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Isabelle Jupin - One of the best experts on this subject based on the ideXlab platform.
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The Ubiquitin-Proteasome System Regulates the Accumulation of Turnip yellow mosaic virus RNA-Dependent RNA Polymerase during Viral Infection.
The Plant cell, 2010Co-Authors: Laurent Camborde, Séverine Planchais, Vincent Tournier, Anna Jakubiec, Gabrièle Drugeon, Emmanuelle Lacassagne, Stéphanie Pflieger, Mélanie Chenon, Isabelle JupinAbstract:Replication of positive-strand RNA viruses, the largest group of plant viruses, is initiated by viral RNA-dependent RNA polymerase (RdRp). Given its essential function in viral replication, understanding the regulation of RdRp is of great importance. Here, we show that Turnip yellow mosaic virus (TYMV) RdRp (termed 66K) is degraded by the proteasome at late time points during viral infection and that the accumulation level of 66K affects viral RNA replication in infected Arabidopsis thaliana cells. We mapped the cis-determinants responsible for 66K degradation within its N-terminal noncatalytic domain, but we conclude that 66K is not a natural N-end rule substrate. Instead, we show that a proposed PEST Sequence within 66K functions as a transferable degradation motif. In addition, several Lys residues that constitute target sites for ubiquitylation were mapped; mutation of these Lys residues leads to stabilization of 66K. Altogether, these results demonstrate that TYMV RdRp is a target of the ubiquitin-proteasome system in plant cells and support the idea that proteasomal degradation may constitute yet another fundamental level of regulation of viral replication.
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Phosphorylation of viral RNA-dependent RNA polymerase and its role in replication of a plus-strand RNA virus.
Journal of Biological Chemistry, 2006Co-Authors: Anna Jakubiec, François Héricourt, Virginie Redeker, Laurent Camborde, Vincent Tournier, Gabrièle Drugeon, Stéphanie Pflieger, Joelle Vinh, Isabelle JupinAbstract:Central to the process of plus-strand RNA virus genome amplification is the viral RNA-dependent RNA polymerase (RdRp). Understanding its regulation is of great importance given its essential function in viral replication and the common architecture and catalytic mechanism of polymerases. Here we show that Turnip yellow mosaic virus (TYMV) RdRp is phosphorylated, when expressed both individually and in the context of viral infection. Using a comprehensive biochemical approach, including metabolic labeling and mass spectrometry analyses, phosphorylation sites were mapped within an N-terminal PEST Sequence and within the highly conserved palm subdomain of RNA polymerases. Systematic mutational analysis of the corresponding residues in a reverse genetic system demonstrated their importance for TYMV infectivity. Upon mutation of the phosphorylation sites, distinct steps of the viral cycle appeared affected, but in contrast to other plus-strand RNA viruses, the interaction between viral replication proteins was unaltered. Our results also highlighted the role of another TYMV-encoded replication protein as an antagonistic protein that may prevent the inhibitory effect of RdRp phosphorylation on viral infectivity. Based on these data, we propose that phosphorylation-dependent regulatory mechanisms are essential for viral RdRp function and virus replication.
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Evidence for phosphorylation and ubiquitinylation of the turnip yellow mosaic virus RNA-dependent RNA polymerase domain expressed in a baculovirus-insect cell system
Biochemical Journal, 2000Co-Authors: François Héricourt, Stéphane Blanc, Virginie Redeker, Isabelle JupinAbstract:All RNA viruses known to date encode an RNA-dependent RNA polymerase (RdRp) that is required for replication of the viral genome. We have expressed and purified the turnip yellow mosaic virus (TYMV) RdRp in insect cells using a recombinant baculovirus, either in its native form, or fused to an hexa-histidine tag. Phosphorylation of the protein was demonstrated by labelling experiments in vivo, as well as phosphatase treatment of the purified protein in vitro. Phospho amino acid analysis and immunoblotting experiments identified serine and threonine residues as being the subject of phosphorylation. Peptide mass mapping using MS analysis of a protein digest revealed that phosphorylation sites are localized within a putative PEST Sequence [a Sequence rich in proline (P), glutamic acid (E), serine (S) and threonine (T) residues] in the N-terminal region of the protein. Using monoclonal antibodies specific for ubiquitin conjugates, we were able to demonstrate that the TYMV RdRp is conjugated to ubiquitin molecules when expressed in insect cells. These observations suggest that the TYMV RdRp may be processed selectively by the ubiquitin/proteasome degradation system upon phosphorylation of the PEST Sequence.
Elizabeth A. Komives - One of the best experts on this subject based on the ideXlab platform.
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The Acidic Residues of the IκBα PEST Sequence are Responsible for “Stripping” NFκB from DNA
Biophysical Journal, 2014Co-Authors: Holly E. Dembinski, Elizabeth A. KomivesAbstract:Nuclear factor kappa B (NFκB) transcription factors are responsible for the regulation of more than 150 target genes, their expression is induced by many classes of stimuli, and NFκBs play essential roles in the healthy regulation of cellular development and proliferation in inflammatory and immune responses. Diseases such as cancer, heart disease, Alzheimer's disease, and AIDS can be attributed to the aberrant regulation of NFκB. The transcriptional activity of NFκB is controlled by its inhibitors, the IκBs. IκBα, in particular, dynamically responds to extracellular stimuli releasing a burst of NFκB that enters the nucleus and activates hundreds of target genes. The transcriptional activation is short-lived, and our lab has been investigating the mechanism of post-induction repression. We previously showed that IκBα actively dissociates or "strips" NFκB from DNA. Analysis of the crystal structures of NFκB (RelA/p50) with DNA and with IκBα shows that the IκBα PEST Sequence, which is rich in glutamate and aspartate residues, forms similar electrostatic contacts to NFκB as the DNA. Given this, we hypothesized that the IκBα PEST Sequence electrostatically repels DNA from NFκB during the stripping process. Here we present fascinating results that show that the individual and collective, conservative mutation of these acidic residues to their amide counterparts does not affect the binding affinities of these mutants to NFκB; however, the mutant in which all five acidic residues are neutralized is incapable of stripping NFκB from DNA and instead forms a stable IκBα-NFκB-DNA ternary complex.
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Exploration of the Role of the IκB PEST Sequence in Stripping NFκB from DNA
Biophysical Journal, 2012Co-Authors: Holly E. Dembinski, Vera Alverdi, Hector M. González, Elizabeth A. KomivesAbstract:Nuclear factor kappa B (NFκB) transcription factors are responsible for the regulation of more than 150 target genes, their expression is induced by many classes of stimuli, and NFκBs play essential roles in the healthy regulation of cellular development and proliferation in inflammatory and immune responses. Diseases such as cancer, heart disease, Alzheimer's disease, and AIDS can be attributed to the wayward regulation of NFκB. The transcriptional activity of NFκB is commanded by its inhibitor—IκB. Two major isoforms of the inhibitor—IκBα and IκBβ—are structurally similar (i.e., crystal structures of the two exhibit a six ankyrin repeat domain followed by a largely unstructured, C-terminal PEST Sequence), yet their inhibitory activities markedly differ. It has been shown that IκBα can actively “strip” NFκB from DNA in vitro, but IκBβ cannot. We are investivating a panel of “PEST swap” mutants to probe the role of the PEST Sequence in the regulation of NFκB. In addition, the role of individual residues within the IκBα PEST Sequence in the “stripping” phenomenon is being analyzed.
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Kinetic enhancement of NF-κB·DNA dissociation by IκBα
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Simon Bergqvist, Gourisankar Ghosh, Vera Alverdi, Benedicte Mengel, Alexander Hoffmann, Elizabeth A. KomivesAbstract:A hallmark of the NF-κB transcription response to inflammatory cytokines is the remarkably rapid rate of robust activation and subsequent signal repression. Although the rapidity of postinduction repression is explained partly by the fact that the gene for IκBα is strongly induced by NF-κB, the newly synthesized IκBα still must enter the nucleus and compete for binding to NF-κB with the very large number of κB sites in the DNA. We present results from real-time binding kinetic experiments, demonstrating that IκBα increases the dissociation rate of NF-κB from the DNA in a highly efficient kinetic process. Analysis of various IκB mutant proteins shows that this process requires the C-terminal PEST Sequence and the weakly folded fifth and sixth ankyrin repeats of IκBα. Mutational stabilization of these repeats reduces the efficiency with which IκBα enhances the dissociation rate.
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Functional Dynamics of the Folded Ankyrin Repeats of IκBα Revealed by Nuclear Magnetic Resonance
Biochemistry, 2009Co-Authors: Carla F. Cervantes, Shih-che Sue, H. Jane Dyson, Phineus R. L. Markwick, J. Andrew Mccammon, Elizabeth A. KomivesAbstract:Inhibition of nuclear factor kappaB (NF-kappaB) is mainly accomplished by IkappaB alpha, which consists of a signal response Sequence at the N-terminus, a six-ankyrin repeat domain (ARD) that binds NF-kappaB, and a C-terminal PEST Sequence. Previous studies with the ARD revealed that the fifth and sixth repeats are only partially folded in the absence of NF-kappaB. Here we report NMR studies of a truncated version of IkappaB alpha, containing only the first four ankyrin repeats, IkappaB alpha(67-206). This four-repeat segment is well-structured in the free state, enabling full resonance assignments to be made. H-D exchange, backbone dynamics, and residual dipolar coupling (RDC) experiments reveal regions of flexibility. In addition, regions consistent with the presence of micro- to millisecond motions occur periodically throughout the repeat structure. Comparison of the RDCs with the crystal structure gave only moderate agreement, but an ensemble of structures generated by accelerated molecular dynamics gave much better agreement with the measured RDCs. The regions showing flexibility correspond to those implicated in entropic compensation for the loss of flexibility in ankyrin repeats 5 and 6 upon binding to NF-kappaB. The regions showing micro- to millisecond motions in the free protein are the ends of the beta-hairpins that directly interact with NF-kappaB in the complex.
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Biophysical characterization of the free IκBα ankyrin repeat domain in solution
Protein science : a publication of the Protein Society, 2004Co-Authors: Carrie H. Croy, Simon Bergqvist, Tom Huxford, Gourisankar Ghosh, Elizabeth A. KomivesAbstract:The crystal structure of IκBα in complex with the transcription factor, nuclear factor κ-B (NF-κB) shows six ankyrin repeats, which are all ordered. Electron density was not observed for most of the residues within the PEST Sequence, although it is required for high-affinity binding. To characterize the folded state of IκBα (67–317) when it is not in complex with NF-κB, we have carried out circular dichroism (CD) spectroscopy, 8-anilino-1-napthalenesulphonic acid (ANS) binding, differential scanning calorimetry, and amide hydrogen/deuterium exchange experiments. The CD spectrum shows the presence of helical structure, consistent with other ankyrin repeat proteins. The large amount of ANS-binding and amide exchange suggest that the protein may have molten globule character. The amide exchange experiments show that the third ankyrin repeat is the most compact, the second and fourth repeats are somewhat less compact, and the first and sixth repeats are solvent exposed. The PEST extension is also highly solvent accessible. Iκ Bα unfolds with a Tm of 42°C, and forms a soluble aggregate that sequesters helical and variable loop parts of the first, fourth, and sixth repeats and the PEST extension. The second and third repeats, which conform most closely to a consensus for stable ankyrin repeats, appear to remain outside of the aggregate. The ramifications of these observations for the biological function of IκBα are discussed.
François Héricourt - One of the best experts on this subject based on the ideXlab platform.
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Phosphorylation of viral RNA-dependent RNA polymerase and its role in replication of a plus-strand RNA virus.
Journal of Biological Chemistry, 2006Co-Authors: Anna Jakubiec, François Héricourt, Virginie Redeker, Laurent Camborde, Vincent Tournier, Gabrièle Drugeon, Stéphanie Pflieger, Joelle Vinh, Isabelle JupinAbstract:Central to the process of plus-strand RNA virus genome amplification is the viral RNA-dependent RNA polymerase (RdRp). Understanding its regulation is of great importance given its essential function in viral replication and the common architecture and catalytic mechanism of polymerases. Here we show that Turnip yellow mosaic virus (TYMV) RdRp is phosphorylated, when expressed both individually and in the context of viral infection. Using a comprehensive biochemical approach, including metabolic labeling and mass spectrometry analyses, phosphorylation sites were mapped within an N-terminal PEST Sequence and within the highly conserved palm subdomain of RNA polymerases. Systematic mutational analysis of the corresponding residues in a reverse genetic system demonstrated their importance for TYMV infectivity. Upon mutation of the phosphorylation sites, distinct steps of the viral cycle appeared affected, but in contrast to other plus-strand RNA viruses, the interaction between viral replication proteins was unaltered. Our results also highlighted the role of another TYMV-encoded replication protein as an antagonistic protein that may prevent the inhibitory effect of RdRp phosphorylation on viral infectivity. Based on these data, we propose that phosphorylation-dependent regulatory mechanisms are essential for viral RdRp function and virus replication.
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Evidence for phosphorylation and ubiquitinylation of the turnip yellow mosaic virus RNA-dependent RNA polymerase domain expressed in a baculovirus-insect cell system
Biochemical Journal, 2000Co-Authors: François Héricourt, Stéphane Blanc, Virginie Redeker, Isabelle JupinAbstract:All RNA viruses known to date encode an RNA-dependent RNA polymerase (RdRp) that is required for replication of the viral genome. We have expressed and purified the turnip yellow mosaic virus (TYMV) RdRp in insect cells using a recombinant baculovirus, either in its native form, or fused to an hexa-histidine tag. Phosphorylation of the protein was demonstrated by labelling experiments in vivo, as well as phosphatase treatment of the purified protein in vitro. Phospho amino acid analysis and immunoblotting experiments identified serine and threonine residues as being the subject of phosphorylation. Peptide mass mapping using MS analysis of a protein digest revealed that phosphorylation sites are localized within a putative PEST Sequence [a Sequence rich in proline (P), glutamic acid (E), serine (S) and threonine (T) residues] in the N-terminal region of the protein. Using monoclonal antibodies specific for ubiquitin conjugates, we were able to demonstrate that the TYMV RdRp is conjugated to ubiquitin molecules when expressed in insect cells. These observations suggest that the TYMV RdRp may be processed selectively by the ubiquitin/proteasome degradation system upon phosphorylation of the PEST Sequence.
Laurent Camborde - One of the best experts on this subject based on the ideXlab platform.
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The Ubiquitin-Proteasome System Regulates the Accumulation of Turnip yellow mosaic virus RNA-Dependent RNA Polymerase during Viral Infection.
The Plant cell, 2010Co-Authors: Laurent Camborde, Séverine Planchais, Vincent Tournier, Anna Jakubiec, Gabrièle Drugeon, Emmanuelle Lacassagne, Stéphanie Pflieger, Mélanie Chenon, Isabelle JupinAbstract:Replication of positive-strand RNA viruses, the largest group of plant viruses, is initiated by viral RNA-dependent RNA polymerase (RdRp). Given its essential function in viral replication, understanding the regulation of RdRp is of great importance. Here, we show that Turnip yellow mosaic virus (TYMV) RdRp (termed 66K) is degraded by the proteasome at late time points during viral infection and that the accumulation level of 66K affects viral RNA replication in infected Arabidopsis thaliana cells. We mapped the cis-determinants responsible for 66K degradation within its N-terminal noncatalytic domain, but we conclude that 66K is not a natural N-end rule substrate. Instead, we show that a proposed PEST Sequence within 66K functions as a transferable degradation motif. In addition, several Lys residues that constitute target sites for ubiquitylation were mapped; mutation of these Lys residues leads to stabilization of 66K. Altogether, these results demonstrate that TYMV RdRp is a target of the ubiquitin-proteasome system in plant cells and support the idea that proteasomal degradation may constitute yet another fundamental level of regulation of viral replication.
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Phosphorylation of viral RNA-dependent RNA polymerase and its role in replication of a plus-strand RNA virus.
Journal of Biological Chemistry, 2006Co-Authors: Anna Jakubiec, François Héricourt, Virginie Redeker, Laurent Camborde, Vincent Tournier, Gabrièle Drugeon, Stéphanie Pflieger, Joelle Vinh, Isabelle JupinAbstract:Central to the process of plus-strand RNA virus genome amplification is the viral RNA-dependent RNA polymerase (RdRp). Understanding its regulation is of great importance given its essential function in viral replication and the common architecture and catalytic mechanism of polymerases. Here we show that Turnip yellow mosaic virus (TYMV) RdRp is phosphorylated, when expressed both individually and in the context of viral infection. Using a comprehensive biochemical approach, including metabolic labeling and mass spectrometry analyses, phosphorylation sites were mapped within an N-terminal PEST Sequence and within the highly conserved palm subdomain of RNA polymerases. Systematic mutational analysis of the corresponding residues in a reverse genetic system demonstrated their importance for TYMV infectivity. Upon mutation of the phosphorylation sites, distinct steps of the viral cycle appeared affected, but in contrast to other plus-strand RNA viruses, the interaction between viral replication proteins was unaltered. Our results also highlighted the role of another TYMV-encoded replication protein as an antagonistic protein that may prevent the inhibitory effect of RdRp phosphorylation on viral infectivity. Based on these data, we propose that phosphorylation-dependent regulatory mechanisms are essential for viral RdRp function and virus replication.
Anna Jakubiec - One of the best experts on this subject based on the ideXlab platform.
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The Ubiquitin-Proteasome System Regulates the Accumulation of Turnip yellow mosaic virus RNA-Dependent RNA Polymerase during Viral Infection.
The Plant cell, 2010Co-Authors: Laurent Camborde, Séverine Planchais, Vincent Tournier, Anna Jakubiec, Gabrièle Drugeon, Emmanuelle Lacassagne, Stéphanie Pflieger, Mélanie Chenon, Isabelle JupinAbstract:Replication of positive-strand RNA viruses, the largest group of plant viruses, is initiated by viral RNA-dependent RNA polymerase (RdRp). Given its essential function in viral replication, understanding the regulation of RdRp is of great importance. Here, we show that Turnip yellow mosaic virus (TYMV) RdRp (termed 66K) is degraded by the proteasome at late time points during viral infection and that the accumulation level of 66K affects viral RNA replication in infected Arabidopsis thaliana cells. We mapped the cis-determinants responsible for 66K degradation within its N-terminal noncatalytic domain, but we conclude that 66K is not a natural N-end rule substrate. Instead, we show that a proposed PEST Sequence within 66K functions as a transferable degradation motif. In addition, several Lys residues that constitute target sites for ubiquitylation were mapped; mutation of these Lys residues leads to stabilization of 66K. Altogether, these results demonstrate that TYMV RdRp is a target of the ubiquitin-proteasome system in plant cells and support the idea that proteasomal degradation may constitute yet another fundamental level of regulation of viral replication.
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Phosphorylation of viral RNA-dependent RNA polymerase and its role in replication of a plus-strand RNA virus.
Journal of Biological Chemistry, 2006Co-Authors: Anna Jakubiec, François Héricourt, Virginie Redeker, Laurent Camborde, Vincent Tournier, Gabrièle Drugeon, Stéphanie Pflieger, Joelle Vinh, Isabelle JupinAbstract:Central to the process of plus-strand RNA virus genome amplification is the viral RNA-dependent RNA polymerase (RdRp). Understanding its regulation is of great importance given its essential function in viral replication and the common architecture and catalytic mechanism of polymerases. Here we show that Turnip yellow mosaic virus (TYMV) RdRp is phosphorylated, when expressed both individually and in the context of viral infection. Using a comprehensive biochemical approach, including metabolic labeling and mass spectrometry analyses, phosphorylation sites were mapped within an N-terminal PEST Sequence and within the highly conserved palm subdomain of RNA polymerases. Systematic mutational analysis of the corresponding residues in a reverse genetic system demonstrated their importance for TYMV infectivity. Upon mutation of the phosphorylation sites, distinct steps of the viral cycle appeared affected, but in contrast to other plus-strand RNA viruses, the interaction between viral replication proteins was unaltered. Our results also highlighted the role of another TYMV-encoded replication protein as an antagonistic protein that may prevent the inhibitory effect of RdRp phosphorylation on viral infectivity. Based on these data, we propose that phosphorylation-dependent regulatory mechanisms are essential for viral RdRp function and virus replication.