The Experts below are selected from a list of 6486 Experts worldwide ranked by ideXlab platform
C. Cheng Kao - One of the best experts on this subject based on the ideXlab platform.
-
The Coronavirus Endoribonuclease Nsp15 Interacts with Retinoblastoma Tumor Suppressor Protein
Journal of virology, 2012Co-Authors: Kanchan Bhardwaj, Pinghua Liu, Julian L. Leibowitz, C. Cheng KaoAbstract:Coronaviruses encode an Endoribonuclease, Nsp15, which has a poorly defined role in infection. Sequence analysis revealed a retinoblastoma protein-binding motif (LXCXE/D) in the majority of the Nsp15 of the severe acute respiratory syndrome coronavirus (SARS-CoV) and its orthologs in the alpha and beta coronaviruses. The Endoribonuclease activity of the SARS-CoV Nsp15 (sNsp15) was stimulated by retinoblastoma protein (pRb) in vitro, and the two proteins can be coimmunoprecipitated from cellular extracts. Mutations in the pRb-binding motif rendered sNsp15 to be differentially modified by ubiquitin in cells, and cytotoxicity was observed upon its expression. Expression of the sNsp15 in cells resulted in an increased abundance of pRb in the cytoplasm, decreased overall levels of pRb, an increased proportion of cells in the S phase of the cell cycle, and an enhanced expression from a promoter normally repressed by pRb. The Endoribonuclease activity of the mouse hepatitis virus (MHV) A59 Nsp15 was also increased by pRb in vitro, and an MHV with mutations in the LXCXE/D-motif, named vLC, exhibited a smaller plaque diameter and reduced the virus titer by ∼1 log. Overexpression of pRb delayed the viral protein production by wild-type MHV but not by vLC. This study reveals that pRb and its interaction with Nsp15 can affect coronavirus infection and adds coronaviruses to a small but growing family of RNA viruses that encode a protein to interact with pRb.
-
The Severe Acute Respiratory Syndrome Coronavirus Nsp15 Protein Is an Endoribonuclease That Prefers Manganese as a Cofactor
Journal of Virology, 2004Co-Authors: Kanchan Bhardwaj, Linda A. Guarino, C. Cheng KaoAbstract:Nonstructural protein 15 (Nsp15) of the severe acute respiratory syndrome coronavirus (SARS-CoV) produced in Escherichia coli has Endoribonuclease activity that preferentially cleaved 5′ of uridylates of RNAs. Blocking either the 5′ or 3′ terminus did not affect cleavage. Double- and single-stranded RNAs were both substrates for Nsp15 but with different kinetics for cleavage. Mn2+ at 2 to 10 mM was needed for optimal Endoribonuclease activity, but Mg2+ and several other divalent metals were capable of supporting only a low level of activity. Concentrations of Mn2+ needed for Endoribonuclease activity induced significant conformation change(s) in the protein, as measured by changes in tryptophan fluorescence. A similar endoribonucleolytic activity was detected for the orthologous protein from another coronavirus, demonstrating that the Endoribonuclease activity of Nsp15 may be common to coronaviruses. This work presents an initial biochemical characterization of a novel coronavirus Endoribonuclease.
-
The Severe Acute Respiratory Syndrome Coronavirus Nsp15 Protein Is an Endoribonuclease That Prefers Manganese as a Cofactor
Journal of Virology, 2004Co-Authors: Kanchan Bhardwaj, Linda A. Guarino, C. Cheng KaoAbstract:Nonstructural protein 15 (Nsp15) of the severe acute respiratory syndrome coronavirus (SARS-CoV) produced in Escherichia coli has Endoribonuclease activity that preferentially cleaved 5′ of uridylates of RNAs. Blocking either the 5′ or 3′ terminus did not affect cleavage. Double- and single-stranded RNAs were both substrates for Nsp15 but with different kinetics for cleavage. Mn2+ at 2 to 10 mM was needed for optimal Endoribonuclease activity, but Mg2+ and several other divalent metals were capable of supporting only a low level of activity. Concentrations of Mn2+ needed for Endoribonuclease activity induced significant conformation change(s) in the protein, as measured by changes in tryptophan fluorescence. A similar endoribonucleolytic activity was detected for the orthologous protein from another coronavirus, demonstrating that the Endoribonuclease activity of Nsp15 may be common to coronaviruses. This work presents an initial biochemical characterization of a novel coronavirus Endoribonuclease.
Kanchan Bhardwaj - One of the best experts on this subject based on the ideXlab platform.
-
The Coronavirus Endoribonuclease Nsp15 Interacts with Retinoblastoma Tumor Suppressor Protein
Journal of virology, 2012Co-Authors: Kanchan Bhardwaj, Pinghua Liu, Julian L. Leibowitz, C. Cheng KaoAbstract:Coronaviruses encode an Endoribonuclease, Nsp15, which has a poorly defined role in infection. Sequence analysis revealed a retinoblastoma protein-binding motif (LXCXE/D) in the majority of the Nsp15 of the severe acute respiratory syndrome coronavirus (SARS-CoV) and its orthologs in the alpha and beta coronaviruses. The Endoribonuclease activity of the SARS-CoV Nsp15 (sNsp15) was stimulated by retinoblastoma protein (pRb) in vitro, and the two proteins can be coimmunoprecipitated from cellular extracts. Mutations in the pRb-binding motif rendered sNsp15 to be differentially modified by ubiquitin in cells, and cytotoxicity was observed upon its expression. Expression of the sNsp15 in cells resulted in an increased abundance of pRb in the cytoplasm, decreased overall levels of pRb, an increased proportion of cells in the S phase of the cell cycle, and an enhanced expression from a promoter normally repressed by pRb. The Endoribonuclease activity of the mouse hepatitis virus (MHV) A59 Nsp15 was also increased by pRb in vitro, and an MHV with mutations in the LXCXE/D-motif, named vLC, exhibited a smaller plaque diameter and reduced the virus titer by ∼1 log. Overexpression of pRb delayed the viral protein production by wild-type MHV but not by vLC. This study reveals that pRb and its interaction with Nsp15 can affect coronavirus infection and adds coronaviruses to a small but growing family of RNA viruses that encode a protein to interact with pRb.
-
Mutational analysis of the SARS virus Nsp15 Endoribonuclease: identification of residues affecting hexamer formation.
Journal of molecular biology, 2005Co-Authors: Linda A. Guarino, Kanchan Bhardwaj, Wen Dong, Jingchuan Sun, Andreas Holzenburg, Cheng KaoAbstract:The severe acute respiratory syndrome (SARS) coronavirus virus non-structural protein 15 is a Mn2+-dependent Endoribonuclease with specificity for cleavage at uridylate residues. To better understand structural and functional characteristics of Nsp15, 22 mutant versions of Nsp15 were produced in Escherichia coli as His-tagged proteins and purified by metal-affinity and ion-exchange chromatography. Nineteen of the mutants were soluble and were analyzed for enzymatic activity. Six mutants, including four at the putative active site, were significantly reduced in Endoribonuclease activity. Two of the inactive mutants had unusual secondary structures compared to the wild-type protein, as measured by circular dichroism spectroscopy. Gel-filtration analysis, velocity sedimentation ultracentrifugation, and native gradient pore electrophoresis all showed that the wild-type protein exists in an equilibrium between hexamers and monomers in solution, with hexamers dominating at micromolar protein concentration, while native gradient pore electrophoresis also revealed the presence of trimers. A mutant in the N terminus of Nsp15 was impaired in hexamer formation and had low Endoribonuclease activity, suggesting that oligomerization is required for Endoribonuclease activity. This idea was supported by titration experiments showing that enzyme activity was strongly concentration-dependent, indicating that oligomeric Nsp15 is the active form. Three-dimensional reconstruction of negatively stained single particles of Nsp15 viewed by transmission electron microscopic analysis suggested that the six subunits were arranged as a dimer of trimers with a number of cavities or channels that may constitute RNA binding sites.
-
The Severe Acute Respiratory Syndrome Coronavirus Nsp15 Protein Is an Endoribonuclease That Prefers Manganese as a Cofactor
Journal of Virology, 2004Co-Authors: Kanchan Bhardwaj, Linda A. Guarino, C. Cheng KaoAbstract:Nonstructural protein 15 (Nsp15) of the severe acute respiratory syndrome coronavirus (SARS-CoV) produced in Escherichia coli has Endoribonuclease activity that preferentially cleaved 5′ of uridylates of RNAs. Blocking either the 5′ or 3′ terminus did not affect cleavage. Double- and single-stranded RNAs were both substrates for Nsp15 but with different kinetics for cleavage. Mn2+ at 2 to 10 mM was needed for optimal Endoribonuclease activity, but Mg2+ and several other divalent metals were capable of supporting only a low level of activity. Concentrations of Mn2+ needed for Endoribonuclease activity induced significant conformation change(s) in the protein, as measured by changes in tryptophan fluorescence. A similar endoribonucleolytic activity was detected for the orthologous protein from another coronavirus, demonstrating that the Endoribonuclease activity of Nsp15 may be common to coronaviruses. This work presents an initial biochemical characterization of a novel coronavirus Endoribonuclease.
-
The Severe Acute Respiratory Syndrome Coronavirus Nsp15 Protein Is an Endoribonuclease That Prefers Manganese as a Cofactor
Journal of Virology, 2004Co-Authors: Kanchan Bhardwaj, Linda A. Guarino, C. Cheng KaoAbstract:Nonstructural protein 15 (Nsp15) of the severe acute respiratory syndrome coronavirus (SARS-CoV) produced in Escherichia coli has Endoribonuclease activity that preferentially cleaved 5′ of uridylates of RNAs. Blocking either the 5′ or 3′ terminus did not affect cleavage. Double- and single-stranded RNAs were both substrates for Nsp15 but with different kinetics for cleavage. Mn2+ at 2 to 10 mM was needed for optimal Endoribonuclease activity, but Mg2+ and several other divalent metals were capable of supporting only a low level of activity. Concentrations of Mn2+ needed for Endoribonuclease activity induced significant conformation change(s) in the protein, as measured by changes in tryptophan fluorescence. A similar endoribonucleolytic activity was detected for the orthologous protein from another coronavirus, demonstrating that the Endoribonuclease activity of Nsp15 may be common to coronaviruses. This work presents an initial biochemical characterization of a novel coronavirus Endoribonuclease.
Linda A. Guarino - One of the best experts on this subject based on the ideXlab platform.
-
Mutational analysis of the SARS virus Nsp15 Endoribonuclease: identification of residues affecting hexamer formation.
Journal of molecular biology, 2005Co-Authors: Linda A. Guarino, Kanchan Bhardwaj, Wen Dong, Jingchuan Sun, Andreas Holzenburg, Cheng KaoAbstract:The severe acute respiratory syndrome (SARS) coronavirus virus non-structural protein 15 is a Mn2+-dependent Endoribonuclease with specificity for cleavage at uridylate residues. To better understand structural and functional characteristics of Nsp15, 22 mutant versions of Nsp15 were produced in Escherichia coli as His-tagged proteins and purified by metal-affinity and ion-exchange chromatography. Nineteen of the mutants were soluble and were analyzed for enzymatic activity. Six mutants, including four at the putative active site, were significantly reduced in Endoribonuclease activity. Two of the inactive mutants had unusual secondary structures compared to the wild-type protein, as measured by circular dichroism spectroscopy. Gel-filtration analysis, velocity sedimentation ultracentrifugation, and native gradient pore electrophoresis all showed that the wild-type protein exists in an equilibrium between hexamers and monomers in solution, with hexamers dominating at micromolar protein concentration, while native gradient pore electrophoresis also revealed the presence of trimers. A mutant in the N terminus of Nsp15 was impaired in hexamer formation and had low Endoribonuclease activity, suggesting that oligomerization is required for Endoribonuclease activity. This idea was supported by titration experiments showing that enzyme activity was strongly concentration-dependent, indicating that oligomeric Nsp15 is the active form. Three-dimensional reconstruction of negatively stained single particles of Nsp15 viewed by transmission electron microscopic analysis suggested that the six subunits were arranged as a dimer of trimers with a number of cavities or channels that may constitute RNA binding sites.
-
The Severe Acute Respiratory Syndrome Coronavirus Nsp15 Protein Is an Endoribonuclease That Prefers Manganese as a Cofactor
Journal of Virology, 2004Co-Authors: Kanchan Bhardwaj, Linda A. Guarino, C. Cheng KaoAbstract:Nonstructural protein 15 (Nsp15) of the severe acute respiratory syndrome coronavirus (SARS-CoV) produced in Escherichia coli has Endoribonuclease activity that preferentially cleaved 5′ of uridylates of RNAs. Blocking either the 5′ or 3′ terminus did not affect cleavage. Double- and single-stranded RNAs were both substrates for Nsp15 but with different kinetics for cleavage. Mn2+ at 2 to 10 mM was needed for optimal Endoribonuclease activity, but Mg2+ and several other divalent metals were capable of supporting only a low level of activity. Concentrations of Mn2+ needed for Endoribonuclease activity induced significant conformation change(s) in the protein, as measured by changes in tryptophan fluorescence. A similar endoribonucleolytic activity was detected for the orthologous protein from another coronavirus, demonstrating that the Endoribonuclease activity of Nsp15 may be common to coronaviruses. This work presents an initial biochemical characterization of a novel coronavirus Endoribonuclease.
-
The Severe Acute Respiratory Syndrome Coronavirus Nsp15 Protein Is an Endoribonuclease That Prefers Manganese as a Cofactor
Journal of Virology, 2004Co-Authors: Kanchan Bhardwaj, Linda A. Guarino, C. Cheng KaoAbstract:Nonstructural protein 15 (Nsp15) of the severe acute respiratory syndrome coronavirus (SARS-CoV) produced in Escherichia coli has Endoribonuclease activity that preferentially cleaved 5′ of uridylates of RNAs. Blocking either the 5′ or 3′ terminus did not affect cleavage. Double- and single-stranded RNAs were both substrates for Nsp15 but with different kinetics for cleavage. Mn2+ at 2 to 10 mM was needed for optimal Endoribonuclease activity, but Mg2+ and several other divalent metals were capable of supporting only a low level of activity. Concentrations of Mn2+ needed for Endoribonuclease activity induced significant conformation change(s) in the protein, as measured by changes in tryptophan fluorescence. A similar endoribonucleolytic activity was detected for the orthologous protein from another coronavirus, demonstrating that the Endoribonuclease activity of Nsp15 may be common to coronaviruses. This work presents an initial biochemical characterization of a novel coronavirus Endoribonuclease.
Graham C Walker - One of the best experts on this subject based on the ideXlab platform.
-
sinorhizobium meliloti ybey is a zinc dependent single strand specific Endoribonuclease that plays an important role in 16s ribosomal rna processing
Nucleic Acids Research, 2020Co-Authors: Vignesh M P Babu, Siva Sankari, James A Budnick, Clayton C Caswell, Graham C WalkerAbstract:: Single-strand specific Endoribonuclease YbeY has been shown to play an important role in the processing of the 3' end of the 16S rRNA in Escherichia coli. Lack of YbeY results in the accumulation of the 17S rRNA precursor. In contrast to a previous report, we show that Sinorhizobium meliloti YbeY exhibits Endoribonuclease activity on single-stranded RNA substrate but not on the double-stranded substrate. This study also identifies the previously unknown metal ion involved in YbeY function to be Zn2+ and shows that the activity of YbeY is enhanced when the occupancy of zinc is increased. We have identified a pre-16S rRNA precursor that accumulates in the S. meliloti ΔybeY strain. We also show that ΔybeY mutant of Brucella abortus, a mammalian pathogen, also accumulates a similar pre-16S rRNA. The pre-16S species is longer in alpha-proteobacteria than in gamma-proteobacteria. We demonstrate that the YbeY from E. coli and S. meliloti can reciprocally complement the rRNA processing defect in a ΔybeY mutant of the other organism. These results establish YbeY as a zinc-dependent single-strand specific Endoribonuclease that functions in 16S rRNA processing in both alpha- and gamma-proteobacteria.
-
elevated levels of era gtpase improve growth 16s rrna processing and 70s ribosome assembly of escherichia coli lacking highly conserved multifunctional ybey Endoribonuclease
Journal of Bacteriology, 2018Co-Authors: Anubrata Ghosal, Vignesh M P Babu, Graham C WalkerAbstract:YbeY is a highly conserved, multifunctional Endoribonuclease that plays a significant role in ribosome biogenesis and has several additional roles. Here we show that overexpression of the conserved GTPase Era in Escherichia coli partially suppresses the growth defect of a ΔybeY strain while improving 16S rRNA processing and 70S ribosome assembly. This suppression requires both the ability of Era to hydrolyze GTP and the function of three exoribonucleases, RNase II, RNase R, and RNase PH, suggesting a model for the action of Era. Overexpression of Vibrio cholerae Era similarly partially suppresses the defects of an E. coli ΔybeY strain, indicating that this property of Era is conserved in bacteria other than E. coliIMPORTANCE This work provides insight into the critical, but still incompletely understood, mechanism of processing of the E. coli 16S rRNA 3' terminus. The highly conserved GTPase Era is known to bind to the precursor of the 16S rRNA near its 3' end. Both the Endoribonuclease YbeY, which binds to Era, and four exoribonucleases have been implicated in this 3'-end processing. The results reported here offer additional insights into the role of Era in 16S rRNA 3'-end maturation and into the relationship between the action of the Endoribonuclease YbeY and that of the four exoribonucleases. This study also hints at why YbeY is essential only in some bacteria and suggests that YbeY could be a target for a new class of antibiotics in these bacteria.
Masayori Inouye - One of the best experts on this subject based on the ideXlab platform.
-
Interference of mRNA function by sequence-specific Endoribonuclease PemK.
The Journal of biological chemistry, 2004Co-Authors: Junjie Zhang, Yonglong Zhang, Ling Zhu, Motoo Suzuki, Masayori InouyeAbstract:In Escherichia coli, programmed cell death is mediated through the system called "addiction module," which consists of a pair of genes encoding a stable toxin and a labile antitoxin. The pemI-pemK system is an addiction module present on plasmid R100. It helps to maintain the plasmid by post-segregational killing in E. coli population. Here we demonstrate that purified PemK, the toxin encoded by the pemI-pemK addiction module, inhibits protein synthesis in an E. coli cell-free system, whereas the addition of PemI, the antitoxin against PemK, resumes the protein synthesis. Further studies reveal that PemK is a sequence-specific Endoribonuclease that cleaves mRNAs to inhibit protein synthesis, whereas PemI blocks the Endoribonuclease activity of PemK. PemK cleaves only single-stranded RNA preferentially at the 5' or 3' side of the A residue in the "UAH" sequences (where H is C, A, or U). Upon induction, PemK cleaves cellular mRNAs to effectively block protein synthesis in E. coli. The pemK homologue genes have been identified on the genomes of a wide range of bacteria. We propose that PemK and its homologues form a novel Endoribonuclease family that interferes with mRNA function by cleaving cellular mRNAs in a sequence-specific manner.
-
mazf cleaves cellular mrnas specifically at aca to block protein synthesis in escherichia coli
Molecular Cell, 2003Co-Authors: Yonglong Zhang, Junjie Zhang, Klaus P Hoeflich, Mitsuhiko Ikura, Guoliang Qing, Masayori InouyeAbstract:Escherichia coli contains operons called "addiction modules," encoding toxin and antitoxin, which are responsible for growth arrest and cell death. Here, we demonstrate that MazF toxin encoded by "mazEF addiction module" is a sequence-specific (ACA) Endoribonuclease functional only for single-stranded RNA. MazF works as a ribonuclease independent of ribosomes, and is, therefore, functionally distinct from RelE, another E. coli toxin, which assists mRNA cleavage at the A site on ribosomes. Upon induction, MazF cleaves whole cellular mRNAs to efficiently block protein synthesis. Purified MazF inhibited protein synthesis in both prokaryotic and eukaryotic cell-free systems. This inhibition was released by MazE, the labile antitoxin against MazF. Thus, MazF functions as a toxic Endoribonuclease to interfere with the function of cellular mRNAs by cleaving them at specific sequences leading to rapid cell growth arrest and cell death. The role of such Endoribonucleases may have broad implication in cell physiology under various growth conditions.