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Adrian Whitehouse - One of the best experts on this subject based on the ideXlab platform.
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Kaposi's Sarcoma-Associated Herpesvirus RTA Promotes Degradation of the Hey1 Repressor Protein through the Ubiquitin Proteasome Pathway
Journal of Virology, 2009Co-Authors: Faye Gould, Sally M Harrison, Eric W Hewitt, Adrian WhitehouseAbstract:The Kaposi's sarcoma-associated herpesvirus (KSHV) replication and transcription activator (RTA) Protein regulates the latent-lytic switch by transactivating a variety of KSHV lytic and cellular promoters. RTA is a novel E3 ubiquitin ligase that targets a number of transcriptional Repressor Proteins for degradation by the ubiquitin proteasome pathway. Herein, we show that RTA interacts with the cellular transcriptional Repressor Protein Hey1. We demonstrate that Hey1 is a target for RTA-mediated ubiquitination and is subsequently degraded by the proteasome. Moreover, a Cys-plus-His-rich region within RTA is important for RTA-mediated degradation of Hey1. We confirm that Hey1 represses the RTA promoter and, furthermore, show that Hey1 binds to the RTA promoter. An interaction was observed between Hey1 and the coRepressor mSin3A, and this interaction was abolished in the presence of RTA. Additionally, mSin3A associated with the RTA promoter in nonreactivated, but not reactivated, BCBL1 cells. Small interfering RNA knockdown of Hey1 in HEK 293T cells latently infected with the recombinant virus rKSHV.219 led to increased levels of RTA expression upon reactivation but was insufficient to induce complete lytic reactivation. These results suggest that other additional transcriptional Repressors are also important in maintenance of KSHV latency. Taken together, our results suggest that Hey1 has a contributory role in the maintenance of KSHV latency and that disruption of the Hey1 repressosome by RTA-targeted degradation may be one step in the mechanism to regulate lytic reactivation.
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Kaposi's Sarcoma-Associated Herpesvirus RTA Promotes Degradation of the Hey1 Repressor Protein through the Ubiquitin Proteasome Pathway
Journal of Virology, 2009Co-Authors: Faye Gould, Sally M Harrison, Eric W Hewitt, Adrian WhitehouseAbstract:The Kaposi's sarcoma-associated herpesvirus (KSHV) replication and transcription activator (RTA) Protein regulates the latent-lytic switch by transactivating a variety of KSHV lytic and cellular promoters. RTA is a novel E3 ubiquitin ligase that targets a number of transcriptional Repressor Proteins for degradation by the ubiquitin proteasome pathway. Herein, we show that RTA interacts with the cellular transcriptional Repressor Protein Hey1. We demonstrate that Hey1 is a target for RTA-mediated ubiquitination and is subsequently degraded by the proteasome. Moreover, a Cys-plus-His-rich region within RTA is important for RTA-mediated degradation of Hey1. We confirm that Hey1 represses the RTA promoter and, furthermore, show that Hey1 binds to the RTA promoter. An interaction was observed between Hey1 and the coRepressor mSin3A, and this interaction was abolished in the presence of RTA. Additionally, mSin3A associated with the RTA promoter in nonreactivated, but not reactivated, BCBL1 cells. Small interfering RNA knockdown of Hey1 in HEK 293T cells latently infected with the recombinant virus rKSHV.219 led to increased levels of RTA expression upon reactivation but was insufficient to induce complete lytic reactivation. These results suggest that other additional transcriptional Repressors are also important in maintenance of KSHV latency. Taken together, our results suggest that Hey1 has a contributory role in the maintenance of KSHV latency and that disruption of the Hey1 repressosome by RTA-targeted degradation may be one step in the mechanism to regulate lytic reactivation.
Sandip Paul - One of the best experts on this subject based on the ideXlab platform.
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The conformational stability of terminal helices of λ-Repressor Protein in aqueous dodine and choline-O-sulfate solutions.
International journal of biological macromolecules, 2019Co-Authors: Srijita Paul, Sandip PaulAbstract:Abstract In this article, we have ventured into the denaturation of fast folding λ 6 - 85 -Repressor Protein at a millimolar concentration of dodine and henceforth, evaluated the candidature of choline-O-sulfate as a protecting osmolyte against it, employing classical molecular dynamics simulations. Our simulation results show that, the terminal helices of λ -Repressor Protein get unfolded in presence of ∼ 15 mM dodine while 0.5 M and higher concentration of COS can prevent this deleterious effect of dodine. Careful analyses of a set of simulations with increasing COS concentration reveals that a higher concentration of COS can provide remarkable stability to the Protein, even slightly better than its native state in water. Different interaction parameters show that in aqueous dodine, both the dodinium and acetate ions interact strongly with the terminal helices to disrupt the structure whereas in presence of COS, due to the preferential interaction of COS with the Protein molecule, dodine molecules get excluded from the Protein surface. In addition, the favorable interaction of COS with dodinium head group, the dodinium ions become less available to the vicinity of Protein surface which also plays an indirect but decisive role to prevent the unfolding of the terminal helical domains of the λ -Repressor Protein.
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The conformational stability of terminal helices of [formula omitted]-Repressor Protein in aqueous dodine and choline-O-sulfate solutions
International Journal of Biological Macromolecules, 2019Co-Authors: Srijita Paul, Sandip PaulAbstract:In this article, we have ventured into the denaturation of fast folding λ6-85-Repressor Protein at a millimolar concentration of dodine and henceforth, evaluated the candidature of choline-O-sulfate as a protecting osmolyte against it, employing classical molecular dynamics simulations. Our simulation results show that, the terminal helices of λ-Repressor Protein get unfolded in presence of ∼15 mM dodine while 0.5 M and higher concentration of COS can prevent this deleterious effect of dodine. Careful analyses of a set of simulations with increasing COS concentration reveals that a higher concentration of COS can provide remarkable stability to the Protein, even slightly better than its native state in water. Different interaction parameters show that in aqueous dodine, both the dodinium and acetate ions interact strongly with the terminal helices to disrupt the structure whereas in presence of COS, due to the preferential interaction of COS with the Protein molecule, dodine molecules get excluded from the Protein surface. In addition, the favorable interaction of COS with dodinium head group, the dodinium ions become less available to the vicinity of Protein surface which also plays an indirect but decisive role to prevent the unfolding of the terminal helical domains of the λ-Repressor Protein.
Faye Gould - One of the best experts on this subject based on the ideXlab platform.
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Kaposi's Sarcoma-Associated Herpesvirus RTA Promotes Degradation of the Hey1 Repressor Protein through the Ubiquitin Proteasome Pathway
Journal of Virology, 2009Co-Authors: Faye Gould, Sally M Harrison, Eric W Hewitt, Adrian WhitehouseAbstract:The Kaposi's sarcoma-associated herpesvirus (KSHV) replication and transcription activator (RTA) Protein regulates the latent-lytic switch by transactivating a variety of KSHV lytic and cellular promoters. RTA is a novel E3 ubiquitin ligase that targets a number of transcriptional Repressor Proteins for degradation by the ubiquitin proteasome pathway. Herein, we show that RTA interacts with the cellular transcriptional Repressor Protein Hey1. We demonstrate that Hey1 is a target for RTA-mediated ubiquitination and is subsequently degraded by the proteasome. Moreover, a Cys-plus-His-rich region within RTA is important for RTA-mediated degradation of Hey1. We confirm that Hey1 represses the RTA promoter and, furthermore, show that Hey1 binds to the RTA promoter. An interaction was observed between Hey1 and the coRepressor mSin3A, and this interaction was abolished in the presence of RTA. Additionally, mSin3A associated with the RTA promoter in nonreactivated, but not reactivated, BCBL1 cells. Small interfering RNA knockdown of Hey1 in HEK 293T cells latently infected with the recombinant virus rKSHV.219 led to increased levels of RTA expression upon reactivation but was insufficient to induce complete lytic reactivation. These results suggest that other additional transcriptional Repressors are also important in maintenance of KSHV latency. Taken together, our results suggest that Hey1 has a contributory role in the maintenance of KSHV latency and that disruption of the Hey1 repressosome by RTA-targeted degradation may be one step in the mechanism to regulate lytic reactivation.
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Kaposi's Sarcoma-Associated Herpesvirus RTA Promotes Degradation of the Hey1 Repressor Protein through the Ubiquitin Proteasome Pathway
Journal of Virology, 2009Co-Authors: Faye Gould, Sally M Harrison, Eric W Hewitt, Adrian WhitehouseAbstract:The Kaposi's sarcoma-associated herpesvirus (KSHV) replication and transcription activator (RTA) Protein regulates the latent-lytic switch by transactivating a variety of KSHV lytic and cellular promoters. RTA is a novel E3 ubiquitin ligase that targets a number of transcriptional Repressor Proteins for degradation by the ubiquitin proteasome pathway. Herein, we show that RTA interacts with the cellular transcriptional Repressor Protein Hey1. We demonstrate that Hey1 is a target for RTA-mediated ubiquitination and is subsequently degraded by the proteasome. Moreover, a Cys-plus-His-rich region within RTA is important for RTA-mediated degradation of Hey1. We confirm that Hey1 represses the RTA promoter and, furthermore, show that Hey1 binds to the RTA promoter. An interaction was observed between Hey1 and the coRepressor mSin3A, and this interaction was abolished in the presence of RTA. Additionally, mSin3A associated with the RTA promoter in nonreactivated, but not reactivated, BCBL1 cells. Small interfering RNA knockdown of Hey1 in HEK 293T cells latently infected with the recombinant virus rKSHV.219 led to increased levels of RTA expression upon reactivation but was insufficient to induce complete lytic reactivation. These results suggest that other additional transcriptional Repressors are also important in maintenance of KSHV latency. Taken together, our results suggest that Hey1 has a contributory role in the maintenance of KSHV latency and that disruption of the Hey1 repressosome by RTA-targeted degradation may be one step in the mechanism to regulate lytic reactivation.
Kathleen S. Matthews - One of the best experts on this subject based on the ideXlab platform.
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Lactose Repressor Protein Modified with Fluorescein Mercuric
2012Co-Authors: Alex A. Burgum, Kathleen S. MatthewsAbstract:The reaction of lac Repressor Protein with fluorescein mercuric acetate has been studied. A maximum of 1.5 cysteine residues/monomer was modified with this reagent; the presence of ligands (inducer, anti-inducer, nonspecific DNA) did not affect ‘the reaction. Modification of the cysteines increased the affinity of the Protein for inducer, but did not affect binding to nonspecific DNA. The operator binding activity, in contrast, was abolished at excesses of reagent 20.75 mercurials/monomer. Reversal of the effects on operator and inducer binding activity could be obtained by addition of dithiothreitol to the modified Repressor Protein. Mapping studies to determine the extent of reaction at each of the cysteines indicated that with excesses of fluorescein mercuric acetate below 0.5 molecules/monomer, cysteine 268 was less reactive than either cysteine 107 or cysteine 140; at the point of maximum reaction, each of the cysteines was approximately 50% reacted. Using 2-bromoacetamido-4-nitrophenol as a selective blocking agent for cysteines 107 and 140 prior to reaction with fluorescein mercuric acetate, the loss of operator binding activity was decreased, while no effect was observed on the increased affinity for inducer noted for mercurial modified Protein. The loss of operator DNA binding activity with simultaneous maintenance of nonspecific DNA binding activity suggests that, while the determinants for binding may overlap, there are also independent determinants. It is of interest to note that loss of operator binding occurred with modification of residues in the core region of the molecule rather than in the NH2 terminus. Perturbations of the Protein structure by inducer, anti-inducer, and nonspecific DNA were reflected in the fluorescein absorbance spectrum. Inducer difference spectra and anti-inducer difference spectra exhibited opposite characteristics; this is consistent with their different effects on the function of the Protein molecule. Nonspecific DNA also perturbed the fluorescein spectrum; part of this difference spectrum involves direct interaction of the fluorescein mercuric acetate with the nucleic acid, but the spectrum also contained components attributable to perturbation of the fluorescein spectrum indirectly through the Protein. This constitutes the first direct spectral evidence for the effect of nonspecific DNA on the Protein structure. It is interesting to note that the spectral changes arise from residues which are in the core region of the molecule * This work was supported by Grant GM 22441-02 from the National Institutes of Health, Grant PCM 77-16634 from the National Science Foundation, and Grant C-576 from the Robert A. Welch Foundation, all to K.S.M. The costs of publication of this article were
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glycine insertion in the hinge region of lactose Repressor Protein alters dna binding
Journal of Biological Chemistry, 1999Co-Authors: Catherine M. Falcon, Kathleen S. MatthewsAbstract:Amino acid alterations were designed at the C terminus of the hinge segment (amino acids approximately 51-59) that links two functional domains within lactose Repressor Protein (LacI). Gly was introduced between Gly(58) and Lys(59) to generate Gly(58+1); Gln(60) was changed to Gly or Pro, and up to three additional glycines were inserted following Gln(60) --> Gly. All mutant Proteins exhibited purification behavior, CD spectra, assembly state, and inducer binding properties similar to wild-type LacI and only small differences in trypsin proteolysis patterns. In contrast, significant differences were observed in DNA binding properties. Gly(58+1) exhibited a decrease of approximately 100-fold in affinity for O(1) operator, and sequential Gly insertion C-terminal to Gln(60) --> Gly resulted in progressively decreased affinity for O(1) operator, approaching nonspecific levels for insertion of >/=2 glycines. Where sufficient affinity for O(1) operator existed, decreased binding to O(1) in the presence of inducer indicated no disruption in the allosteric response for these Proteins. Collectively, these results indicate that flexibility and/or spacing between the core and N-terminal domains did not significantly affect folding or assembly, but these alterations in the hinge domain profoundly altered affinity of the lactose Repressor Protein for its wild-type target sequence.
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Glycine Insertion in the Hinge Region of Lactose Repressor Protein Alters DNA Binding
Journal of Biological Chemistry, 1999Co-Authors: Catherine M. Falcon, Kathleen S. MatthewsAbstract:Abstract Amino acid alterations were designed at the C terminus of the hinge segment (amino acids ∼51–59) that links two functional domains within lactose Repressor Protein (LacI). Gly was introduced between Gly58 and Lys59 to generate Gly58+1; Gln60 was changed to Gly or Pro, and up to three additional glycines were inserted following Gln60 → Gly. All mutant Proteins exhibited purification behavior, CD spectra, assembly state, and inducer binding properties similar to wild-type LacI and only small differences in trypsin proteolysis patterns. In contrast, significant differences were observed in DNA binding properties. Gly58+1 exhibited a decrease of ∼100-fold in affinity for O1 operator, and sequential Gly insertion C-terminal to Gln60 → Gly resulted in progressively decreased affinity for O1operator, approaching nonspecific levels for insertion of ≥2 glycines. Where sufficient affinity for O1 operator existed, decreased binding to O1 in the presence of inducer indicated no disruption in the allosteric response for these Proteins. Collectively, these results indicate that flexibility and/or spacing between the core and N-terminal domains did not significantly affect folding or assembly, but these alterations in the hinge domain profoundly altered affinity of the lactose Repressor Protein for its wild-type target sequence.
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Thermodynamic analysis of unfolding and dissociation in lactose Repressor Protein.
Biochemistry, 1999Co-Authors: Jennifer K. Barry, Kathleen S. MatthewsAbstract:Lactose Repressor Protein, regulator of lac enzyme expression in Escherichia coli, maintains its structure and function at extremely low Protein concentrations (
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Lactose Repressor Protein: Functional Properties and Structure
Progress in nucleic acid research and molecular biology, 1998Co-Authors: Kathleen S. Matthews, Jeffry C. NicholsAbstract:The lactose Repressor Protein (LacI), the prototype for genetic regulatory Proteins, controls expression of lactose metabolic genes by binding to its cognate operator sequences in E. coli DNA. Inducer binding elicits a conformational change that diminishes affinity for operator sequences with no effect on nonspecific binding. The release of operator is followed by synthesis of mRNA encoding the enzymes for lactose utilization. Genetic, chemical and physical studies provided detailed insight into the function of this Protein prior to the recent completion of X-ray crystallographic structures. The structural information can now be correlated with the phenotypic data for numerous mutants. These structures also provide the opportunity for physical and chemical studies on mutants designed to examine various aspects of lac Repressor structure and function. In addition to providing insight into Protein structure-function correlations, LacI has been utilized in a wide variety of applications both in prokaryotic gene expression and in eukaryotic gene regulation and studies of mutagenesis. © 1998 Academic Press
Eric B. Kmiec - One of the best experts on this subject based on the ideXlab platform.
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The glucocorticoid receptor precludes the binding of a transcriptional Repressor Protein to the long terminal repeat of the mouse mammary tumor virus
Molecular and Cellular Biochemistry, 1993Co-Authors: Shanzhang Ye, Eric B. KmiecAbstract:The long terminal repeat (LTR) of the mouse mammary tumor virus was used as a template to examine the dual binding parameters of the glucocorticoid-receptor (GR) and a Repressor Protein termed Inhibitory Factor 1 (IF1). The roceptor binds specifically to the glucocorticoid response element and precludes the binding of IF1 to its juxtaposed binding site within the LTR. When the two DNA targets are separated by the insertion of an additional 52 base pairs, coincident binding of both Proteins is observed. Gel retention assays reveal three distinct nucleoProtein complexes. The first complex consists of the receptor and the LTR, the second is comprised of IF1 and DNA and the third is a multiProtein-DNA complex consisting of the GR, IF1 and DNA, migrating at a higher molecular weight position. The inhibition of IF1 binding by the presence of prebound GR leads to the repression of transcription of juxtaposed genes. The GR may act to block access of a sequence, used by the cell to titrate Repressor Proteins and facilitate the onset of gene expression. (Mol Cell Biochem122: 25–37, 1993)
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The glucocorticoid receptor precludes the binding of a transcriptional Repressor Protein to the long terminal repeat of the mouse mammary tumor virus.
Molecular and cellular biochemistry, 1993Co-Authors: Eric B. KmiecAbstract:The long terminal repeat (LTR) of the mouse mammary tumor virus was used as a template to examine the dual binding parameters of the glucocorticoid-receptor (GR) and a Repressor Protein termed Inhibitory Factor 1 (IF1). The receptor binds specifically to the glucocorticoid response element and precludes the binding of IF1 to its juxtaposed binding site within the LTR. When the two DNA targets are separated by the insertion of an additional 52 base pairs, coincident binding of both Proteins is observed. Gel retention assays reveal three distinct nucleoProtein complexes. The first complex consists of the receptor and the LTR, the second is comprised of IF1 and DNA and the third is a multiProtein-DNA complex consisting of the GR, IF1 and DNA, migrating at a higher molecular weight position. The inhibition of IF1 binding by the presence of prebound GR leads to the repression of transcription of juxtaposed genes. The GR may act to block access of a sequence, used by the cell to titrate Repressor Proteins and facilitate the onset of gene expression.