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Thomas Dobner – One of the best experts on this subject based on the ideXlab platform.
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A Role for E1B-AP5 in ATR Signaling Pathways during Adenovirus Infection
Journal of virology, 2008Co-Authors: Andrew N. Blackford, Rachel Bruton, Orkide Dirlik, Grant S. Stewart, A. Malcolm R. Taylor, Thomas Dobner, Roger J. A. Grand, Andrew S. TurnellAbstract:E1B-55K-associated protein 5 (E1B-AP5) is a cellular, heterogeneous nuclear ribonucleoprotein that is targeted by adenovirus (Ad) E1B-55K during infection. The function of E1B-AP5 during infection, however, remains largely unknown. Given the role of E1B-55K targets in the DNA damage response, we examined whether E1B-AP5 function was integral to these pathways. Here, we show a novel role for E1B-AP5 as a key regulator of ATR signaling pathways activated during Ad infection. E1B-AP5 is recruited to viral replication centers during infection, where it colocalizes with ATR-interacting protein (ATRIP) and the ATR substrate replication protein A 32 (RPA32). Indeed, E1B-AP5 associates with ATRIP and RPA complex component RPA70 in both uninfected and Ad-infected cells. Additionally, glutathione S-transferase pull-downs show that E1B-AP5 associates with RPA components RPA70 and RPA32 directly in vitro. E1B-AP5 is required for the ATR-dependent phosphorylation of RPA32 during infection and contributes to the Ad-induced phosphorylation of Smc1 and H2AX. In this regard, it is interesting that Ad5 and Ad12 differentially promote the phosphorylation of RPA32, Rad9, and Smc1 during infection such that Ad12 promotes a significant phosphorylation of RPA32 and Rad9, whereas Ad5 only weakly promotes RPA32 phosphorylation and does not induce Rad9 phosphorylation. These data suggest that Ad5 and Ad12 have evolved different strategies to regulate DNA damage signaling pathways during infection in order to promote viral replication. Taken together, our results define a role for E1B-AP5 in ATR signaling pathways activated during infection. This might have broader implications for the regulation of ATR activity during cellular DNA replication or in response to DNA damage.
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The interaction of the hnRNP family member E1B-AP5 with p53.
FEBS letters, 2005Co-Authors: Paola M. Barral, Andrew S. Turnell, Thomas Dobner, Andre Rusch, Phillip H. Gallimore, Philip J. Byrd, Roger J. A. GrandAbstract:Adenovirus early region 1B-associated protein 5, E1B-AP5, a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family, was originally isolated on the basis of its ability to bind to the adenovirus 5 early region1B55K protein. Here, it has been demonstrated that E1B-AP5 interacts with mutant and wild-type p53 from human cells in pull-down assays using GST-E1B-AP5. This interaction has been confirmed by co-immunoprecipitation studies and pull-down experiments with in vitro translated E1B-AP5 and GST-p53. The binding site for E1B-AP5 has been mapped to the C-terminal region of p53. In reciprocal experiments, it has been shown that several regions of E1B-AP5 bound to p53 although it is probable that a major site of interaction is located between amino acids 395 and 732 of E1B-AP5. In reporter assays, E1B-AP5 inhibited p53 transcriptional activity although not as efficiently as the Ad5E1B55K protein. Transfection of E1B-AP5 into human tumour cells affected the cellular response to UV radiation, such that, although p53 expression was induced, little change in the level of p53-inducible genes could be observed.
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Regulation of transcription by the heterogeneous nuclear ribonucleoprotein E1B-AP5 is mediated by complex formation with the novel bromodomain-containing protein BRD7.
Biochemical Journal, 2003Co-Authors: Julia Kzhyshkowska, Andre Rusch, Hans Wolf, Thomas DobnerAbstract:E1B-AP5 was initially identified as a target of the early adenovirus E1B-55 kDa protein during the course of lytic infection. E1B-AP5 belongs to the heterogeneous nuclear ribonucleoprotein family and was demonstrated to be involved in mRNA processing and transport [Gabler, Schutt, Groitl, Wolf, Shenk and Dobner (1998) J. Virol. 72, 7960-7971]. In the present paper, we demonstrate that E1B-AP5 differentially regulates basic and ligand-dependent transcription. We found that E1B-AP5 represses basic transcription driven by several virus and cellular promoters, and mapped the repression activity to the N-terminal part of the protein. In contrast with basic repression, E1B-AP5 activated the glucocorticoid-dependent promoter in the absence of dexamethasone, but did not contribute to the dexamethasone-induced activation. Mutant analysis indicated the presence of an additional cellular factor that modulates E1B-AP5 transcriptional activity. Using yeast two-hybrid screening, we identified a novel chromatin-associated bromodomain-containing protein, BRD7, as an E1B-AP5 interaction partner. We confirmed E1B-AP5-BRD7 complex formation in vivo and in vitro. We found that, although BRD7 binds to histones H2A, H2B, H3 and H4 through its bromodomain, this domain was not necessary for the interaction with E1B-AP5. Indeed, the triple complex formation of E1B-AP5, BRD7 and histones was demonstrated. Disruption of the E1B-AP5-BRD7 complex increased E1B-AP5 repression activity for basic transcription and converted it from being an activator of the hormone-dependent promoter into being a strong repressor. We conclude that complex formation between BRD7 and E1B-AP5 links chromatin events with mRNA processing at the level of transcriptional regulation.
Richard G. M. Morris – One of the best experts on this subject based on the ideXlab platform.
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n methyl d aspartate receptors learning and memory chronic intraventricular infusion of the nmda receptor antagonist d AP5 interacts directly with the neural mechanisms of spatial learning
European Journal of Neuroscience, 2013Co-Authors: Richard G. M. Morris, Robert Steele, Jeanne E. Bell, Stephen J. MartinAbstract:Three experiments were conducted to contrast the hypothesis that hippocampal N-methyl-d-aspartate (NMDA) receptors participate directly in the mechanisms of hippocampus-dependent learning with an alternative view that apparent impairments of learning induced by NMDA receptor antagonists arise because of drug-induced neuropathological and/or sensorimotor disturbances. In experiment 1, rats given a chronic i.c.v. infusion of d-AP5 (30 mm) at 0.5 μL/h were selectively impaired, relative to aCSF-infused animals, in place but not cued navigation learning when they were trained during the 14-day drug infusion period, but were unimpaired on both tasks if trained 11 days after the minipumps were exhausted. d-AP5 caused sensorimotor disturbances in the spatial task, but these gradually worsened as the animals failed to learn. Histological assessment of potential neuropathological changes revealed no abnormalities in d-AP5-treated rats whether killed during or after chronic drug infusion. In experiment 2, a deficit in spatial learning was also apparent in d-AP5-treated rats trained on a spatial reference memory task involving two identical but visible platforms, a task chosen and shown to minimise sensorimotor disturbances. HPLC was used to identify the presence of d-AP5 in selected brain areas. In Experiment 3, rats treated with d-AP5 showed a delay-dependent deficit in spatial memory in the delayed matching-to-place protocol for the water maze. These data are discussed with respect to the learning mechanism and sensorimotor accounts of the impact of NMDA receptor antagonists on brain function. We argue that NMDA receptor mechanisms participate directly in spatial learning.
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N‐methyl‐d‐aspartate receptors, learning and memory: chronic intraventricular infusion of the NMDA receptor antagonist d‐AP5 interacts directly with the neural mechanisms of spatial learning
The European journal of neuroscience, 2013Co-Authors: Richard G. M. Morris, Robert Steele, Jeanne E. Bell, Stephen J. MartinAbstract:Three experiments were conducted to contrast the hypothesis that hippocampal N-methyl-d-aspartate (NMDA) receptors participate directly in the mechanisms of hippocampus-dependent learning with an alternative view that apparent impairments of learning induced by NMDA receptor antagonists arise because of drug-induced neuropathological and/or sensorimotor disturbances. In experiment 1, rats given a chronic i.c.v. infusion of d-AP5 (30 mm) at 0.5 μL/h were selectively impaired, relative to aCSF-infused animals, in place but not cued navigation learning when they were trained during the 14-day drug infusion period, but were unimpaired on both tasks if trained 11 days after the minipumps were exhausted. d-AP5 caused sensorimotor disturbances in the spatial task, but these gradually worsened as the animals failed to learn. Histological assessment of potential neuropathological changes revealed no abnormalities in d-AP5-treated rats whether killed during or after chronic drug infusion. In experiment 2, a deficit in spatial learning was also apparent in d-AP5-treated rats trained on a spatial reference memory task involving two identical but visible platforms, a task chosen and shown to minimise sensorimotor disturbances. HPLC was used to identify the presence of d-AP5 in selected brain areas. In Experiment 3, rats treated with d-AP5 showed a delay-dependent deficit in spatial memory in the delayed matching-to-place protocol for the water maze. These data are discussed with respect to the learning mechanism and sensorimotor accounts of the impact of NMDA receptor antagonists on brain function. We argue that NMDA receptor mechanisms participate directly in spatial learning.
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Delay-dependent impairment of a matching-to-place task with chronic and intrahippocampal infusion of the NMDA-antagonist D-AP5.
Hippocampus, 1999Co-Authors: Robert Steele, Richard G. M. MorrisAbstract:We investigated the role of NMDA receptors in memory encoding and retrieval. A delayed matching-to-place (DMP) paradigm in the watermaze was used to examine 1-trial spatial memory in rats. Over periods of up to 21 days, 4 daily trials were given to an escape platform hidden in a new location each day, with the memory interval (ITI) varying from 15 sec to 2 hours between trials 1 and 2, but always at 15 sec for the remaining ITIs. Using chronic i.c.v. infusions of D-AP5, acute intrahippocampal infusions, ibotenate hippocampus + dentate lesions and relevant aCSF or sham surgery control groups, we established: (1) the DMP task is hippocampal-dependent; (2) D-AP5 causes a delay-dependent impairment of memory in which the Groups x Delay interaction was significant on two separate measures of performance; (3) this memory impairment also occurs with acute intrahippocampal infusions; (4) the impairment occurs irrespective of whether the animals stay in or are removed from the training context during the memory delay interval; and (5) D-AP5 affects neither the retrieval of information about the spatial layout of the environment, nor memory of where the escape platform had been located on the last day before the start of chronic D-AP5 infusion. LTP in vivo in the dentate gyrus was blocked in the chronically-infused D-AP5 rats and HPLC measurements at sacrifice revealed appropriate intrahippocampal levels. Acute intrahippocampal infusion of radiolabelled D-AP5 revealed relatively restricted diffusion and was used to estimate whole-tissue hippocampal drug concentrations. These results indicate that (1) short-term memory for spatial information is independent of NMDA receptors; (2) the rapid consolidation of spatial information into long-term memory requires activation of hippocampal NMDA receptors; (3) NMDA receptors are not involved in memory retrieval; and (4) the delay-related effects of NMDA receptor antagonists on performance of this task cannot be explained in terms of sensorimotor disturbances. The findings relate to the idea that hippocampal synaptic plasticity is involved in event-memory (Morris and Frey, Phil Trans R Soc Lond B 1997;352:1489–1503) and to a computational model of one-trial DMP performance of Foster et al. (unpublished data). Hippocampus 9:118–136, 1999. © 1999 Wiley-Liss, Inc.
Roger J. A. Grand – One of the best experts on this subject based on the ideXlab platform.
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A Role for E1B-AP5 in ATR Signaling Pathways during Adenovirus Infection
Journal of virology, 2008Co-Authors: Andrew N. Blackford, Rachel Bruton, Orkide Dirlik, Grant S. Stewart, A. Malcolm R. Taylor, Thomas Dobner, Roger J. A. Grand, Andrew S. TurnellAbstract:E1B-55K-associated protein 5 (E1B-AP5) is a cellular, heterogeneous nuclear ribonucleoprotein that is targeted by adenovirus (Ad) E1B-55K during infection. The function of E1B-AP5 during infection, however, remains largely unknown. Given the role of E1B-55K targets in the DNA damage response, we examined whether E1B-AP5 function was integral to these pathways. Here, we show a novel role for E1B-AP5 as a key regulator of ATR signaling pathways activated during Ad infection. E1B-AP5 is recruited to viral replication centers during infection, where it colocalizes with ATR-interacting protein (ATRIP) and the ATR substrate replication protein A 32 (RPA32). Indeed, E1B-AP5 associates with ATRIP and RPA complex component RPA70 in both uninfected and Ad-infected cells. Additionally, glutathione S-transferase pull-downs show that E1B-AP5 associates with RPA components RPA70 and RPA32 directly in vitro. E1B-AP5 is required for the ATR-dependent phosphorylation of RPA32 during infection and contributes to the Ad-induced phosphorylation of Smc1 and H2AX. In this regard, it is interesting that Ad5 and Ad12 differentially promote the phosphorylation of RPA32, Rad9, and Smc1 during infection such that Ad12 promotes a significant phosphorylation of RPA32 and Rad9, whereas Ad5 only weakly promotes RPA32 phosphorylation and does not induce Rad9 phosphorylation. These data suggest that Ad5 and Ad12 have evolved different strategies to regulate DNA damage signaling pathways during infection in order to promote viral replication. Taken together, our results define a role for E1B-AP5 in ATR signaling pathways activated during infection. This might have broader implications for the regulation of ATR activity during cellular DNA replication or in response to DNA damage.
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The interaction of the hnRNP family member E1B-AP5 with p53.
FEBS letters, 2005Co-Authors: Paola M. Barral, Andrew S. Turnell, Thomas Dobner, Andre Rusch, Phillip H. Gallimore, Philip J. Byrd, Roger J. A. GrandAbstract:Adenovirus early region 1B-associated protein 5, E1B-AP5, a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family, was originally isolated on the basis of its ability to bind to the adenovirus 5 early region1B55K protein. Here, it has been demonstrated that E1B-AP5 interacts with mutant and wild-type p53 from human cells in pull-down assays using GST-E1B-AP5. This interaction has been confirmed by co-immunoprecipitation studies and pull-down experiments with in vitro translated E1B-AP5 and GST-p53. The binding site for E1B-AP5 has been mapped to the C-terminal region of p53. In reciprocal experiments, it has been shown that several regions of E1B-AP5 bound to p53 although it is probable that a major site of interaction is located between amino acids 395 and 732 of E1B-AP5. In reporter assays, E1B-AP5 inhibited p53 transcriptional activity although not as efficiently as the Ad5E1B55K protein. Transfection of E1B-AP5 into human tumour cells affected the cellular response to UV radiation, such that, although p53 expression was induced, little change in the level of p53-inducible genes could be observed.