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Current Search:
'Biochemistry' in all fields;
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Displaying Results 1 - 25 of 301 on page 1 of 13
Marked
Mark
Interleukin-1 signal transduction: increased GTP binding and hydrolysis in membranes of a murine thymoma line (EL4).
(1990)
O'NEILL, LUKE ANTHONY JOHN
Interleukin-1 signal transduction: increased GTP binding and hydrolysis in membranes of a murine thymoma line (EL4).
(1990)
O'NEILL, LUKE ANTHONY JOHN
Abstract:
The post-receptor events which follow the binding of interleukin 1 (IL1) to cells are unclear. The present studies provide evidence for the activation of a guanine nucleotide binding protein (G protein) by IL1 in the membranes of an IL1 receptor-rich strain (NOB-1) of the EL4 murine thymoma line. IL1 alpha and beta increased the binding of the GTP analogue [35S]guanosine 5'-[gamma-thiol]trisphosphate (GTP gamma S) to membranes prepared from these cells. By 1 min after addition of IL1 there was a 2-fold enhancement in binding which was dose dependent in the range 0.1-100 ng/ml. A qualitatively similar result was obtained with IL1 beta although it was 10 times less potent. Specific neutralizing antisera to IL1 alpha and IL1 beta abolished the response. Experiments in which the concentration of [35S]GTP gamma S was varied revealed that IL1 increased the affinity of the binding sites for [35S]GTP gamma S and not their number. IL1 alpha was shown to stimulate GTPase activity in the ...
http://hdl.handle.net/2262/33728
Marked
Mark
Interleukin 1 induces NF-kappa B through its type I but not its type II receptor in lymphocytes.
(1992)
O'NEILL, LUKE ANTHONY JOHN
Interleukin 1 induces NF-kappa B through its type I but not its type II receptor in lymphocytes.
(1992)
O'NEILL, LUKE ANTHONY JOHN
Abstract:
peer-reviewed
It is not known whether one or both of the interleukin 1 (IL1) receptors mediates the induction of the DNA-binding protein NF-kappa B. Nuclear extracts of the murine lines EL4.NOB.1 and 70Z/3, which bear the type I (80 kDa) and type II (67 kDa) IL1 receptor, respectively, were analyzed by an electrophoretic mobility shift assay. A 265-base pair sequence of the human serum amyloid A gene or a synthetic oligonucleotide each containing the NF-kappa B site were used as the DNA probes. IL1 induction of NF-kappa B was rapid (optimal at 15-30 min) and transient in both cell types. The IL1 receptor antagonist (IL1ra), which binds strongly to the type I receptor, inhibited the NF-kappa B response in both cell lines. IL1ra did not bind to the type II receptor on 70Z/3 cells as judged by competition for binding with 125I-IL1 alpha. When 125I-IL1ra binding to 70Z/3 cells was measured, a small number (10/cell) of high affinity sites (Kd = 5 x 10(-12) M) were detected. These wer...
http://hdl.handle.net/2262/33729
Marked
Mark
Cellular proliferation and activation of NF kappa B are induced by autocrine production of tumor necrosis factor alpha in the human T lymphoma line HuT 78
(1995)
O'NEILL, LUKE ANTHONY JOHN
Cellular proliferation and activation of NF kappa B are induced by autocrine production of tumor necrosis factor alpha in the human T lymphoma line HuT 78
(1995)
O'NEILL, LUKE ANTHONY JOHN
Abstract:
Tumor necrosis factor (TNF) is a pleiotropic cytokine which has both cytotoxic and proliferative effects. HuT 78, a T-cell line derived from a Sezary lymphoma, is resistant to the cytotoxic effects of TNF, suggesting that TNF may be a growth factor for this cell line. The aim of this study was to determine whether autocrine TNF production could function as a growth factor for HuT 78. Resting HuT 78 and K-4 cells, a protein kinase C-beta-deficient clone of HuT 78, both produced significant amounts of TNF compared with Jurkat cells. Thymidine incorporation by HuT 78 and K-4 cells was inhibited by 90.5 and 73.2%, respectively, with addition of a neutralizing monoclonal antibody to TNF alpha, suggesting that TNF is an autocrine growth factor for these cells. HuT 78 and K-4 cells also expressed high levels of constitutively active NF kappa B, unlike Jurkat cells, which expressed high levels only upon activation with TNF or phorbol 12-myristate 13-acetate. p50 was the major component in t...
http://hdl.handle.net/2262/33731
Marked
Mark
Studies into the effect of the tyrosine kinase inhibitor herbimycin A on NF-kappa B activation in T lymphocytes. Evidence for covalent modification of the p50 subunit.
(1995)
O'NEILL, LUKE ANTHONY JOHN
Studies into the effect of the tyrosine kinase inhibitor herbimycin A on NF-kappa B activation in T lymphocytes. Evidence for covalent modification of the p50 subunit.
(1995)
O'NEILL, LUKE ANTHONY JOHN
Abstract:
peer-reviewed
The tyrosine kinase inhibitor herbimycin A was found to block NF-kappa B stimulation in response to interleukin-1 and phorbol 12-myristate 13-acetate in EL4.NOB-1 thymoma cells and phorbol 12-myristate 13-acetate in Jurkat T lymphoma cells. The effect appeared not to involve inhibition of tyrosine kinase activation as neither interleukin-1 nor phorbol 12-myristate 13-acetate induced major changes in tyrosine phosphorylation in EL4.NOB-1 or Jurkat cells, respectively. Herbimycin A did not interfere with I kappa B-alpha degradation, and in unstimulated cells, it modified NF-kappa B prior to chemical dissociation with sodium deoxycholate. Because herbimycin A is thiol-reactive, we suspected that the target was the p50 subunit of NF-kappa B, which has a key thiol at cysteine 62. Herbimycin A inhibited DNA binding when added to nuclear extracts prepared from stimulated cells, which were shown to contain high levels of p50. Incubation of herbimycin A with 2-mercaptoethan...
http://hdl.handle.net/2262/33733
Marked
Mark
Daunorubicin activates NFkappaB and induces kappaB-dependent gene expression in HL-60 promyelocytic and Jurkat T lymphoma cells
(1997)
O'NEILL, LUKE ANTHONY JOHN
Daunorubicin activates NFkappaB and induces kappaB-dependent gene expression in HL-60 promyelocytic and Jurkat T lymphoma cells
(1997)
O'NEILL, LUKE ANTHONY JOHN
Abstract:
peer-reviewed
The anthracycline antibiotic, daunorubicin, can induce programmed cell death (apoptosis) in cells. Recent work suggests that this event is mediated by ceramide via enhanced ceramide synthase activity. Since the generation of ceramide has been directly linked with the activation of the transcription factor, NFkappaB, this was investigated as a novel target for the action of daunorubicin. Here we describe how treatment of HL-60 promyelocytes and Jurkat T lymphoma cells with daunorubicin results in the activation of the transcription factor NFkappaB. The effect of daunorubicin was evident following 1-2 h treatment, which was in contrast to the time course of activation obtained with the cytokine, tumor necrosis factor, where NFkappaB activation was detected within minutes of cellular stimulation. Activated complexes were shown to contain predominantly p50 and p65/RelA subunit components. Daunorubicin also induced IkappaB degradation and increased the expression of an ...
http://hdl.handle.net/2262/33735
Marked
Mark
Ceramide activates NFkappaB by inducing the processing of p105
(1998)
O'NEILL, LUKE ANTHONY JOHN
Ceramide activates NFkappaB by inducing the processing of p105
(1998)
O'NEILL, LUKE ANTHONY JOHN
Abstract:
peer-reviewed
The role of ceramide as a second messenger in tumor necrosis factor (TNF)-mediated signal transduction has been much debated. It is supported by recent reports describing an expanding number of potential targets for this lipid, but is opposed by those describing how ceramide is not necessary for many TNF-mediated cellular events. In this paper, we directly compare the effects of the cell-permeable ceramide analogue, N-acetylsphingosine (C2-ceramide), with TNF, on NFkappaB function, a transcription factor whose activation is central to many TNF-mediated effects. We describe how C2-ceramide failed to drive kappaB-linked chloramphenicol acetyltransferase gene expression in either HL60 promyelocytic or Jurkat T lymphoma cells. Furthermore, it had no effect on TNF-mediated transcription of this reporter gene. However, electrophoretic mobility shift analysis following cell stimulation with this ceramide analogue revealed a dose-responsive activation of NFkappaB, which wa...
http://hdl.handle.net/2262/33708
Marked
Mark
Rac1 regulates interleukin 1-induced nuclear factor kappaB activation in an inhibitory protein kappaBalpha-independent manner by enhancing the ability of the p65 subunit to transactivate gene expression
(2000)
O'NEILL, LUKE ANTHONY JOHN
Rac1 regulates interleukin 1-induced nuclear factor kappaB activation in an inhibitory protein kappaBalpha-independent manner by enhancing the ability of the p65 subunit to transactivate gene expression
(2000)
O'NEILL, LUKE ANTHONY JOHN
Abstract:
peer-reviewed
We have examined the involvement of Rac1 in nuclear factor kappaB (NFkappaB) activation by interleukin 1 (IL1). IL1 induced a rapid and sustained activation of Rac1 in the thymoma cell line EL4.NOB-1. Transient transfection with dominant negative RacN17 inhibited IL1-induced kappaB-dependent reporter gene expression but not IkappaBalpha degradation, whereas constitutively active RacV12 potentiated kappaB-dependent reporter gene expression in response to IL1 but had no effects on its own. Using porcine aortic endothelial cells stably transfected with RacV12 or RacN17 under the control of an inducible promoter, we confirmed that RacV12 did not affect IkappaBalpha degradation, nor did RacN17 inhibit the IL1-induced response. RacV12 was also unable to induce nuclear translocation of NFkappaB. These effects suggested a role for Rac1 in p65-mediated transactivation of NFkappaB, independent of IkappaBalpha regulation. In support of this we found that IL1 activated a pathw...
http://hdl.handle.net/2262/33706
Marked
Mark
Divergent roles for Ras and Rap in the activation of p38 mitogen-activated protein kinase by interleukin-1
(2000)
O'NEILL, LUKE ANTHONY JOHN; PALSSON, EVA
Divergent roles for Ras and Rap in the activation of p38 mitogen-activated protein kinase by interleukin-1
(2000)
O'NEILL, LUKE ANTHONY JOHN; PALSSON, EVA
Abstract:
peer-reviewed
We have found that lethal toxin from Clostridium sordellii, which specifically inactivates the low molecular weight G proteins Ras, Rap, and Rac, inhibits the activation of p38 mitogen-activated protein kinase (MAPK) by interleukin-1 (IL-1) in EL4.NOB-1 cells and primary fibroblasts. The target protein involved appeared to be Ras, because transient transfections with dominant negative RasN17 inhibited p38 MAPK activation by IL-1. Furthermore, transfections of cells with constitutively active RasVHa-activated p38 MAPK. Further evidence for Ras involvement came from the observation that IL-1 caused a rapid activation of Ras in the cells and from the inhibitory effects of the Ras inhibitors manumycin A and damnacanthal. Toxin B from Clostridium difficile, which inactivates Rac, Cdc42, and Rho, was without effect. Dominant negative versions of Rac (RacN17) or Rap (Rap1AN17) did not inhibit the response. Intriguingly, transfection of cells with dominant negative Rap1AN1...
http://hdl.handle.net/2262/33704
Marked
Mark
Topoisomerase II is required for mitoxantrone to signal nuclear factor kappa B activation in HL60 cells
(2000)
O'NEILL, LUKE ANTHONY JOHN
Topoisomerase II is required for mitoxantrone to signal nuclear factor kappa B activation in HL60 cells
(2000)
O'NEILL, LUKE ANTHONY JOHN
Abstract:
peer-reviewed
Topoisomerase II is a target for a number of chemotherapeutic agents used in the treatment of cancer. Its essential physiological role in modifying the topology of DNA involves the generation of transient double-strand breaks. Anti-cancer drugs, such as mitoxantrone, that target this enzyme interrupt its catalytic cycle and give rise to persistent double strand breaks, which may be lethal to a cell. We investigated the role of such lesions in signaling the activation of the transcription factor nuclear factor kappaB (NFkappaB) by this drug. Mitoxantrone activated NFkappaB and stimulated IkappaBalpha degradation in the promyelocytic leukemia cell line HL60 but not in the variant cells, HL60/MX2 cells, which lack the beta isoform of topoisomerase II and express a truncated alpha isoform that results in an altered subcellular distribution. Treatment of sensitive HL60 cells with mitoxantrone led to a depletion of both isoforms, suggesting the stabilization of transient...
http://hdl.handle.net/2262/33700
Marked
Mark
Internalin B activates nuclear factor-kappa B via Ras, phosphoinositide 3-kinase, and Akt
(2001)
O'NEILL, LUKE ANTHONY JOHN
Internalin B activates nuclear factor-kappa B via Ras, phosphoinositide 3-kinase, and Akt
(2001)
O'NEILL, LUKE ANTHONY JOHN
Abstract:
peer-reviewed
Internalin B (InlB), a 630-amino acid protein loosely attached to the surface of Listeria monocytogenes, participates in the entry of the bacterium into mammalian cells. This process requires the activation of phosphoinositide (PI) 3-kinase by InlB. Previously, we demonstrated that InlB activates the transcription factor Nuclear Factor-kappaB in murine J774 macrophage-like cells, an event that also requires PI 3-kinase. Here we have further investigated this phenomenon. InlB activated the small G-protein Ras in J774 cells. Inhibition of Ras with the farnesyltransferase inhibitor manumycin A inhibited NF-kappaB activation and the recruitment of the p85 subunit of PI 3-kinase, implying that Ras is required for PI 3-kinase activation. InlB also activated the PI 3-kinase downstream effector, Akt, as assessed by increased phosphorylation of Akt on serine 473. Transfection of Hep2 cells with dominant negative Ras N17 or dominant negative Akt inhibited the induction of a ...
http://hdl.handle.net/2262/33697
Marked
Mark
Ras participates in the activation of p38 MAPK by interleukin-1 by associating with IRAK, IRAK2, TRAF6, and TAK-1
(2002)
O'NEILL, LUKE ANTHONY JOHN
Ras participates in the activation of p38 MAPK by interleukin-1 by associating with IRAK, IRAK2, TRAF6, and TAK-1
(2002)
O'NEILL, LUKE ANTHONY JOHN
Abstract:
peer-reviewed
Interleukin-1 (IL-1) activates p38 MAP kinase via the small G protein Ras, and this activity can be down-regulated by another small G protein Rap. Here we have further investigated the role of Ras and Rap in p38 MAPK activation by IL-1. Transient transfection of cells with constitutively active forms of the known IL-1 signaling components MyD88, IRAK, and TRAF-6, or the upstream kinases MKK6 and MKK3, activated p38 MAPK. Dominant negative forms of these were found to inhibit activation of p38 MAPK by IL-1. Dominant negative RasN17 blocked the effect of the active forms of all but MKK3 and MKK6, indicating that Ras lies downstream of TRAF-6 but upstream of MKK3 and MKK6 on the pathway. Furthermore, the activation of p38 MAPK caused by overexpressing active RasVHa could not be inhibited using dominant negative mutants of MyD88, IRAK, or IRAK-2, or TRAF6, but could be inhibited by dominant negative MKK3 or MKK6. In the same manner, the inhibitory effect of Rap on the ...
http://hdl.handle.net/2262/33695
Marked
Mark
Bruton's tyrosine kinase is a Toll/interleukin-1 receptor domain-binding protein that participates in nuclear factor kappaB activation by Toll-like receptor 4
(2003)
O'NEILL, LUKE ANTHONY JOHN
Bruton's tyrosine kinase is a Toll/interleukin-1 receptor domain-binding protein that participates in nuclear factor kappaB activation by Toll-like receptor 4
(2003)
O'NEILL, LUKE ANTHONY JOHN
Abstract:
peer-reviewed
In this study we have identified members of the Toll-like receptor (TLR) family (namely, TLRs 4, 6, 8, and 9) as proteins to which the intracellular protein tyrosine kinase, Bruton's tyrosine kinase (Btk), binds. Detailed analysis of the interaction between Btk and TLR8 demonstrates that the presence of both Box 2 and 3 motifs in the Toll/interleukin-1 receptor domain was required for the interaction. Furthermore, co-immunoprecipitation experiments revealed that Btk can also interact with key proteins involved in TLR4 signal transduction, namely, MyD88, Mal (MyD88 adapter-like protein), and interleukin-1 receptor-associated kinase-1, but not TRAF-6. The ability of Btk to interact with TLR4 and Mal suggests a role for Btk in lipopolysaccharide (LPS) signal transduction. Stimulation of the human monocytic cell line THP-1 with LPS resulted in an increase in the level of tyrosine phosphorylation of Btk (indicative of activation). The autokinase activity of Btk was...
http://hdl.handle.net/2262/33693
Marked
Mark
Interferon regulatory factor-3-mediated activation of the interferon-sensitive response element by Toll-like receptor (TLR) 4 but not TLR3 requires the p65 subunit of NF-kappa
(2003)
O'NEILL, LUKE ANTHONY JOHN
Interferon regulatory factor-3-mediated activation of the interferon-sensitive response element by Toll-like receptor (TLR) 4 but not TLR3 requires the p65 subunit of NF-kappa
(2003)
O'NEILL, LUKE ANTHONY JOHN
Abstract:
peer-reviewed
Interferon regulatory factor (IRF) 3 is a transcription factor that binds the interferon-sensitive response element (ISRE) and is activated by Toll-like receptor 3 (TLR3) and TLR4. We have found that a dominant negative form of IκB kinase 2 and a mutant form of IκB, which acts as a super-repressor of NF-κB, blocked activation of the ISRE by the TLR4 ligand lipopolysaccharide but not the TLR3 ligand poly(I-C). TLR4 failed to activate the ISRE in mouse embryonic fibroblasts bearing a targeted deletion of p65, whereas the response to TLR3 in these cells was normal. The p65 subunit of NF-κB was detected in the lipopolysaccharide-activated but not poly(I-C)-activated ISRE-binding complex. Finally, p65 promoted transactivation of gene expression by IRF-3. These results therefore indicate that IRF-3-mediated activation of the ISRE by TLR4 but not TLR3 requires the p65 subunit of NF-κB.
http://hdl.handle.net/2262/33691
Marked
Mark
The murine IRAK2 gene encodes four alternatively spliced isoforms, two of which are inhibitory
(2004)
O'NEILL, LUKE ANTHONY JOHN
The murine IRAK2 gene encodes four alternatively spliced isoforms, two of which are inhibitory
(2004)
O'NEILL, LUKE ANTHONY JOHN
Abstract:
peer-reviewed
The interleukin-1 receptor-associated kinases (IRAKs) are important downstream signaling components of Toll-like receptors (TLRs). To date, four mammalian IRAKs have been found, namely IRAK-1, IRAK-2, IRAK-4, and IRAK-M. Herein, we show a detailed analysis of the genomic region encompassing the murine Irak2 gene and the molecular cloning of four isoforms of Irak2 (designated Irak2a, Irak2b, Irak2c, and Irak2d) generated by alternative splicing at the 5′-end of the gene. This alternative splicing has direct effects on the expression of the N-terminal death domain and/or inter-domain. No evidence of similar alternative splicing was found for the human IRAK2 gene. When overexpressed, Irak2a and Irak2b potentiated NF-κB activation by lipopolysaccharide. Importantly, Irak2c and Irak2d were inhibitory. The promoter for Irak2c differed from that of the other Irak2 isoforms in that it contained putative NF-κB binding sites. Lipopolysaccharide induced the expression of Irak...
http://hdl.handle.net/2262/33688
Marked
Mark
Crystal structure of the Toll/interleukin-1 receptor domain of human IL-1RAPL
(2004)
O'NEILL, LUKE ANTHONY JOHN
Crystal structure of the Toll/interleukin-1 receptor domain of human IL-1RAPL
(2004)
O'NEILL, LUKE ANTHONY JOHN
Abstract:
peer-reviewed
The Toll/interleukin-1 receptor (TIR) domain is conserved in the intracellular regions of Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs) as well as in several cytoplasmic adapter molecules. This domain has crucial roles in signal transduction by these receptors for host immune response. Here we report the crystal structure at 2.3-Å resolution of the TIR domain of human IL-1RAPL, the first structure of a TIR domain of the IL-1R superfamily. There are large structural differences between this TIR domain and that of TLR1 and TLR2. Helix αD in IL-1RAPL is almost perpendicular to its equivalent in TLR1 or TLR2. The BB loop contains a hydrogen bond unique to IL-1RAPL between Thr residues at the 8th and 10th positions. The structural and sequence diversity among these domains may be important for specificity in the signal transduction by these receptors. A dimer of the TIR domain of IL-1RAPL is observed in the crystal, although this domain is monomeric in...
http://hdl.handle.net/2262/33687
Marked
Mark
Mal interacts with tumor necrosis factor receptor-associated factor (TRAF)-6 to mediate NF-kappaB activation by toll-like receptor (TLR)-2 and TLR4
(2004)
O'NEILL, LUKE ANTHONY JOHN
Mal interacts with tumor necrosis factor receptor-associated factor (TRAF)-6 to mediate NF-kappaB activation by toll-like receptor (TLR)-2 and TLR4
(2004)
O'NEILL, LUKE ANTHONY JOHN
Abstract:
peer-reviewed
The Toll-interleukin-1 receptor domain-containing adapter Mal (MyD88 adapter-like protein) is involved in Toll-like receptor (TLR)-2 and TLR4 signal transduction. However, no studies have yet identified a function for Mal distinct from the related adapter MyD88. In this study, we have identified a putative TRAF6 interaction site in Mal but not in MyD88 and we demonstrate that Mal can be co-immunoprecipitated with TRAF6. Overexpression of MalE190A, which contains a mutation within the TRAF6-binding motif, failed to induce the expression of an NF-κB-dependent reporter gene, p65-mediated transactivation of gene expression, or activation of Jun N-terminal kinase or p42/p44 MAP kinase, which are induced with wild type Mal. MalE190A inhibited TLR2- and TLR4-mediated activation of NF-κB. These results identify a specific role for Mal in TLR-mediated signaling in regulating NF-κB-dependent gene transcription via its interaction with TRAF6.
http://hdl.handle.net/2262/33686
Marked
Mark
Human tribbles, a protein family controlling mitogen-activated protein kinase cascades
(2004)
O'NEILL, LUKE ANTHONY JOHN
Human tribbles, a protein family controlling mitogen-activated protein kinase cascades
(2004)
O'NEILL, LUKE ANTHONY JOHN
Abstract:
peer-reviewed
Control of mitogen-activated protein kinase (MAPK) cascades is central to regulation of many cellular responses. We describe here human tribbles homologues (Htrbs) that control MAPK activity. MAPK kinases interact with Trbs and regulate their steady state levels. Further, Trbs selectively regulate the activation of extracellular signal-regulated kinases, c-Jun NH2-terminal kinases, and p38 MAPK with different relative levels of activity for the three classes of MAPK observed depending on the level of Trb expression. These results suggest that Trbs control both the extent and the specificity of MAPK kinase activation of MAPK.
http://hdl.handle.net/2262/33449
Marked
Mark
Schlafen-1 causes a cell cycle arrest by inhibiting induction of cyclin D1
(2005)
O'NEILL, LUKE ANTHONY JOHN
Schlafen-1 causes a cell cycle arrest by inhibiting induction of cyclin D1
(2005)
O'NEILL, LUKE ANTHONY JOHN
Abstract:
peer-reviewed
Schlafen-1 (Slfn-1), the prototypic member of the Schlafen family of proteins, was described as an inducer of growth arrest in T-lymphocytes and causes a cell cycle arrest in NIH3T3 fibroblasts prior to the G1/S transition. How Slfn-1 exerts its effects on the cell cycle is not currently known. We report that synchronized murine fibroblasts expressing Slfn-1 do not exit G1 when stimulated with fetal calf serum, platelet-derived growth factor BB (PDGF-BB) or epidermal growth factor (EGF). The induction of cyclin D1 by these stimuli was blocked in the presence of Slfn-1 as were all downstream cell cycle processes. Overexpression of cyclin D1 in growth-arrested, Slfn-1-expressing cells induced an increase in cell growth consistent with this protein being the biological target of Slfn-1. Activation of the mitogen-activated protein kinase pathway by EGF or phorbol 12-myristate 13-acetate was unaffected by Slfn-1 expression. PDGF signaling was, however, almost completely...
http://hdl.handle.net/2262/33446
Marked
Mark
IkappaB kinase epsilon interacts with p52 and promotes transactivation via p65
(2006)
O'NEILL, LUKE ANTHONY JOHN
IkappaB kinase epsilon interacts with p52 and promotes transactivation via p65
(2006)
O'NEILL, LUKE ANTHONY JOHN
Abstract:
peer-reviewed
The members of the NF-κB transcription factor family are key regulators of gene expression in the immune response. Different combinations of NF-κB subunits not only diverge in timing to induce transcription but also recognize varying sequences of the NF-κB-binding site of their target genes. The p52 subunit is generated as a result of processing of NF-κB2 p100. Here, we demonstrate that the non-canonical IκB kinase ϵ (IKKϵ) directly interacts with p100. In a transactivation assay, IKKϵ promoted the ability of p52 to transactivate gene expression. This effect was indirect, requiring p65, which was shown to be part of the IKKϵ-p52 complex and to be phosphorylated by IKKϵ. These novel interactions reveal a hitherto unknown function of IKKϵ in the regulation of the alternative NF-κB activation pathway involving p52 and p65.
http://hdl.handle.net/2262/33441
Marked
Mark
Mass Spectrometric Analysis of the Endogenous Type I Interleukin-1 (IL-1) Receptor Signaling Complex Formed after IL-1 Binding Identifies IL-1RAcP, MyD88, and IRAK-4 as the Stable Components
(2007)
O'NEILL, LUKE ANTHONY JOHN
Mass Spectrometric Analysis of the Endogenous Type I Interleukin-1 (IL-1) Receptor Signaling Complex Formed after IL-1 Binding Identifies IL-1RAcP, MyD88, and IRAK-4 as the Stable Components
(2007)
O'NEILL, LUKE ANTHONY JOHN
Abstract:
peer-reviewed
We investigated the composition of the endogenous ligand-bound type I interleukin-1 (IL-1) receptor (IL-1RI) signaling complex using immunoprecipitation and tandem mass spectrometry. Three proteins with approximate molecular masses of 60 (p60), 36 (p36), and 90 kDa (p90) became phosphorylated after treatment with IL-1. Phosphorylation in vitro of p60 has been reported previously, but its identity was unknown. We showed using tandem mass spectrometry that p60 is identical to interleukin-1 receptor-associated kinase (IRAK)-4. MS also enabled detection of IL-1, IL-1RI, IL-1 receptor accessory protein (IL-1RAcP), and myeloid differentiation primary response protein 88 (MyD88) in the complex. The p60 protein (IRAK-4) was the earliest component of the complex to be phosphorylated. Phosphorylated IRAK-4 from the receptor complex migrated more slowly in SDS-PAGE than its unphosphorylated form as did recombinant IRAK-4 autophosphorylated in vitro. Phosphorylation was restri...
http://hdl.handle.net/2262/33436
Marked
Mark
Tyrosine phosphorylation of MyD88 adapter-like (Mal) is critical for signal transduction and blocked in endotoxin tolerance
(2008)
O'NEILL, LUKE ANTHONY JOHN
Tyrosine phosphorylation of MyD88 adapter-like (Mal) is critical for signal transduction and blocked in endotoxin tolerance
(2008)
O'NEILL, LUKE ANTHONY JOHN
Abstract:
peer-reviewed
Toll-like receptor 4 (TLR4) recognition of lipopolysaccharide triggers signalosome assembly among TLR4, sorting (e.g. MyD88 adapter-like (Mal)) and signaling (e.g. MyD88) adapters, initiating recruitment and activation of kinases, activation of transcription factors, and production of inflammatory mediators. In this study we examined whether tyrosine phosphorylation of Mal regulates its interactions with TLR4, MyD88, interleukin-1 (IL-1) receptor-associated kinase (IRAK)-2, and tumor necrosis factor receptor-associated factor (TRAF)-6 and is important for signaling. Overexpression of wild-type Mal in human embryonic kidney 293T cells induced its constitutive tyrosine phosphorylation and led to activation of p38, NF-κB, and IL-8 gene expression. Mutagenesis of Tyr-86, Tyr-106, and Tyr-159 residues within the Toll-IL-1 receptor domain impaired Mal tyrosine phosphorylation, interactions with Bruton tyrosine kinase, phosphorylation of p38, and NF-κB activation. Lipopol...
http://hdl.handle.net/2262/33431
Marked
Mark
Queuosine formation in eukaryotic tRNA occurs via a mitochondrial localized heteromeric transglycoslase.
(2009)
KELLY, VINCENT PATRICK
Queuosine formation in eukaryotic tRNA occurs via a mitochondrial localized heteromeric transglycoslase.
(2009)
KELLY, VINCENT PATRICK
Abstract:
peer-reviewed
tRNA guanine transglycosylase (TGT) enzymes are responsible for the formation of queuosine in the anticodon loop (position 34) of tRNAAsp, tRNAAsn, tRNAHis, and tRNATyr; an almost universal event in eubacterial and eukaryotic species. Despite extensive characterization of the eubacterial TGT the eukaryotic activity has remained undefined. Our search of mouse EST and cDNA data bases identified a homologue of the Escherichia coli TGT and three spliced variants of the queuine tRNA guanine transglycosylase domain containing 1 (QTRTD1) gene. QTRTD1 variant_1 (Qv1) was found to be the predominant adult form. Functional cooperativity of TGT and Qv1 was suggested by their coordinate mRNA expression in Northern blots and from their association in vivo by immunoprecipitation. Neither TGT nor Qv1 alone could complement a tgt mutation in E. coli. However, transglycosylase activity could be obtained when the proteins were combined in vitro. Confocal and immunoblot analysis sugg...
http://hdl.handle.net/2262/31426
Marked
Mark
Platelet factor 4 impairs the anticoagulant activity of activated protein C.
(2009)
HARMON, SHONA; PRESTON, ROGER; JOHNSON, JENNIFER; O'DONNELL, JAMES; NI AINLE, FION...
Platelet factor 4 impairs the anticoagulant activity of activated protein C.
(2009)
HARMON, SHONA; PRESTON, ROGER; JOHNSON, JENNIFER; O'DONNELL, JAMES; NI AINLE, FIONNUALA
Abstract:
Platelet factor 4 (PF4) is an abundant platelet -granule chemokine released following platelet activation. PF4 interacts with thrombomodulin and the -carboxyglutamic acid (Gla) domain of protein C, thereby enhancing activated protein C (APC) generation by the thrombin-thrombomodulin complex. However, the protein C Gla domain not only mediates protein C activation in vivo, but also plays a critical role in modulating the diverse functional properties of APC once generated. In this study we demonstrate that PF4 significantly inhibits APC anticoagulant activity. PF4 inhibited both protein S-dependentAPC anticoagulant function in plasma and protein S-dependent factor Va (FVa) proteolysis 3- to 5-fold, demonstrating that PF4 impairs protein S cofactor enhancement of APC anticoagulant function. Using recombinant factor Va variants FVa-R506Q/ R679Q and FVa-R306Q/R679Q, PF4 was shown to impair APC proteolysis of FVa at position Arg306 by 3-fold both in the presence and absence of protein ...
http://hdl.handle.net/2262/41143
Marked
Mark
Characterization of two protein disulphide iosmerases from the endocytic pathway of Trypanosoma brucei
(2005)
NOLAN, DEREK
Characterization of two protein disulphide iosmerases from the endocytic pathway of Trypanosoma brucei
(2005)
NOLAN, DEREK
Abstract:
Proteins from the endocytic pathway in bloodstream forms of Trypanosome brucei are modified by the addition of linear poly-N-acetyllactosamine side chains, which permits their isolation by tomato lectin affinity chromatography. Antibodies against this tomato lectin binding fraction were employed to screen a cDNA expression library from bloodstream forms of T. brucei. Two cDNAs were prominent among those selected. These cDNAs coded for two putative protein disulfide isomerases (PDIs) that respectively contained one and two double-cysteine redox-active sites and corresponded to a single domain PDI and a class 1 PDI. Assays of the purified recombinant proteins demonstrated that both proteins possess isomerase activity, but only the single domain PDI had a reducing activity. These PDIs possess a number of unusual features that distinguish them from previously characterized PDIs. The expression of both is developmentally regulated, they both co-localize with markers of the endocytic path...
http://hdl.handle.net/2262/36527
Marked
Mark
Study of the Efficacy, Biodistribution, and Safety Profile of Therapeutic Gutless Adenovirus Vectors as a Prelude to a Phase I Clinical Trial for Glioblastoma
(2010)
Muhammad, AKM Ghulam; Puntel, Mariana; Candolfi, Marianela; Salem, A; Yagiz, Kader; Far...
Study of the Efficacy, Biodistribution, and Safety Profile of Therapeutic Gutless Adenovirus Vectors as a Prelude to a Phase I Clinical Trial for Glioblastoma
(2010)
Muhammad, AKM Ghulam; Puntel, Mariana; Candolfi, Marianela; Salem, A; Yagiz, Kader; Farrokhi, C; Kroeger, Kurt M; Xiong, Weidong; Curtin, James; Liu, Chunyan; Lawrence, K; Bondale, Niyati; Lerner, Jonathan; Baker, G; Foulad, David; Pechnick, Robert; Palmer, Donna; Ng, Philip; Lowenstein, Pedro; Castro, Maria
Abstract:
Glioblastoma multiforme (GBM) is the most common and most aggressive primary brain tumor in humans. Systemic immunity against gene therapy vectors has been shown to hamper therapeutic efficacy; however, helper-dependent high-capacity adenovirus (HC-Ad) vectors elicit sustained transgene expression, even in the presence of systemic anti-adenoviral immunity. We engineered HC-Ads encoding the conditional cytotoxic herpes simplex type 1 thymidine kinase (TK) and the immunostimulatory cytokine fms-like tyrosine kinase ligand 3 (Flt3L). Flt3L expression is under the control of the regulatable Tet-ON system. In anticipation of a phase I clinical trial for GBM, we assessed the therapeutic efficacy, biodistribution, and clinical and neurotoxicity with escalating doses of HC-Ad-TetOn-Flt3L + HC-Ad-TK in rats. Intratumoral administration of these therapeutic HC-Ads in rats bearing large intracranial GBMs led to long-term survival in ~70% of the animals and development of antiglioma immunologic...
http://arrow.dit.ie/scschbioart/53
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