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Nuchprayoon, I., S. Meyers, L. M. Scott, J. Suzow, S. Hiebert i A. D. Friedman. "PEBP2/CBF, the murine homolog of the human myeloid AML1 and PEBP2 beta/CBF beta proto-oncoproteins, regulates the murine myeloperoxidase and neutrophil elastase genes in immature myeloid cells." Molecular and Cellular Biology 14, nr 8 (sierpień 1994): 5558–68. http://dx.doi.org/10.1128/mcb.14.8.5558.
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The myeloperoxidase (MPO) and neutrophil elastase genes are expressed specifically in immature myeloid cells. The integrity of a polyomavirus enhancer core sequence, 5'-AACCACA-3', is critical to the activity of the murine MPO proximal enhancer. This element binds two species, myeloid nuclear factors 1 alpha and 1 beta (MyNF1 alpha and -beta), present in 32D cl3 myeloid cell nuclear extracts. The levels of the MyNF1s increase during early 32D cl3 cell granulocytic differentiation. Both MyNF1 alpha and -beta supershift with an antiserum raised by using a peptide derived from the N terminus of polyomavirus enhancer-binding protein 2/core-binding factor (PEBP2/CBF) alpha subunit. The specific peptide inhibits these supershifts. In vitro-translated PEBP2/CBF DNA-binding domain binds the murine MPO PEBP2/CBF site. An alternate PEBP2/CBF consensus site, 5'-GACCGCA-3', but not a simian virus 40 enhancer core sequence, 5'-TTCCACA-3', binds the MyNF1s in vitro and activates a minimal murine MPO-thymidine kinase promoter in vivo. The murine neutrophil elastase gene 100-bp 5'-flanking sequences contain several functional elements, including potential binding sites for PU.1, C/EBP, c-Myb, and PEBP2/CBF. The functional element 5'-GGCCACA-3' located at positions -66 to 72 differs from the PEBP2/CBF consensus (5'-PuACCPuCA-3') only by an A-to-G transition at position 2. This DNA element binds MyNF1 alpha and -beta weakly. The N terminis of two PEBP2/CBF alpha subunit family members, PEBP2 alpha A and PEBP2 alpha B (murine AML1), are nearly identical, and 32D c13 cl3 cells contain both corresponding mRNAs. Since t(8;21), t(3;21), and inv(16), associated with myeloid leukemias, disrupt subunits of PEBP2/CBF, we speculate that the resulting oncoproteins, AML1-ETO, AML1-EAP, AML1-Evi1, and CBF beta-MYH11, inhibit early myeloid differentiation.
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Nuchprayoon, I., S. Meyers, L. M. Scott, J. Suzow, S. Hiebert i A. D. Friedman. "PEBP2/CBF, the murine homolog of the human myeloid AML1 and PEBP2 beta/CBF beta proto-oncoproteins, regulates the murine myeloperoxidase and neutrophil elastase genes in immature myeloid cells". Molecular and Cellular Biology 14, nr 8 (sierpień 1994): 5558–68. http://dx.doi.org/10.1128/mcb.14.8.5558-5568.1994.
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The myeloperoxidase (MPO) and neutrophil elastase genes are expressed specifically in immature myeloid cells. The integrity of a polyomavirus enhancer core sequence, 5'-AACCACA-3', is critical to the activity of the murine MPO proximal enhancer. This element binds two species, myeloid nuclear factors 1 alpha and 1 beta (MyNF1 alpha and -beta), present in 32D cl3 myeloid cell nuclear extracts. The levels of the MyNF1s increase during early 32D cl3 cell granulocytic differentiation. Both MyNF1 alpha and -beta supershift with an antiserum raised by using a peptide derived from the N terminus of polyomavirus enhancer-binding protein 2/core-binding factor (PEBP2/CBF) alpha subunit. The specific peptide inhibits these supershifts. In vitro-translated PEBP2/CBF DNA-binding domain binds the murine MPO PEBP2/CBF site. An alternate PEBP2/CBF consensus site, 5'-GACCGCA-3', but not a simian virus 40 enhancer core sequence, 5'-TTCCACA-3', binds the MyNF1s in vitro and activates a minimal murine MPO-thymidine kinase promoter in vivo. The murine neutrophil elastase gene 100-bp 5'-flanking sequences contain several functional elements, including potential binding sites for PU.1, C/EBP, c-Myb, and PEBP2/CBF. The functional element 5'-GGCCACA-3' located at positions -66 to 72 differs from the PEBP2/CBF consensus (5'-PuACCPuCA-3') only by an A-to-G transition at position 2. This DNA element binds MyNF1 alpha and -beta weakly. The N terminis of two PEBP2/CBF alpha subunit family members, PEBP2 alpha A and PEBP2 alpha B (murine AML1), are nearly identical, and 32D c13 cl3 cells contain both corresponding mRNAs. Since t(8;21), t(3;21), and inv(16), associated with myeloid leukemias, disrupt subunits of PEBP2/CBF, we speculate that the resulting oncoproteins, AML1-ETO, AML1-EAP, AML1-Evi1, and CBF beta-MYH11, inhibit early myeloid differentiation.
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Steensma, David P., Richard J. Gibbons, Ruben A. Mesa, Ayalew Tefferi i Douglas R. Higgs. "Somatic Point Mutations in RUNX1/CBFA2/AML1 Are Common in High-Risk Myelodysplastic Syndrome, but Not in Myelofibrosis with Myeloid Metaplasia." Blood 104, nr 11 (16.11.2004): 2438. http://dx.doi.org/10.1182/blood.v104.11.2438.2438.
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Abstract Introduction: The core-binding factor subunit RUNX1/CBFA2/AML1 (Runt-related transcription factor 1, Core-binding factor alpha 2 subunit, Oncogene AML1) is critical for generation of hematopoietic stem cells during embryogenesis and for normal megakaryopoiesis in adults. RUNX1/CBFA2/AML1 has long been recognized as an oncogene, and is frequently involved in leukemia-associated translocations that create aberrant fusion proteins (e.g., AML1-ETO). Recently, acquired somatic point mutations in the RUNX1/CBFA2/AML1 gene have been described in a subset of patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). These point mutations are associated with the loss of normal RUNX1 trans-activation potential and/or altered binding to the non-DNA-binding subunit of core-binding factor, CBFB. Given the importance of core-binding factor in megakaryocytic differentiation and platelet production and the central role of megakaryocytes in the pathophysiology of myelofibrosis with myeloid metaplasia (MMM), and in light of the myelofibrotic phenotype of GATA-1low mice (GATA-1 and RUNX1 are co-expressed and co-operate in megakaryocytic differentiation), we hypothesized that RUNX1 gene mutations might be common in MMM. In addition, we wanted to know whether patients with MDS-associated acquired alpha thalassemia (i.e., ATMDS), a special MDS subgroup with a very high incidence of point mutations in the X-linked ATRX gene, have an especially high incidence of RUNX1 mutations that might account for the more severe hematopoietic defect found in these patients compared with boys with inherited ATR-X syndrome (detailed in Steensma et al, Blood2004;103:2019). Methods and Results: We analyzed samples from 26 well-characterized patients with MMM and from 52 with MDS (11 RA, 3 RARS, 8 RCMD, 3 RAEB-1, 7 RAEB-2, 2 MDS in transition to AML-M6, 14 atypical/unclassifable MDS, 4 MDS subtype unknown - including 18 with ATMDS) for RUNX1 point mutations by denaturing high-performance liquid chromatography (DHPLC) followed by subcloning and sequencing of samples exhibiting heteroduplex peaks. We found 5 RUNX1 mutations in MDS patients (9.6%), all of whom had RAEB-2 (4 patients) or a history of treated AML (1 patient), but detected no mutations in MMM patients. All patients with RUNX1 mutations progressed to overt leukemia within 1 year. ATMDS patients did not have an increased risk of RUNX1 point mutations (2/18 patients with mutations, 11.1%) when compared with MDS without thalassemia (3/34 patients, 8.8%; p=0.58). Additionally, 1 MDS patient had a clonally restricted mutation in GATA-1 (c.1066 C>T) (also screened by DHPLC), but this is not predicted to change the GATA-1 amino acid sequence and is likely a random event reflecting the genomic instability characteristic of MDS. Conclusion: RUNX1 point mutations are common in high-risk MDS, but not in MMM, and that ATMDS patients do not have a risk of RUNX1 mutations in excess of that expected for MDS in general.
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Hamey, Fiona K., Sonia Nestorowa, Sarah J. Kinston, David G. Kent, Nicola K. Wilson i Berthold Göttgens. "Reconstructing blood stem cell regulatory network models from single-cell molecular profiles". Proceedings of the National Academy of Sciences 114, nr 23 (5.06.2017): 5822–29. http://dx.doi.org/10.1073/pnas.1610609114.
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Adult blood contains a mixture of mature cell types, each with specialized functions. Single hematopoietic stem cells (HSCs) have been functionally shown to generate all mature cell types for the lifetime of the organism. Differentiation of HSCs toward alternative lineages must be balanced at the population level by the fate decisions made by individual cells. Transcription factors play a key role in regulating these decisions and operate within organized regulatory programs that can be modeled as transcriptional regulatory networks. As dysregulation of single HSC fate decisions is linked to fatal malignancies such as leukemia, it is important to understand how these decisions are controlled on a cell-by-cell basis. Here we developed and applied a network inference method, exploiting the ability to infer dynamic information from single-cell snapshot expression data based on expression profiles of 48 genes in 2,167 blood stem and progenitor cells. This approach allowed us to infer transcriptional regulatory network models that recapitulated differentiation of HSCs into progenitor cell types, focusing on trajectories toward megakaryocyte–erythrocyte progenitors and lymphoid-primed multipotent progenitors. By comparing these two models, we identified and subsequently experimentally validated a difference in the regulation of nuclear factor, erythroid 2 (Nfe2) and core-binding factor, runt domain, alpha subunit 2, translocated to, 3 homolog (Cbfa2t3h) by the transcription factor Gata2. Our approach confirms known aspects of hematopoiesis, provides hypotheses about regulation of HSC differentiation, and is widely applicable to other hierarchical biological systems to uncover regulatory relationships.
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Qi, Xiangjun, Hongbin Xu, Peng Zhang, Guoming Chen, Zhiqiang Chen, Caishan Fang i Lizhu Lin. "Investigating the Mechanism of Scutellariae barbata Herba in the Treatment of Colorectal Cancer by Network Pharmacology and Molecular Docking". Evidence-Based Complementary and Alternative Medicine 2021 (2.08.2021): 1–18. http://dx.doi.org/10.1155/2021/3905367.
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Background. Colorectal cancer (CRC) is one of the most common gastrointestinal tumors, which accounts for approximately 10% of all diagnosed cancers and cancer deaths worldwide per year. Scutellariae barbatae Herba (SBH) is one of the most frequently used traditional Chinese medicine (TCM) in the treatment of CRC. Although many experiments have been carried out to explain the mechanisms of SBH, the mechanisms of SBH have not been illuminated fully. Thus, we constructed a network pharmacology and molecular docking to investigate the mechanisms of SBH. Methods. We adopted active constituent prescreening, target predicting, protein-protein interaction (PPI) analysis, Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, differentially expressed gene analysis, and molecular docking to establish a system pharmacology database of SBH against CRC. Results. A total of 64 active constituents of SBH were obtained and 377 targets were predicted, and the result indicated that quercetin, luteolin, wogonin, and apigenin were the main active constituents of SBH. Glucocorticoid receptor (NR3C1), pPhosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (PIK3CA), cellular tumor antigen p53 (TP53), transcription factor AP-1 (JUN), mitogen-activated protein kinase 1 (MAPK1), Myc protooncogene protein (MYC), cyclin-dependent kinase 1 (CDK1), and broad substrate specificity ATP-binding cassette transporter ABCG2 (ABCG2) were the major targets of SBH in the treatment of CRC. GO analysis illustrated that the core biological process regulated by SBH was the regulation of the cell cycle. Thirty pathways were presented and 8 pathways related to CRC were involved. Molecular docking presented the binding details of 3 key targets with 6 active constituents. Conclusions. The mechanisms of SBH against CRC depend on the synergistic effect of multiple active constituents, multiple targets, and multiple pathways.
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Cammenga, Jorg, Gabriela Putz, Birte Niebuhr, Stefan Horn, Ulla Bergholz, Frank Buchholz i Carol Stocking. "The Role of RUNX1 DNA-Binding Mutations in Acute Myeloid Leukemia." Blood 106, nr 11 (16.11.2005): 1373. http://dx.doi.org/10.1182/blood.v106.11.1373.1373.
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Abstract The RUNX1 gene encodes an alpha subunit of the core-binding factor (CBF), an important heterodimeric transcription factor in hematopoietic ontogeny and development, and is one of the most frequently disrupted genes in acute leukemia. In addition to its involvement in several translocations, the RUNX1 gene is often subject to deletions or point mutations in acute myelogenous leukemia (AML). Interestingly, in addition to complete loss-of-function mutations, many of the alterations involve missense point mutations within the Runt domain that disrupt DNA binding activity (DB-mutants). In vitro assays have suggested that these DB mutants have a dominant-negative (DN) activity, presumably due to their ability to bind and sequester CBF beta but inability to bind DNA. A strict correlation between the type of mutation and its monoallelic or biallelic incidence is not apparent even though a DN mutant should only affect one allele while a loss of function mutation should affect both alleles. It has been hypothesized that loss of one allele (haploinsufficiency) is sufficient for loss of tumor suppressor activity but the relative high incidence of specific DB mutations suggests a more complex scenario. We thus sought to determine if expression of DB mutants in murine bone marrow (BM) resulted in a similar phenotype as the loss of Runx1, or if these mutations are associated with a gain-of-function. Two RUNX1-DB mutants were thus evaluated using the established retroviral transduction/transplantation mouse model. Between 3 and 6 months after transplantation, peripheral blood, spleen and BM cells were analyzed. Long-term repopulating cells expressing RUNX1 DB-mutants were able to contribute normally to both myeloid and lymphoid compartments, although a disproportionate increase in the B-cell compartment was observed in 3 out of 10 mice. Surprisingly, and inconsistent with a DN activity, disruption of normal T-cell or megakaryocytic development was not observed in the mice, in contrast to Runx1−/+ mice. Significantly, however, replating assays in vitro demonstrated that RUNX1-DB mutants lead to a significant increase in self-renewal activity, in contrast to BM cells of floxed Runx1 mice expressing the Cre recombinase, which showed a less dramatic effect on self-renewal. Colonies derived from CFU-Cs expressing RUNX1-DB mutants were composed of dysplastic granulocytic and monocytic cells, with an increasing number of immature blasts after multiple replatings (>7), whereas residual colonies from Runx1fl/− BM receiving CRE showed a different morphology with more mature cells. Thus our data suggest that RUNX1-DB mutants do not act in a dominant negative fashion to inhibit normal RUNX1 function, but impart a gain-of-function that results in impaired myeloid differentiation and increased self-renewal potential, consistent with its association with AML.
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Fang, Yue, Xinquan Liu i Jing Su. "Network Pharmacology Analysis of Traditional Chinese Medicine Formula Shuang Di Shou Zhen Tablets Treating Nonexudative Age-Related Macular Degeneration". Evidence-Based Complementary and Alternative Medicine 2021 (24.03.2021): 1–14. http://dx.doi.org/10.1155/2021/6657521.
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Objective. To analyze the pharmacological mechanism of the treatment of dry age-related macular degeneration (dry AMD) based on a network pharmacological approach of Shuang Di Shou Zhen Tablets (SDSZT) and to provide a new reference for the current lack of effective treatment of dry AMD. Methods. The main chemical constituents and their targets of Rehmanniae Radix Praeparata, Ligustrum lucidum, Mori Fructus, Paeonia albiflora, Rhizoma Dioscoreae, Alisma orientale, Schisandra chinensis, Radix Polygoni Multiflori Preparata, Ophiopogon japonicus, and Radix Rehmanniae were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and Traditional Chinese Medicine Integrated Database (TCMID). The active ingredients of traditional Chinese medicine were screened according to Absorption, Distribution, Metabolism, and Excretion (ADME), the gene names of the targets of each active ingredient were obtained from the Uniprot database, the main targets of dry AMD were obtained from GeneCards and DisGeNET database, and the protein interaction analysis was performed on the String database. The Metascape database was used to analyze the “drug-component-target” and the biological processes and networks involved, and then, Cytoscape 3.8.1 was used to construct the “ SDSZT component-dry AMD target-pathway” network. Results. The main active ingredients of SDSZT for dry AMD treatment are quercetin, kaempferol, luteolin, β-glutamine, β-carotene, etc. And, the core targets are RAC-alpha serine/threonine-protein kinase (AKT1), prostaglandin G/H synthase 1 (PTGS1), tumor necrosis factor (TNF), transcription factor AP-1 (JUN), apoptosis regulator Bcl-2 (BCL2), caspase-3 (CASP3), phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (PIK3CG), androgen receptor (AR), apoptosis regulator BAX (BAX), etc. The biological pathways for the treatment of age-related macular degeneration by SDSZT mainly act on pathways in cancer, fluid shear stress and atherosclerosis, and TNF signaling pathway, and the main function of SDSZT is to regulate intracellular cytokine receptor binding. Conclusion. This study initially reveals the multiconstituent, multitarget, and multipathway mechanism of action of SDSZT in the treatment of dry AMD and provides the basis for the clinical application of SDSZT.
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Burda, Pavel, Nikola Curik, Juraj Kokavec, Dana Mikulenkova, Arthur Skoultchi, Jiri Zavadil i Tomas Stopka. "PU.1 Relieves Its GATA-1-Mediated Repression near Cebpa and Cbfb During Transdifferentiation of Murine Erythroleukemia - Tool of Inducing Leukemic Blasts to Differentiate." Blood 114, nr 22 (20.11.2009): 547. http://dx.doi.org/10.1182/blood.v114.22.547.547.
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Abstract Abstract 547 Transcription factors GATA-1 and PU.1 interact on DNA to block transcriptional programs of undesired lineage during hematopoietic commitment. Murine erythroleukemia (MEL) cells that co-express GATA-1 and PU.1 are blocked at the blast stage but respond to down-regulation of PU.1 or up-regulation of GATA-1 by inducing terminal erythroid differentiation. To test whether GATA-1 blocks PU.1 in MEL cells we have conditionally activated a transgenic PU.1 protein fused with the estrogen receptor ligand-binding domain (PUER), resulting in activation of a myeloid transcriptional program. Gene expression arrays identified components of the PU.1-dependent transcriptome negatively regulated by GATA-1 in MEL cells, including CCAAT/enhancer binding protein (C/EBP) alpha (Cebpa) and Core-binding factor, beta subunit (Cbfb) that encode two key hematopoietic transcription factors. Inhibition of GATA-1 by siRNA resulted in derepression of PU.1 target genes. Chromatin immunoprecipitation and reporter assays identified PU.1 motif sequences near Cebpa and Cbfb co-occupied by PU.1 and GATA-1 in the leukemic blasts. Substantial derepression of Cebpa and Cbfb is achieved in MEL cells by either activation of PU.1 or knockdown of GATA-1. Furthermore, transcriptional regulation of these loci by manipulating the levels of PU.1 and GATA-1 involves quantitative increases in a transcriptionally active chromatin mark: acetylation of histone H3K9. Collectively, we demonstrate that either activation of PU.1 or inhibition of GATA-1 efficiently reverse the transcriptional block imposed by GATA-1 and lead to activation of a myeloid transcriptional program directed by PU.1. The mechanism of PU.1 and GATA-1 in leukemic state and upon leukemia differentiation involves the following putative steps: at myeloid genes such as Cebpa, PU.1 binds directly to DNA but is repressed by GATA1 that binds directly to PU.1 molecules on DNA. Activation of PU.1-ER and stable levels of GATA-1 create excess of availabel PU.1, which is not paired by availabel GATA-1 on DNA, allowing thus gene activation. Similarly, on erythroid genes such as Nfe2, GATA1 is bound to DNA, but is repressed by PU.1 that binds to this GATA-1 molecule. Activation of GATA1-ER creates an excess of availabel GATA-1 which is not paired on DNA by availabel PU.1, also allowing gene activation. Our mechanistic study implicates that transcription factor manipulation, such as inhibition of GATA-1 or activation of PU.1 in erythroleukemias, may represent an efficient tool of inducing leukemic blasts to differentiate. (Grants NR9021-4, 10310-3, 2B06077) Disclosures: No relevant conflicts of interest to declare.
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Stopka, Tomas, Pavel Burda, Petra Basova, Karin Vargova, Nikola Curik, Anna Jonasova i Marek Trneny. "5-Azacytidine and G-CSF Derepressed Chromatin Structure of PU.1 and Its Targets Cebpa and Cbfb In Myelodysplastic Syndrome (MDS)". Blood 116, nr 21 (19.11.2010): 124. http://dx.doi.org/10.1182/blood.v116.21.124.124.
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Abstract Abstract 124 The myelodysplastic syndrome (MDS) represents a heterogeneous disorder characterized by ineffective hematopoiesis and evolution to acute myelogenous leukemia that is strikingly refractory to current therapeutic approaches. Novel epigenetic drugs including DNA-methyltransferase inhibitor 5-Azacitidine (5-AZA, Vidaza) are currently considered to improve clinical response in patients with MDS. MDS is characterized by abnormal differentiation and blocked maturation responsive to 5-AZA, therefore we studied major regulator of hematopoietic differentiation, transcription factor PU.1 as a candidate target of the epigenetic therapy. Transcription factor PU.1 represents very important myelo-lymphoid regulator of differentiation. PU.1 expression is regulated by Upstream Regulatory Element (URE) and its deletion in mouse caused downregulation of PU.1 leading to acute leukemia (Rosenbauer 2004). Our laboratory recently demonstrated that PU.1 in murine acute leukemic cells binds and promotes derepression of CCAAT/enhancer binding protein (C/EBP) alpha (Cebpa) and Core-binding factor, beta subunit (Cbfb) (Burda 2009) that encode two key hematopoietic transcription factors involved in myeloid differentiation. Furthermore, transcriptional regulation through PU.1 binding sites of Cebpa and Cbfb loci involves quantitative increases in a transcriptionally active chromatin mark: acetylation of histone H3 lysine K9. Others reported that Cebpa expression is augmented by G-CSF (Dahl 2003). To determine if 5-AZA regulates PU.1 and its targets we determined their expression and chromatin structure following the 5-AZA treatment in MDS patient-derived blasts and in cell lines derived from MDS (MOLM-13, OCI-M2, SKM-1) and AML (K562). Our data provide evidence that in the chosen cell lines and in so far limited number of patients-derived cells (N=4) the gene expression of PU.1 and its direct targets Cebpa and Cbfb is stimulated by 5-AZA and this effect is further enhanced by G-CSF. Furthermore, marks of activated chromatin structure including histone H3K9 hyperacetylation and H3K4 hypermethylation are increased at the URE of the PU.1 gene again documenting its transcriptional activation. Conversely, levels of H3K9 methylation at URE are significantly reduced upon 5-AZA treatment documenting 5-AZA stimulates loss of repressive chromatin structure near PU.1 gene. These observations are currently compared with responsiveness of the patients to 5-AZA in vivo and expanded to larger set of patients. Our data collectively supports importance of the chromatin structure upstream of PU.1 gene and of its direct targets Cebpa and Cbfb in patients with MDS that may add to better understanding of effectiveness of epigenetic therapy in MDS. (Grants # IGA 10310-3, MSMT 2B06077, SVV-2010-254260507, MPO FR-TI2/509, GAUK 251135 82210). Disclosures: No relevant conflicts of interest to declare.
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Kutny, Matthew A., Todd A. Alonzo, Robert B. Gerbing, Kristen Miller, Phoenix Ho, Nyla A. Heerema, Beverly Lange, Alan S. Gamis i Soheil Meshinchi. "RUNX1 Mutations in Pediatric AML: A Report From the Children's Oncology Group." Blood 114, nr 22 (20.11.2009): 2614. http://dx.doi.org/10.1182/blood.v114.22.2614.2614.
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Abstract Abstract 2614 Poster Board II-590 The RUNX1 gene (previously AML1) encodes the alpha subunit of core binding factor (CBFα), which is implicated in normal and malignant hematopoiesis. Translocations involving RUNX1 have been associated with favorable prognosis in acute leukemias (e.g., t(8;21) in AML and t(12;21) in ALL). Point mutations of RUNX1 have been identified in exon 3 and exon 8 of the RUNX1 gene in a subset of AML patients and are most closely associated with MDS associated AML and AML subtype M0. We evaluated the prevalence and prognostic significance of RUNX1 mutations in pediatric patients with de novo AML treated on pediatric AML trials CCG-2941 and CCG-2961. Initial evaluation of exon 3 and exon 8 of the RUNX1 gene was conducted on a cohort of 100 randomly selected patient specimens. In this initial analysis, we identified 4 missense mutations of exon 3 that caused a change in codon 56. No exon 8 mutations were identified. Subsequent molecular genotyping of the remaining 484 patient specimens were limited to exon 3. Of the 584 diagnostic specimens tested, a missense mutation of exon 3 was detected in 19 patients (3.3%). All detected mutations occurred at nucleotide 167 (T to C) causing a leucine to serine substitution at codon 56 (L56S). Additionally, two patients had a synonymous mutation (G to A at nucleotide 183, P61P) and were considered wild type. Demographics, laboratory characteristics and clinical outcome were compared between those with and without RUNX1 mutations. There was no significant difference with regard to age, gender or race between these groups. Those with RUNX1 mutations had a significantly lower prevalence of organomegaly (16% vs. 42%, p=0.02) and significantly higher rate of extramedullary disease (chloroma 26% vs. 10%, p=0.04). There was no significant difference in association with other known cytogenetic abnormalities or risk groupings (standard, low, or high risk grouping on these studies). t(8;21) translocations were detected in 22% of those with RUNX1 mutations compared to 16% in those without mutation (p=0.6), and there was no RUNX1 mutation detected in patients with inv(16). Of the 85 patients with FLT3/ITD, NPM1 or CEBPA mutations, only 3 patients had a concomitant RUNX1 mutation (2 with FLT3/ITD and 1 with NPM1 mutation). Remission induction rate was compared between patients with and without RUNX1 mutations. Those with RUNX1 mutations had a similar CR rate to those without mutations (89% vs. 79%, p=0.4). Overall survival from remission for patients with and without RUNX1 mutation was 39 ± 25% and 60 ± 5% respectively (p=0.1) with a corresponding disease-free survival of 34 ± 24% vs. 49 ± 5% (p=0.25). RUNX1 mutations may represent a biologically distinct group that presents in 3.3% of pediatric AML patients across different morphologic and cytogenetic populations. Alterations of RUNX1 affect signal transduction pathways and may be exploited in defining a population for directed and risk based therapy. Disclosures: No relevant conflicts of interest to declare.
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Scott, C. D., i R. C. Baxter. "Synthesis of the acid-labile subunit of the growth-hormone-dependent insulin-like-growth-factor-binding protein complex by rat hepatocytes in culture". Biochemical Journal 275, nr 2 (15.04.1991): 441–46. http://dx.doi.org/10.1042/bj2750441.
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Insulin-like growth factors (IGFs) circulate predominantly in a growth-hormone-dependent ternary complex of 125-150 kDa. This study investigates the production of the alpha-subunit of this complex, an acid-labile glycoprotein without intrinsic IGF-binding activity, which binds to the IGF-binding protein IGFBP-3 in the presence of IGFs. Medium conditioned by primary cultures of rat hepatocytes produced alpha-subunit with similar complex-forming activity to purified rat serum alpha-subunit. Bovine growth hormone stimulated hepatocyte production of both IGF-I and alpha-subunit. IGF-I tracer bound to pure rat IGFBP-3 was converted from approx. 60 kDa to 150 kDa by serum alpha-subunit, whole rat serum or rat hepatocyte culture medium; this converting activity was destroyed by transient acidification. In contrast, IGF-I bound to hepatocyte-medium IGF-binding proteins could not be converted into a high-molecular-mass from by purified rat serum alpha-subunit. Rat serum and hepatocyte-medium alpha-subunit appeared identical by electrophoretic analysis, since reaction of either with cross-linked IGF-I.IGFBP-3 tracer resulted in bands of molecular mass 130 kDa and 160 kDa, probably representing intact and partially deglycosylated complexes. However, IGF-binding proteins in rat serum and hepatocyte medium were different, in that affinity labelling of medium binding proteins, depleted of endogenous IGFs, showed no evidence of the 50-60 kDa cluster of bands characteristic of rat serum IGFBP-3. We conclude that rat hepatocytes in primary culture produce alpha-subunit similar to that in rat serum; however, alpha-subunit is unable to form ternary complexes with hepatocyte IGF-binding proteins, since cultured hepatocytes do not secrete IGFBP-3.
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Baxter, R. C. "Glycosaminoglycans inhibit formation of the 140 kDa insulin-like growth factor-binding protein complex". Biochemical Journal 271, nr 3 (1.11.1990): 773–77. http://dx.doi.org/10.1042/bj2710773.
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The 140 kDa insulin-like growth factor (IGF)-binding protein complex in human serum consists of three subunits: an acid-labile, non-IGF-binding glycoprotein (alpha-subunit), an IGF-binding glycoprotein known as BP-53 or IGFBP-3 (beta-subunit), and IGF-I or IGF-II (gamma-subunit). This study investigates the regulation, by salt and glycosaminoglycans, of ternary (alpha-beta-gamma) complex formation, measured by incubating radioiodinated alpha-subunit with a mixture of IGF-I and IGFBP-3 and precipitating bound radioactivity with an anti-IGFBP-3 antiserum. Increasing NaCl concentrations progressively decreased ternary complex formation without any effect on binary (beta-gamma) complex formation. In 0.15 M-NaCl, the association constant for the ternary complex was 0.318 +/- 0.092 nM-1, 100-fold lower than that for the binary complex. Glycosaminoglycans also inhibited ternary complex formation without affecting the binary complex. Heparin [50% inhibition at 0.27 +/- 0.08 units/ml (1.5 +/- 0.4 micrograms/ml)] was more potent than heparan sulphate (50% inhibition at 15 +/- 7 micrograms/ml), with chondroitin sulphate even less potent. The inhibition by heparin was due principally to a decrease in binding affinity, from 0.604 +/- 0.125 to 0.151 +/- 0.024 nM-1 in the presence of 0.25 units of heparin/ml, with a slight decrease in the number of apparent binding sites from 1.05 +/- 0.08 to 0.85 +/- 0.15 mol of alpha-subunit bound/mol of beta-subunit. Since the ternary IGF-binding protein complex cannot cross the capillary barrier, it is proposed that a decrease in the affinity of the complex, mediated by circulating or cell-associated glycosaminoglycans, may be important in the passage of IGFs and IGFBP-3 to the tissues.
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Park, Kyung-Ran, Joon-Yeop Lee, Myounglae Cho, Jin-Tae Hong i Hyung-Mun Yun. "Paeonolide as a Novel Regulator of Core-Binding Factor Subunit Alpha-1 in Bone-Forming Cells". International Journal of Molecular Sciences 22, nr 9 (6.05.2021): 4924. http://dx.doi.org/10.3390/ijms22094924.
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Paeonia suffruticosa has been extensively used as a traditional medicine with various beneficial effects; paeonolide (PALI) was isolated from its dried roots. This study aimed to investigate the novel effects and mechanisms of PALI in pre-osteoblasts. Here, cell viability was evaluated using an MTT assay. Early and late osteoblast differentiation was examined by analyzing the activity of alkaline phosphatase (ALP) and by staining it with Alizarin red S (ARS). Cell migration was assessed using wound healing and Boyden chamber assays. Western blot and immunofluorescence analyses were used to examine the intracellular signaling pathways and differentiation proteins. PALI (0.1, 1, 10, 30, and 100 μM) showed no cytotoxic or proliferative effects in pre-osteoblasts. In the absence of cytotoxicity, PALI (1, 10, and 30 μM) promoted wound healing and transmigration during osteoblast differentiation. ALP staining demonstrated that PALI (1, 10, and 30 μM) promoted early osteoblast differentiation in a dose-dependent manner, and ARS staining showed an enhanced mineralized nodule formation, a key indicator of late osteoblast differentiation. Additionally, low concentrations of PALI (1 and 10 μM) increased the bone morphogenetic protein (BMP)–Smad1/5/8 and Wnt–β-catenin pathways in osteoblast differentiation. Particularly, PALI (1 and 10 μM) increased the phosphorylation of ERK1/2 compared with BMP2 treatment, an FDA-approved drug for bone diseases. Furthermore, PALI-mediated early and late osteoblast differentiation was abolished in the presence of the ERK1/2 inhibitor U0126. PALI-induced RUNX2 (Cbfa1) expression and nuclear localization were also attenuated by blocking the ERK1/2 pathway during osteoblast differentiation. We suggest that PALI has biologically novel activities, such as enhanced osteoblast differentiation and bone mineralization mainly through the intracellular ERK1/2-RUNX2 signaling pathway, suggesting that PALI might have therapeutic action and aid the treatment and prevention of bone diseases, such as osteoporosis and periodontitis.
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Beghini, Alessandro. "Core Binding Factor Leukemia: Chromatin Remodeling Moves Towards Oncogenic Transcription". Cancers 11, nr 12 (7.12.2019): 1973. http://dx.doi.org/10.3390/cancers11121973.
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Acute myeloid leukemia (AML), the most common acute leukemia in adults, is a heterogeneous malignant clonal disorder arising from multipotent hematopoietic progenitor cells characterized by genetic and concerted epigenetic aberrations. Core binding factor-Leukemia (CBFL) is characterized by the recurrent reciprocal translocations t(8;21)(q22;q22) or inv(16)(p13;q22) that, expressing the distinctive RUNX1-RUNX1T1 (also known as Acute myeloid leukemia1-eight twenty-one, AML1-ETO or RUNX1/ETO) or CBFB-MYH11 (also known as CBFβ-SMMHC) translocation product respectively, disrupt the essential hematopoietic function of the CBF. In the past decade, remarkable progress has been achieved in understanding the structure, three-dimensional (3D) chromosomal topology, and disease-inducing genetic and epigenetic abnormalities of the fusion proteins that arise from disruption of the CBF subunit alpha and beta genes. Although CBFLs have a relatively good prognosis compared to other leukemia subtypes, 40–50% of patients still relapse, requiring intensive chemotherapy and allogenic hematopoietic cell transplantation (alloHCT). To provide a rationale for the CBFL-associated altered hematopoietic development, in this review, we summarize the current understanding on the various molecular mechanisms, including dysregulation of Wnt/β-catenin signaling as an early event that triggers the translocations, playing a pivotal role in the pathophysiology of CBFL. Translation of these findings into the clinical setting is just beginning by improvement in risk stratification, MRD assessment, and development of targeted therapies.
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McGlade, C. J., C. Ellis, M. Reedijk, D. Anderson, G. Mbamalu, A. D. Reith, G. Panayotou, P. End, A. Bernstein i A. Kazlauskas. "SH2 domains of the p85 alpha subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors." Molecular and Cellular Biology 12, nr 3 (marzec 1992): 991–97. http://dx.doi.org/10.1128/mcb.12.3.991.
Pełny tekst źródłaStreszczenie:
The binding of cytoplasmic signaling proteins such as phospholipase C-gamma 1 and Ras GTPase-activating protein to autophosphorylated growth factor receptors is directed by their noncatalytic Src homology region 2 (SH2) domains. The p85 alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase, which associates with several receptor protein-tyrosine kinases, also contains two SH2 domains. Both p85 alpha SH2 domains, when expressed individually as fusion proteins in bacteria, bound stably to the activated beta receptor for platelet-derived growth factor (PDGF). Complex formation required PDGF stimulation and was dependent on receptor tyrosine kinase activity. The bacterial p85 alpha SH2 domains recognized activated beta PDGF receptor which had been immobilized on a filter, indicating that SH2 domains contact autophosphorylated receptors directly. Several receptor tyrosine kinases within the PDGF receptor subfamily, including the colony-stimulating factor 1 receptor and the Steel factor receptor (Kit), also associate with PI 3-kinase in vivo. Bacterially expressed SH2 domains derived from the p85 alpha subunit of PI 3-kinase bound in vitro to the activated colony-stimulating factor 1 receptor and to Kit. We infer that the SH2 domains of p85 alpha bind to high-affinity sites on these receptors, whose creation is dependent on receptor autophosphorylation. The SH2 domains of p85 are therefore primarily responsible for the binding of PI 3-kinase to activated growth factor receptors.
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McGlade, C. J., C. Ellis, M. Reedijk, D. Anderson, G. Mbamalu, A. D. Reith, G. Panayotou, P. End, A. Bernstein i A. Kazlauskas. "SH2 domains of the p85 alpha subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors". Molecular and Cellular Biology 12, nr 3 (marzec 1992): 991–97. http://dx.doi.org/10.1128/mcb.12.3.991-997.1992.
Pełny tekst źródłaStreszczenie:
The binding of cytoplasmic signaling proteins such as phospholipase C-gamma 1 and Ras GTPase-activating protein to autophosphorylated growth factor receptors is directed by their noncatalytic Src homology region 2 (SH2) domains. The p85 alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase, which associates with several receptor protein-tyrosine kinases, also contains two SH2 domains. Both p85 alpha SH2 domains, when expressed individually as fusion proteins in bacteria, bound stably to the activated beta receptor for platelet-derived growth factor (PDGF). Complex formation required PDGF stimulation and was dependent on receptor tyrosine kinase activity. The bacterial p85 alpha SH2 domains recognized activated beta PDGF receptor which had been immobilized on a filter, indicating that SH2 domains contact autophosphorylated receptors directly. Several receptor tyrosine kinases within the PDGF receptor subfamily, including the colony-stimulating factor 1 receptor and the Steel factor receptor (Kit), also associate with PI 3-kinase in vivo. Bacterially expressed SH2 domains derived from the p85 alpha subunit of PI 3-kinase bound in vitro to the activated colony-stimulating factor 1 receptor and to Kit. We infer that the SH2 domains of p85 alpha bind to high-affinity sites on these receptors, whose creation is dependent on receptor autophosphorylation. The SH2 domains of p85 are therefore primarily responsible for the binding of PI 3-kinase to activated growth factor receptors.
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Rapoport, AP, S. Luhowskyj, P. Doshi i JF DiPersio. "Mutational analysis of the alpha subunit of the human interleukin-3 receptor". Blood 87, nr 1 (1.01.1996): 112–22. http://dx.doi.org/10.1182/blood.v87.1.112.112.
Pełny tekst źródłaStreszczenie:
Abstract The alpha subunit of the human interleukin-3 receptor (IL-3R alpha) is a 70-kD glycoprotein member of the hematopoietin receptor superfamily. This protein associates with a beta subunit common to the receptors for IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF) to form a high-affinity receptor for IL-3. To identify regions of IL-3R alpha critical for ligand binding and receptor function, cDNAs encoding mutant receptors were generated and expressed in COS cells along with the beta subunit. Mutant receptors lacking almost the entire cytoplasmic domain of IL-3R alpha [IL-3R alpha(CD)] or carrying a substitution of trp for leu in the membrane proximal leu-ser-x-trp-ser (LSXWS) box bound 125I-IL-3 with nearly the same affinity as wild-type IL-3R alpha. In contrast, a mutant lacking the entire “LSXWS” motif failed to bind 125I-IL-3 with high affinity despite showing surface expression. In addition, hybrid receptors composed of the first 104 amino acids (aa) of IL-3R alpha joined to aa 118 through 400 of the alpha subunit of the GM-CSF receptor (GM-R alpha) [IL-3R alpha/GM-R alpha] or the first 118 aa of GM-R alpha joined to aa 104 through 378 of IL-3R alpha [GM-R alpha/IL-3R alpha] failed to bind 125I-IL-3 in the presence of the beta subunit. A third hybrid receptor composed of the first 281 residues of IL-3R alpha fused to residues 306 through 379 of GM-R alpha [IL-3R alpha/GM-R alpha-DS] also failed to bind 125I-IL-3 in the presence of the beta subunit but, in contrast to the IL-3R alpha/GM- R alpha hybrid, demonstrated weak surface expression. Mutant receptors lacking the N-terminal 30 aa and the N-terminal 9 aa also did not bind 125I-IL-3 with high affinity, although both were expressed on the cell surface. These data suggest that although the cytoplasmic domain and the leucine residue of the “LSXWS” box are not critical for ligand binding or beta-subunit association, the “LSXWS” motif and amino-terminal sequences are important for these functions.
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Rapoport, AP, S. Luhowskyj, P. Doshi i JF DiPersio. "Mutational analysis of the alpha subunit of the human interleukin-3 receptor". Blood 87, nr 1 (1.01.1996): 112–22. http://dx.doi.org/10.1182/blood.v87.1.112.bloodjournal871112.
Pełny tekst źródłaStreszczenie:
The alpha subunit of the human interleukin-3 receptor (IL-3R alpha) is a 70-kD glycoprotein member of the hematopoietin receptor superfamily. This protein associates with a beta subunit common to the receptors for IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF) to form a high-affinity receptor for IL-3. To identify regions of IL-3R alpha critical for ligand binding and receptor function, cDNAs encoding mutant receptors were generated and expressed in COS cells along with the beta subunit. Mutant receptors lacking almost the entire cytoplasmic domain of IL-3R alpha [IL-3R alpha(CD)] or carrying a substitution of trp for leu in the membrane proximal leu-ser-x-trp-ser (LSXWS) box bound 125I-IL-3 with nearly the same affinity as wild-type IL-3R alpha. In contrast, a mutant lacking the entire “LSXWS” motif failed to bind 125I-IL-3 with high affinity despite showing surface expression. In addition, hybrid receptors composed of the first 104 amino acids (aa) of IL-3R alpha joined to aa 118 through 400 of the alpha subunit of the GM-CSF receptor (GM-R alpha) [IL-3R alpha/GM-R alpha] or the first 118 aa of GM-R alpha joined to aa 104 through 378 of IL-3R alpha [GM-R alpha/IL-3R alpha] failed to bind 125I-IL-3 in the presence of the beta subunit. A third hybrid receptor composed of the first 281 residues of IL-3R alpha fused to residues 306 through 379 of GM-R alpha [IL-3R alpha/GM-R alpha-DS] also failed to bind 125I-IL-3 in the presence of the beta subunit but, in contrast to the IL-3R alpha/GM- R alpha hybrid, demonstrated weak surface expression. Mutant receptors lacking the N-terminal 30 aa and the N-terminal 9 aa also did not bind 125I-IL-3 with high affinity, although both were expressed on the cell surface. These data suggest that although the cytoplasmic domain and the leucine residue of the “LSXWS” box are not critical for ligand binding or beta-subunit association, the “LSXWS” motif and amino-terminal sequences are important for these functions.
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Calvete, J. J., A. Henschen i J. González-Rodríguez. "Assignment of disulphide bonds in human platelet GPIIIa. A disulphide pattern for the β-subunits of the integrin family". Biochemical Journal 274, nr 1 (15.02.1991): 63–71. http://dx.doi.org/10.1042/bj2740063.
Pełny tekst źródłaStreszczenie:
Integrins are cell-surface heterodimers formed by the association of one alpha- and one beta-subunit. Glycoprotein IIIa (GPIIIa or beta 3 subunit) is the common beta-subunit of the beta 3 subfamily of integrins, which, when associated with glycoprotein IIb (GPIIb), constitutes the receptor for fibrinogen and other adhesive proteins at the platelet surface (the GPIIb-IIIa complex) and, when associated with the alpha v-subunit, constitutes the vitronectin receptor present in several cell types. Protein chemical analysis of GPIIIa allows us to define the following structural domains: the cysteine-rich and proteinase-resistant N-terminal domain (GPIIIa 1-62); the adhesive-protein-binding domain (GPIIIa 101-422); the cysteine-rich and proteinase-resistant core (GPIIIa 423-622); and the C-terminal domain comprising an extracellular subdomain (GPIIIa 623-692), a transmembrane subdomain (GPIIIa 693-721), and a cytoplasmic subdomain (GPIIIa 722-762). We also assign unambiguously the disulphide bonds within the N-terminal, the fibrinogen-binding and the C-terminal domains, and the two long-range disulphide bonds which join the N-terminus to the proteinase-resistant core (Cys5-Cys435) and the fibrinogen-binding domain to the extracellular side of the C-terminal domain (Cys406-Cys655). In addition, we propose three alternative models for the arrangement of the disulphide bonds within the core and of the disulphide bonds joining the core to the extracellular side of the C-terminal domain, consistent with our experimental findings, favouring temporarily that which imposes less steric hindrance for the formation of these disulphide bonds. On the basis of this information and on the highly conserved overall structure observed in the beta-subunits of the integrin family known so far, except in beta 4, we propose to extend the cysteine-pairing pattern and the structural domains outlined here for GPIIIa to all the beta-subunits of the integrin family.
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Goldfeld, A. E., E. Tsai, R. Kincaid, P. J. Belshaw, S. L. Schrieber, J. L. Strominger i A. Rao. "Calcineurin mediates human tumor necrosis factor alpha gene induction in stimulated T and B cells." Journal of Experimental Medicine 180, nr 2 (1.08.1994): 763–68. http://dx.doi.org/10.1084/jem.180.2.763.
Pełny tekst źródłaStreszczenie:
The tumor necrosis factor alpha (TNF-alpha) gene is rapidly transcribed in activated T cells via a calcium-dependent pathway that does not require de novo protein synthesis, but is completely blocked by the immunosuppressive drugs cyclosporin A (CsA) and FK506. Here we show that calcineurin phosphatase activity is both necessary and sufficient for TNF-alpha gene transcription in T cells, and identify the factor that binds to the kappa 3 element of the TNF-alpha gene promoter as the target for calcineurin action. The ability of analogues of CsA and FK506 to block calcineurin phosphatase activity correlates completely with their ability to inhibit induction of TNF-alpha mRNA, induction of a TNF-alpha promoter reporter plasmid in transiently transfected T cells, and induction of the kappa 3 binding factor in an electrophoretic mobility shift assay. Moreover, a cDNA encoding the constitutively active form of calcineurin is sufficient to activate the TNF-alpha promoter and the kappa 3 element. TNF-alpha gene transcription is also highly inducible, CsA-sensitive, and protein synthesis-independent in B cells stimulated through their surface immunoglobulin receptors. Using the panel of CsA and FK506 analogues, we show that calcineurin participates in the induction of TNF-alpha transcription in activated B cells. These results extend our previous demonstration that the kappa 3 binding factor is related to NFATp, the preexisting subunit of nuclear factor of activated T cells, and suggest that calcineurin-mediated modification of the kappa 3 binding factor in T cells is of key importance in the induction of TNF-alpha transcription.
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Lagna, G., R. Kovelman, J. Sukegawa i R. G. Roeder. "Cloning and characterization of an evolutionarily divergent DNA-binding subunit of mammalian TFIIIC." Molecular and Cellular Biology 14, nr 5 (maj 1994): 3053–64. http://dx.doi.org/10.1128/mcb.14.5.3053.
Pełny tekst źródłaStreszczenie:
Transcription factor IIIC (TFIIIC) is required for the assembly of a preinitiation complex on 5S RNA, tRNA, and adenovirus VA RNA genes and contains two separable components, TFIIIC1 and TFIIIC2. TFIIIC2 binds to the 3' end of the internal control region of the VAI RNA gene and contains five polypeptides ranging in size from 63 to 220 kDa; the largest of these directly contacts DNA. Here we describe the cloning of cDNAs encoding all (rat) or part (human) of the 220-kDa subunit (TFIIIC alpha). Surprisingly, TFIIIC alpha has no homology to any of the yeast TFIIIC subunits already cloned, suggesting a significant degree of evolutionary divergence for RNA polymerase III factors. Antibodies raised against the N terminus of recombinant human TFIIIC alpha specifically inhibit binding of natural TFIIIC to DNA. Furthermore, immunodepletion assays indicate that TFIIIC alpha is absolutely required for RNA polymerase III transcription of 5S RNA, tRNA, and VAI RNA genes but not for the 7SK RNA and U6 small nuclear RNA genes. Transcription from the tRNA and VAI RNA genes in TFIIIC-depleted nuclear extracts can be restored by addition of purified TFIIIC. In contrast, restoration of 5S RNA gene transcription requires readdition of both TFIIIC and TFIIIA, indicating a promoter-independent interaction between these factors. Immunoprecipitation experiments demonstrate a tight association of all five polypeptides previously identified in the TFIIIC2 fraction, confirming the multisubunit structure of the human factor.
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Lagna, G., R. Kovelman, J. Sukegawa i R. G. Roeder. "Cloning and characterization of an evolutionarily divergent DNA-binding subunit of mammalian TFIIIC". Molecular and Cellular Biology 14, nr 5 (maj 1994): 3053–64. http://dx.doi.org/10.1128/mcb.14.5.3053-3064.1994.
Pełny tekst źródłaStreszczenie:
Transcription factor IIIC (TFIIIC) is required for the assembly of a preinitiation complex on 5S RNA, tRNA, and adenovirus VA RNA genes and contains two separable components, TFIIIC1 and TFIIIC2. TFIIIC2 binds to the 3' end of the internal control region of the VAI RNA gene and contains five polypeptides ranging in size from 63 to 220 kDa; the largest of these directly contacts DNA. Here we describe the cloning of cDNAs encoding all (rat) or part (human) of the 220-kDa subunit (TFIIIC alpha). Surprisingly, TFIIIC alpha has no homology to any of the yeast TFIIIC subunits already cloned, suggesting a significant degree of evolutionary divergence for RNA polymerase III factors. Antibodies raised against the N terminus of recombinant human TFIIIC alpha specifically inhibit binding of natural TFIIIC to DNA. Furthermore, immunodepletion assays indicate that TFIIIC alpha is absolutely required for RNA polymerase III transcription of 5S RNA, tRNA, and VAI RNA genes but not for the 7SK RNA and U6 small nuclear RNA genes. Transcription from the tRNA and VAI RNA genes in TFIIIC-depleted nuclear extracts can be restored by addition of purified TFIIIC. In contrast, restoration of 5S RNA gene transcription requires readdition of both TFIIIC and TFIIIA, indicating a promoter-independent interaction between these factors. Immunoprecipitation experiments demonstrate a tight association of all five polypeptides previously identified in the TFIIIC2 fraction, confirming the multisubunit structure of the human factor.
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Hajra, A., P. P. Liu, N. A. Speck i F. S. Collins. "Overexpression of core-binding factor alpha (CBF alpha) reverses cellular transformation by the CBF beta-smooth muscle myosin heavy chain chimeric oncoprotein." Molecular and Cellular Biology 15, nr 9 (wrzesień 1995): 4980–89. http://dx.doi.org/10.1128/mcb.15.9.4980.
Pełny tekst źródłaStreszczenie:
A fusion between the transcription factor core-binding factor beta (CBF beta; also known as PEBP2 beta) and the tail region of smooth muscle myosin heavy chain (SMMHC) is generated by an inversion of chromosome 16 [inv(16) (p13q22)] associated with the M4Eo subtype of acute myeloid leukemia. We have previously shown that this CBF beta-SMMHC chimeric protein can transform NIH 3T3 cells and that this process requires regions of the chimeric protein necessary for association with the CBF alpha subunit. In this study, we show that NIH 3T3 cells overexpressing murine Cbf alpha 2 (also known as Aml1) cannot be transformed by CBF beta-SMMHC and that overexpression of Cbf alpha 2 in cells previously transformed by CBF beta-SMMHC reverts the cells to a less transformed phenotype. Cbf alpha 2 overexpression does not cause any gross morphological changes to NIH 3T3 cells but does result in increased CBF activity, as indicated by electrophoretic mobility shift assays and transactivation of reporter constructs. Cells transformed by CBF beta-SMMHC lack normal CBF-DNA complexes and have decreased levels of transactivation. Reversion of CBF beta-SMMHC transformation by Cbf alpha 2 is associated with a restoration of normal CBF-DNA complexes and transactivation activity. A Cbf alpha 2 mutant lacking transactivation properties does not transform cells when overexpressed, nor does it protect cells from CBF beta-SMMHC transformation. These results suggest that CBF beta-SMMHC interferes with the normal function of CBF and that this interference is necessary but not sufficient for cellular transformation.
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Watanabe, Y., T. Kitamura, K. Hayashida i A. Miyajima. "Monoclonal antibody against the common beta subunit (beta c) of the human interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony- stimulating factor receptors shows upregulation of beta c by IL-1 and tumor necrosis factor-alpha". Blood 80, nr 9 (1.11.1992): 2215–20. http://dx.doi.org/10.1182/blood.v80.9.2215.2215.
Pełny tekst źródłaStreszczenie:
Abstract High-affinity receptors for human granulocyte macrophage colony- stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 are composed of two distinct subunits, alpha and beta. Each receptor has its own ligand-specific alpha subunit, and the three receptors share the common beta subunit, beta c. Using a transfectant of NIH3T3 cells expressing the high-affinity human GM-CSF receptor, monoclonal antibodies (MoAbs) against beta c were generated. These MoAbs specifically bound to cells bearing beta c and immunoprecipitated the beta c protein of 120 Kd. Using these MoAbs, expression of beta c was examined. It is known that IL-1 augments the proliferative response of a human factor-dependent hematopoietic cell line TF-1 to either GM-CSF, IL-3, or IL-5, and that it upregulates the high-affinity receptors for GM-CSF, IL-3, and IL-5. Antibody binding and immunoprecipitation demonstrated that IL-1 increased cell surface expression of beta c. This enhancement by IL-1 was accompanied by an increased level of beta c mRNA. In addition, we found that tumor necrosis factor-alpha (TNF- alpha) also increased the expression of beta c, although it did not augment the proliferative response of TF-1 to GM-CSF, IL-3, and IL-5.
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Watanabe, Y., T. Kitamura, K. Hayashida i A. Miyajima. "Monoclonal antibody against the common beta subunit (beta c) of the human interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony- stimulating factor receptors shows upregulation of beta c by IL-1 and tumor necrosis factor-alpha". Blood 80, nr 9 (1.11.1992): 2215–20. http://dx.doi.org/10.1182/blood.v80.9.2215.bloodjournal8092215.
Pełny tekst źródłaStreszczenie:
High-affinity receptors for human granulocyte macrophage colony- stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 are composed of two distinct subunits, alpha and beta. Each receptor has its own ligand-specific alpha subunit, and the three receptors share the common beta subunit, beta c. Using a transfectant of NIH3T3 cells expressing the high-affinity human GM-CSF receptor, monoclonal antibodies (MoAbs) against beta c were generated. These MoAbs specifically bound to cells bearing beta c and immunoprecipitated the beta c protein of 120 Kd. Using these MoAbs, expression of beta c was examined. It is known that IL-1 augments the proliferative response of a human factor-dependent hematopoietic cell line TF-1 to either GM-CSF, IL-3, or IL-5, and that it upregulates the high-affinity receptors for GM-CSF, IL-3, and IL-5. Antibody binding and immunoprecipitation demonstrated that IL-1 increased cell surface expression of beta c. This enhancement by IL-1 was accompanied by an increased level of beta c mRNA. In addition, we found that tumor necrosis factor-alpha (TNF- alpha) also increased the expression of beta c, although it did not augment the proliferative response of TF-1 to GM-CSF, IL-3, and IL-5.
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Kitamura, T., i A. Miyajima. "Functional reconstitution of the human interleukin-3 receptor". Blood 80, nr 1 (1.07.1992): 84–90. http://dx.doi.org/10.1182/blood.v80.1.84.84.
Pełny tekst źródłaStreszczenie:
Abstract The high-affinity receptors for human interleukin-3 (IL-3), GM-CSF, and IL-5 are composed of alpha and beta subunits. The alpha subunits are primary ligand binding proteins specific for each ligand, whereas the three human receptors share a common beta subunit (beta c). In contrast to humans mice have two closely related genes, AIC2A and AIC2B, which are homologous to human beta c. The AIC2A gene encodes a low-affinity murine IL-3 binding protein, and the AIC2B protein is the beta subunit shared between murine GM-CSF receptors (mGMR) and IL-5 receptors (mIL- 5R). To examine the function of these receptor components, we established various stable transfectants of murine IL-2-dependent CTLL- 2 cells. CTLL-2 transfectants expressing both the alpha and beta subunits of the human IL-3 receptor (hIL-3R) proliferated in response to physiologic concentrations of hIL-3. Coexpression of hIL-3R alpha with AIC2B but not with AIC2A in CTLL-2 cells conferred a growth response to hIL-3. Although CTLL-2 transfectants expressing hIL-3R alpha alone did not proliferate in the presence of hIL-3, hIL-3- responsive sublines were repeatedly isolated. These sublines expressed endogenous AIC2B but not AIC2A. These results indicate that human beta c is essential for hIL-3 signaling and that AIC2B is a murine equivalent of human beta c. We also showed that hIL-3 and hGM-CSF induced tyrosine phosphorylation of several proteins in CTLL transfectants, similar to those observed in human factor-dependent TF-1 cells stimulated with hIL-3 and hGM-CSF.
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Kitamura, T., i A. Miyajima. "Functional reconstitution of the human interleukin-3 receptor". Blood 80, nr 1 (1.07.1992): 84–90. http://dx.doi.org/10.1182/blood.v80.1.84.bloodjournal80184.
Pełny tekst źródłaStreszczenie:
The high-affinity receptors for human interleukin-3 (IL-3), GM-CSF, and IL-5 are composed of alpha and beta subunits. The alpha subunits are primary ligand binding proteins specific for each ligand, whereas the three human receptors share a common beta subunit (beta c). In contrast to humans mice have two closely related genes, AIC2A and AIC2B, which are homologous to human beta c. The AIC2A gene encodes a low-affinity murine IL-3 binding protein, and the AIC2B protein is the beta subunit shared between murine GM-CSF receptors (mGMR) and IL-5 receptors (mIL- 5R). To examine the function of these receptor components, we established various stable transfectants of murine IL-2-dependent CTLL- 2 cells. CTLL-2 transfectants expressing both the alpha and beta subunits of the human IL-3 receptor (hIL-3R) proliferated in response to physiologic concentrations of hIL-3. Coexpression of hIL-3R alpha with AIC2B but not with AIC2A in CTLL-2 cells conferred a growth response to hIL-3. Although CTLL-2 transfectants expressing hIL-3R alpha alone did not proliferate in the presence of hIL-3, hIL-3- responsive sublines were repeatedly isolated. These sublines expressed endogenous AIC2B but not AIC2A. These results indicate that human beta c is essential for hIL-3 signaling and that AIC2B is a murine equivalent of human beta c. We also showed that hIL-3 and hGM-CSF induced tyrosine phosphorylation of several proteins in CTLL transfectants, similar to those observed in human factor-dependent TF-1 cells stimulated with hIL-3 and hGM-CSF.
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Guo, Yalin, Laleh Talebian, Ivan Maillard, Caroline L. Speck, Warren S. Pear, Ellen V. Rothenberg i Nancy A. Speck. "Reduction of Core Binding Factor beta (CBFβ) Dosage Blocks T Cell Development." Blood 106, nr 11 (16.11.2005): 2714. http://dx.doi.org/10.1182/blood.v106.11.2714.2714.
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Abstract Core binding factors (CBFs) are heterodimers consisting of a DNA binding subunit (Runx1, Runx2, or Runx3) and a non-DNA binding CBFβ subunit. CBFβ increases the affinity of the Runx subunits for DNA. Embryos deficient for Runx1 or CBFβ die at midgestation with a complete failure of definitive hematopoiesis due to a block in hematopoietic stem cell (HSC) emergence. To examine the role of core binding factors at later stages of hematopoiesis, we generated a hypomorphic Cbfb allele (Cbfbrss), that when carried over a Cbfb null allele (Cbfbrss/−) results in a 3-4 fold reduction in CBFβ protein levels. Although HSCs emerge in Cbfbrss/− animals, fetal T cell development is severely impaired. Here we examined the T cell developmental block in more detail by culturing fetal liver cells from Cbfbrss/− animals on OP9 stromal cells that express the Notch ligand Delta-like-1 (DL1) (Schmitt and Zúñiga-Pflücker, Immunity17: 749, 2002). Fetal livers (E14.5) from Cbfbrss/− animals contained normal numbers of both c-kit+Sca-1+lin- cells and c-kit+IL7r+ lymphoid progenitors. Lin- fetal liver progenitors cultured on OP9-DL1 cells in the presence of IL-7 and Flt3L displayed a growth disadvantage relative to wild type cells, and a block at the double negative 1 (DN1, CD44+ CD25−) stage of T cell development. The T cell defect could be rescued by retroviral transduction of the CBFβ heterodimerization domain into lin- fetal liver cells, but not by a G61A/N104A mutant that cannot bind the Runx subunits. Genes whose expression was decreased in DN1 cells purified from the OP9-DL1 cultures included CD3 and the early T cell transcription factors GATA3 and TCF. Although expression of several Notch pathway genes (Notch1, Hes-1/5, Deltex-1) was mildly decreased, Notch signals were clearly transduced, suggesting that Notch signaling was intact. These data demonstrate that reduced CBFβ levels impair the differentiation of stem cells/progenitors into T cells at the earliest stage of T cell development. This in vitro model will be useful for characterizing the molecular circuitry involving CBFβ in T cell development, and for identifying CBFβ protein partners.
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Jaffray, E., K. M. Wood i R. T. Hay. "Domain organization of I kappa B alpha and sites of interaction with NF-kappa B p65." Molecular and Cellular Biology 15, nr 4 (kwiecień 1995): 2166–72. http://dx.doi.org/10.1128/mcb.15.4.2166.
Pełny tekst źródłaStreszczenie:
The DNA-binding activity and cellular distribution of the transcription factor NF-kappa B are regulated by the inhibitor protein I kappa B alpha. I kappa B alpha belongs to a family of proteins that contain multiple repeats of a 30- to 35-amino-acid sequence that was initially recognized in the erythrocyte protein ankyrin. Partial proteolysis has been used to study the domain structure of I kappa B alpha and to determine the sites at which it interacts with NF-kappa B. The data reveal a tripartite structure for I kappa B alpha in which a central, protease-resistant domain composed of five ankyrin repeats is flanked by an unstructured N-terminal extension and a compact, highly acidic C-terminal domain that is connected to the core of the protein by a flexible linker. Functional analysis of V8 cleavage products indicates that I kappa B alpha molecules lacking the N-terminal region can interact with and inhibit the DNA-binding activity of the p65 subunit of NF-kappa B, whereas I kappa B alpha molecules which lack both the N- and C-terminal regions are incapable of doing so. Protease cleavage of the N terminus of I kappa B alpha was unaffected by the presence of the p65 subunit of NF-kappa B, whereas bound p65 blocked cleavage of the flexible linker connecting the C-terminal domain to the ankyrin repeat-containing core of the protein. This linker region is highly conserved within the human, rat, pig, and chicken homologs of I kappa B alpha, and while it has been suggested that it represents a sixth ankyrin repeat, it is also likely that this is a flexible region of the protein that interacts with NF-kappa B.
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Hel, Z., E. Skamene i D. Radzioch. "Two distinct regions in the 3' untranslated region of tumor necrosis factor alpha mRNA form complexes with macrophage proteins." Molecular and Cellular Biology 16, nr 10 (październik 1996): 5579–90. http://dx.doi.org/10.1128/mcb.16.10.5579.
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The production of tumor necrosis factor alpha (TNF-alpha), a key proinflammatory cytokine essential for the function of the immune system, is regulated at both the transcriptional and posttranscriptional levels. In this report, we focus on the interaction of TNF-alpha mRNA with macrophage proteins, likely mediators of its post-transcriptional control. Mapping of murine TNF-alpha mRNA by using a combination of RNase protection and RNA gel shift assays revealed that two distinct sites within the 3' untranslated region (3'-UTR) engage in the formation of four major RNA-protein complexes, while no protein binding to the 5'-UTR or coding sequences was detected. The protein-binding site of three RNA-protein complexes, A, B, and C, is positioned between bases 1291 and 1320 inside the AU-rich sequence, a region previously shown to be crucial for both translational repression and lipopolysaccharide inducibility of TNF-alpha. An additional protein complex (complex D) whose binding to the TNF-alpha 3'-UTR was independent of the presence of AU-rich sequences was identified. At least six protein species with apparent molecular masses of 48, 52, 54, 81, 101, and 150 kDa are in direct contact with TNF-alpha mRNA. The RNA-binding proteins are differentially distributed in the cell: complexes A and D are present predominantly in the cytosol, while complexes B and C are found in the nucleus and associated with particulate cytoplasmic fractions. Cytosolic complex A displays comparatively high specificity for TNF-alpha mRNA, while the binding of complexes B and C to TNF-alpha mRNA is readily competed for by other AU-rich sequence-containing RNAs. In summary, these findings demonstrate that two regions of the TNF-alpha mRNA molecule interact with macrophage RNA-binding protein complexes that differ in their core protein composition, cellular distribution, and affinity to TNF-alpha mRNA.
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Weiss, M., C. Yokoyama, Y. Shikama, C. Naugle, B. Druker i CA Sieff. "Human granulocyte-macrophage colony-stimulating factor receptor signal transduction requires the proximal cytoplasmic domains of the alpha and beta subunits". Blood 82, nr 11 (1.12.1993): 3298–306. http://dx.doi.org/10.1182/blood.v82.11.3298.3298.
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Abstract Human granulocyte-macrophage colony-stimulating factor (GM-CSF) controls the production, maturation, and function of cells in multiple hematopoietic lineages. These effects are mediated by a cell-surface receptor (GM-R) composed of alpha and beta subunits, each containing 378 and 881 amino acids, respectively. Whereas the alpha subunit exists as several isoforms that bind GM-CSF with low affinity, the beta common subunit (beta c) does not bind GM-CSF itself, but acts as a high- affinity converter for GM-CSF, interleukin-3 (IL-3), and IL-5 receptor alpha subunits. The cytoplasmic region of GM-R alpha consists of a membrane-proximal conserved region shared by the alpha 1 and alpha 2 isoforms and a C-terminal variable region that is divergent between alpha 1 and alpha 2. The cytoplasmic region of beta c contains membrane proximal serine and acidic domains. To investigate the amino acid sequences that influence signal transduction by this receptor complex, we constructed a series of cytoplasmic truncation mutants of the alpha 2 and beta subunits. To study these truncations, we stably transfected the IL-3-dependent murine cell line Ba/F3 with wild-type or mutant cDNAs. We found that the wild-type and mutant alpha subunits conferred similar low-affinity binding sites for human GM-CSF to Ba/F3, and the wild-type or mutant beta subunit converted some of these sites to high- affinity; the cytoplasmic domain of beta was unnecessary for this high- affinity conversion. Proliferation assays showed that the membrane- proximal conserved region of GM-R alpha and the serine-acidic domain of beta c are required for both cell proliferation and ligand-dependent phosphorylation of a 93-kD cytoplasmic protein. We suggest that these regions may represent an important signal transduction motif present in several cytokine receptors.
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Weiss, M., C. Yokoyama, Y. Shikama, C. Naugle, B. Druker i CA Sieff. "Human granulocyte-macrophage colony-stimulating factor receptor signal transduction requires the proximal cytoplasmic domains of the alpha and beta subunits". Blood 82, nr 11 (1.12.1993): 3298–306. http://dx.doi.org/10.1182/blood.v82.11.3298.bloodjournal82113298.
Pełny tekst źródłaStreszczenie:
Human granulocyte-macrophage colony-stimulating factor (GM-CSF) controls the production, maturation, and function of cells in multiple hematopoietic lineages. These effects are mediated by a cell-surface receptor (GM-R) composed of alpha and beta subunits, each containing 378 and 881 amino acids, respectively. Whereas the alpha subunit exists as several isoforms that bind GM-CSF with low affinity, the beta common subunit (beta c) does not bind GM-CSF itself, but acts as a high- affinity converter for GM-CSF, interleukin-3 (IL-3), and IL-5 receptor alpha subunits. The cytoplasmic region of GM-R alpha consists of a membrane-proximal conserved region shared by the alpha 1 and alpha 2 isoforms and a C-terminal variable region that is divergent between alpha 1 and alpha 2. The cytoplasmic region of beta c contains membrane proximal serine and acidic domains. To investigate the amino acid sequences that influence signal transduction by this receptor complex, we constructed a series of cytoplasmic truncation mutants of the alpha 2 and beta subunits. To study these truncations, we stably transfected the IL-3-dependent murine cell line Ba/F3 with wild-type or mutant cDNAs. We found that the wild-type and mutant alpha subunits conferred similar low-affinity binding sites for human GM-CSF to Ba/F3, and the wild-type or mutant beta subunit converted some of these sites to high- affinity; the cytoplasmic domain of beta was unnecessary for this high- affinity conversion. Proliferation assays showed that the membrane- proximal conserved region of GM-R alpha and the serine-acidic domain of beta c are required for both cell proliferation and ligand-dependent phosphorylation of a 93-kD cytoplasmic protein. We suggest that these regions may represent an important signal transduction motif present in several cytokine receptors.
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Miyajima, I., L. Levitt, T. Hara, MA Bedell, NG Copeland, NA Jenkins i A. Miyajima. "The murine interleukin-3 receptor alpha subunit gene: chromosomal localization, genomic structure, and promoter function". Blood 85, nr 5 (1.03.1995): 1246–53. http://dx.doi.org/10.1182/blood.v85.5.1246.bloodjournal8551246.
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The interleukin-3 receptor (IL-3R) is composed of alpha and beta subunits, members of the class I cytokine receptor family. Here we describe isolation and characterization of the chromosomal gene for the mouse IL-3R alpha subunit (mIL-3R alpha). Whereas the human IL-3R alpha gene is tightly linked with the granulocyte-macrophage colony- stimulating factor receptor alpha subunit (GM-CSFR alpha) gene in the pseudoautosomal region of the X and Y chromosomes, the mIL-3R alpha gene (II3ra) is located in the proximal region of mouse chromosome 14, separated from the mouse GM-CSFR alpha gene, which is on chromosome 19. The mIL-3R alpha gene spans about 10 kb and is divided into 12 exons. All the exon-intron boundaries possess the splicing junction consensus sequences (52GT-AG352), and the whole genomic structure is similar to those of the previously reported class I cytokine receptor genes. There are two major transcription initiation sites that are located at 215 and 188 nucleotides upstream of the initiator codon. The promoter region is GC-rich and contains potential binding sites for GATA, Ets, c- myb,, Sp1, Ap-2, and G-C boxes, but not a typical TATA or CAAT sequence. A fusion gene containing 0.8 kb of the 52 noncoding sequence linked to the firefly luciferase gene directed the transcription in mouse mast cells but not in fibroblasts or T cells, suggesting that this promoter functions in a cell type-specific manner. Further sequential deletion of the 52 region suggests two potential regulatory regions for transcription of the mIL-3R alpha gene.
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Bhutada, A., i F. Ismail-Beigi. "Serum and growth factor induction of Na(+)-K(+)-ATPase subunit mRNAs in Clone 9 cells: role of protein kinase C". American Journal of Physiology-Cell Physiology 261, nr 4 (1.10.1991): C699—C707. http://dx.doi.org/10.1152/ajpcell.1991.261.4.c699.
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In a previous study, we found that addition of serum to confluent Clone 9 cells, a nontransformed rat liver cell line, increased the abundance of mRNA alpha 1 and mRNA beta 1 at 3 h by 2- and 2.7-fold, respectively [Bhutada et al. Am. J. Physiol. 258 (Cell Physiol. 27): C1044-C1050, 1990]. We now report that exposure of these cells to 160 nM 12-O-tetradecanoylphorbol 13-acetate (TPA) for 6 h increases mRNA alpha 1 and mRNA beta 1 by 1.7 +/- 0.2- and 2.1 +/- 0.3-fold, respectively. Incubation in the presence of 160 nM TPA for 24 h reduced high-affinity phorbol dibutyrate-binding sites [dissociation constant (Kd) = 5 nM; maximum binding (Bmax) = 1.2 pmol/mg protein] to undetectable levels. In such cells, exposure to 10% serum for 6 h still resulted in two- and fourfold increment in mRNA alpha 1 and mRNA beta 1 abundances, respectively, while further addition of TPA to these protein kinase C (PKC)-depleted cells resulted in no change in the subunit mRNA abundances. The increments in mRNA alpha 1 content in response to 10% serum and 160 nM TPA at 6 h were additive, whereas the increments in mRNA beta 1 were not. The following agents increased mRNA alpha 1 and mRNA beta 1 abundance in both control and PKC-depleted cells: epidermal growth factor, platelet-derived growth factor, basic fibroblast growth factor, insulin, dexamethasone, and hypothyroid calf serum. In contrast, N6,2'-O-dibutyryl-adenosine 3',5'-cyclic monophosphate and aldosterone had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)
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Wang, S., Q. Wang, B. E. Crute, I. N. Melnikova, S. R. Keller i N. A. Speck. "Cloning and characterization of subunits of the T-cell receptor and murine leukemia virus enhancer core-binding factor." Molecular and Cellular Biology 13, nr 6 (czerwiec 1993): 3324–39. http://dx.doi.org/10.1128/mcb.13.6.3324.
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Moloney murine leukemia virus causes thymic leukemias when injected into newborn mice. A major determinant of the thymic disease specificity of Moloney virus genetically maps to the conserved viral core motif in the Moloney virus enhancer. Point mutations introduced into the core site significantly shifted the disease specificity of the Moloney virus from thymic leukemia to erythroid leukemia (N.A. Speck, B. Renjifo, E. Golemis, T.N. Fredrickson, J.W. Hartley, and N. Hopkins, Genes Dev. 4:233-242, 1990). We previously reported the purification of core-binding factors (CBF) from calf thymus nuclei (S. Wang and N.A. Speck, Mol. Cell. Biol. 12:89-102, 1992). CBF binds to core sites in murine leukemia virus and T-cell receptor enhancers. Affinity-purified CBF contains multiple polypeptides. In this study, we sequenced five tryptic peptides from two of the bovine CBF proteins and isolated three cDNA clones from a mouse thymus cDNA library encoding three of the tryptic peptides from the bovine proteins. The cDNA clones, which we call CBF beta p22.0, CBF beta p21.5, and CBF beta p17.6, encode three highly related but distinct proteins with deduced molecular sizes of 22.0, 21.5, and 17.6 kDa that appear to be translated from multiply spliced mRNAs transcribed from the same gene. CBF beta p22.0, CBF beta p21.5, and CBF beta p17.6 do not by themselves bind the core site. However, CBF beta p22.0 and CBF beta p21.5 form a complex with DNA-binding CBF alpha subunits and as a result decrease the rate of dissociation of the CBF protein-DNA complex. Association of the CBF beta subunits does not extend the phosphate contacts in the binding site. We propose that CBF beta is a non-DNA-binding subunit of CBF and does not contact DNA directly.
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Wang, S., Q. Wang, B. E. Crute, I. N. Melnikova, S. R. Keller i N. A. Speck. "Cloning and characterization of subunits of the T-cell receptor and murine leukemia virus enhancer core-binding factor". Molecular and Cellular Biology 13, nr 6 (czerwiec 1993): 3324–39. http://dx.doi.org/10.1128/mcb.13.6.3324-3339.1993.
Pełny tekst źródłaStreszczenie:
Moloney murine leukemia virus causes thymic leukemias when injected into newborn mice. A major determinant of the thymic disease specificity of Moloney virus genetically maps to the conserved viral core motif in the Moloney virus enhancer. Point mutations introduced into the core site significantly shifted the disease specificity of the Moloney virus from thymic leukemia to erythroid leukemia (N.A. Speck, B. Renjifo, E. Golemis, T.N. Fredrickson, J.W. Hartley, and N. Hopkins, Genes Dev. 4:233-242, 1990). We previously reported the purification of core-binding factors (CBF) from calf thymus nuclei (S. Wang and N.A. Speck, Mol. Cell. Biol. 12:89-102, 1992). CBF binds to core sites in murine leukemia virus and T-cell receptor enhancers. Affinity-purified CBF contains multiple polypeptides. In this study, we sequenced five tryptic peptides from two of the bovine CBF proteins and isolated three cDNA clones from a mouse thymus cDNA library encoding three of the tryptic peptides from the bovine proteins. The cDNA clones, which we call CBF beta p22.0, CBF beta p21.5, and CBF beta p17.6, encode three highly related but distinct proteins with deduced molecular sizes of 22.0, 21.5, and 17.6 kDa that appear to be translated from multiply spliced mRNAs transcribed from the same gene. CBF beta p22.0, CBF beta p21.5, and CBF beta p17.6 do not by themselves bind the core site. However, CBF beta p22.0 and CBF beta p21.5 form a complex with DNA-binding CBF alpha subunits and as a result decrease the rate of dissociation of the CBF protein-DNA complex. Association of the CBF beta subunits does not extend the phosphate contacts in the binding site. We propose that CBF beta is a non-DNA-binding subunit of CBF and does not contact DNA directly.
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Takaki, S., Y. Murata, T. Kitamura, A. Miyajima, A. Tominaga i K. Takatsu. "Reconstitution of the functional receptors for murine and human interleukin 5." Journal of Experimental Medicine 177, nr 6 (1.06.1993): 1523–29. http://dx.doi.org/10.1084/jem.177.6.1523.
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The murine interleukin 5 receptor (mIL-5R) is composed of two distinct subunits, alpha and beta. The alpha subunit (mIL-5R alpha) specifically binds IL-5 with low affinity. The beta subunit (mIL-5R beta) does not bind IL-5 by itself, but forms the high-affinity receptor with mIL-5R alpha. mIL-5R beta has been revealed to be the mIL-3R-like protein, AIC2B which is shared with receptors for IL-3 and granulocyte/macrophage colony-stimulating factor. We demonstrated here the reconstitution of the functional receptors for murine and human IL-5 on the mouse IL-2-dependent cell line, CTLL-2. CTLL-2 was transfected with the cDNAs for mIL-5R alpha and/or AIC2B. Only CTLL-2 transfectant expressing both mIL-5R alpha and AIC2B expressed the high-affinity receptor and proliferated in response to murine IL-5. Then CTLL-2 was transfected with the cDNAs for hIL-5R alpha and/or KH97 (beta c), the human homologue of AIC2B. Though beta c did not contribute much to binding affinity of hIL-5R, only CTLL-2 transfectant expressing both hIL-5R alpha and beta c proliferated in response to human IL-5. These results showed that the beta subunit is indispensable in IL-5 signal transduction. We further investigated the function of IL-5-specific alpha subunit in transmitting IL-5 signals. Mutant mIL-5R alpha, which lacks its whole cytoplasmic domain, was transfected into mouse IL-3-dependent cell line, FDC-P1 expressing AIC2B intrinsically. The resulting transfectant did not respond to IL-5, though the transfectant expressed the high-affinity IL-5R, indicating that the cytoplasmic portion of the alpha subunit also has some important role in IL-5-mediated signal transduction.
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O'Brien, R. M., E. L. Noisin, A. Suwanichkul, T. Yamasaki, P. C. Lucas, J. C. Wang, D. R. Powell i D. K. Granner. "Hepatic nuclear factor 3- and hormone-regulated expression of the phosphoenolpyruvate carboxykinase and insulin-like growth factor-binding protein 1 genes." Molecular and Cellular Biology 15, nr 3 (marzec 1995): 1747–58. http://dx.doi.org/10.1128/mcb.15.3.1747.
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The rate of transcription of the hepatic phosphoenolpyruvate carboxykinase (PEPCK) and insulin-like growth factor-binding protein 1 (IGFBP-1) genes is stimulated by glucocorticoids and inhibited by insulin. In both cases, the effect of insulin is dominant, since it suppresses both basal and glucocorticoid-stimulated PEPCK or IGFBP-1 gene transcription. Analyses of both promoters by transfection of PEPCK or IGFBP-1-chloramphenicol acetyltransferase fusion genes into rat hepatoma cells has led to the identification of insulin response sequences (IRSs) in both genes. The core IRS, T(G/A)TTTTG, is the same in both genes, but the PEPCK promoter has a single copy of this element whereas the IGFBP-1 promoter has two copies arranged as an inverted palindrome. The IGFBP-1 IRS and PEPCK IRS both bind the alpha and beta forms of hepatic nuclear factor 3 (HNF-3), although the latter does so with a sixfold-lower relative affinity. Both the PEPCK and the IGFBP-1 IRSs also function as accessory factor binding sites required for the full induction of gene transcription by glucocorticoids. A combination of transient transfection and DNA binding studies suggests that HNF-3 is the accessory factor that supports glucocorticoid-induced gene transcription. In both genes, the HNF-3 binding site overlaps the IRS core motif(s). A model in which insulin is postulated to mediate its negative effect on glucocorticoid-induced PEPCK and IGFBP-1 gene transcription indirectly by inhibiting HNF-3 action is proposed.
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Cockell, M., D. Stolarczyk, S. Frutiger, G. J. Hughes, O. Hagenbüchle i P. K. Wellauer. "Binding sites for hepatocyte nuclear factor 3 beta or 3 gamma and pancreas transcription factor 1 are required for efficient expression of the gene encoding pancreatic alpha-amylase." Molecular and Cellular Biology 15, nr 4 (kwiecień 1995): 1933–41. http://dx.doi.org/10.1128/mcb.15.4.1933.
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Efficient expression of genes under the control of alpha-amylase 2 5'-flanking sequences in exocrine pancreatic cells requires, in addition to the pancreas transcription factor 1 binding site (M. Cockell, B.J. Stevenson, M. Strubin, O. Hagenbüchle, and P. K. Wellauer, Mol. Cell. Biol. 9:2464-2476, 1989), another cis-acting element at positions -60 to -86. This DNA element, which contains an AT-rich core, site for nuclear proteins present not only in the pancreas but also in other tissues and cell lines derived from the endoderm. Purification of binding activities from pancreatic cells by DNA affinity chromatography reveals several distinct proteins ranging in size from 45 to 54 kDa (p45, p47/48, and p54). All of these proteins interact with the specific DNA sequence upon renaturation in vitro. Protein sequencing, electrophoretic mobility shift assay, and immunoblot analyses identify p54 and p47/48 as members of the hepatocyte nuclear factor 3 (HNF3 [forkhead]) family of transcription factors. p54 belongs to the subfamily of HNF3 beta proteins, while p47/48 binding activity includes HNF3 gamma. The cDNAs for two HNF3 beta proteins differing only in N-terminal amino acid sequences were isolated from a pancreatic cDNA library. The mRNAs encoding the two protein species accumulate to different steady-state levels in poly(A)+ RNA of pancreatic cells. Our results support a model by which the pancreas-specific expression of the alpha-amylase gene is mediated by a combination of cell-specific and cell lineage-specific transcription factors.
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Muraoka, A., M. Kaise, Y. J. Guo, J. Yamada, I. Song, J. DelValle, A. Todisco i T. Yamada. "Canine H(+)-K(+)-ATPase alpha-subunit gene promoter: studies with canine parietal cells in primary culture". American Journal of Physiology-Gastrointestinal and Liver Physiology 271, nr 6 (1.12.1996): G1104—G1113. http://dx.doi.org/10.1152/ajpgi.1996.271.6.g1104.
Pełny tekst źródłaStreszczenie:
H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) is the principal enzyme responsible for the process of gastric acid secretion. This enzyme is expressed in a cell-type-specific manner in gastric parietal cells. To explore the mechanisms regulating its expression, we transfected differentiated canine parietal cells in primary culture with H(+)-K(+)-ATPase-luciferase reporter genes and assessed transcriptional activities. Deletional analysis of the 5'-flanking region of this gene demonstrated a remarkable increment in transcriptional activity associated with a segment between bases -54 to -45 (5' GCTCCGCCTC 3') relative to the transcriptional initiation site. Gel shift assays with competition and supershift analysis demonstrated that this segment is specifically bound by the transcription factor Sp1. A point mutation, eliminating Sp1 binding, diminished basal transcriptional activity by 80%, indicating that this Sp1 binding site is important for constitutive transcriptional activity. Although these studies indicate that Sp1 is required to maintain a high concentration of the H(+)-K(+)-ATPase gene in the parietal cell, its cell-type-specific expression must rely on other elements because Sp1 is a ubiquitously expressed transcription factor.
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Gout, I., R. Dhand, G. Panayotou, M. J. Fry, I. Hiles, M. Otsu i M. D. Waterfield. "Expression and characterization of the p85 subunit of the phosphatidylinositol 3-kinase complex and a related p85β protein by using the baculovirus expression system". Biochemical Journal 288, nr 2 (1.12.1992): 395–405. http://dx.doi.org/10.1042/bj2880395.
Pełny tekst źródłaStreszczenie:
PtdIns 3-kinase associates with certain activated protein-tyrosine kinase receptors and with the pp60c-src/polyoma middle-T complex, suggesting that the enzyme is involved in growth regulation. The purified PtdIns 3-kinase appears to have two subunits, of 85 kDa and 110 kDa. Structural analysis at protein and cDNA levels revealed two forms of the 85 kDa subunit, one which associates with PtdIns 3-kinase activity termed p85 alpha, and a protein of unknown function, p85 beta. Both 85 kDa proteins contain src-homology regions 2 and 3 (SH2 and SH3), but lack enzymic activity, suggesting that they may be regulatory subunits of PtdIns 3-kinase. To probe their structure and function further, p85 alpha and p85 beta have been expressed and purified in large amounts from insect cells by using baculovirus vectors. Specific antisera detect p85 alpha, but not p85 beta, associated with PtdIns 3-kinase activity in various cell types. Co-expression studies in insect cells have shown that p85 alpha and p85 beta are substrates for the protein-tyrosine kinases of epidermal growth factor, colony-stimulating factor 1 and c-erbB2 receptors and the src family kinase p59c-fyn. Both p85 alpha and p85 beta form tight complexes with these protein-tyrosine kinases as measured by immunoprecipitation and kinase assays in vitro. The specificity of binding of free p85 is less restricted than that of p85 in the active PtdIns 3-kinase complex with the 110 kDa protein. The relevance of these results to growth-factor-induced PtdIns 3-kinase activation is discussed.
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Cooper, J. A., i A. Kashishian. "In vivo binding properties of SH2 domains from GTPase-activating protein and phosphatidylinositol 3-kinase." Molecular and Cellular Biology 13, nr 3 (marzec 1993): 1737–45. http://dx.doi.org/10.1128/mcb.13.3.1737.
Pełny tekst źródłaStreszczenie:
We have used a transient expression system and mutant platelet-derived growth factor (PDGF) receptors to study the binding specificities of the Src homology 2 (SH2) regions of the Ras GTPase-activator protein (GAP) and the p85 alpha subunit of phosphatidylinositol 3-kinase (PI3 kinase). A number of fusion proteins, each tagged with an epitope allowing recognition by a monoclonal antibody, were expressed at levels comparable to those of endogenous GAP. Fusion proteins containing the central SH2-SH3-SH2 region of GAP or the C-terminal region of p85 alpha, which includes two SH2 domains, bound to PDGF receptors in response to PDGF stimulation. Both fusion proteins showed the same requirements for tyrosine phosphorylation sites in the PDGF receptor as the full-length proteins from which they were derived, i.e., binding of the GAP fusion protein was reduced by mutation of Tyr-771, and binding of the p85 fusion protein was reduced by mutation of Tyr-740, Tyr-751, or both residues. Fusion proteins containing single SH2 domains from either GAP or p85 alpha did not bind detectably to PDGF receptors in this system, suggesting that two SH2 domains in a single polypeptide cooperate to raise the affinity of binding. The sequence specificities of individual SH2 domains were deduced from the binding properties of fusion proteins containing one SH2 domain from GAP and another from p85. The results suggest that the C-terminal GAP SH2 domain specifies binding to Tyr-771, the C-terminal p85 alpha SH2 domain binds to either Tyr-740 or Tyr-751, and each protein's N-terminal SH2 domain binds to unidentified phosphorylation sites.(ABSTRACT TRUNCATED AT 250 WORDS)
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Cooper, J. A., i A. Kashishian. "In vivo binding properties of SH2 domains from GTPase-activating protein and phosphatidylinositol 3-kinase". Molecular and Cellular Biology 13, nr 3 (marzec 1993): 1737–45. http://dx.doi.org/10.1128/mcb.13.3.1737-1745.1993.
Pełny tekst źródłaStreszczenie:
We have used a transient expression system and mutant platelet-derived growth factor (PDGF) receptors to study the binding specificities of the Src homology 2 (SH2) regions of the Ras GTPase-activator protein (GAP) and the p85 alpha subunit of phosphatidylinositol 3-kinase (PI3 kinase). A number of fusion proteins, each tagged with an epitope allowing recognition by a monoclonal antibody, were expressed at levels comparable to those of endogenous GAP. Fusion proteins containing the central SH2-SH3-SH2 region of GAP or the C-terminal region of p85 alpha, which includes two SH2 domains, bound to PDGF receptors in response to PDGF stimulation. Both fusion proteins showed the same requirements for tyrosine phosphorylation sites in the PDGF receptor as the full-length proteins from which they were derived, i.e., binding of the GAP fusion protein was reduced by mutation of Tyr-771, and binding of the p85 fusion protein was reduced by mutation of Tyr-740, Tyr-751, or both residues. Fusion proteins containing single SH2 domains from either GAP or p85 alpha did not bind detectably to PDGF receptors in this system, suggesting that two SH2 domains in a single polypeptide cooperate to raise the affinity of binding. The sequence specificities of individual SH2 domains were deduced from the binding properties of fusion proteins containing one SH2 domain from GAP and another from p85. The results suggest that the C-terminal GAP SH2 domain specifies binding to Tyr-771, the C-terminal p85 alpha SH2 domain binds to either Tyr-740 or Tyr-751, and each protein's N-terminal SH2 domain binds to unidentified phosphorylation sites.(ABSTRACT TRUNCATED AT 250 WORDS)
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Li, Xianjing, Duowei Wang, Zhen Chen, Ermei Lu, Zhuo Wang, Jingjing Duan, Wei Tian i in. "Gαi1and Gαi3regulate macrophage polarization by forming a complex containing CD14 and Gab1". Proceedings of the National Academy of Sciences 112, nr 15 (30.03.2015): 4731–36. http://dx.doi.org/10.1073/pnas.1503779112.
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Heterotrimeric G proteins have been implicated in Toll-like receptor 4 (TLR4) signaling in macrophages and endothelial cells. However, whether guanine nucleotide-binding protein G(i) subunit alpha-1 and alpha-3 (Gαi1/3) are required for LPS responses remains unclear, and if so, the underlying mechanisms need to be studied. In this study, we demonstrated that, in response to LPS, Gαi1/3form complexes containing the pattern recognition receptor (PRR) CD14 and growth factor receptor binding 2 (Grb2)-associated binding protein (Gab1), which are required for activation of PI3K-Akt signaling. Gαi1/3deficiency decreased LPS-induced TLR4 endocytosis, which was associated with decreased phosphorylation of IFN regulatory factor 3 (IRF3). Gαi1/3knockdown in bone marrow-derived macrophage cells (Gαi1/3KD BMDMs) exhibited an M2-like phenotype with significantly suppressed production of TNF-α, IL-6, IL-12, and NO in response to LPS. The altered polarization coincided with decreased Akt activation. Further, Gαi1/3deficiency caused LPS tolerance in mice. In vitro studies revealed that, in LPS-tolerant macrophages, Gαi1/3were down-regulated partially by the proteasome pathway. Collectively, the present findings demonstrated that Gαi1/3can interact with CD14/Gab1, which modulates macrophage polarization in vitro and in vivo.
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Benjamin, Bradley, Ana M. Sanchez, Angad Garg, Beate Schwer i Stewart Shuman. "Structure-function analysis of fission yeast cleavage and polyadenylation factor (CPF) subunit Ppn1 and its interactions with Dis2 and Swd22". PLOS Genetics 17, nr 3 (12.03.2021): e1009452. http://dx.doi.org/10.1371/journal.pgen.1009452.
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Fission yeast Cleavage and Polyadenylation Factor (CPF), a 13-subunit complex, executes the cotranscriptional 3’ processing of RNA polymerase II (Pol2) transcripts that precedes transcription termination. The three-subunit DPS sub-complex of CPF, consisting of a PP1-type phosphoprotein phosphatase Dis2, a WD-repeat protein Swd22, and a putative phosphatase regulatory factor Ppn1, associates with the CPF core to form the holo-CPF assembly. Here we probed the functional, physical, and genetic interactions of DPS by focusing on the Ppn1 subunit, which mediates association of DPS with the core. Transcriptional profiling by RNA-seq defined limited but highly concordant sets of protein-coding genes that were dysregulated in ppn1Δ, swd22Δ and dis2Δ cells, which included the DPSΔ down-regulated phosphate homeostasis genes pho1 and pho84 that are controlled by lncRNA-mediated transcriptional interference. Essential and inessential modules of the 710-aa Ppn1 protein were defined by testing the effects of Ppn1 truncations in multiple genetic backgrounds in which Ppn1 is required for growth. An N-terminal 172-aa disordered region was dispensable and its deletion alleviated hypomorphic phenotypes caused by deleting C-terminal aa 640–710. A TFIIS-like domain (aa 173–330) was not required for viability but was important for Ppn1 activity in phosphate homeostasis. Distinct sites within Ppn1 for binding to Dis2 (spanning Ppn1 aa 506 to 532) and Swd22 (from Ppn1 aa 533 to 578) were demarcated by yeast two-hybrid assays. Dis2 interaction-defective missense mutants of full-length Ppn1 (that retained Swd22 interaction) were employed to show that binding to Dis2 (or its paralog Sds21) was necessary for Ppn1 biological activity. Ppn1 function was severely compromised by missense mutations that selectively affected its binding to Swd22.
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Gruel, Y., E. Brojer, D. J. Nugent i T. J. Kunicki. "Further characterization of the thrombasthenia-related idiotype OG. Antiidiotype defines a novel epitope(s) shared by fibrinogen B beta chain, vitronectin, and von Willebrand factor and required for binding to beta 3." Journal of Experimental Medicine 180, nr 6 (1.12.1994): 2259–67. http://dx.doi.org/10.1084/jem.180.6.2259.
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A patient (OG) with Glanzmann thrombasthenia became refractory to platelet transfusion after the production of an immunoglobulin G (IgG) isoantibody (Ab1) specific for the integrin subunit beta 3. To determine the frequency at which the OG idiotype is found in the general population and in immune-mediated disease states, we developed a rabbit polyclonal antibody (Ab2) specific for affinity-purified OG anti-beta 3 Fab. The binding of Ab2 to Ab1 is inhibited by purified alpha IIb beta 3. Ab2 als binds to IgG specific for alpha IIb beta 3 obtained from one nonrelated Glanzmann thrombasthenia patient ES who has developed isoantibodies of similar specificity. On the other hand, Ab2 does not recognize alpha IIb beta 3-specific antibodies produced by two Glanzmann thrombasthenia patients, AF and LUC, who have developed isoantibodies with specificities distinct from that of the OG isoantibody. Moreover, Ab2 does not recognize alpha IIb beta 3-specific antibodies developed by three representative patients with (autoimmune) thrombocytopenic purpura or six representative patients with alloimmune thrombocytopenias, nor does it bind to IgG from any of 13 nonimmunized individuals. We have found that Ab2 also binds to selected protein ligands of alpha IIb beta 3 namely, fibrinogen, vitronectin, and von Willebrand factor, but not to other protein ligands or control proteins, such a fibronectin, type I collagen, and albumin. The epitope(s) recognized by Ab2 on each adhesive protein are either very similar or identical since each protein can inhibit the binding of Ab2 to any of the other proteins. The epitope on fibrinogen recognized by Ab2 resides in the B beta chain, and is likely contained within the first 42 amino acids from the NH2 terminus. Since OG IgG inhibits fibrinogen binding to alpha IIb beta 3, the specificity of the OG idiotype defines a novel binding motif for the integrin alpha IIb beta 3 that is shared by fibrinogen, vitronectin, and von Willebrand factor, but distinct from previously described RGD-containing sites on the fibrinogen, A alpha chain or the fibrinogen gamma chain COOH-terminal decapeptide site. Our findings reported here represent an excellent example of molecular mimicry in which an antigen-selected, IgG inhibitor of alpha IIb beta 3 function shares a novel recognition sequence common to three physiologic protein ligands of that receptor.
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Denis, C., JA Williams, X. Lu, D. Meyer i D. Baruch. "Solid-phase von Willebrand factor contains a conformationally active RGD motif that mediates endothelial cell adhesion through the alpha v beta 3 receptor". Blood 82, nr 12 (15.12.1993): 3622–30. http://dx.doi.org/10.1182/blood.v82.12.3622.3622.
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Abstract The interaction of von Willebrand factor (vWF) with the alpha v beta 3 integrin of human umbilical vein endothelial cells is dependent on the RGD sequence present at residues 1744–1746 of the mature vWF subunit. We compared vWF and its two dimeric fragments, SpIII (residues 1–1365) and SpII (residues 1366–2050), as adhesion substrates. Solid-phase vWF and SpII supported endothelial cell adhesion, whereas SpIII, which contains the glycoprotein (GP) Ib binding domain, did not. Soluble SpII inhibited adhesion to immobilized ligands, whereas soluble vWF did not, suggesting that exposure of the cell attachment domain involves a conformational modification of vWF. Dendroaspin and albolabrin, two RGD- containing peptides of the disintegrin family, were potent inhibitors of cell adhesion to vWF (IC50 approximately 15 nmol/L). Complete inhibition of endothelial cell adhesion to vWF was obtained in the presence of F(ab')2 of monoclonal antibody 9 to vWF, which blocks vWF binding to platelet GPIIb/IIIa. In contrast, monoclonal antibody 713 to vWF, which blocks its binding to platelet GPIb, did not inhibit cell adhesion to vWF. These results indicate that endothelial cell adhesion to vWF is mediated by an RGD-dependent interaction with alpha v beta 3, but does not seem to involve a GPIb-like receptor, and show the importance of the conformation of the RGD sequence.
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Denis, C., JA Williams, X. Lu, D. Meyer i D. Baruch. "Solid-phase von Willebrand factor contains a conformationally active RGD motif that mediates endothelial cell adhesion through the alpha v beta 3 receptor". Blood 82, nr 12 (15.12.1993): 3622–30. http://dx.doi.org/10.1182/blood.v82.12.3622.bloodjournal82123622.
Pełny tekst źródłaStreszczenie:
The interaction of von Willebrand factor (vWF) with the alpha v beta 3 integrin of human umbilical vein endothelial cells is dependent on the RGD sequence present at residues 1744–1746 of the mature vWF subunit. We compared vWF and its two dimeric fragments, SpIII (residues 1–1365) and SpII (residues 1366–2050), as adhesion substrates. Solid-phase vWF and SpII supported endothelial cell adhesion, whereas SpIII, which contains the glycoprotein (GP) Ib binding domain, did not. Soluble SpII inhibited adhesion to immobilized ligands, whereas soluble vWF did not, suggesting that exposure of the cell attachment domain involves a conformational modification of vWF. Dendroaspin and albolabrin, two RGD- containing peptides of the disintegrin family, were potent inhibitors of cell adhesion to vWF (IC50 approximately 15 nmol/L). Complete inhibition of endothelial cell adhesion to vWF was obtained in the presence of F(ab')2 of monoclonal antibody 9 to vWF, which blocks vWF binding to platelet GPIIb/IIIa. In contrast, monoclonal antibody 713 to vWF, which blocks its binding to platelet GPIb, did not inhibit cell adhesion to vWF. These results indicate that endothelial cell adhesion to vWF is mediated by an RGD-dependent interaction with alpha v beta 3, but does not seem to involve a GPIb-like receptor, and show the importance of the conformation of the RGD sequence.
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Fry, M. J., G. Panayotou, R. Dhand, F. Ruiz-Larrea, I. Gout, O. Nguyen, S. A. Courtneidge i M. D. Waterfield. "Purification and characterization of a phosphatidylinositol 3-kinase complex from bovine brain by using phosphopeptide affinity columns". Biochemical Journal 288, nr 2 (1.12.1992): 383–93. http://dx.doi.org/10.1042/bj2880383.
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Specific phosphorylated tyrosine residues in the kinase insert region of the human platelet-derived-growth-factor beta-receptor mediate the formation of multienzyme complexes with this receptor. When phosphorylated, tyrosine residue 751 within the kinase insert region mediates binding of PtdIns 3-kinase to this receptor. A 17-amino-acid peptide containing this tyrosine residue was synthesized, phosphorylated by using epidermal-growth-factor receptor and then coupled to an Actigel matrix. The tyrosine-751 phosphopeptide column is used here as a final affinity step in the purification of the PtdIns 3-kinase from bovine brain to apparent homogeneity. The active resin-bound PtdIns 3-kinase is composed of two polypeptides, p110 and p85, which are elutable with SDS-containing buffers and detectable by silver staining of polyacrylamide gels. The 85 kDa protein is shown to be identical with the recently cloned p85 alpha. Phosphotyrosine is demonstrated to be an essential part of the structure required for binding of both of these proteins and PtdIns 3-kinase activity to this peptide. The active PtdIns 3-kinase complex from bovine brain, but not recombinant p85 subunits, shows specificity for binding to phosphopeptides containing a YXXM consensus sequence. Neither PtdIns 3-kinase activity, nor the complex of p85 and 110 kDa proteins, binds to several other phosphopeptide affinity columns lacking this sequence motif. The selectivity of binding of baculovirus-expressed free p85 alpha subunit of bovine brain PtdIns 3-kinase, the closely related protein p85 beta and purified bovine brain PtdIns 3-kinase to these and other phosphopeptide columns is examined.
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McLeish, K. R., J. B. Klein, T. Schepers i G. Sonnenfeld. "Modulation of transmembrane signalling in HL-60 granulocytes by tumour necrosis factor-α". Biochemical Journal 279, nr 2 (15.10.1991): 455–60. http://dx.doi.org/10.1042/bj2790455.
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Differentiated HL-60 granulocytes were used to study the mechanism by which tumour necrosis factor-alpha (TNF) enhances responses to N-formyl-methionyl-leucylphenylalanine (FMLP). Cultivation of differentiated HL-60 cells with 100 units of TNF/ml for 24 h resulted in a 3-fold increase in superoxide release and 4-fold increase in prostaglandin E2 production on stimulation with 1 microM-FMLP. On the other hand, cultivation with TNF failed to increase phorbol diester stimulation of superoxide release. Formyl-peptide-receptor expression determined on isolated membranes from cells cultivated with TNF (TNF-M) was increased by 50% compared with membranes from control cells (NM). Similarly, FMLP binding to intact HL-60 cells was increased by cultivation with TNF. Guanine-nucleotide-binding proteins (G-protein) levels were not different between TNF-M and NM, as determined by pertussis-toxin-catalysed ADP-ribosylation and by immunoblotting with antisera recognizing alpha i2 subunit. Binding of guanosine 5′-[gamma-thio]triphosphate and GTP hydrolysis stimulated by FMLP were enhanced by about 50% in TNF-M. The efficiency of G-protein activation by formyl-peptide receptors did not differ between TNF-M and NM. TNF regulates expression of formyl-peptide receptors independently of G-protein levels. The regulation of receptor expression is one mechanism by which TNF enhances cell responses to formylated peptides.