keywords><keywords scheme="MESH" qualifier="enzymologie" xml:lang="fr"><term>Coronavirus du syndrome respiratoire du Moyen-Orient</term></keywords><keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>Middle East Respiratory Syndrome Coronavirus</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Middle East Respiratory Syndrome Coronavirus</term><term>Peptide Hydrolases</term><term>Recombinant Proteins</term><term>Viral Nonstructural Proteins</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Coronavirus du syndrome respiratoire du Moyen-Orient</term><term>Peptide hydrolases</term><term>Protéines recombinantes</term><term>Protéines virales non structurales</term></keywords><keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" qualifier="isolement et purification" xml:lang="fr"><term>Protéines recombinantes</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Peptide Hydrolases</term><term>RNA Replicase</term><term>Recombinant Proteins</term><term>Viral Nonstructural Proteins</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Peptide hydrolases</term><term>Protéines recombinantes</term><term>Protéines virales non structurales</term><term>RNA replicase</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Cell Line</term><term>Cloning, Molecular</term><term>Gene Expression</term><term>Humans</term><term>Kinetics</term><term>Protein Structure, Quaternary</term><term>Substrate Specificity</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Cinétique</term><term>Clonage moléculaire</term><term>Expression des gènes</term><term>Humains</term><term>Lignée cellulaire</term><term>Peptide hydrolases</term><term>Protéines recombinantes</term><term>Protéines virales non structurales</term><term>RNA replicase</term><term>Spécificité du substrat</term><term>Structure quaternaire des protéines</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The papain-like protease (PLpro) domain from the deadly Middle East respiratory syndrome coronavirus (MERS-CoV) was overexpressed and purified. MERS-CoV PLpro constructs with and without the putative ubiquitin-like (UBL) domain at the N terminus were found to possess protease, deubiquitinating, deISGylating, and interferon antagonism activities in transfected HEK293T cells. The quaternary structure and substrate preferences of MERS-CoV PLpro were determined and compared to those of severe acute respiratory syndrome coronavirus (SARS-CoV) PLpro, revealing prominent differences between these closely related enzymes. Steady-state kinetic analyses of purified MERS-CoV and SARS-CoV PLpros uncovered significant differences in their rates of hydrolysis of 5-aminomethyl coumarin (AMC) from C-terminally labeled peptide, ubiquitin, and ISG15 substrates, as well as in their rates of isopeptide bond cleavage of K48- and K63-linked polyubiquitin chains. MERS-CoV PLpro was found to have 8-fold and 3,500-fold higher catalytic efficiencies for hydrolysis of ISG15-AMC than for hydrolysis of the Ub-AMC and Z-RLRGG-AMC substrates, respectively. A similar trend was observed for SARS-CoV PLpro, although it was much more efficient than MERS-CoV PLpro toward ISG15-AMC and peptide-AMC substrates. MERS-CoV PLpro was found to process K48- and K63-linked polyubiquitin chains at similar rates and with similar debranching patterns, producing monoubiquitin species. However, SARS-CoV PLpro much preferred K48-linked polyubiquitin chains to K63-linked chains, and it rapidly produced di-ubiquitin molecules from K48-linked chains. Finally, potent inhibitors of SARS-CoV PLpro were found to have no effect on MERS-CoV PLpro. A homology model of the MERS-CoV PLpro structure was generated and compared to the X-ray structure of SARS-CoV PLpro to provide plausible explanations for differences in substrate and inhibitor recognition.</div></front></TEI><affiliations><list><country><li>États-Unis</li></country><region><li>Illinois</li><li>Indiana</li></region></list><tree><country name="États-Unis"><region name="Indiana"><name sortKey="Baez Santos, Yahira M" sort="Baez Santos, Yahira M" uniqKey="Baez Santos Y" first="Yahira M" last="Báez-Santos">Yahira M. Báez-Santos</name></region><name sortKey="Baker, Susan" sort="Baker, Susan" uniqKey="Baker S" first="Susan" last="Baker">Susan Baker</name><name sortKey="Deng, Xufang" sort="Deng, Xufang" uniqKey="Deng X" first="Xufang" last="Deng">Xufang Deng</name><name sortKey="Mesecar, Andrew D" sort="Mesecar, Andrew D" uniqKey="Mesecar A" first="Andrew D" last="Mesecar">Andrew D. Mesecar</name><name sortKey="Mielech, Anna M" sort="Mielech, Anna M" uniqKey="Mielech A" first="Anna M" last="Mielech">Anna M. Mielech</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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