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Spyros Artavanistsakonas - One of the best experts on this subject based on the ideXlab platform.
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in vivo mapping of Notch pathway activity in normal and stress hematopoiesis
Cell Stem Cell, 2013Co-Authors: Philmo Oh, Camille Lobry, Anastasia Tikhonova, Evangelia Loizou, Jan Manent, Ben Van Handel, Sherif Ibrahim, Jeffrey Greve, Hanna K A Mikkola, Spyros ArtavanistsakonasAbstract:Summary Accumulating evidence suggests that Notch signaling is active at multiple points during hematopoiesis. Until recently, the majority of such studies focused on Notch signaling in lymphocyte differentiation and knowledge of individual Notch Receptor roles has been limited due to a paucity of genetic tools available. In this manuscript we generate and describe animal models to identify and fate-map stem and progenitor cells expressing each Notch Receptor, delineate Notch pathway activation, and perform in vivo gain- and loss-of-function studies dissecting Notch signaling in early hematopoiesis. These models provide comprehensive genetic maps of lineage-specific Notch Receptor expression and activation in hematopoietic stem and progenitor cells. Moreover, they establish a previously unknown role for Notch signaling in the commitment of blood progenitors toward the erythrocytic lineage and link Notch signaling to optimal organismal response to stress erythropoiesis.
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in vivo analysis of the Notch Receptor s1 cleavage
PLOS ONE, 2009Co-Authors: Robert J Lake, Spyros Artavanistsakonas, Lisa Grimm, Alexey Veraksa, Andrew BanosAbstract:A ligand-independent cleavage (S1) in the extracellular domain of the mammalian Notch Receptor results in what is considered to be the canonical heterodimeric form of Notch on the cell surface. The in vivo consequences and significance of this cleavage on Drosophila Notch signaling remain unclear and contradictory. We determined the cleavage site in Drosophila and examined its in vivo function by a transgenic analysis of Receptors that cannot be cleaved. Our results demonstrate a correlation between loss of cleavage and loss of in vivo function of the Notch Receptor, supporting the notion that S1 cleavage is an in vivo mechanism of Notch signal control.
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the drosophila melanogaster suppressor of deltex gene a regulator of the Notch Receptor signaling pathway is an e3 class ubiquitin ligase
Genetics, 1999Co-Authors: Michael Cornell, Spyros Artavanistsakonas, Dana A P Evans, Robert S Mann, Maggy Fostier, M Flasza, M Monthatong, Martin BaronAbstract:During development, the Notch Receptor regulates many cell fate decisions by a signaling pathway that has been conserved during evolution. One positive regulator of Notch is Deltex, a cytoplasmic, zinc finger domain protein, which binds to the intracellular domain of Notch. Phenotypes resulting from mutations in deltex resemble loss-of-function Notch phenotypes and are suppressed by the mutation Suppressor of deltex [Su(dx)]. Homozygous Su(dx) mutations result in wing-vein phenotypes and interact genetically with Notch pathway genes. We have previously defined Su(dx) genetically as a negative regulator of Notch signaling. Here we present the molecular identification of the Su(dx) gene product. Su(dx) belongs to a family of E3 ubiquitin ligase proteins containing membrane-targeting C2 domains and WW domains that mediate protein-protein interactions through recognition of proline-rich peptide sequences. We have identified a seven-codon deletion in a Su(dx) mutant allele and we show that expression of Su(dx) cDNA rescues Su(dx) mutant phenotypes. Overexpression of Su(dx) also results in ectopic vein differentiation, wing margin loss, and wing growth phenotypes and enhances the phenotypes of loss-of-function mutations in Notch, evidence that supports the conclusion that Su(dx) has a role in the downregulation of Notch signaling.
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Notch signaling cell fate control and signal integration in development
Science, 1999Co-Authors: Spyros Artavanistsakonas, Matthew D. Rand, Robert J LakeAbstract:Notch signaling defines an evolutionarily ancient cell interaction mechanism, which plays a fundamental role in metazoan development. Signals exchanged between neighboring cells through the Notch Receptor can amplify and consolidate molecular differences, which eventually dictate cell fates. Thus, Notch signals control how cells respond to intrinsic or extrinsic developmental cues that are necessary to unfold specific developmental programs. Notch activity affects the implementation of differentiation, proliferation, and apoptotic programs, providing a general developmental tool to influence organ formation and morphogenesis.
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human ligands of the Notch Receptor
American Journal of Pathology, 1999Co-Authors: Grace E Gray, Robert S Mann, Efthimios Mitsiadis, Domingos Henrique, Maria Louisa Carcangiu, Amy Banks, John Leiman, David C Ward, David Ishhorowitz, Spyros ArtavanistsakonasAbstract:During development, the Notch signaling pathway is essential for the appropriate differentiation of many cell types in organisms across the phylogenetic scale, including humans. Notch signaling is also implicated in human diseases, including a leukemia and two hereditary syndromes known as Alagille and CADASIL. To generate tools for pursuing the role of the Notch pathway in human disease and development, we have cloned and analyzed the expression of three human homologues of the Notch ligands Delta and Serrate, human Jagged1 ( HJ1 ), human Jagged2 ( HJ2 ), and human Delta1 ( H-Delta-1 ), and determined their chromosomal localizations. We have also raised antibodies to HJ1, and used these antibodies in conjunction with in situ hybridization to examine the expression of these ligands in normal and cancerous cervical tissue. We find that, as reported previously for Notch, the ligands are up-regulated in certain neoplastic tissues. This observation is consistent with the notion that Notch signaling is an important element in these pathogenic conditions, raising the possibility that modulation of Notch activity could be used to influence the fate of the cells and offering a conceivable therapeutic avenue.
Christel Brou - One of the best experts on this subject based on the ideXlab platform.
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the ubiquitin specific protease 12 usp12 is a negative regulator of Notch signaling acting on Notch Receptor trafficking toward degradation
Journal of Biological Chemistry, 2012Co-Authors: Julien Moretti, Patricia Chastagner, Alain Israel, Chihchao Liang, Martin A Cohn, Christel BrouAbstract:Notch signaling is critical for development and adult tissue physiology, controlling cell fate in a context-dependent manner. Upon ligand binding, the transmembrane Notch Receptor undergoes two ordered proteolytic cleavages releasing Notch intracellular domain, which regulates the transcription of Notch target genes. The strength of Notch signaling is of crucial importance and depends notably on the quantity of Notch Receptor at the cell surface. Using an shRNA library screen monitoring Notch trafficking and degradation in the absence of ligand, we identified mammalian USP12 and its Drosophila melanogaster homolog as novel negative regulators of Notch signaling. USP12 silencing specifically interrupts Notch trafficking to the lysosomes and, as a consequence, leads to an increased amount of Receptor at the cell surface and to a higher Notch activity. At the biochemical level, USP12 with its activator UAF1 deubiquitinate the nonactivated form of Notch in cell culture and in vitro. These results characterize a new level of conserved regulation of Notch signaling by the ubiquitin system.
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the translation initiation factor 3f eif3f exhibits a deubiquitinase activity regulating Notch activation
PLOS Biology, 2010Co-Authors: Julien Moretti, Patricia Chastagner, Alain Israel, Stefano Gastaldello, Sara Farrah Heuss, Annette M G Dirac, Rene Bernards, Maria G Masucci, Christel BrouAbstract:Activation of the mammalian Notch Receptor after ligand binding relies on a succession of events including metalloprotease-cleavage, endocytosis, monoubiquitination, and eventually processing by the gamma-secretase, giving rise to a soluble, transcriptionally active molecule. The Notch1 Receptor was proposed to be monoubiquitinated before its gamma-secretase cleavage; the targeted lysine has been localized to its submembrane domain. Investigating how this step might be regulated by a deubiquitinase (DUB) activity will provide new insight for understanding Notch Receptor activation and downstream signaling. An immunofluorescence-based screening of an shRNA library allowed us to identify eIF3f, previously known as one of the subunits of the translation initiation factor eIF3, as a DUB targeting the activated Notch Receptor. We show that eIF3f has an intrinsic DUB activity. Knocking down eIF3f leads to an accumulation of monoubiquitinated forms of activated Notch, an effect counteracted by murine WT eIF3f but not by a catalytically inactive mutant. We also show that eIF3f is recruited to activated Notch on endocytic vesicles by the putative E3 ubiquitin ligase Deltex1, which serves as a bridging factor. Finally, catalytically inactive forms of eIF3f as well as shRNAs targeting eIF3f repress Notch activation in a coculture assay, showing that eIF3f is a new positive regulator of the Notch pathway. Our results support two new and provocative conclusions: (1) The activated form of Notch needs to be deubiquitinated before being processed by the gamma-secretase activity and entering the nucleus, where it fulfills its transcriptional function. (2) The enzyme accounting for this deubiquitinase activity is eIF3f, known so far as a translation initiation factor. These data improve our knowledge of Notch signaling but also open new avenues of research on the Zomes family and the translation initiation factors.
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aip4 itch regulates Notch Receptor degradation in the absence of ligand
PLOS ONE, 2008Co-Authors: Patricia Chastagner, Alain Israel, Christel BrouAbstract:Background: The regulation of Notch signaling heavily relies on ubiquitination events. Drosophila Su(dx), a member of the HECT family of ubiquitin-ligases, has been described as a negative regulator of Notch signaling, acting on the postendocytic sorting of Notch. The mammalian ortholog of Su(dx), Itch/AIP4, has been shown to have multiple substrates, including Notch, but the precise events regulated by Itch/AIP4 in the Notch pathway have not been identified yet. Methodology/Principal Findings: Using Itch-/- fibroblasts expressing the Notch1 Receptor, we show that Itch is not necessary for Notch activation, but rather for controlling the degradation of Notch in the absence of ligand. Itch is indeed required after the early steps of Notch endocytosis to target it to the lysosomes where it is degraded. Furthermore Itch/AIP4 catalyzes Notch polyubiquitination through unusual K29-linked chains. We also demonstrate that although Notch is associated with Itch/AIP4 in cells, their interaction is not detectable in vitro and thus requires either a post-translational modification, or a bridging factor that remains to be identified. Conclusions/Significance: Taken together our results identify a specific step of Notch regulation in the absence of any activation and underline differences between mammalian and Drosophila Notch pathways.
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monoubiquitination and endocytosis direct γ secretase cleavage of activated Notch Receptor
Journal of Cell Biology, 2004Co-Authors: Neetu Guptarossi, Odile Lebail, Frederique Logeat, Patricia Chastagner, Annie Olry, Alain Israel, Christel BrouAbstract:Activation of mammalian Notch Receptor by its ligands induces TNFα-converting enzyme–dependent ectodomain shedding, followed by intramembrane proteolysis due to presenilin (PS)-dependent γ-secretase activity. Here, we demonstrate that a new modification, a monoubiquitination, as well as clathrin-dependent endocytosis, is required for γ-secretase processing of a constitutively active Notch derivative, ΔE, which mimics the TNFα-converting enzyme–processing product. PS interacts with this modified form of ΔE, ΔEu. We identified the lysine residue targeted by the monoubiquitination event and confirmed its importance for activation of Notch Receptor by its ligand, Delta-like 1. We propose a new model where monoubiquitination and endocytosis of Notch are a prerequisite for its PS-dependent cleavage, and discuss its relevance for other γ-secretase substrates.
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the Notch1 Receptor is cleaved constitutively by a furin like convertase
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Frederique Logeat, Odile Lebail, Christel Brou, Christine Bessia, Sophie Jarriault, Nabil G Seidah, Alain IsraelAbstract:The Notch Receptor, which is involved in numerous cell fate decisions in invertebrates and vertebrates, is synthesized as a 300-kDa precursor molecule (p300). We show here that proteolytic processing of p300 is an essential step in the formation of the biologically active Receptor because only the cleaved fragments are present at the cell surface. Our results confirm and extend recent reports indicating that the Notch Receptor exists at the plasma membrane as a heterodimeric molecule, but disagree as to the nature of the protease that is responsible for the cleavage that takes place in the extracellular region. We report here that constitutive processing of murine Notch1 involves a furin-like convertase. We show that the calcium ionophore A23187 and the α1-antitrypsin variant, α 1-PDX, a known inhibitor of furin-like convertases, inhibit p300 processing. When expressed in the furin-deficient Lovo cell line, p300 is not processed. In vitro digestion of a recombinant Notch-derived substrate with purified furin allowed mapping of the processing site to the carboxyl side of the sequence RQRR (amino acids 1651–1654). Mutation of these four amino acids (and of two secondary dibasic furin sites located nearby) completely abolished processing of the Notch1 Receptor.
Juan Carlos Zunigapflucker - One of the best experts on this subject based on the ideXlab platform.
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Notch promotes survival of pre t cells at the beta selection checkpoint by regulating cellular metabolism
Nature Immunology, 2005Co-Authors: Maria Ciofani, Juan Carlos ZunigapfluckerAbstract:Notch signals are necessary for the functional outcomes of T cell Receptor beta-selection, including differentiation, proliferation and rescue from apoptosis. The mechanism underlying this requirement for T cell development is unknown. Here we show that Notch Receptor and Delta-like 1 ligand interactions promoted the survival of CD4(-)CD8(-) pre-T cells through the maintenance of cell size, glucose uptake and metabolism. Furthermore, the trophic effects of Notch signaling were mediated by the pathway of phosphatidylinositol-3-OH kinase and the kinase Akt, such that expression of active Atk overcame the requirement for Notch in beta-selection. Collectively, our results demonstrate involvement of Notch Receptor-ligand interactions in the regulation of cellular metabolism, thus enabling the autonomous signaling capacity of the pre-T cell Receptor complex.
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maintenance of t cell specification and differentiation requires recurrent Notch Receptor ligand interactions
Journal of Experimental Medicine, 2004Co-Authors: Thomas M Schmitt, Maria Ciofani, Howard T Petrie, Juan Carlos ZunigapfluckerAbstract:Notch signaling has been shown to play a pivotal role in inducing T lineage commitment. However, T cell progenitors are known to retain other lineage potential long after the first point at which Notch signaling is required. Thus, additional requirements for Notch signals and the timing of these events relative to intrathymic differentiation remain unknown. Here, we address this issue by culturing subsets of CD4 CD8 double negative (DN) thymocytes on control stromal cells or stromal cells expressing Delta-like 1 (Dll1). All DN subsets were found to require Notch signals to differentiate into CD4+ CD8+ T cells. Using clonal analyses, we show that CD44+ CD25+ (DN2) cells, which appeared committed to the T cell lineage when cultured on Dll1-expressing stromal cells, nonetheless gave rise to natural killer cells with a progenitor frequency similar to that of CD44+ CD25− (DN1) thymocytes when Notch signaling was absent. These data, together with the observation that Dll1 is expressed on stromal cells throughout the thymic cortex, indicates that Notch Receptor–ligand interactions are necessary for induction and maintenance of T cell lineage specification at both the DN1 and DN2 stages of T cell development, suggesting that the Notch-induced repression of the B cell fate is temporally separate from Notch-induced commitment to the T lineage.
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obligatory role for cooperative signaling by pre tcr and Notch during thymocyte differentiation
Journal of Immunology, 2004Co-Authors: Maria Ciofani, Thomas M Schmitt, Amelia Ciofani, Alison M Michie, Nicolas Cuburu, Anne Aublin, Janet L Maryanski, Juan Carlos ZunigapfluckerAbstract:The first checkpoint during T cell development, known as β selection, requires the successful rearrangement of the TCR-β gene locus. Notch signaling has been implicated in various stages during T lymphopoiesis. However, it is unclear whether Notch Receptor-ligand interactions are necessary during β selection. Here, we show that pre-TCR signaling concurrent with Notch Receptor and Delta-like-1 ligand interactions are required for the survival, proliferation, and differentiation of mouse CD4 − CD8 − thymocytes to the CD4 + CD8 + stage. Furthermore, we address the minimal signaling requirements underlying β selection and show a hierarchical positioning of key proximal signaling molecules. Collectively, our results demonstrate an essential role for Notch Receptor-ligand interactions in enabling the autonomous signaling capacity of the pre-TCR complex.
Bernhard M Degnan - One of the best experts on this subject based on the ideXlab platform.
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the expression of delta ligands in the sponge amphimedon queenslandica suggests an ancient role for Notch signaling in metazoan development
Evodevo, 2012Co-Authors: Gemma S Richards, Bernhard M DegnanAbstract:Background Intercellular signaling via the Notch pathway regulates cell fate, patterning, differentiation and proliferation, and is essential for the proper development of bilaterians and cnidarians. To investigate the origins of the Notch pathway, we are studying its deployment in a representative of an early branching lineage, the poriferan Amphimedon queenslandica. The A. queenslandica genome encodes a single Notch Receptor and five membrane-bound Delta ligands, as well as orthologs of many genes that enact and regulate canonical Notch signaling events in other animals.
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The expression of Delta ligands in the sponge Amphimedon queenslandica suggests an ancient role for Notch signaling in metazoan development
EvoDevo, 2012Co-Authors: Gemma S Richards, Bernhard M DegnanAbstract:Background Intercellular signaling via the Notch pathway regulates cell fate, patterning, differentiation and proliferation, and is essential for the proper development of bilaterians and cnidarians. To investigate the origins of the Notch pathway, we are studying its deployment in a representative of an early branching lineage, the poriferan Amphimedon queenslandica . The A. queenslandica genome encodes a single Notch Receptor and five membrane-bound Delta ligands, as well as orthologs of many genes that enact and regulate canonical Notch signaling events in other animals. Methods In the present report we analyze the structure of the five A. queenslandica Deltas using bioinformatic methods, and characterize their developmental expression via whole mount in situ hybridization and histological staining. Results Sequence analysis of the A. queenslandica Delta ligands highlights the conservation of their extracellular domains. This contrasts with the divergence of their intracellular regions, each of which is predicted to bear a unique repertoire of protein interaction motifs. In keeping with this diversity, these ligands are expressed differentially and dynamically throughout A. queenslandica embryogenesis, both in cell type specific patterns and broader regional domains. Notably, this expression coincides with the development of the photosensitive larval pigment ring, the non-ciliated cuboidal cells located at the anterior pole of the larva, and the intraepithelial flask cells and globular cells that are presumed to have sensory and/or secretory roles. Conclusions Based on the dynamic and complex patterns of expression of these Delta ligands and the Notch Receptor, we propose that the Notch signaling pathway is involved in regulating the development of diverse cell types in A. queenslandica . From these observations we infer that Notch signaling is a conserved feature of metazoan development, ancestrally contributing to cell determination, patterning and differentiation processes.
Russell R Lonser - One of the best experts on this subject based on the ideXlab platform.
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Notch Receptor and effector expression in von hippel lindau disease associated central nervous system hemangioblastomas
Journal of Neurosurgery, 2011Co-Authors: Marsha J Merrill, Nancy A Edwards, Russell R LonserAbstract:Object Central nervous system hemangioblastomas are the most common manifestation of von Hippel-Lindau (VHL) disease, an autosomal dominant tumor suppressor syndrome that results in loss of VHL protein function and continuous upregulation of hypoxia-inducible factors. These tumors are composed of neoplastic stromal cells and abundant vasculature. Stromal cells express markers consistent with multipotent embryonically arrested hemangioblasts, which are precursors for hematopoietic and vascular lineages. Notch Receptors are transmembrane signaling molecules that regulate multiple developmental processes including hematopoiesis and vasculogenesis. To investigate the importance of Notch signaling in the development of VHL disease–associated CNS hemangioblastomas, the authors examined the presence of the four Notch Receptors and downstream Notch effectors in this setting. Methods The authors used surgical specimens obtained from confirmed VHL-associated hemangioblastomas. Immunohistochemical analysis for the f...
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Notch Receptor and effector expression in von Hippel-Lindau disease–associated central nervous system hemangioblastomas
Journal of Neurosurgery, 2011Co-Authors: Marsha J Merrill, Nancy A Edwards, Russell R LonserAbstract:Object Central nervous system hemangioblastomas are the most common manifestation of von Hippel-Lindau (VHL) disease, an autosomal dominant tumor suppressor syndrome that results in loss of VHL protein function and continuous upregulation of hypoxia-inducible factors. These tumors are composed of neoplastic stromal cells and abundant vasculature. Stromal cells express markers consistent with multipotent embryonically arrested hemangioblasts, which are precursors for hematopoietic and vascular lineages. Notch Receptors are transmembrane signaling molecules that regulate multiple developmental processes including hematopoiesis and vasculogenesis. To investigate the importance of Notch signaling in the development of VHL disease–associated CNS hemangioblastomas, the authors examined the presence of the four Notch Receptors and downstream Notch effectors in this setting. Methods The authors used surgical specimens obtained from confirmed VHL-associated hemangioblastomas. Immunohistochemical analysis for the f...
