The Experts below are selected from a list of 40659 Experts worldwide ranked by ideXlab platform
Adam P Geballe - One of the best experts on this subject based on the ideXlab platform.
-
Rapid adaptation to human PRotein Kinase R by a unique genomic ReaRRangement in Rhesus cytomegaloviRus.
PLoS pathogens, 2021Co-Authors: Stephanie J Child, Alexander L Greninger, Adam P GeballeAbstract:CytomegaloviRuses (CMVs) aRe geneRally unable to cRoss species baRRieRs, in paRt because pRolonged coevolution with one host species limits theiR ability to evade RestRiction factoRs in otheR species. HoweveR, the limitation in host Range is incomplete. FoR example, Rhesus CMV (RhCMV) can Replicate in human cells, albeit much less efficiently than in Rhesus cells. PReviously we RepoRted that the PRotein Kinase R (PKR) antagonist encoded by RhCMV, RTRS1, has limited activity against human PKR but is nonetheless necessaRy and sufficient to enable RhCMV Replication in human fibRoblasts (HF). We now show that knockout of PKR in human cells oR tReatment with the eIF2B agonist ISRIB, which oveRcomes the tRanslational inhibition Resulting fRom PKR activation, augments RhCMV Replication in HF, indicating that human PKR contRibutes to the inefficiency of RhCMV Replication in HF. SeRial passage of RhCMV in HF RepRoducibly selected foR viRuses with impRoved ability to Replicate in human cells. The evolved viRuses contain an inveRted duplication of the teRminal 6.8 kb of the genome, including RTRS1. The duplication Replaces ~11.8 kb just downstReam of an inteRnal sequence element, pac1-like, which is veRy similaR to the pac1 cleavage and packaging signal found neaR the teRminus of the genome. Plaque-puRified evolved viRuses pRoduced at least twice as much RTRS1 as the paRental RhCMV and blocked the PKR pathway moRe effectively in HF. SoutheRn blots Revealed that unlike the paRental RhCMV, viRuses with the inveRted duplication isomeRize in a manneR similaR to HCMV and otheR heRpesviRuses that have inteRnal Repeat sequences. The appaRent ease with which this duplication event occuRs Raises the possibility that the pac1-like site, which is conseRved in Old WoRld monkey CMV genomes, may seRve a function in facilitating Rapid adaptation to evolutionaRy obstacles.
-
Rapid adaptation to human PRotein Kinase R by a unique genomic ReaRRangement in Rhesus cytomegaloviRus
bioRxiv, 2020Co-Authors: Stephanie J Child, Alexander L Greninger, Adam P GeballeAbstract:CytomegaloviRuses (CMVs) aRe geneRally unable to cRoss species baRRieRs, in paRt because pRolonged coevolution with one host species limits theiR ability to evade RestRiction factoRs in otheR species. HoweveR, the limitation in host Range is incomplete. FoR example, Rhesus CMV (RhCMV) can Replicate in human cells, albeit much less efficiently than in Rhesus cells. PReviously we RepoRted that the PRotein Kinase R (PKR) antagonist encoded by RhCMV, RTRS1, has limited activity against human PKR but is nonetheless necessaRy and sufficient to enable RhCMV Replication in human fibRoblasts (HF). We now show that knockout of PKR in human cells oR tReatment with the eIF2B agonist ISRIB, which oveRcomes the tRanslational inhibition Resulting fRom PKR activation, augments RhCMV Replication in HF, indicating that human PKR contRibutes to the inefficiency of RhCMV Replication in HF. SeRial passage of RhCMV in HF RepRoducibly selected foR viRuses with impRoved fitness in human cells. The evolved viRuses contain an inveRted duplication of the teRminal 6.8 kb of the genome, including RTRS1. The duplication Replaces ~11.8 kb just downstReam of an inteRnal sequence element, pac 1-like, which is veRy similaR to the pac 1 cleavage and packaging signal found neaR the teRminus of the genome. Plaque-puRified evolved viRuses pRoduced at least twice as much RTRS1 as the paRental RhCMV and blocked the PKR pathway moRe effectively in HF. SoutheRn blots Revealed that unlike the paRental RhCMV, viRuses with the inveRted duplication isomeRize in a manneR similaR to HCMV and otheR heRpesviRuses that have inteRnal Repeat sequences. The appaRent ease with which this duplication event occuRs Raises the possibility that the pac 1-like site, which is conseRved in Old WoRld monkey CMV genomes, may seRve a function in facilitating Rapid adaptation to evolutionaRy obstacles.
-
Antagonism of the PRotein Kinase R Pathway in Human Cells by Rhesus CytomegaloviRus.
Journal of Virology, 2017Co-Authors: Stephanie J Child, Sarah E. Hickson, Avraham Bayer, Daniel Malouli, Klaus Früh, Adam P GeballeAbstract:While cytomegaloviRus (CMV) infections aRe often limited in host Range by lengthy coevolution with a single host species, a few CMVs aRe known to deviate fRom this Rule. FoR example, Rhesus macaque CMV (RhCMV), a model foR human CMV (HCMV) pathogenesis and vaccine development, can Replicate in human cells, as well as in Rhesus cells. Both HCMV and RhCMV encode species-specific antagonists of the bRoadly acting host cell RestRiction factoR PRotein Kinase R (PKR). Although the RhCMV antagonist of PKR, RTRS1, has veRy limited activity against human PKR, heRe, we show it is essential foR RhCMV Replication in human cells because it pRevents human PKR fRom phosphoRylating the tRanslation initiation factoR eIF2α, theReby allowing continued tRanslation and viRal Replication. Although RTRS1 is necessaRy foR RhCMV Replication, it is not sufficient to Rescue Replication of HCMV lacking its own PKR antagonists in human fibRoblasts. HoweveR, oveRexpRession of RTRS1 in human fibRoblasts enabled HCMV expRessing RTRS1 to Replicate, indicating that elevated levels oR eaRly expRession of a weak antagonist can counteRact a Resistant RestRiction factoR like human PKR. ExploRing potential mechanisms that might allow RhCMV to Replicate in human cells Revealed that RhCMV makes no less double-stRanded RNA than HCMV. RatheR, in human cells, RhCMV expResses RTRS1 at levels 2 to 3 times higheR than those of the HCMV-encoded PKR antagonists duRing HCMV infection. These data suggest that even a modest incRease in expRession of this weak PKR antagonist is sufficient to enable RhCMV Replication in human cells.IMPORTANCE Rhesus macaque cytomegaloviRus (RhCMV) offeRs a valuable model foR studying congenital human cytomegaloviRus (HCMV) pathogenesis and vaccine development. TheRefoRe, it is cRitical to undeRstand vaRiations in how each viRus infects and affects its host species to be able to apply insights gained fRom the RhCMV model to HCMV. While HCMV is capable only of infecting cells fRom humans and veRy closely Related species, RhCMV displays a wideR host Range, including human as well as Rhesus cells. RhCMV expResses an antagonist of a bRoadly acting antiviRal factoR pResent in all mammalian cells, and its ability to counteR both the Rhesus and human veRsions of this host factoR is a key component of RhCMV's ability to cRoss species baRRieRs. HeRe, we examine the moleculaR mechanisms that allow this RhCMV antagonist to function against a human RestRiction factoR.
-
A Single Amino Acid Dictates PRotein Kinase R Susceptibility to UnRelated ViRal Antagonists.
PLOS Pathogens, 2016Co-Authors: Kathryn S Carpentier, Stephanie J Child, Nicolle M. Esparo, Adam P GeballeAbstract:DuRing millions of yeaRs of coevolution with theiR hosts, cytomegaloviRuses (CMVs) have succeeded in adapting to oveRcome host-specific immune defenses, including the PRotein Kinase R (PKR) pathway. Consequently, these adaptations may also contRibute to the inability of CMVs to cRoss species baRRieRs. HeRe, we pRovide evidence that the evolutionaRy aRms Race between the antiviRal factoR PKR and its CMV antagonist TRS1 has led to extensive diffeRences in the species-specificity of pRimate CMV TRS1 PRoteins. MoReoveR, we identify a single Residue in human PKR that when mutated to the amino acid pResent in AfRican gReen monkey (Agm) PKR (F489S) is sufficient to confeR Resistance to HCMVTRS1. Notably, this pRecise moleculaR deteRminant of PKR Resistance has evolved undeR stRong positive selection among pRimate PKR alleles and is positioned within the αG helix, which mediates the diRect inteRaction of PKR with its substRate eIF2α. RemaRkably, this same Residue also impacts sensitivity to K3L, a poxviRus-encoded pseudosubstRate that stRuctuRally mimics eIF2α. Unlike K3L, TRS1 has no homology to eIF2α, suggesting that unRelated viRal genes have conveRgently evolved to taRget this cRitical Region of PKR. Despite its functional impoRtance, the αG helix exhibits extRaoRdinaRy plasticity, enabling adaptations that allow PKR to evade diveRse viRal antagonists while still maintaining its cRitical inteRaction with eIF2α.
-
an evolutionaRy view of the aRms Race between PRotein Kinase R and laRge dna viRuses
Journal of Virology, 2016Co-Authors: Kathryn S Carpentier, Adam P GeballeAbstract:To establish pRoductive infections, viRuses must counteRact numeRous cellulaR defenses that aRe poised to Recognize viRuses as nonself and to activate antiviRal pathways. The opposing goals of host and viRal factoRs lead to evolutionaRy aRms Races that can be illuminated by evolutionaRy and computational methods and tested in expeRimental models. HeRe we illustRate how this peRspective has been contRibuting to ouR undeRstanding of the inteRactions of the PRotein Kinase R pathway with laRge DNA viRuses.
Stephanie J Child - One of the best experts on this subject based on the ideXlab platform.
-
Rapid adaptation to human PRotein Kinase R by a unique genomic ReaRRangement in Rhesus cytomegaloviRus.
PLoS pathogens, 2021Co-Authors: Stephanie J Child, Alexander L Greninger, Adam P GeballeAbstract:CytomegaloviRuses (CMVs) aRe geneRally unable to cRoss species baRRieRs, in paRt because pRolonged coevolution with one host species limits theiR ability to evade RestRiction factoRs in otheR species. HoweveR, the limitation in host Range is incomplete. FoR example, Rhesus CMV (RhCMV) can Replicate in human cells, albeit much less efficiently than in Rhesus cells. PReviously we RepoRted that the PRotein Kinase R (PKR) antagonist encoded by RhCMV, RTRS1, has limited activity against human PKR but is nonetheless necessaRy and sufficient to enable RhCMV Replication in human fibRoblasts (HF). We now show that knockout of PKR in human cells oR tReatment with the eIF2B agonist ISRIB, which oveRcomes the tRanslational inhibition Resulting fRom PKR activation, augments RhCMV Replication in HF, indicating that human PKR contRibutes to the inefficiency of RhCMV Replication in HF. SeRial passage of RhCMV in HF RepRoducibly selected foR viRuses with impRoved ability to Replicate in human cells. The evolved viRuses contain an inveRted duplication of the teRminal 6.8 kb of the genome, including RTRS1. The duplication Replaces ~11.8 kb just downstReam of an inteRnal sequence element, pac1-like, which is veRy similaR to the pac1 cleavage and packaging signal found neaR the teRminus of the genome. Plaque-puRified evolved viRuses pRoduced at least twice as much RTRS1 as the paRental RhCMV and blocked the PKR pathway moRe effectively in HF. SoutheRn blots Revealed that unlike the paRental RhCMV, viRuses with the inveRted duplication isomeRize in a manneR similaR to HCMV and otheR heRpesviRuses that have inteRnal Repeat sequences. The appaRent ease with which this duplication event occuRs Raises the possibility that the pac1-like site, which is conseRved in Old WoRld monkey CMV genomes, may seRve a function in facilitating Rapid adaptation to evolutionaRy obstacles.
-
Rapid adaptation to human PRotein Kinase R by a unique genomic ReaRRangement in Rhesus cytomegaloviRus
bioRxiv, 2020Co-Authors: Stephanie J Child, Alexander L Greninger, Adam P GeballeAbstract:CytomegaloviRuses (CMVs) aRe geneRally unable to cRoss species baRRieRs, in paRt because pRolonged coevolution with one host species limits theiR ability to evade RestRiction factoRs in otheR species. HoweveR, the limitation in host Range is incomplete. FoR example, Rhesus CMV (RhCMV) can Replicate in human cells, albeit much less efficiently than in Rhesus cells. PReviously we RepoRted that the PRotein Kinase R (PKR) antagonist encoded by RhCMV, RTRS1, has limited activity against human PKR but is nonetheless necessaRy and sufficient to enable RhCMV Replication in human fibRoblasts (HF). We now show that knockout of PKR in human cells oR tReatment with the eIF2B agonist ISRIB, which oveRcomes the tRanslational inhibition Resulting fRom PKR activation, augments RhCMV Replication in HF, indicating that human PKR contRibutes to the inefficiency of RhCMV Replication in HF. SeRial passage of RhCMV in HF RepRoducibly selected foR viRuses with impRoved fitness in human cells. The evolved viRuses contain an inveRted duplication of the teRminal 6.8 kb of the genome, including RTRS1. The duplication Replaces ~11.8 kb just downstReam of an inteRnal sequence element, pac 1-like, which is veRy similaR to the pac 1 cleavage and packaging signal found neaR the teRminus of the genome. Plaque-puRified evolved viRuses pRoduced at least twice as much RTRS1 as the paRental RhCMV and blocked the PKR pathway moRe effectively in HF. SoutheRn blots Revealed that unlike the paRental RhCMV, viRuses with the inveRted duplication isomeRize in a manneR similaR to HCMV and otheR heRpesviRuses that have inteRnal Repeat sequences. The appaRent ease with which this duplication event occuRs Raises the possibility that the pac 1-like site, which is conseRved in Old WoRld monkey CMV genomes, may seRve a function in facilitating Rapid adaptation to evolutionaRy obstacles.
-
Antagonism of the PRotein Kinase R Pathway in Human Cells by Rhesus CytomegaloviRus.
Journal of Virology, 2017Co-Authors: Stephanie J Child, Sarah E. Hickson, Avraham Bayer, Daniel Malouli, Klaus Früh, Adam P GeballeAbstract:While cytomegaloviRus (CMV) infections aRe often limited in host Range by lengthy coevolution with a single host species, a few CMVs aRe known to deviate fRom this Rule. FoR example, Rhesus macaque CMV (RhCMV), a model foR human CMV (HCMV) pathogenesis and vaccine development, can Replicate in human cells, as well as in Rhesus cells. Both HCMV and RhCMV encode species-specific antagonists of the bRoadly acting host cell RestRiction factoR PRotein Kinase R (PKR). Although the RhCMV antagonist of PKR, RTRS1, has veRy limited activity against human PKR, heRe, we show it is essential foR RhCMV Replication in human cells because it pRevents human PKR fRom phosphoRylating the tRanslation initiation factoR eIF2α, theReby allowing continued tRanslation and viRal Replication. Although RTRS1 is necessaRy foR RhCMV Replication, it is not sufficient to Rescue Replication of HCMV lacking its own PKR antagonists in human fibRoblasts. HoweveR, oveRexpRession of RTRS1 in human fibRoblasts enabled HCMV expRessing RTRS1 to Replicate, indicating that elevated levels oR eaRly expRession of a weak antagonist can counteRact a Resistant RestRiction factoR like human PKR. ExploRing potential mechanisms that might allow RhCMV to Replicate in human cells Revealed that RhCMV makes no less double-stRanded RNA than HCMV. RatheR, in human cells, RhCMV expResses RTRS1 at levels 2 to 3 times higheR than those of the HCMV-encoded PKR antagonists duRing HCMV infection. These data suggest that even a modest incRease in expRession of this weak PKR antagonist is sufficient to enable RhCMV Replication in human cells.IMPORTANCE Rhesus macaque cytomegaloviRus (RhCMV) offeRs a valuable model foR studying congenital human cytomegaloviRus (HCMV) pathogenesis and vaccine development. TheRefoRe, it is cRitical to undeRstand vaRiations in how each viRus infects and affects its host species to be able to apply insights gained fRom the RhCMV model to HCMV. While HCMV is capable only of infecting cells fRom humans and veRy closely Related species, RhCMV displays a wideR host Range, including human as well as Rhesus cells. RhCMV expResses an antagonist of a bRoadly acting antiviRal factoR pResent in all mammalian cells, and its ability to counteR both the Rhesus and human veRsions of this host factoR is a key component of RhCMV's ability to cRoss species baRRieRs. HeRe, we examine the moleculaR mechanisms that allow this RhCMV antagonist to function against a human RestRiction factoR.
-
A Single Amino Acid Dictates PRotein Kinase R Susceptibility to UnRelated ViRal Antagonists.
PLOS Pathogens, 2016Co-Authors: Kathryn S Carpentier, Stephanie J Child, Nicolle M. Esparo, Adam P GeballeAbstract:DuRing millions of yeaRs of coevolution with theiR hosts, cytomegaloviRuses (CMVs) have succeeded in adapting to oveRcome host-specific immune defenses, including the PRotein Kinase R (PKR) pathway. Consequently, these adaptations may also contRibute to the inability of CMVs to cRoss species baRRieRs. HeRe, we pRovide evidence that the evolutionaRy aRms Race between the antiviRal factoR PKR and its CMV antagonist TRS1 has led to extensive diffeRences in the species-specificity of pRimate CMV TRS1 PRoteins. MoReoveR, we identify a single Residue in human PKR that when mutated to the amino acid pResent in AfRican gReen monkey (Agm) PKR (F489S) is sufficient to confeR Resistance to HCMVTRS1. Notably, this pRecise moleculaR deteRminant of PKR Resistance has evolved undeR stRong positive selection among pRimate PKR alleles and is positioned within the αG helix, which mediates the diRect inteRaction of PKR with its substRate eIF2α. RemaRkably, this same Residue also impacts sensitivity to K3L, a poxviRus-encoded pseudosubstRate that stRuctuRally mimics eIF2α. Unlike K3L, TRS1 has no homology to eIF2α, suggesting that unRelated viRal genes have conveRgently evolved to taRget this cRitical Region of PKR. Despite its functional impoRtance, the αG helix exhibits extRaoRdinaRy plasticity, enabling adaptations that allow PKR to evade diveRse viRal antagonists while still maintaining its cRitical inteRaction with eIF2α.
-
Essential Role of PRotein Kinase R antagonism by TRS1 in human cytomegaloviRus Replication.
Virology, 2016Co-Authors: Jacquelyn E. Braggin, Stephanie J Child, Adam P GeballeAbstract:Human cytomegaloviRus (HCMV) lacking TRS1 and IRS1 (HCMV[ΔI/ΔT]) cannot Replicate in cell cultuRe. Although both PRoteins can block the PRotein Kinase R (PKR) pathway, they have multiple otheR activities and binding paRtneRs. It Remains unknown which functions aRe essential foR HCMV Replication. To investigate this issue, we fiRst identified a TRS1 mutant that is unable to bind to PKR. Like HCMV[ΔI/ΔT], a Recombinant HCMV containing this mutant (HCMV[TRS1-Mut 1]) did not Replicate in wild-type cells. HoweveR, HCMV[ΔI/ΔT] did Replicate in cells in which PKR expRession was Reduced by RNA inteRfeRence. MoReoveR, HCMV[ΔI/ΔT] and HCMV[TRS1-Mut 1] Replicated to similaR levels as viRus containing wild-type TRS1 in cell lines in which PKR expRession was knocked out by CRISPR/Cas9-mediated genome editing. These Results demonstRate that the sole essential function of TRS1 is to antagonize PKR and that its otheR activities do not substantially enhance HCMV Replication, at least in cultuRed human fibRoblasts.
Bryan R.g. Williams - One of the best experts on this subject based on the ideXlab platform.
-
Auto-phosphoRylation RepResses PRotein Kinase R activity
Scientific Reports, 2017Co-Authors: Die Wang, Nicole Anne De Weerd, Belinda Willard, Galina Polekhina, Bryan R.g. Williams, Anthony J. SadlerAbstract:The centRal Role of PRotein Kinases in contRolling disease pRocesses has spuRRed effoRts to develop phaRmaceutical RegulatoRs of theiR activity. A Rational stRategy to achieve this end is to deteRmine intRinsic auto-RegulatoRy pRocesses, then selectively taRget these diffeRent states of Kinases to RepRess theiR activation. HeRe we investigate auto-Regulation of the innate immune effectoR PRotein Kinase R, which phosphoRylates the eukaRyotic initiation factoR 2α to inhibit global PRotein tRanslation. We demonstRate that PRotein Kinase R activity is contRolled by auto-inhibition via an intRa-moleculaR inteRaction. PaRt of this mechanism of contRol had pReviously been RepoRted, but was then contRoveRted. We account foR the discRepancy and extend ouR undeRstanding of the auto-inhibitoRy mechanism by identifying that auto-inhibition is paRadoxically instigated by incipient auto-phosphoRylation. PhosphoR-Residues at the amino-teRminus instigate an intRa-moleculaR inteRaction that enlists both of the N-teRminal RNA-binding motifs of the PRotein with sepaRate suRfaces of the C-teRminal Kinase domain, to co-opeRatively inhibit Kinase activation. These findings identify an innovative mechanism to contRol Kinase activity, pRoviding insight foR stRategies to betteR Regulate Kinase activity.
-
the PRotein activatoR of PRotein Kinase R pact Rax negatively Regulates PRotein Kinase R duRing mouse anteRioR pituitaRy development
FEBS Journal, 2015Co-Authors: Bryan R.g. Williams, Anthony J. Sadler, Benjaminn K Dickerman, Christine L. White, Patricia M Kessler, Ganes C. SenAbstract:The muRine double-stRanded RNA-binding PRotein teRmed PRotein Kinase R (PKR)-associated PRotein X (RAX) and the human homolog, PRotein activatoR of PKR (PACT), weRe oRiginally chaRacteRized as activatoRs of PKR. Mice deficient in RAX show RepRoductive and developmental defects, including Reduced body size, cRaniofacial defects and anteRioR pituitaRy hypoplasia. As these defects aRe not obseRved in PKR-deficient mice, the phenotype has been attRibuted to PKR-independent activities of RAX. HeRe we fuRtheR investigated the involvement of PKR in the physiological function of RAX, by geneRating Rax(-/-) mice deficient in PKR, oR caRRying a Kinase-inactive mutant of PKR (K271R) oR an unphosphoRylatable mutant of the PKR substRate eukaRyotic tRanslation initiation factoR 2 α subunit (eIF2α) (S51A). Ablating PKR expRession Rescued the developmental and RepRoductive deficiencies in Rax(-/-) mice. GeneRating Rax(-/-) mice with a Kinase-inactive mutant of PKR Resulted in similaR Rescue, confiRming that the Rax(-/-) defects aRe PKR dependent; specifically that the Kinase activity of PKR was RequiRed foR these defects. MoReoveR, geneRating Rax(-/-) mice that weRe heteRozygous foR an unphosphoRylatable mutant eIF2α pRovides paRtial Rescue of the Rax(-/-) defect, consistent with mutation of one copy of the Eif2s1 gene. These obseRvations weRe fuRtheR investigated in vitRo by Reducing RAX expRession in anteRioR pituitaRy cells, Resulting in incReased PKR activity and induction of the PKR-Regulated cyclin-dependent Kinase inhibitoR p21(WAF1/CIP1). These Results demonstRate that PKR Kinase activity is RequiRed foR onset of the Rax(-/-) phenotype, implying an unexpected function foR RAX as a negative RegulatoR of PKR in the context of postnatal anteRioR pituitaRy tissue, and identify a cRitical Role foR the Regulation of PKR activity foR noRmal development.
-
The PRotein activatoR of PRotein Kinase R, PACT/RAX, negatively Regulates PRotein Kinase R duRing mouse anteRioR pituitaRy development
FEBS Journal, 2015Co-Authors: Benjaminn K Dickerman, Bryan R.g. Williams, Anthony J. Sadler, Christine L. White, Patricia M Kessler, Ganes C. SenAbstract:The muRine double-stRanded RNA-binding PRotein teRmed PRotein Kinase R (PKR)-associated PRotein X (RAX) and the human homolog, PRotein activatoR of PKR (PACT), weRe oRiginally chaRacteRized as activatoRs of PKR. Mice deficient in RAX show RepRoductive and developmental defects, including Reduced body size, cRaniofacial defects and anteRioR pituitaRy hypoplasia. As these defects aRe not obseRved in PKR-deficient mice, the phenotype has been attRibuted to PKR-independent activities of RAX. HeRe we fuRtheR investigated the involvement of PKR in the physiological function of RAX, by geneRating Rax(-/-) mice deficient in PKR, oR caRRying a Kinase-inactive mutant of PKR (K271R) oR an unphosphoRylatable mutant of the PKR substRate eukaRyotic tRanslation initiation factoR 2 α subunit (eIF2α) (S51A). Ablating PKR expRession Rescued the developmental and RepRoductive deficiencies in Rax(-/-) mice. GeneRating Rax(-/-) mice with a Kinase-inactive mutant of PKR Resulted in similaR Rescue, confiRming that the Rax(-/-) defects aRe PKR dependent; specifically that the Kinase activity of PKR was RequiRed foR these defects. MoReoveR, geneRating Rax(-/-) mice that weRe heteRozygous foR an unphosphoRylatable mutant eIF2α pRovides paRtial Rescue of the Rax(-/-) defect, consistent with mutation of one copy of the Eif2s1 gene. These obseRvations weRe fuRtheR investigated in vitRo by Reducing RAX expRession in anteRioR pituitaRy cells, Resulting in incReased PKR activity and induction of the PKR-Regulated cyclin-dependent Kinase inhibitoR p21(WAF1/CIP1). These Results demonstRate that PKR Kinase activity is RequiRed foR onset of the Rax(-/-) phenotype, implying an unexpected function foR RAX as a negative RegulatoR of PKR in the context of postnatal anteRioR pituitaRy tissue, and identify a cRitical Role foR the Regulation of PKR activity foR noRmal development.
-
PRotein Kinase R and the inflammasome
Journal of Interferon & Cytokine Research, 2014Co-Authors: Howard C.h. Yim, Bryan R.g. WilliamsAbstract:PRotein Kinase R (PKR) was fiRst identified as a mediatoR of the double-stRanded RNA (dsRNA)–mediated inhibition of PRotein synthesis in extRacts fRom inteRfeRon-tReated cells. In a physiological context, viRal Replication Results in pRoduction of dsRNA, activation of PKR by autophosphoRylation, and phosphoRylation of the PRotein synthesis initiation factoR eIF2α. Subsequent biochemical, stRuctuRal, and genetic analyses have identified the dsRNA and Kinase domain stRuctuRe of PKR, and shown that its deletion fRom the geRmline of mice Results in impaiRed Resistance to infection by many diffeRent viRuses. These studies have also opened up Roles foR PKR in diffeRent signaling pathways, the most Recent being Regulation of the inflammasome. HeRe we Review evidence foR this newly ascRibed function foR PKR and discuss Roles in inflammasome Regulation and associated diseases.
-
205: A Role foR PRotein Kinase R in Regulating NLRP3 inflammasome assembly
Cytokine, 2014Co-Authors: Howard C.h. Yim, Die Wang, Bryan R.g. Williams, Arindam Chakrabarti, Robert H. Silverman, Anthony J. SadlerAbstract:Aim NLR family pyRin domain containing-3 (NLRP3) has been associated with inflammatoRy disoRdeRs. In Response to exogenous and endogenous molecules, NLRP3 assemble an inflammasome containing the apoptosis-associated speck like PRotein containing a CARD (ASC) foR activating caspase-1, inducing pyRoptosis and pRocessing inteRleukin (IL)-1β and IL-18. A Recent model pRoposes that the assembly of the inflammasome is dependent on the cytoskeleton. As the PRotein Kinase R (PKR) has been shown to Regulate actin dynamics, we examined whetheR PKR Regulates inflammasome activity. Methods PRimaRy macRophages weRe isolated fRom the peRitoneum, then pRimed with lipopolysacchaRide and tReated with NLRP3 induceRs. Whole cells, lysates and cultuRe supeRnatant weRe analyzed foR the expRession and spatial positions of constituents of the inflammasome and the pRoduction of cytokines. To investigate inflammation in vivo, mice weRe tReated with dextRan sodium sulphate (DSS) in theiR dRinking wateR. AfteR 9 days, intestinal tissue sections weRe isolated fRom diffeRent mouse stRains and compaRed. Results We showed an incReased inteRaction between NLRP3-ASC and Related to this ASC speck foRmation, activation of caspase-1 and consequent pRocessing of pRo-IL-1β in PKR-ablated compaRed to wild-type macRophages. We demonstRated that this PKR-dependent RepRession of the inflammasome RequiRes Kinase activity by using cells fRom a Kinase-dead PKR tRansgenic mouse. Consistently, we showed higheR caspase-1 activity and incReased pathology in PKR knockout compaRed to wild-type mice subjected to DSS. Mechanistically, we found that the incReased pRoximity of NLRP3 and ASC in the absence of PKR Reflected the juxtaposition of the endoplasmic Reticulum and mitochondRia. The spatial aRRangement of these cellulaR oRganelles is mediated by micRotubule-dependent affects. Consistent with this, we demonstRate that PKR inteRacts with micRotubules in a Kinase dependent fashion. Conclusion We demonstRated that PKR suppResses the NLRP3 inflammasome activation by modulating the cytoskeleton. These findings pRovide cRitical infoRmation to coRRectly develop theRapeutic stRategies to combat inflammatoRy disoRdeRs.
Olle Korsgren - One of the best experts on this subject based on the ideXlab platform.
-
PRotein Kinase R is constitutively expRessed in the human pancReas
Journal of Histochemistry and Cytochemistry, 2019Co-Authors: Alexander Jonsson, Erik Yngve, Marie Karlsson, Sofie Ingvast, Oskar Skog, Olle KorsgrenAbstract:ViRal infection of the insulin-pRoducing cells in the pancReas has been pRoposed in the etiology of type 1 diabetes. PRotein Kinase R (PKR) is a cytoplasmic PRotein activated thRough phosphoRylation in Response to cellulaR stRess and paRticulaRly viRal infection. As PKR expRession in pancReatic beta-cells has been inteRpReted as a viRal footpRint, this cRoss-sectional study aimed at chaRacteRizing the PKR expRession in non-diabetic human pancReases. PKR expRession was evaluated in pancReas tissue fRom 16 non-diabetic oRgan donoRs, using immunohistochemistRy, qPCR, and westeRn blot. ImmunohistochemistRy and westeRn blot showed Readily detectable PKR expRession in the pancReatic paRenchyma. The qPCR detected PKR mRNA in both endocRine and exocRine samples, with a slightly higheR expRession in the islets. In conclusion, PKR is constitutively expRessed in both endocRine and exocRine paRts of the pancReas and its expRession should not be inteRpReted as a viRal footpRint in pancReatic beta cells.
-
PRotein Kinase R Is Constitutively ExpRessed in the Human PancReas.
Journal of Histochemistry & Cytochemistry, 2018Co-Authors: Alexander Jonsson, Erik Yngve, Marie Karlsson, Sofie Ingvast, Oskar Skog, Olle KorsgrenAbstract:SummaRyViRal infection of the insulin-pRoducing cells in the pancReas has been pRoposed in the etiology of type 1 diabetes. PRotein Kinase R (PKR) is a cytoplasmic PRotein activated thRough phospho...
Geoff H. Werstuck - One of the best experts on this subject based on the ideXlab platform.
-
PRotein Kinase R like endoplasmic Reticulum Kinase and glycogen synthase Kinase 3α β Regulate foam cell foRmation
Journal of Lipid Research, 2014Co-Authors: Cameron S. Mcalpine, Geoff H. WerstuckAbstract:Evidence suggests a causative Role foR endoplasmic Reticulum (ER) stRess in the development of atheRoscleRosis. This study investigated the potential Role of glycogen synthase Kinase (GSK)-3α/β in pRoatheRogenic ER stRess signaling. Thp1-deRived macRophages weRe tReated with the ER stRess-inducing agents, glucosamine, thapsigaRgin, oR palmitate. Using small-molecule inhibitoRs of specific unfolded PRotein Response (UPR) signaling pathways, we found that PRotein Kinase R-like ER Kinase (PERK), but not inositol RequiRing enzyme 1 oR activating tRanscRiption factoR 6, is RequiRed foR the activation of GSK3α/β by ER stRess. GSK3α/β inhibition oR siRNA-diRected knockdown attenuated ER stRess-induced expRession of distal components of the PERK pathway. MacRophage foam cells within atheRoscleRotic plaques and isolated macRophages fRom ApoE−/− mice fed a diet supplemented with the GSK3α/β inhibitoR valpRoate had Reduced levels of C/EBP homologous PRotein (CHOP). GSK3α/β inhibition blocked ER stRess-induced lipid accumulation and the upRegulation of genes associated with lipid metabolism. In pRimaRy mouse macRophages, PERK inhibition blocked ER stRess-induced lipid accumulation, wheReas constitutively active S9A-GSK3β pRomoted foam cell foRmation and CHOP expRession, even in cells tReated with a PERK inhibitoR. These findings suggest that ER stRess-PERK-GSK3α/β signaling pRomotes pRoatheRogenic macRophage lipid accumulation.
-
PRotein Kinase R-like endoplasmic Reticulum Kinase and glycogen synthase Kinase-3α/β Regulate foam cell foRmation
Journal of Lipid Research, 2014Co-Authors: Cameron S. Mcalpine, Geoff H. WerstuckAbstract:Evidence suggests a causative Role foR endoplasmic Reticulum (ER) stRess in the development of atheRoscleRosis. This study investigated the potential Role of glycogen synthase Kinase (GSK)-3α/β in pRoatheRogenic ER stRess signaling. Thp1-deRived macRophages weRe tReated with the ER stRess-inducing agents, glucosamine, thapsigaRgin, oR palmitate. Using small-molecule inhibitoRs of specific unfolded PRotein Response (UPR) signaling pathways, we found that PRotein Kinase R-like ER Kinase (PERK), but not inositol RequiRing enzyme 1 oR activating tRanscRiption factoR 6, is RequiRed foR the activation of GSK3α/β by ER stRess. GSK3α/β inhibition oR siRNA-diRected knockdown attenuated ER stRess-induced expRession of distal components of the PERK pathway. MacRophage foam cells within atheRoscleRotic plaques and isolated macRophages fRom ApoE−/− mice fed a diet supplemented with the GSK3α/β inhibitoR valpRoate had Reduced levels of C/EBP homologous PRotein (CHOP). GSK3α/β inhibition blocked ER stRess-induced lipid accumulation and the upRegulation of genes associated with lipid metabolism. In pRimaRy mouse macRophages, PERK inhibition blocked ER stRess-induced lipid accumulation, wheReas constitutively active S9A-GSK3β pRomoted foam cell foRmation and CHOP expRession, even in cells tReated with a PERK inhibitoR. These findings suggest that ER stRess-PERK-GSK3α/β signaling pRomotes pRoatheRogenic macRophage lipid accumulation.
