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Keith N Leppard - One of the best experts on this subject based on the ideXlab platform.
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promyelocytic leukemia Protein Isoform ii inhibits infection by human adenovirus type 5 through effects on hsp70 and the interferon response
Journal of General Virology, 2016Co-Authors: Zeenah Weheed Atwan, Jordan Wright, Andrew Woodman, Keith N LeppardAbstract:Promyelocytic leukemia (PML) Proteins have been implicated in antiviral responses but PML and associated Proteins are also suggested to support virus replication. One Isoform, PML-II, is required for efficient transcription of interferon and interferon-responsive genes. . We therefore investigated the PML-II contribution to human adenovirus 5 (Ad5) infection, using shRNA-mediated knock-down. HelaΔII cells showed a 2 - 3 fold elevation in Ad5 yield, reflecting an increase in late gene expression. This increase was found to be due in part to the reduced innate immune response consequent upon PML-II depletion. However the effect was minor because the viral E4 Orf3 Protein targets and inactivates this PML-II function. The major benefit to Ad5 in HelaΔII cells was exerted via an increase in HSP70; depletion of HSP70 completely reversed this replicative advantage. Increased Ad5 late gene expression was not due either to the previously described inhibition of inflammatory responses by HSP70 or to effects of HSP70 on major late promoter or L4 promoter activity but might be linked to an observed increase in E1B 55K, as this Protein is known to be required for efficient late gene expression. The induction of HSP70 by PML-II removal was specific for the HSPA1B gene among the HSP70 gene family and thus was not the consequence of a general stress response. Taken together, these data show that PML-II, through its various actions, has an overall negative effect on the Ad5 life-cycle.
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promyelocytic leukemia Protein Isoform ii promotes transcription factor recruitment to activate interferon beta and interferon responsive gene expression
Molecular and Cellular Biology, 2015Co-Authors: Yixiang Chen, Jordan Wright, Xueqiong Meng, Keith N LeppardAbstract:To trigger type I interferon (IFN) responses, pattern recognition receptors activate signaling cascades that lead to transcription of IFN and IFN-stimulated genes (ISGs). The promyelocytic leukemia (PML) Protein has been implicated in these responses, although its role has not been defined. Here, we show that PML Isoform II (PML-II) is specifically required for efficient induction of IFN-β transcription and of numerous ISGs, acting at the point of transcriptional complex assembly on target gene promoters. PML-II associated with specific transcription factors NF-κB and STAT1, as well as the coactivator CREB-binding Protein (CBP), to facilitate transcriptional complex formation. The absence of PML-II substantially reduced binding of these factors and IFN regulatory factor 3 (IRF3) to IFN-β or ISGs promoters and sharply reduced gene activation. The unique C-terminal domain of PML-II was essential for its activity, while the N-terminal RBCC motif common to all PML Isoforms was dispensable. We propose a model in which PML-II contributes to the transcription of multiple genes via the association of its C-terminal domain with relevant transcription complexes, which promotes the stable assembly of these complexes at promoters/enhancers of target genes, and that in this way PML-II plays a significant role in the development of type I IFN responses.
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interaction of the adenovirus type 5 e4 orf3 Protein with promyelocytic leukemia Protein Isoform ii is required for nd10 disruption
Journal of Virology, 2006Co-Authors: Anne Hoppe, Stephanie J Beech, John Dimmock, Keith N LeppardAbstract:Nuclear domain 10 (ND10s), or promyelocytic leukemia Protein (PML) nuclear bodies, are spherical nuclear structures that require PML Proteins for their formation. Many viruses target these structures during infection. The E4 Orf3 Protein of adenovirus 5 (Ad5) rearranges ND10s, causing PML to colocalize with Orf3 in nuclear tracks or fibers. There are six different PML Isoforms (I to VI) present at ND10s, all sharing a common N terminus but with structural differences at their C termini. In this study, PML II was the only one of these six Isoforms that was found to interact directly and specifically with Ad5 E4 Orf3 in vitro and in vivo; these results define a new Orf3 activity. Three of a series of 18 mutant Orf3 Proteins were unable to interact with PML II; these were also unable to cause ND10 rearrangement. Moreover, in PML-null cells that contained neoformed ND10s comprising a single PML Isoform, only ND10s formed of PML II were rearranged by Orf3. These data show that the interaction between Orf3 and PML II is necessary for ND10 rearrangement to occur. Finally, Orf3 was shown to self-associate in vitro. This activity was absent in mutant Orf3 Proteins that were unable to form tracks and to bind PML II. Thus, Orf3 oligomerization may mediate the formation of nuclear tracks in vivo and may also be important for PML II binding.
Sherry Ogg - One of the best experts on this subject based on the ideXlab platform.
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expression of novel fusion antiviral Proteins ricin a chain pokeweed antiviral Proteins rta paps in escherichia coli and their inhibition of Protein synthesis and of hepatitis b virus in vitro
BMC Biotechnology, 2018Co-Authors: Yasser Hassan, Sherry OggAbstract:Ricin A chain (RTA) and Pokeweed antiviral Proteins (PAPs) are plant-derived N-glycosidase ribosomal-inactivating Proteins (RIPs) isolated from Ricinus communis and Phytolacca Americana respectively. This study was to investigate the potential production amenability and sub-toxic antiviral value of novel fusion Proteins between RTA and PAPs (RTA-PAPs). In brief, RTA-Pokeweed antiviral Protein Isoform 1 from seeds (RTA-PAPS1) was produced in an E. coli in vivo expression system, purified from inclusion bodies using gel filtration chromatography and Protein synthesis inhibitory activity assayed by comparison to the production of a control Protein Luciferase. The antiviral activity of the RTA-PAPS1 against Hepatitis B virus (HBV) in HepAD38 cells was then determined using a dose response assay by quantifying supernatant HBV DNA compared to control virus infected HepAD38 cells. The cytotoxicity in HepAD38 cells was determined by measuring cell viability using a tetrazolium dye uptake assay. The fusion Protein was further optimized using in silico tools, produced in an E. coli in vivo expression system, purified by a three-step process from soluble lysate and confirmed in a Protein synthesis inhibition activity assay. Results showed that RTA-PAPS1 could effectively be recovered and purified from inclusion bodies. The refolded Protein was bioactive with a 50% Protein synthesis inhibitory concentration (IC50) of 0.06 nM (3.63 ng/ml). The results also showed that RTA-PAPS1 had a synergetic activity against HBV with a half-maximal response concentration value (EC50) of 0.03 nM (1.82 ng/ml) and a therapeutic index of > 21,818 with noticeable steric hindrance. Results also showed that the optimized Protein ricin A chain mutant-Pokeweed antiviral Protein Isoform 1 from leaves (RTAM-PAP1) could be recovered and purified from soluble lysates with gain of function on Protein synthesis inhibition activity, with an IC50 of 0.03 nM (1.82 ng/ml), and with minimal, if any, steric hindrance. Collectively, our results demonstrate that RTA-PAPs are amenable to effective production and purification in native form, possess significant gain of function on Protein synthesis inhibition and anti-HBV activities in vitro with a high therapeutic index and, thus, merit further development as potential potent antiviral agents against chronic HBV infection to be used as a standalone or in combination with existent therapies.
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Expression of novel fusion antiviral Proteins ricin a chain-pokeweed antiviral Proteins (RTA-PAPs) in Escherichia coli and their inhibition of Protein synthesis and of hepatitis B virus in vitro
BMC, 2018Co-Authors: Yasser Hassan, Sherry OggAbstract:Abstract Background Ricin A chain (RTA) and Pokeweed antiviral Proteins (PAPs) are plant-derived N-glycosidase ribosomal-inactivating Proteins (RIPs) isolated from Ricinus communis and Phytolacca Americana respectively. This study was to investigate the potential production amenability and sub-toxic antiviral value of novel fusion Proteins between RTA and PAPs (RTA-PAPs). In brief, RTA-Pokeweed antiviral Protein Isoform 1 from seeds (RTA-PAPS1) was produced in an E. coli in vivo expression system, purified from inclusion bodies using gel filtration chromatography and Protein synthesis inhibitory activity assayed by comparison to the production of a control Protein Luciferase. The antiviral activity of the RTA-PAPS1 against Hepatitis B virus (HBV) in HepAD38 cells was then determined using a dose response assay by quantifying supernatant HBV DNA compared to control virus infected HepAD38 cells. The cytotoxicity in HepAD38 cells was determined by measuring cell viability using a tetrazolium dye uptake assay. The fusion Protein was further optimized using in silico tools, produced in an E. coli in vivo expression system, purified by a three-step process from soluble lysate and confirmed in a Protein synthesis inhibition activity assay. Results Results showed that RTA-PAPS1 could effectively be recovered and purified from inclusion bodies. The refolded Protein was bioactive with a 50% Protein synthesis inhibitory concentration (IC50) of 0.06 nM (3.63 ng/ml). The results also showed that RTA-PAPS1 had a synergetic activity against HBV with a half-maximal response concentration value (EC50) of 0.03 nM (1.82 ng/ml) and a therapeutic index of > 21,818 with noticeable steric hindrance. Results also showed that the optimized Protein ricin A chain mutant-Pokeweed antiviral Protein Isoform 1 from leaves (RTAM-PAP1) could be recovered and purified from soluble lysates with gain of function on Protein synthesis inhibition activity, with an IC50 of 0.03 nM (1.82 ng/ml), and with minimal, if any, steric hindrance. Conclusions Collectively, our results demonstrate that RTA-PAPs are amenable to effective production and purification in native form, possess significant gain of function on Protein synthesis inhibition and anti-HBV activities in vitro with a high therapeutic index and, thus, merit further development as potential potent antiviral agents against chronic HBV infection to be used as a standalone or in combination with existent therapies
Yasser Hassan - One of the best experts on this subject based on the ideXlab platform.
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expression of novel fusion antiviral Proteins ricin a chain pokeweed antiviral Proteins rta paps in escherichia coli and their inhibition of Protein synthesis and of hepatitis b virus in vitro
BMC Biotechnology, 2018Co-Authors: Yasser Hassan, Sherry OggAbstract:Ricin A chain (RTA) and Pokeweed antiviral Proteins (PAPs) are plant-derived N-glycosidase ribosomal-inactivating Proteins (RIPs) isolated from Ricinus communis and Phytolacca Americana respectively. This study was to investigate the potential production amenability and sub-toxic antiviral value of novel fusion Proteins between RTA and PAPs (RTA-PAPs). In brief, RTA-Pokeweed antiviral Protein Isoform 1 from seeds (RTA-PAPS1) was produced in an E. coli in vivo expression system, purified from inclusion bodies using gel filtration chromatography and Protein synthesis inhibitory activity assayed by comparison to the production of a control Protein Luciferase. The antiviral activity of the RTA-PAPS1 against Hepatitis B virus (HBV) in HepAD38 cells was then determined using a dose response assay by quantifying supernatant HBV DNA compared to control virus infected HepAD38 cells. The cytotoxicity in HepAD38 cells was determined by measuring cell viability using a tetrazolium dye uptake assay. The fusion Protein was further optimized using in silico tools, produced in an E. coli in vivo expression system, purified by a three-step process from soluble lysate and confirmed in a Protein synthesis inhibition activity assay. Results showed that RTA-PAPS1 could effectively be recovered and purified from inclusion bodies. The refolded Protein was bioactive with a 50% Protein synthesis inhibitory concentration (IC50) of 0.06 nM (3.63 ng/ml). The results also showed that RTA-PAPS1 had a synergetic activity against HBV with a half-maximal response concentration value (EC50) of 0.03 nM (1.82 ng/ml) and a therapeutic index of > 21,818 with noticeable steric hindrance. Results also showed that the optimized Protein ricin A chain mutant-Pokeweed antiviral Protein Isoform 1 from leaves (RTAM-PAP1) could be recovered and purified from soluble lysates with gain of function on Protein synthesis inhibition activity, with an IC50 of 0.03 nM (1.82 ng/ml), and with minimal, if any, steric hindrance. Collectively, our results demonstrate that RTA-PAPs are amenable to effective production and purification in native form, possess significant gain of function on Protein synthesis inhibition and anti-HBV activities in vitro with a high therapeutic index and, thus, merit further development as potential potent antiviral agents against chronic HBV infection to be used as a standalone or in combination with existent therapies.
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Expression of novel fusion antiviral Proteins ricin a chain-pokeweed antiviral Proteins (RTA-PAPs) in Escherichia coli and their inhibition of Protein synthesis and of hepatitis B virus in vitro
BMC, 2018Co-Authors: Yasser Hassan, Sherry OggAbstract:Abstract Background Ricin A chain (RTA) and Pokeweed antiviral Proteins (PAPs) are plant-derived N-glycosidase ribosomal-inactivating Proteins (RIPs) isolated from Ricinus communis and Phytolacca Americana respectively. This study was to investigate the potential production amenability and sub-toxic antiviral value of novel fusion Proteins between RTA and PAPs (RTA-PAPs). In brief, RTA-Pokeweed antiviral Protein Isoform 1 from seeds (RTA-PAPS1) was produced in an E. coli in vivo expression system, purified from inclusion bodies using gel filtration chromatography and Protein synthesis inhibitory activity assayed by comparison to the production of a control Protein Luciferase. The antiviral activity of the RTA-PAPS1 against Hepatitis B virus (HBV) in HepAD38 cells was then determined using a dose response assay by quantifying supernatant HBV DNA compared to control virus infected HepAD38 cells. The cytotoxicity in HepAD38 cells was determined by measuring cell viability using a tetrazolium dye uptake assay. The fusion Protein was further optimized using in silico tools, produced in an E. coli in vivo expression system, purified by a three-step process from soluble lysate and confirmed in a Protein synthesis inhibition activity assay. Results Results showed that RTA-PAPS1 could effectively be recovered and purified from inclusion bodies. The refolded Protein was bioactive with a 50% Protein synthesis inhibitory concentration (IC50) of 0.06 nM (3.63 ng/ml). The results also showed that RTA-PAPS1 had a synergetic activity against HBV with a half-maximal response concentration value (EC50) of 0.03 nM (1.82 ng/ml) and a therapeutic index of > 21,818 with noticeable steric hindrance. Results also showed that the optimized Protein ricin A chain mutant-Pokeweed antiviral Protein Isoform 1 from leaves (RTAM-PAP1) could be recovered and purified from soluble lysates with gain of function on Protein synthesis inhibition activity, with an IC50 of 0.03 nM (1.82 ng/ml), and with minimal, if any, steric hindrance. Conclusions Collectively, our results demonstrate that RTA-PAPs are amenable to effective production and purification in native form, possess significant gain of function on Protein synthesis inhibition and anti-HBV activities in vitro with a high therapeutic index and, thus, merit further development as potential potent antiviral agents against chronic HBV infection to be used as a standalone or in combination with existent therapies
Francis S. Collins - One of the best experts on this subject based on the ideXlab platform.
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a lamin a Protein Isoform overexpressed in hutchinson gilford progeria syndrome interferes with mitosis in progeria and normal cells
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Ka Cao, Michael R. Erdos, Karima Djabali, Ia C Capell, Francis S. CollinsAbstract:Hutchinson–Gilford progeria syndrome (HGPS) is a rare genetic disorder characterized by dramatic premature aging. Classic HGPS is caused by a de novo point mutation in exon 11 (residue 1824, C → T) of the LMNA gene, activating a cryptic splice donor and resulting in a mutant lamin A (LA) Protein termed “progerin/LAΔ50” that lacks the normal cleavage site to remove a C-terminal farnesyl group. During interphase, irreversibly farnesylated progerin/LAΔ50 anchors to the nuclear membrane and causes characteristic nuclear blebbing. Progerin/LAΔ50's localization and behavior during mitosis, however, are completely unknown. Here, we report that progerin/LAΔ50 mislocalizes into insoluble cytoplasmic aggregates and membranes during mitosis and causes abnormal chromosome segregation and binucleation. These phenotypes are largely rescued with either farnesyltransferase inhibitors or a farnesylation-incompetent mutant progerin/LAΔ50. Furthermore, we demonstrate that small amounts of progerin/LAΔ50 exist in normal fibroblasts, and a significant percentage of these progerin/LAΔ50-expressing normal cells are binucleated, implicating progerin/LAΔ50 as causing similar mitotic defects in the normal aging process. Our findings present evidence of mitotic abnormality in HGPS and may shed light on the general phenomenon of aging.
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A lamin A Protein Isoform overexpressed in Hutchinson-Gilford progeria syndrome interferes with mitosis in progeria and normal cells.
Proceedings of the National Academy of Sciences, 2007Co-Authors: Kan Cao, Michael R. Erdos, Karima Djabali, Brian C. Capell, Francis S. CollinsAbstract:Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder characterized by dramatic premature aging. Classic HGPS is caused by a de novo point mutation in exon 11 (residue 1824, C --> T) of the LMNA gene, activating a cryptic splice donor and resulting in a mutant lamin A (LA) Protein termed "progerin/LADelta50" that lacks the normal cleavage site to remove a C-terminal farnesyl group. During interphase, irreversibly farnesylated progerin/LADelta50 anchors to the nuclear membrane and causes characteristic nuclear blebbing. Progerin/LADelta50's localization and behavior during mitosis, however, are completely unknown. Here, we report that progerin/LADelta50 mislocalizes into insoluble cytoplasmic aggregates and membranes during mitosis and causes abnormal chromosome segregation and binucleation. These phenotypes are largely rescued with either farnesyltransferase inhibitors or a farnesylation-incompetent mutant progerin/LADelta50. Furthermore, we demonstrate that small amounts of progerin/LADelta50 exist in normal fibroblasts, and a significant percentage of these progerin/LADelta50-expressing normal cells are binucleated, implicating progerin/LADelta50 as causing similar mitotic defects in the normal aging process. Our findings present evidence of mitotic abnormality in HGPS and may shed light on the general phenomenon of aging.
Yao-shen Chen - One of the best experts on this subject based on the ideXlab platform.
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increased 14 3 3β and γ Protein Isoform expressions in parasitic eosinophilic meningitis caused by angiostrongylus cantonensis infection in mice
PLOS ONE, 2019Co-Authors: Hung-chin Tsai, Yu-hsin Chen, Chuan-min Yen, Susan Shin-jung Lee, Yao-shen ChenAbstract:The 14-3-3 Proteins are cerebrospinal fluid (CSF) markers of neuronal damage during infectious meningitis and Creutzfeldt-Jakob disease. Little is known about dynamic changes in the individual Isoforms in response to parasitic eosinophilic meningitis. The purposes of this study were to determine the 14-3-3 Protein Isoform patterns, examine the kinetics and correlate the severity of blood brain barrier (BBB) damage with the expressions of these markers in mice with eosinophilic meningitis. Mice were orally infected with 50 A. cantonensis L3 via an oro-gastric tube and sacrificed every week for 3 consecutive weeks after infection. The Evans blue method and BBB junctional Protein expressions were used to measure changes in the BBB. Hematoxylin and eosin staining was used to analyze pathological changes in the mice brains following 1–3 weeks of infection with A. cantonensis. The levels of 14-3-3 Protein Isoforms in serum/CSF and brain homogenates were analyzed by Western blot, and immunohistochemistry (IHC) was used to explore the different Isoform distributions of 14-3-3 Proteins and changes in BBB junctional Proteins in the mice brain meninges. Dexamethasone was injected intraperitoneally from the seventh day post infection (dpi) until the end of the study (21 dpi) to study the changes in BBB junctional Proteins. The amounts of Evans blue, tight junction and 14-3-3 Protein Isoforms in the different groups of mice were compared using the nonparametric Kruskal-Wallis test. There were significant increases in 14-3-3 Protein Isoforms β and γ in the CSF in the second and third weeks after infection compared to the controls and first week of infection, which were correlated with the severity of BBB damage in brain histology, and Evans blue extravasation. Using IHC to assess the distribution of 14-3-3 Protein Isoforms and changes in BBB junctional Proteins in the mice brain meninges, the expressions of Isoforms β, γ, e, and θ and junctional Proteins occludin and claudin-5 in the brain meninges increased over a 3-week period after infection compared to the controls and 1 week after infection. The administration of dexamethasone decreased the expressions of BBB junctional Proteins occludin and claudin-5 in the mice brain meninges. Our findings support that 14-3-3 Proteins β and γ can potentially be used as a CSF marker of neuronal damage in parasitic eosinophilic meningitis caused by A. cantonensis.
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Increased 14-3-3β and γ Protein Isoform expressions in parasitic eosinophilic meningitis caused by Angiostrongylus cantonensis infection in mice
2019Co-Authors: Hung-chin Tsai, Yu-hsin Chen, Chuan-min Yen, Susan Shin-jung Lee, Yao-shen ChenAbstract:The 14-3-3 Proteins are cerebrospinal fluid (CSF) markers of neuronal damage during infectious meningitis and Creutzfeldt-Jakob disease. Little is known about dynamic changes in the individual Isoforms in response to parasitic eosinophilic meningitis. The purposes of this study were to determine the 14-3-3 Protein Isoform patterns, examine the kinetics and correlate the severity of blood brain barrier (BBB) damage with the expressions of these markers in mice with eosinophilic meningitis.Mice were orally infected with 50 A. cantonensis L3 via an oro-gastric tube and sacrificed every week for 3 consecutive weeks after infection. The Evans blue method and BBB junctional Protein expressions were used to measure changes in the BBB. Hematoxylin and eosin staining was used to analyze pathological changes in the mice brains following 1–3 weeks of infection with A. cantonensis. The levels of 14-3-3 Protein Isoforms in serum/CSF and brain homogenates were analyzed by Western blot, and immunohistochemistry (IHC) was used to explore the different Isoform distributions of 14-3-3 Proteins and changes in BBB junctional Proteins in the mice brain meninges. Dexamethasone was injected intraperitoneally from the seventh day post infection (dpi) until the end of the study (21 dpi) to study the changes in BBB junctional Proteins. The amounts of Evans blue, tight junction and 14-3-3 Protein Isoforms in the different groups of mice were compared using the nonparametric Kruskal-Wallis test.There were significant increases in 14-3-3 Protein Isoforms β and γ in the CSF in the second and third weeks after infection compared to the controls and first week of infection, which were correlated with the severity of BBB damage in brain histology, and Evans blue extravasation. Using IHC to assess the distribution of 14-3-3 Protein Isoforms and changes in BBB junctional Proteins in the mice brain meninges, the expressions of Isoforms β, γ, ε, and θ and junctional Proteins occludin and claudin-5 in the brain meninges increased over a 3-week period after infection compared to the controls and 1 week after infection. The administration of dexamethasone decreased the expressions of BBB junctional Proteins occludin and claudin-5 in the mice brain meninges.Our findings support that 14-3-3 Proteins β and γ can potentially be used as a CSF marker of neuronal damage in parasitic eosinophilic meningitis caused by A. cantonensis.
