Academic literature on the topic 'Core Binding Factor alpha Subunits'
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Journal articles on the topic "Core Binding Factor alpha Subunits"
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Nuchprayoon, I., S. Meyers, L. M. Scott, J. Suzow, S. Hiebert, and 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, no. 8 (August 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, and 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, no. 8 (August 1994): 5558–68. http://dx.doi.org/10.1128/mcb.14.8.5558-5568.1994.
Full textAbstract:
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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Wang, S., Q. Wang, B. E. Crute, I. N. Melnikova, S. R. Keller, and 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, no. 6 (June 1993): 3324–39. http://dx.doi.org/10.1128/mcb.13.6.3324.
Full textAbstract:
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, and 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, no. 6 (June 1993): 3324–39. http://dx.doi.org/10.1128/mcb.13.6.3324-3339.1993.
Full textAbstract:
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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Zaiman, A. L., A. F. Lewis, B. E. Crute, N. A. Speck, and J. Lenz. "Transcriptional activity of core binding factor-alpha (AML1) and beta subunits on murine leukemia virus enhancer cores." Journal of virology 69, no. 5 (1995): 2898–906. http://dx.doi.org/10.1128/jvi.69.5.2898-2906.1995.
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Sun, Ge, Chunyu Wang, Shengli Wang, Hongmiao Sun, Kai Zeng, Renlong Zou, Lin Lin, et al. "An H3K4me3 reader, BAP18 as an adaptor of COMPASS-like core subunits co-activates ERα action and associates with the sensitivity of antiestrogen in breast cancer." Nucleic Acids Research 48, no. 19 (September 28, 2020): 10768–84. http://dx.doi.org/10.1093/nar/gkaa787.
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Abstract Estrogen receptor alpha (ERα) signaling pathway is essential for ERα-positive breast cancer progression and endocrine therapy resistance. Bromodomain PHD Finger Transcription Factor (BPTF) associated protein of 18kDa (BAP18) has been recognized as a crucial H3K4me3 reader. However, the whole genomic occupation of BAP18 and its biological function in breast cancer is still elusive. Here, we found that higher expression of BAP18 in ERα-positive breast cancer is positively correlated with poor prognosis. ChIP-seq analysis further demonstrated that the half estrogen response elements (EREs) and the CCCTC binding factor (CTCF) binding sites are the significant enrichment sites found in estrogen-induced BAP18 binding sites. Also, we provide the evidence to demonstrate that BAP18 as a novel co-activator of ERα is required for the recruitment of COMPASS-like core subunits to the cis-regulatory element of ERα target genes in breast cancer cells. BAP18 is recruited to the promoter regions of estrogen-induced genes, accompanied with the enrichment of the lysine 4-trimethylated histone H3 tail (H3K4me3) in the presence of E2. Furthermore, BAP18 promotes cell growth and associates the sensitivity of antiestrogen in ERα-positive breast cancer. Our data suggest that BAP18 facilitates the association between ERα and COMPASS-like core subunits, which might be an essential epigenetic therapeutic target for breast cancer.
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Beghini, Alessandro. "Core Binding Factor Leukemia: Chromatin Remodeling Moves Towards Oncogenic Transcription." Cancers 11, no. 12 (December 7, 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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Fischer, G., C. Schmidt, J. Opitz, Z. Cully, K. Kühn, and E. Pöschl. "Identification of a novel sequence element in the common promoter region of human collagen type IV genes, involved in the regulation of divergent transcription." Biochemical Journal 292, no. 3 (June 15, 1993): 687–95. http://dx.doi.org/10.1042/bj2920687.
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The expression of the heterotrimeric collagen IV molecule alpha 1(IV)2 alpha 2(IV) is essential for the structural integrity and functional properties of all basement membranes. The two genes COL4A1 and COL4A2 that code for the subunits are found closely linked on chromosome 13 in a head-to-head arrangement and are transcribed in divergent directions. We have identified a novel trans-acting factor that binds in vitro to a unique homopyrimidine/homopurine stretch within the shared promoter region of the two collagen IV genes. Additional binding sites have been identified within the first introns of both genes and the consensus sequence CCCTYCCCC for efficient binding has been deduced; the factor was named therefore ‘CTC-binding factor’ or ‘CTCBF’. Mutations in the binding site of CTC-binding factor within the promoter inhibited binding in vitro and resulted in reduced transcription from both genes. The effect of mutations on the transcription of COL4A2 is more pronounced than on the transcription of COL4A1. CTC-binding factor is a nuclear factor that binds dominantly in vitro to the collagen IV promoter and is involved in regulating the expression of both collagen IV genes.
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Park, Kyung-Ran, Joon-Yeop Lee, Myounglae Cho, Jin-Tae Hong, and 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, no. 9 (May 6, 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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Weiss, Andy, J. Antonio Ibarra, Jessica Paoletti, Ronan K. Carroll, and Lindsey N. Shaw. "The δ Subunit of RNA Polymerase Guides Promoter Selectivity and Virulence in Staphylococcus aureus." Infection and Immunity 82, no. 4 (February 3, 2014): 1424–35. http://dx.doi.org/10.1128/iai.01508-14.
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ABSTRACTIn Gram-positive bacteria, and particularly theFirmicutes, the DNA-dependent RNA polymerase (RNAP) complex contains an additional subunit, termed the δ factor, or RpoE. This enigmatic protein has been studied for more than 30 years for various organisms, but its function is still not well understood. In this study, we investigated its role in the major human pathogenStaphylococcus aureus. We showed conservation of important structural regions of RpoE inS. aureusand other species and demonstrated binding to core RNAP that is mediated by the β and/or β′ subunits. To identify the impact of the δ subunit on transcription, we performed transcriptome sequencing (RNA-seq) analysis and observed 191 differentially expressed genes in therpoEmutant. Ontological analysis revealed, quite strikingly, that many of the downregulated genes were known virulence factors, while several mobile genetic elements (SaPI5 and prophage ϕSA3usa) were strongly upregulated. Phenotypically, therpoEmutant had decreased accumulation and/or activity of a number of key virulence factors, including alpha toxin, secreted proteases, and Panton-Valentine leukocidin (PVL). We further observed significantly decreased survival of the mutant in whole human blood, increased phagocytosis by human leukocytes, and impaired virulence in a murine model of infection. Collectively, our results demonstrate that the δ subunit of RNAP is a critical component of theS. aureustranscription machinery and plays an important role during infection.
Dissertations / Theses on the topic "Core Binding Factor alpha Subunits"
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Pande, Sandhya. "Regulation of Runx Proteins in Human Cancers: A Dissertation." eScholarship@UMMS, 2011. https://escholarship.umassmed.edu/gsbs_diss/559.
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Runt related transcription factors (Runx) play an important role in mammalian development by regulating the expression of key genes involved in cell proliferation, differentiation and growth. The work described in this thesis details the mechanisms by which the activity of two members of this family are regulated in human cells. Chapter One provides a brief introduction of Runx transcription factors.
Chapter Two describes the regulation of Runx2 protein by the PI3 kinase/Akt pathway in human breast cancer cells. The PI3 kinase/Akt pathway is one of the major signal transduction pathways through which growth factors influence cell proliferation and survival. It is also one of the most frequently dysregulated pathways in human cancers. We identify Runx2 protein, a key regulator of breast cancer invasion as a novel substrate of Akt kinase and map residues of Runx2 that are phosphorylated by Akt in breast cancer cells. Our results show that phosphorylation by Akt increases the binding of Runx2 protein to its target gene promoters and we identify the phosphorylation events that enhance DNA binding of Runx2. Our work establishes Runx2 protein as a critical effecter downstream of Akt that regulates breast cancer invasion.
In Chapter Three we describe the subnuclear localization of the tumor suppressor protein Runx3 during interphase and mitosis. We find that similar to other Runx family members, Runx3 protein resides in nuclear matrix associated foci during interphase. We delineate a subnuclear targeting signal that directs Runx3 to these nuclear matrix associated foci. Our work establishes that this association of Runx3 protein with the nuclear matrix plays a vital role in regulating its transcriptional activity.
Chromatin immunoprecipitation results show that Runx3 occupies rRNA promoters during interphase. We also find that Runx3 remains associated with chromosomes during mitosis and localizes with nucleolar organizing regions (NORs), reflecting an interaction with epigenetic potential.
This thesis provides novel insights into various mechanisms by which cells regulate the activity of Runx proteins.
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LeBlanc, Kimberly T. "Runx Expression in Normal and Osteoarthritic Cartilage: Possible Functions of Runx Proteins in Chondrocytes: A Dissertation." eScholarship@UMMS, 2002. http://escholarship.umassmed.edu/gsbs_diss/655.
Full textAbstract:
The Runx family of transcription factors supports cell fate determination, cell cycle regulation, global protein synthesis control, and genetic as well as epigenetic regulation of target genes. Runx1, which is essential for hematopoiesis; Runx2, which is required for osteoblast differentiation; and Runx3, which is involved in neurologic and gut development; are expressed in the growth plate during chondrocyte maturation, and in the chondrocytes of permanent cartilage structures. While Runx2 is known to control genes that contribute to chondrocyte hypertrophy, the functions of Runx1 and Runx3 during chondrogenesis and in cartilage tissue have been less well studied.
The goals of this project were to characterize expression of Runx proteins in articular cartilage and differentiating chondrocytes and to determine the contribution of Runx1 to osteoarthritis (OA). Here, the expression pattern of Runx1 and Runx2 was characterized in normal bovine articular cartilage. Runx2 is expressed at higher levels in deep zone chondrocytes, while Runx1 is primarily expressed in superficial zone chondrocytes, which is the single cell layer that lines the surface of articular cartilage. Based on this finding, the hypothesis was tested that Runx1 is involved in osteoarthritis, which is a disease characterized by degradation of articular cartilage and changes in chondrocytes. These studies showed that Runx1 is upregulated in articular cartilage explants in response to mechanical compression. Runx1 was also expressed in chondrocytes found at the periphery of OA lesions in the articular cartilage of mice that underwent an OA-inducing surgery. Runx1 was also upregulated in cartilage explants of human osteoarthritic knees, and IHC data showed that Runx1 is mainly expressed in chondrocyte “clones” characteristic of OA.
To ascertain the potential function of the upregulation of Runx1 in these cartilage stress conditions and disease states, the hypothesis was tested that Runx1 is upregulated in very specific chondrocyte populations in response to the cartilage damage in osteoarthritis. These studies addressed the properties of these cells that related to functions in cell growth and differentiation. In both the surface layer of normal articular cartilage, and in OA cartilage, Runx1 expression by IF co-localized with markers of mesenchymal progenitor cells, as well as markers of proliferation Ki-67 and PCNA. This finding indicated that Runx1 is found in a population of cells that represent a proliferative population of mesenchymal progenitor cells in osteoarthritis.
To further address Runx1 function and identify downstream targets of Runx proteins, a promoter analysis of genes that are known to be either downregulated or upregulated during chondrocyte maturation was done. These studies found that many of these genes have 1 or more Runx binding sites within 2kb of their transcription start site, indicating that they are potential downstream Runx target genes.
Lastly, some preliminary experiments were done to characterize novel roles of Runx proteins in the chondrocyte. Runx proteins have been shown to epigenetically regulate their target genes by remaining bound to them throughout mitosis, “poising” them for transcription upon exit from mitosis. The hypothesis that Runx proteins also function by remaining bound to their target genes throughout mitosis in chondrocytes was tested. It was demonstrated by immunofluorescense imaging of Runx proteins on metaphase chromosomes of ATDC5 cells, that Runx2 remains bound to chromosomes during mitosis.
Cell proliferation and hypertrophy are both linked to increases in protein synthesis. Runx factors, which regulate rates of global protein synthesis, are expressed in both proliferating and hypertrophic chondrocytes. Thus, it was hypothesized that Runx proteins regulate rates of global protein synthesis during chondrocyte maturation. These studies showed that the overexpression of Runx proteins in a chondrocyte cell line (ATDC5) did not affect protein synthesis rates or levels of protein synthesis machinery. Additionally, Runx proteins did not affect proliferation rates in this chondrocyte cell line.
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LeBlanc, Kimberly T. "Runx Expression in Normal and Osteoarthritic Cartilage: Possible Functions of Runx Proteins in Chondrocytes: A Dissertation." eScholarship@UMMS, 2013. https://escholarship.umassmed.edu/gsbs_diss/655.
Full textAbstract:
The Runx family of transcription factors supports cell fate determination, cell cycle regulation, global protein synthesis control, and genetic as well as epigenetic regulation of target genes. Runx1, which is essential for hematopoiesis; Runx2, which is required for osteoblast differentiation; and Runx3, which is involved in neurologic and gut development; are expressed in the growth plate during chondrocyte maturation, and in the chondrocytes of permanent cartilage structures. While Runx2 is known to control genes that contribute to chondrocyte hypertrophy, the functions of Runx1 and Runx3 during chondrogenesis and in cartilage tissue have been less well studied.
The goals of this project were to characterize expression of Runx proteins in articular cartilage and differentiating chondrocytes and to determine the contribution of Runx1 to osteoarthritis (OA). Here, the expression pattern of Runx1 and Runx2 was characterized in normal bovine articular cartilage. Runx2 is expressed at higher levels in deep zone chondrocytes, while Runx1 is primarily expressed in superficial zone chondrocytes, which is the single cell layer that lines the surface of articular cartilage. Based on this finding, the hypothesis was tested that Runx1 is involved in osteoarthritis, which is a disease characterized by degradation of articular cartilage and changes in chondrocytes. These studies showed that Runx1 is upregulated in articular cartilage explants in response to mechanical compression. Runx1 was also expressed in chondrocytes found at the periphery of OA lesions in the articular cartilage of mice that underwent an OA-inducing surgery. Runx1 was also upregulated in cartilage explants of human osteoarthritic knees, and IHC data showed that Runx1 is mainly expressed in chondrocyte “clones” characteristic of OA.
To ascertain the potential function of the upregulation of Runx1 in these cartilage stress conditions and disease states, the hypothesis was tested that Runx1 is upregulated in very specific chondrocyte populations in response to the cartilage damage in osteoarthritis. These studies addressed the properties of these cells that related to functions in cell growth and differentiation. In both the surface layer of normal articular cartilage, and in OA cartilage, Runx1 expression by IF co-localized with markers of mesenchymal progenitor cells, as well as markers of proliferation Ki-67 and PCNA. This finding indicated that Runx1 is found in a population of cells that represent a proliferative population of mesenchymal progenitor cells in osteoarthritis.
To further address Runx1 function and identify downstream targets of Runx proteins, a promoter analysis of genes that are known to be either downregulated or upregulated during chondrocyte maturation was done. These studies found that many of these genes have 1 or more Runx binding sites within 2kb of their transcription start site, indicating that they are potential downstream Runx target genes.
Lastly, some preliminary experiments were done to characterize novel roles of Runx proteins in the chondrocyte. Runx proteins have been shown to epigenetically regulate their target genes by remaining bound to them throughout mitosis, “poising” them for transcription upon exit from mitosis. The hypothesis that Runx proteins also function by remaining bound to their target genes throughout mitosis in chondrocytes was tested. It was demonstrated by immunofluorescense imaging of Runx proteins on metaphase chromosomes of ATDC5 cells, that Runx2 remains bound to chromosomes during mitosis.
Cell proliferation and hypertrophy are both linked to increases in protein synthesis. Runx factors, which regulate rates of global protein synthesis, are expressed in both proliferating and hypertrophic chondrocytes. Thus, it was hypothesized that Runx proteins regulate rates of global protein synthesis during chondrocyte maturation. These studies showed that the overexpression of Runx proteins in a chondrocyte cell line (ATDC5) did not affect protein synthesis rates or levels of protein synthesis machinery. Additionally, Runx proteins did not affect proliferation rates in this chondrocyte cell line.
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Dobson, Jason R. "Nuclear Organization in Breast Cancer: A Dissertation." eScholarship@UMMS, 2013. https://escholarship.umassmed.edu/gsbs_diss/650.
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The nuclear matrix (NM) is a fibrogranular network of ribonucleoproteins upon which transcriptional complexes and regulatory genomic sequences are organized. A hallmark of cancer is the disorganization of nuclear architecture; however, the extent to which the NM is involved in malignancy is not well studied.
The RUNX1 and RUNX2 proteins form complexes within the NM to promote hematopoiesis and osteoblastogenesis, respectively at the transcriptional level. RUNX1 and RUNX2 are both expressed in breast cancer cells (BrCCs); however, their genome-wide BrCC functions are unknown. RUNX1 and RUNX2 activate many tumor suppressor pathways in blood and bone lineages, respectively, including attenuation of protein synthesis and cell growth via suppression of ribosomal RNA (rRNA) transcription, which appears contrary to Runx-expression in highly proliferative BrCCs. To define roles for RUNX1 and RUNX2 in BrCC phenotype, we examined the involvement of RUNX1 and RUNX2 in rRNA transcription and generated a genome-wide model for RUNX1 and RUNX2-binding and transcriptional regulation. To validate gene expression patterns identified in our screen, we developed a Real-Time qPCR primer design program, which allows rapid, high-throughput design of primer pairs (FoxPrimer). In BrCCs, RUNX1 and RUNX2 regulate genes that promote invasiveness and do not affect rRNA transcription, protein synthesis, or cell growth. We have characterized in vitro functions of Runx proteins in BrCCs; however, the relationships between Runx expression and diagnostic/prognostic markers of breast cancer (BrCa) in patients are not well studied. Immunohistochemical detection of RUNX1 and RUNX2 in BrCa tissue microarrays reveals RUNX1 expression is associated with early, smaller tumors that are ER+ (estrogen receptor), HER2+, p53-, and correlated with androgen receptor (AR) expression; RUNX2 expression is associated with late-stage, larger tumors that are HER2+. These results show that the functions and expression patterns of NM-associated RUNX1 and RUNX2 are context-sensitive, which suggests potential disease-specific roles.
Two functionally disparate genomic sequence types bind to the NM: matrix associated regions (MARs) are functionally associated with transcriptional repression and scaffold associated regions (SARs) are functionally associated with actively expressed genes. It is unknown whether malignant nuclear disorganization affects the functions of MARs/SARs in BrCC. We have refined a method to isolate nuclear matrix associated DNA (NM-DNA) from a structurally preserved NM and applied this protocol to normal mammary epithelial cells and BrCCs. To define transcriptional functions for NM-DNA, we developed a computational algorithm (PeaksToGenes), which statistically tests the associations of experimentally-defined NM-DNA regions and ChIP-seq-defined positional enrichment of several histone marks with transcriptome-wide gene expression data. In normal mammary epithelial cells, NM-DNA is enriched in both MARs and SARs, and the positional enrichment patterns of MARs and SARs are strongly associated with gene expression patterns, suggesting functional roles. In contrast, the BrCCs are significantly enriched in the silencing mark H3K27me3, and the NM-DNA is enriched in MARs and depleted of SARs. The MARs/SARs in the BrCCs are only weakly associated with gene expression patterns, suggesting that loss of normal DNA-matrix associations accompanies the disease state. Our results show that structural preservation of the in situ NM allows isolation of both MARs and SARs, and further demonstrate that in a disorganized, cancerous nucleus, normal transcriptional functions of NM-DNA are disrupted.
Our studies on nuclear organization in BrCC, show that the disorganized phenotype of the cancer cell nucleus is accompanied by deregulated transcriptional functions of two constituents of the NM. These results reinforce the role of the NM as an important structure-function component of gene expression regulation.
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Dobson, Jason R. "Nuclear Organization in Breast Cancer: A Dissertation." eScholarship@UMMS, 2004. http://escholarship.umassmed.edu/gsbs_diss/650.
Full textAbstract:
The nuclear matrix (NM) is a fibrogranular network of ribonucleoproteins upon which transcriptional complexes and regulatory genomic sequences are organized. A hallmark of cancer is the disorganization of nuclear architecture; however, the extent to which the NM is involved in malignancy is not well studied.
The RUNX1 and RUNX2 proteins form complexes within the NM to promote hematopoiesis and osteoblastogenesis, respectively at the transcriptional level. RUNX1 and RUNX2 are both expressed in breast cancer cells (BrCCs); however, their genome-wide BrCC functions are unknown. RUNX1 and RUNX2 activate many tumor suppressor pathways in blood and bone lineages, respectively, including attenuation of protein synthesis and cell growth via suppression of ribosomal RNA (rRNA) transcription, which appears contrary to Runx-expression in highly proliferative BrCCs. To define roles for RUNX1 and RUNX2 in BrCC phenotype, we examined the involvement of RUNX1 and RUNX2 in rRNA transcription and generated a genome-wide model for RUNX1 and RUNX2-binding and transcriptional regulation. To validate gene expression patterns identified in our screen, we developed a Real-Time qPCR primer design program, which allows rapid, high-throughput design of primer pairs (FoxPrimer). In BrCCs, RUNX1 and RUNX2 regulate genes that promote invasiveness and do not affect rRNA transcription, protein synthesis, or cell growth. We have characterized in vitro functions of Runx proteins in BrCCs; however, the relationships between Runx expression and diagnostic/prognostic markers of breast cancer (BrCa) in patients are not well studied. Immunohistochemical detection of RUNX1 and RUNX2 in BrCa tissue microarrays reveals RUNX1 expression is associated with early, smaller tumors that are ER+ (estrogen receptor), HER2+, p53-, and correlated with androgen receptor (AR) expression; RUNX2 expression is associated with late-stage, larger tumors that are HER2+. These results show that the functions and expression patterns of NM-associated RUNX1 and RUNX2 are context-sensitive, which suggests potential disease-specific roles.
Two functionally disparate genomic sequence types bind to the NM: matrix associated regions (MARs) are functionally associated with transcriptional repression and scaffold associated regions (SARs) are functionally associated with actively expressed genes. It is unknown whether malignant nuclear disorganization affects the functions of MARs/SARs in BrCC. We have refined a method to isolate nuclear matrix associated DNA (NM-DNA) from a structurally preserved NM and applied this protocol to normal mammary epithelial cells and BrCCs. To define transcriptional functions for NM-DNA, we developed a computational algorithm (PeaksToGenes), which statistically tests the associations of experimentally-defined NM-DNA regions and ChIP-seq-defined positional enrichment of several histone marks with transcriptome-wide gene expression data. In normal mammary epithelial cells, NM-DNA is enriched in both MARs and SARs, and the positional enrichment patterns of MARs and SARs are strongly associated with gene expression patterns, suggesting functional roles. In contrast, the BrCCs are significantly enriched in the silencing mark H3K27me3, and the NM-DNA is enriched in MARs and depleted of SARs. The MARs/SARs in the BrCCs are only weakly associated with gene expression patterns, suggesting that loss of normal DNA-matrix associations accompanies the disease state. Our results show that structural preservation of the in situ NM allows isolation of both MARs and SARs, and further demonstrate that in a disorganized, cancerous nucleus, normal transcriptional functions of NM-DNA are disrupted.
Our studies on nuclear organization in BrCC, show that the disorganized phenotype of the cancer cell nucleus is accompanied by deregulated transcriptional functions of two constituents of the NM. These results reinforce the role of the NM as an important structure-function component of gene expression regulation.
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Madera, Dmitri. "Cooperating Events in Core Binding Factor Leukemia Development: A Dissertation." eScholarship@UMMS, 2011. https://escholarship.umassmed.edu/gsbs_diss/532.
Full textAbstract:
Leukemia is a hematopoietic cancer that is characterized by the abnormal differentiation and proliferation of hematopoietic cells. It is ranked 7th by death rate among cancer types in USA, even though it is not one of the top 10 cancers by incidence (USCS, 2010). This indicates an urgent need for more effective treatment strategies. In order to design the new ways of prevention and treatment of leukemia, it is important to understand the molecular mechanisms involved in development of the disease.
In this study, we investigated mechanisms involved in the development of acute myeloid leukemia (AML) that is associated with CBF fusion genes. The RUNX1 and CBFB genes that encode subunits of a transcriptional regulator complex CBF, are mutated in a subset (20 – 25%) of AML cases. As a result of these mutations, fusion genes called CBFB-MYH11 and RUNX1-ETO arise. The chimeric proteins encoded by the fusion genes provide block in proliferation for myeloid progenitors, but are not sufficient for AML development. Genetic studies have indicated that activation of cytokine receptor signaling is a major oncogenic pathway that cooperates in leukemia development. The main goal of my work was to determine a role of two factors that regulate cytokine signaling activity, the microRNA cluster miR-17-92 and the thrombopoietin receptor MPL, in their potential cooperation with the CBF fusions in AML development.
We determined that the miR-17-92 miRNA cluster cooperates with Cbfb-MYH11 in AML development in a mouse model of human CBFB-MYH11 AML. We found that the miR-17-92 cluster downregulates Pten and activates the PI3K/Akt pathway in the leukemic blasts. We also demonstrated that miR-17-92 provides an anti-apoptotic effect in the leukemic cells, but does not seem to affect proliferation. The anti-apoptotic effect was mainly due to activity of miR-17 and miR-20a, but not miR-19a and miR-19b.
Our second study demonstrated that wild type Mpl cooperated with RUNX1-ETO fusion in development of AML in mice. Mpl induced PI3K/Akt, Ras/Raf/Erk and Jak2/Stat5 signaling pathways in the AML cells. We showed that PIK3/Akt pathway plays a role in AML development both in vitro and in vivo by increasing survival of leukemic cells. The levels of MPL transcript in the AML samples correlated with their response to thrombopoietin (THPO). Moreover, we demonstrated that MPL provides pro-proliferative effect for the leukemic cells, and that the effect can be abrogated with inhibitors of PI3K/AKT and MEK/ERK pathways.
Taken together, these data confirm important roles for the PI3K/AKT and RAS/RAF/MEK pathways in the pathogenesis of AML, identifies two novel genes that can serve as secondary mutations in CBF fusions-associated AML, and in general expands our knowledge of mechanisms of leukemogenesis.
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Montelius, Andreas. "Role of transcription factors in sensory neuron specification /." Stockholm, 2007. http://diss.kib.ki.se/2007/978-91-7357-115-9/.
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Barutcu, Ahmet Rasim. "Characterization of Higher-order Chromatin Structure in Bone Differentiation and Breast Cancer: A Dissertation." eScholarship@UMMS, 2016. https://escholarship.umassmed.edu/gsbs_diss/827.
Full textAbstract:
Higher-order genome organization is important for the regulation of gene expression by bringing different cis-regulatory elements and promoters in proximity. The establishment and maintenance of long-range chromatin interactions occur in response to cellular and environmental cues with the binding of transcription factors and chromatin modifiers. Understanding the organization of the nucleus in differentiation and cancer has been a long standing challenge and is still not well-understood. In this thesis, I explore the dynamic changes in the higher-order chromatin structure in bone differentiation and breast cancer. First, we show dynamic chromatin contact between a distal regulatory element and the promoter of Runx2 gene, which encodes the Runtrelated transcription factor 2 (RUNX2) that is essential for bone development. Next, via using a genome-wide approach, we show that breast cancer cells have altered long-range chromatin contacts among small, gene-rich chromosomes and at telomeres when compared with mammary epithelial cells. Furthermore, we assess the changes in nuclear structure and gene expression of breast cancer cells following Runt-related transcription factor 1 (RUNX1) deficiency, an event frequently observed in breast cancer. Finally, I present the role of the central ATPase subunit of the SWI/SNF complex, SMARCA4 (BRG1), in mediating nuclear structure and gene expression. Taken together, the research presented in this thesis reveals novel insight and paradigm for the dynamic changes in disease and differentiation, as well as uncovers previously unidentified roles for two chromatin regulatory proteins, RUNX1 and SMARCA4.
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Theriault, Francesca M. "Regulation of neuronal diversity in the mammalian nervous system." Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=103300.
Full textAbstract:
To acquire its characteristic structural and functional complexity, the mammalian nervous system must undergo several critical developmental processes. One such process requires factors that regulate the decision of dividing progenitors to leave the cell cycle and activate the neuronal differentiation program. It is shown in this thesis that the murine runt-related gene Runx1 is expressed in proliferating cells on the basal side of the murine olfactory epithelium. Disruption of Runx1 function in vivo does not result in a change in the quantity of progenitors but leads to a decrease in precursor number and an increase in differentiated ORNs. These effects result in premature and ectopic ORN differentiation. Further, exogenous Runx1 expression in cultured olfactory neural progenitors causes an expansion of the mitotic cell population. In agreement with these findings, exogenous Runx1 expression also promotes cortical neural progenitor cell proliferation without inhibiting neuronal differentiation. These effects appear to involve transcriptional repression mechanisms. Consistent with this possibility, Runx1 represses transcription driven by the promoter of the cell cycle inhibitor p21Cip1 in cortical progenitors. Taken together, these findings suggest a previously unrecognized role for Runx1 in coordinating the proliferation and neuronal differentiation of selected populations of neural progenitors/precursors.Another significant step in the development of the mammalian nervous system is the acquisition of distinctive neuronal traits. This thesis also shows that Runx1 is expressed in selected populations of postmitotic neurons of the murine embryonic central and peripheral nervous systems. In embryos lacking Runx1 activity, hindbrain branchiovisceral motor neuron precursors of the cholinergie lineage are correctly specified but then fail to enter successive stages of differentiation and undergo increased cell death resulting in neuronal loss in the mantle layer. Runx1 inactivation also leads to a loss of selected sensory neurons in trigeminal and vestibulocochlear ganglia. These findings uncover previously unrecognized roles for Runx1 in the regulation of neuronal subtype specification.
This thesis thus presents a novel factor which functions at several steps in the development of the mammalian nervous system and adds to the growing body of work on the processes involved in elaborating such a complex and vital structure.
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Gutierrez, Gallegos Soraya Elisa. "Mechanisms Contributing to Transcriptional Regulation and Chromatin Remodeling of the Bone Specific Osteocalcin Gene." eScholarship@UMMS, 2002. https://escholarship.umassmed.edu/gsbs_diss/12.
Full textAbstract:
Activation of tissue-specific genes is a tightly controlled process that normally involves the combined action of several transcription factors and transcriptional co-regulators. The bone-specific osteoca1cin gene (OC) has been used as a prototype to study both tissue-specific and hormonal responsiveness. In this study we have examined the role of Runx2, VDR and C/EBP factors in the regulation of OC gene transcription. Contributions of the Runx and VDRE motifs to OC promoter activity were addressed by introducing point mutations within the context of the rat (-1.1 kb) osteocalcin promoter fused to a CAT-reporter gene. The functional significance of these mutations was assayed following transient transfection and after genomic integration in ROS 17/2.8 osteoblastic cell lines. Furthermore, we tested the effect of these mutations on the chromatin organization of the OC promoter. Our data show that all three Runx sites are required for maximal activation of the OC promoter and that the distal sites contribute significantly to the basal activity. Strikingly, mutation of the three Runx sites abrogates responsiveness of the OC promoter to vitamin D; this loss is also observed when only the Runx sites flanking the VDRE are mutated. Chromatin changes that result in the appearance of DNase I hypersensitive sites during activation of the OC gene are well documented. Mutation of the three Runx sites results in altered chromatin structure as reflected by absence of DNase I hypersensitive sites at the vitamin D response element and over the proximal, tissue-specific basal promoter. These data are consistent with the critical role of Runx2 in osteoblast maturation and bone development.
Mutation of the VDRE resulted in a complete loss of vitamin D responsiveness; however, this mutant promoter exhibited increased basal activity. The two DNase I hypersensitive sites characteristic of the transcriptionally active OC gene in osteoblastics cells were not altered upon mutation of the VDRE element, although restriction enzyme accessibility in the proximal promoter region was decreased. We also found an increased level of histone H3 acetylation at the VDRE mutant promoter in comparison to the endogenous gene. Thus binding of VDR to OC promoter is required to achieve a normal transcriptional regulation and chromatin structure of the OC gene.
Although Runx2 is considered a master gene for bone development and osteoblast differentiation, it is noteworthy that osteoblast-specific transcription of the rat OC promoter occurs even in the absence of Runx sites. Therefore, other transcription factor(s) should be able to drive OC expression. We characterized a C/EBP enhancer element in the proximal promoter of the rat osteoca1cin gene that resides in close proximity to a Runx element, essential for tissue-specific activation. We find that C/EBPβ or δ and Runx2 factors interact together in a synergistic manner to enhance OC transcription in cell culture systems. Mutational analysis demonstrated that this synergism is mediated through the C/EBP responsive element in the OC promoter and requires a direct interaction between Runx2 and C/EBPβ or δ.
Taken together, our findings strongly support a mechanism in which combinatorial interaction of Runx2, VDR, C/EBPβ or δ and probably other transcription factors are needed for regulating OC expression. In this process Runx factors not only act as simple transcriptional trans activators but also by facilitating modifications in promoter architecture and maintaining an active conformation of the target gene promoter.
Book chapters on the topic "Core Binding Factor alpha Subunits"
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"Core-Binding Factor Subunit Alpha-3." In Encyclopedia of Signaling Molecules, 1198. Cham: Springer International Publishing, 2018. http://dx.doi.org/10.1007/978-3-319-67199-4_100819.
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"Core-Binding Factor, Runt Domain, Alpha Subunit 3." In Encyclopedia of Signaling Molecules, 1198. Cham: Springer International Publishing, 2018. http://dx.doi.org/10.1007/978-3-319-67199-4_100820.
Full textConference papers on the topic "Core Binding Factor alpha Subunits"
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Plow, E. F., G. A. Marguerie, and M. H. Ginsberg. "RECOGNITION SPECIFICITY OFADHESION RECEPTORS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643728.
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The cytoadhesins are a broadly distributedfamily of structurally, immunologicallyand functionally related cell surface molecules which participate in cellular adhesivereactions. The members of this family are heterodimeric in structure and consist of similar, if not identical, beta subunitsand related, but not identical, alpha subunits. A common functional property of the cytoadhesins is that they possess an arginyl-glycyl-aspartic acid (RGD) recognition specificity which mediates their capacity to act as receptors for adhesive proteins.The above assigned characteristics of thecytoadhesin family are based upon immunochemical analyses, primary structural determinations, deduced amino acid sequences fromcDNA clones and from dissectionof the recognition specificity of specificfamily members for their adhesive proteins.Platelet GPIIb-IIIa, the prototype of thecytoadhesin family, exhibits a very relaxed recognition specificity which permits itto interact with at least four distinct RGD-containing proteins: fibrinogen (Fg), fibronectin, von Willebrand Factor and vitronectin. RGD-containing peptidesas well as peptides corresponding in structure to the extreme carboxyl terminus of the gamma chain of Fg inhibit the binding of this adhesive protein set to GPIIb-IIIa.Both peptide sets interact with GPIIb-IIIaat either the same set of sites or intercommunicating sites. Nevertheless, using crosslinking reagents, certain RGD and gamma chain peptides can be crosslinked to different subunits of GPIIb-IIIa. This raises the possibility that a binding pocket for the adhesive proteins on GPIIb-IIIa may be directly comprised or situated in close proximity to both subunits of the cytoadhesin. Endothelial cells also express a cytoadhesin which is capable of binding Fg. Although both RGD and gamma chain peptides inhibit this interaction, the finerecognition specificity of the platelet and endothelial cell cytoadhesin for Fg are distinguishable. Thus, by combining very similar if not the same beta subunits with different alpha subunits, the RGD specificity of the cytoadhesins may bemodulated to achieve selectivity in the recognition of adhesive proteins.
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Dahiback, Bjorn, Ake Lundwall, Andreas Hillarp, Johan Malm, and Johan Stenflo. "STRUCTURE AND FUNCTION OF VITAMIN K-DEPENDENT PROTEIN S, a cofactor to activated protein C which also interacts with the complement protein C4b-binding protein." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642960.
Full textAbstract:
Protein S is a single chain (Mr 75.000) plasma protein. It is a cofactor to activated protein C (APC) in the regulation of coagulation factors Va and Villa. It has high affinity for negatively charged phospolipids and it forms a 1:1 complex with APC on phospholipid surfaces, platelets and on endothelial cells. Patients with heterozygous protein S deficiency have a high incidence of thrombosis. Protein S is cleaved by thrombin, which leads to a loss of calcium binding sites and of APC cofactor activity. Protein S has two to three high affinity (KD 20uM) calcium binding sites - unrelated to the Gla-region - that are unaffected by the thrombin cleavage. In human plasma protein S (25 mg/liter) circulates in two forms; free (approx. 40%) and in a 1:1 noncovalent complex (KD 1× 10-7M) with the complement protein C4b-binding protein (C4BP). C4BP (Mr 570.000) is composed of seven identical 70 kDa subunits that are linked by disulfide bonds. When visualized by electron microscopy, C4BP has a spiderlike structure with the single protein S binding site located close to the central core and one C4b-binding site on each of the seven tentacles. When bound to C4BP, protein S looses its APC cofactor activity, whereas the function-of C4BP is not directly affected by the protein S binding. Chymotrypsin cleaves each of the seven C4BP subunits close to the central core which results in the liberation of multiple 48 kDa “tentacte” fragments and the formation of a 160 kDa central core fragment. We have successfully isolated a 160 kDa central core fragment with essentially intact protein S binding ability.The primary structure of both bovine and human protein S has been determined and found to contain 635 and 634 amino acids, respectively, with 82 % homology to each other. Four different regions were distinguished; the N-terminal Gla-domain (position 1-45) was followed by a region which has two thrombin-sensitive bonds positioned within a disulfide loop. Position 76 to 244 was occupied by four repeats homologous to the epidermal growth factor (EGF) precursor. In the first EGF-domain a modified aspartic acid was identified at position 95, B-hydroxaspartic acid (Hya), and in corresponding positions in the three following EGF-domains (positions 136,178 and 217) we found B-hydroxyasparagine (Hyn). Hyn has not previously been identified in proteins. The C-terminal half of protein S (from position 245) shows no homology to the serine proteases but instead to human Sexual Hormon Binding Globulin (SHBG)(see separate abstract). To study the structure-function relationship we made eighteen monoclonal antibodies to human protein S. The effects of the monoclonals on the C4BP-protein S interaction and on the APC cofactor activity were analysed. Eight of the antibodies were calciumdependent, four of these were against the Gla-domain, two against the thrombin sensitive portion and two against the region bearing the high affinity calcium binding sites. Three of the monoclonals were dependent on the presence of chelating agents, EDTA or EGTA, and were probably directed against the high affinity calcium binding region. Three other monoclonals inhibited the protein S-C4BP interaction. At present, efforts are made to localize the epitopes to gain information about functionally important regions of protein S.
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Berndt, M. C., X. Du, L. Beutler, W. J. Booth, and P. A. Castaldi. "LOCALIZATION OF FUNCTIONAL DOMAINS ON HUMAN PLATELET GP Ib-IX COMPLEX BY EPITOPE ANALYSIS WITH MONOCLONAL ANTIBODIES." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642923.
Full textAbstract:
There is now considerable evidence that glycoprotein (GP) Ib plays an important functional role in the von Willebrand factor (vWF)-dependent adhesion of platelets to exposed vascular subendothelium and in the a-thrombin activation of platelets, and that GP IX is important for quinine/quinidine drug-dependent antibody platelet recognition. GP Ib (Mr = 170 KD) consists of two disulfide-linked subunits, Iba (Mr = 135 KD) and Ibβ (Mr = 25 KD), and exists as a heterodimer complex with GP IX (Mr = 22 KD). In this study we have used a panel of 10 antiGP Ib-IX complex monoclonal antibodies to define the functional domains on this complex. Immunoprecipitation of trypsin-treated GP Ib-IX complex revealed that the monoclonal antibodies mapped into three distinct groups: FMC 25, AK 1 and SZ 1, epitopes on the membrane-associated fragment (GP IX and an ≃25 KD remnant of the α-chain disulfide linked to the β-chain); AK 3 and WM 23, epitopes on the central macroglycopeptide core (90 KD); AN 51, SZ 2, AK 2, AP 1 and HIP 1, epitopes on peptide tail (45 KD). Crossblocking studies indicated that with the exception of AK 1 and SZ 1, the monoclonal antibodies were directed against distinct epitopes. All five monoclonal antibodies directed against the peptide tail region blocked ristocetin-dependent vWF-platelet interaction whereas the other five monoclonal antibodies were without effect, indicating that the 45 KD peptide tail region at the plasma end of the α-chain of GP Ib contained the vWF binding domain. Similarly, only the three monoclonal antibodies directed against the membrane-associated region interfered with drug-dependent antibody-platelet interaction.By western blot analysis, α-thrombin bound to the 45 KD peptide tail region. However, only AP 1 interfered significantly with the α-thrombin-dependent aggregation of platelets. This panel of epitope-defined monoclonal antibodies should be of value in further defining the structure-function relationships of this important membrane complex.
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Berndt, M. "STRUCTURE AND FUNCTION OF THE GLYCOPROTEIN Ib-IX COMPLEX." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643729.
Full textAbstract:
At high shear flow, the adhesion of platelets to the exposed vascular subendothelium requires von Willebrand factor (vWF) and is dependent upon a specific platelet membrane adhesion receptor, the human platelet membrane glycoprotein (GP) Ib-IX complex. Recent evidence suggests that vWFbinding to the GP Ib-IX complex plays an important role in other key aspects of hemostasis and thrombosis such as shear-induced platelet aggregation and the interaction of platelets with fibrin.Studies in our laboratory with a seriesof murine monoclonal antibodies directed against epitopes on GP lb, GP IX, or against complex-specificepitopes indicate that GP lb and GP IX exist in the intact platelet membrane as a native heterodimer complex(-25,000 copies/platelet). By analysis onSDS-polyacrylamide gels, GP lb has an apparent molecular weight of 170,000 and cnsists of two disulfide-linked subunits, GP Iba (Mr = 135,000) and GP Ibβ (Mr = 25,000),whilst GP IX has an equivalent molecularweight under both nonreducing and reducing conditions (Mr = 22,000).The ±-chain ofGP lb has a central macroglycopeptide core (Mr =90,000) which is highly glycosylated. At each end of themacroglycopeptide region is a domainsensitive to proteolytic cleavage. Cleavage at the end proximal to the platelet membrane, e.g. by calpain, Serratia marcescens metalloprotease and trypsin, generates two fragments :a Mr =130,000 highly glycosylated fragment termed glycocalicin anda membrane-associated region consisting ofa Mr -25,000 fragment that remains disulfide-linkedto GP Ibβ and associated with GP IX. In resting platelets, the membrane-associated region spans the lipid bilayer linking the GP Ib-IX complex to the platelet endoskeleton via actin-binding protein. This membrane-associated region also contains the domain(s) recognized by quinine/quinidine drug-dependent antibodies. Cleavage at the plasma end of the macroglycopeptide, e.g. by human leukocyte elastase, generates a poorly glycosylated Mr = 45,000 fragment of GP Ibα (peptide tail region) and a heavily glycosylated Mr = 100,000 fragment that remains disulfide-linked to GP Ibg and associated with GP IX. Platelets lacking the N-terminal peptide tail region of GP Iba fail to agglutinate with ristocetin and vWF and show a delayed response to a-thrombin.Polyclonal and monoclonal antibodies against this region also inhibit both these platelet responses suggesting that the peptide tail region contains the binding sites for both α-thrombinand vWF. Rotary shadowingelectron microscopy of purified GP Ib-IX complex shows the structure to be highlyasymmetric with each complex existing asa flexible rod with a globular domain at each end. The overall length of the complexwas =60 nm.The smaller globular domain (peptidetail region) has a diameter of =9nm; the larger globular domain (membrane-associated region), a diameter of =16 nmWe have recently examined whetherthe human platelet GP Ib-IX complex is the receptor for the ristocetin-dependent binding of vWF by reconstitution with the purified components using a solid-phasebead assay. Our approach was to indirectlybind and orientate the GP Ib-IX complex onthe beads via a monoclonal antibody directed against the membrane-associated region of the complex (FMC 25, epitope on GP IX).Immunobeads were chosen as the insoluble matrix because they are uniform in size (=10μm in diameter), impermeable,specifically designed for the coupling of IgG, and because, like platelets, the beads have a net negative charge atneutral pH.Specific binding of 125I-labelled human vWF tothe GP Ib-IX complex-coated immunobeads was strictly ristocetin-dependent with maximal binding occurring atristocetin concentrations >1 mg/ml. Ristocetin-dependent specificbinding of 125I-labelled vWF was saturable.Scatchardanalysis revealed a single classof binding sites for vWF with purified GP Ib-IX complex.Monoclonal antibodies against the Mr = 45,000 peptide tail region ofGP lb which stronglyinhibitthe ristocetin-dependent binding ofvWF toplatelets also strongly inhibited the ristocetin-dependent binding of vWFto the GP Ib-IX coated beads. Monoclonalantibody against either themacroglycopeptide or membrane-associated regions of the GPIb-IX complex did not inhibit the ristocetin-dependent binding of vWF to platelets or to the GP Ib-IX complex-coated beads. Similar functional correlations were obtained with anti-vWFmonoclonal antibodies. The reconstitutiondata therefore confirm the functional roleof the GP Ib-IX complex as a major plateletvWF receptor. The region ofthe vWF molecule involved in binding to the GP Ib-IX complex has been localized toa Mr =50,000 domain towards theN-terminal end of the vWF subunit. The reconstitution assay should prove useful in the further definition of active peptides of vWF that bind tothe human platelet GP Ib-IX complex.