TspanC8 tetraspanins regulate ADAM10/Kuzbanian trafficking and promote Notch activation in flies and mammals

The metalloprotease ADAM10/Kuzbanian catalyzes the ligand-dependent ectodomain shedding of Notch receptors and activates Notch. Here, we show that the human tetraspanins of the evolutionary conserved TspanC8 subfamily (Tspan5, Tspan10, Tspan14, Tspan15, Tspan17, and Tspan33) directly interact with ADAM10, regulate its exit from the endoplasmic reticulum, and that four of them regulate ADAM10 surface expression levels. In an independent RNAi screen in Drosophila, two TspanC8 genes were identified as Notch regulators. Functional analysis of the three Drosophila TspanC8 genes (Tsp3A, Tsp86D, and Tsp26D) indicated that these genes act redundantly to promote Notch signaling. During oogenesis, TspanC8 genes were up-regulated in border cells and regulated Kuzbanian distribution, Notch activity, and cell migration. Furthermore, the human TspanC8 tetraspanins Tspan5 and Tspan14 positively regulated ligand-induced ADAM10-dependent Notch1 signaling. We conclude that TspanC8 tetraspanins have a conserved function in the regulation of ADAM10 trafficking and activity, thereby positively regulating Notch receptor activation.

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Notch activation inhibits AML growth and survival: a potential therapeutic approach

Journal of Experimental Medicine ◽

10.1084/jem.20121527 ◽

2013 ◽

Vol 210 (2) ◽

pp. 321-337 ◽

Cited By ~ 87

Author(s):

Sankaranarayanan Kannan ◽

Robert M. Sutphin ◽

Mandy G. Hall ◽

Leonard S. Golfman ◽

Wendy Fang ◽

...

Keyword(s):

Therapeutic Approach ◽

Growth Arrest ◽

B Cell Lymphoma ◽

Receptor Activation ◽

Notch Receptor ◽

Myelogenous Leukemia ◽

Growth And Survival ◽

Notch Receptors ◽

Notch Activation

Although aberrant Notch activation contributes to leukemogenesis in T cells, its role in acute myelogenous leukemia (AML) remains unclear. Here, we report that human AML samples have robust expression of Notch receptors; however, Notch receptor activation and expression of downstream Notch targets are remarkably low, suggesting that Notch is present but not constitutively activated in human AML. The functional role of these Notch receptors in AML is not known. Induced activation through any of the Notch receptors (Notch1–4), or through the Notch target Hairy/Enhancer of Split 1 (HES1), consistently leads to AML growth arrest and caspase-dependent apoptosis, which are associated with B cell lymphoma 2 (BCL2) loss and enhanced p53/p21 expression. These effects were dependent on the HES1 repressor domain and were rescued through reexpression of BCL2. Importantly, activated Notch1, Notch2, and HES1 all led to inhibited AML growth in vivo, and Notch inhibition via dnMAML enhanced proliferation in vivo, thus revealing the physiological inhibition of AML growth in vivo in response to Notch signaling. As a novel therapeutic approach, we used a Notch agonist peptide that led to significant apoptosis in AML patient samples. In conclusion, we report consistent Notch-mediated growth arrest and apoptosis in human AML, and propose the development of Notch agonists as a potential therapeutic approach in AML.

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Current Views on the Roles of O-Glycosylation in Controlling Notch-Ligand Interactions

Biomolecules ◽

10.3390/biom11020309 ◽

2021 ◽

Vol 11 (2) ◽

pp. 309

Author(s):

Wataru Saiki ◽

Chenyu Ma ◽

Tetsuya Okajima ◽

Hideyuki Takeuchi

Keyword(s):

Notch Signaling ◽

100Th Anniversary ◽

Notch Receptor ◽

Notch Receptors ◽

Evolutionarily Conserved ◽

Future Challenges ◽

Ligand Interactions ◽

Epidermal Growth ◽

Notch Activation ◽

Human Specific

The 100th anniversary of Notch discovery in Drosophila has recently passed. The Notch is evolutionarily conserved from Drosophila to humans. The discovery of human-specific Notch genes has led to a better understanding of Notch signaling in development and diseases and will continue to stimulate further research in the future. Notch receptors are responsible for cell-to-cell signaling. They are activated by cell-surface ligands located on adjacent cells. Notch activation plays an important role in determining the fate of cells, and dysregulation of Notch signaling results in numerous human diseases. Notch receptors are primarily activated by ligand binding. Many studies in various fields including genetics, developmental biology, biochemistry, and structural biology conducted over the past two decades have revealed that the activation of the Notch receptor is regulated by unique glycan modifications. Such modifications include O-fucose, O-glucose, and O-N-acetylglucosamine (GlcNAc) on epidermal growth factor-like (EGF) repeats located consecutively in the extracellular domain of Notch receptors. Being fine-tuned by glycans is an important property of Notch receptors. In this review article, we summarize the latest findings on the regulation of Notch activation by glycosylation and discuss future challenges.

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Notch Subunit Heterodimerization and Prevention of Ligand-Independent Proteolytic Activation Depend, Respectively, on a Novel Domain and the LNR Repeats

Molecular and Cellular Biology ◽

10.1128/mcb.24.21.9265-9273.2004 ◽

2004 ◽

Vol 24 (21) ◽

pp. 9265-9273 ◽

Cited By ~ 126

Author(s):

Cheryll Sanchez-Irizarry ◽

Andrea C. Carpenter ◽

Andrew P. Weng ◽

Warren S. Pear ◽

Jon C. Aster ◽

...

Keyword(s):

Cell Surface ◽

Target Genes ◽

Receptor Activation ◽

Notch Receptor ◽

Gamma Secretase ◽

Prior Work ◽

Proteolytic Activation ◽

Transmembrane Receptors ◽

Notch Receptors ◽

Proteolytic Cleavages

ABSTRACT Notch proteins are transmembrane receptors that participate in a highly conserved signaling pathway that regulates morphogenesis in metazoans. Newly synthesized Notch receptors are proteolytically cleaved during transit to the cell surface, creating heterodimeric mature receptors comprising noncovalently associated extracellular (NEC) and transmembrane (NTM) subunits. Ligand binding activates Notch by inducing two successive proteolytic cleavages, catalyzed by metalloproteases and gamma-secretase, respectively, that permit the intracellular portion of NTM to translocate to the nucleus and activate transcription of target genes. Prior work has shown that the presence of NEC prevents ligand-independent activation of NTM, but the mechanisms involved are poorly understood. Here, we define the roles of two regions at the C-terminal end of NEC that participate in maintaining the integrity of resting Notch receptors through distinct mechanisms. The first region, a hydrophobic, previously uncharacterized portion of NEC, is sufficient to form stable complexes with the extracellular portion of NTM. The second region, consisting of the three Lin12/Notch repeats, is not needed for heterodimerization but acts to protect NTM from ligand-independent cleavage by metalloproteases. Together, these two contiguous regions of NEC impose crucial restraints that prevent premature Notch receptor activation.

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Notch ligand activity is modulated by glycosphingolipid membrane composition in Drosophila melanogaster

The Journal of Cell Biology ◽

10.1083/jcb.200907116 ◽

2010 ◽

Vol 188 (4) ◽

pp. 581-594 ◽

Cited By ~ 39

Author(s):

Sophie Hamel ◽

Jacques Fantini ◽

François Schweisguth

Keyword(s):

Protein Interactions ◽

Receptor Activation ◽

Notch Receptor ◽

Binding Motif ◽

Membrane Composition ◽

Notch Receptors ◽

Notch Ligands ◽

Ligand Activity ◽

Signaling Activity

Endocytosis of the transmembrane ligands Delta (Dl) and Serrate (Ser) is required for the proper activation of Notch receptors. The E3 ubiquitin ligases Mindbomb1 (Mib1) and Neuralized (Neur) regulate the ubiquitination of Dl and Ser and thereby promote both ligand endocytosis and Notch receptor activation. In this study, we identify the α1,4-N-acetylgalactosaminyltransferase-1 (α4GT1) gene as a gain of function suppressor of Mib1 inhibition. Expression of α4GT1 suppressed the signaling and endocytosis defects of Dl and Ser resulting from the inhibition of mib1 and/or neur activity. Genetic and biochemical evidence indicate that α4GT1 plays a regulatory but nonessential function in Notch signaling via the synthesis of a specific glycosphingolipid (GSL), N5, produced by α4GT1. Furthermore, we show that the extracellular domain of Ser interacts with GSLs in vitro via a conserved GSL-binding motif, raising the possibility that direct GSL–protein interactions modulate the endocytosis of Notch ligands. Together, our data indicate that specific GSLs modulate the signaling activity of Notch ligands.

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The Role of Intracellular Trafficking of Notch Receptors in Ligand-Independent Notch Activation

Biomolecules ◽

10.3390/biom11091369 ◽

2021 ◽

Vol 11 (9) ◽

pp. 1369 ◽

Cited By ~ 1

Author(s):

Judith Hounjet ◽

Marc Vooijs

Keyword(s):

Notch Signaling ◽

Intracellular Trafficking ◽

Current Knowledge ◽

Unmet Need ◽

Receptor Activation ◽

Notch Receptor ◽

Comprehensive Overview ◽

Normal Tissues ◽

Notch Receptors

Aberrant Notch signaling has been found in a broad range of human malignancies. Consequently, small molecule inhibitors and antibodies targeting Notch signaling in human cancers have been developed and tested; however, these have failed due to limited anti-tumor efficacy because of dose-limiting toxicities in normal tissues. Therefore, there is an unmet need to discover novel regulators of malignant Notch signaling, which do not affect Notch signaling in healthy tissues. This review provides a comprehensive overview of the current knowledge on the role of intracellular trafficking in ligand-independent Notch receptor activation, the possible mechanisms involved, and possible therapeutic opportunities for inhibitors of intracellular trafficking in Notch targeting.

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Xylosyl Extension of O-Glucose Glycans on the Extracellular Domain of NOTCH1 and NOTCH2 Regulates Notch Cell Surface Trafficking

Cells ◽

10.3390/cells9051220 ◽

2020 ◽

Vol 9 (5) ◽

pp. 1220 ◽

Cited By ~ 4

Author(s):

Yusuke Urata ◽

Wataru Saiki ◽

Yohei Tsukamoto ◽

Hiroaki Sago ◽

Hideharu Hibi ◽

...

Keyword(s):

Cell Surface ◽

Consensus Sequence ◽

Surface Expression ◽

Notch Receptor ◽

Cell Surface Expression ◽

Type I ◽

Wild Type ◽

Double Knockout ◽

Notch Receptors

Biochemical and genetic studies have indicated that O-linked glycosylation such as O-glucose (Glc), fucose (Fuc), and N-acetylglucosamine (GlcNAc) is critical for Notch signaling; however, it is not fully understood how O-glycans regulate the Notch receptor function. Notch receptors are type-I transmembrane proteins with large extracellular domains (ECD), containing 29–36 epidermal growth factor-like (EGF) repeats. Here, we analyzed O-Glc glycans on NOTCH1 and NOTCH2 expressed in HEK293T cells using an Orbitrap Fusion mass spectrometer and successfully revealed the structures and stoichiometries of all 17 EGF repeats of NOTCH1 with the O-Glc consensus sequence (C1-X-S-X-(P/A)-C2), and 16 out of 17 EGF repeats of NOTCH2 with the same consensus sequence. High levels of O-Glc attachment and xylosyl elongation were detected on most NOTCH1 and NOTCH2 EGF repeats. When both glucoside xylosyltransferases, GXYLT1 and GXYLT2, responsible for the xylosyl elongation of O-glucose, were genetically deleted, the expression of endogenous NOTCH1 and NOTCH2 on the surface of HEK293T cells did not change, but the cell surface expression of overexpressed NOTCH1 and NOTCH2 decreased compared with that in the wild type cells. In vitro secretion assays consistently showed a reduced secretion of both the NOTCH1 and NOTCH2 ECDs in GXYLT1 and GXYLT2 double knockout cells compared with the wild type cells, suggesting a significant role of the elongation of O-Glc glycans on the Notch ECDs in the quality control of Notch receptors.

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Notch Signaling By Either Notch1 or Notch2 Mediates Expansion of AGM-Derived Long-Term HSC Populations in Vitro

Blood ◽

10.1182/blood.v124.21.2897.2897 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2897-2897

Author(s):

Brandon K Hadland ◽

Barbara Varnum-Finney ◽

Randall T Moon ◽

Michael Gustave Poulos ◽

Jason M. Butler ◽

...

Keyword(s):

Early Stage ◽

Fetal Liver ◽

Notch Pathway ◽

Receptor Activation ◽

Notch Receptor ◽

Gamma Secretase ◽

Hematopoietic Stem ◽

Notch Activation

Abstract Long-term, adult-engrafting hematopoietic stem cells (HSC) first emerge from hemogenic endothelial (HE) precursors in the context of embryonic arterial vessels such as the dorsal aorta of the AGM (Aorta-Gonad-Mesonephros region), a process which requires Notch1 receptor signaling. However, a possible subsequent role for Notch receptor activation during the unique period of substantial HSC expansion in embryonic development remains less well defined. Here, we show that endothelial cells derived from the murine embryonic AGM region (AGM-EC) or fetal liver (FL-EC) provide an in vitro substrate for generation/maturation of HSC from HE/HSC-precursor populations derived from early stage murine embryos, which lack adult-engraftment capacity prior to co-culture. Notably, these EC substrates, endogenously expressing Notch ligands of the Jagged and Delta families, also support subsequent numerical expansion of AGM-derived HSC, expressing Notch1 and Notch2 receptors, as determined by limit dilution transplantation analysis. Consistent with a requirement for Notch activation in this process, phenotypic HSC expansion during EC co-culture is blocked by gamma-secretase mediated Notch pathway inhibition. Furthermore, we show that in vitro Notch activation by immobilized ligand Delta1, together with cytokines and inhibition of the TGF-beta pathway, is sufficient to increase the number of AGM-derived HSC in the absence of EC stroma. Expansion of phenotypic hematopoietic stem/progenitor cells generated by culture on Delta1 is inhibited by antibody-mediated blockade of the combination of Notch1 and Notch2, but not by either Notch1 or Notch2 alone. Consistent with this, Notch receptor-specific activation by either immobilized Notch1 or Notch2 antibody is sufficient to support AGM-derived HSC in vitro in preliminary experiments. Altogether, these studies suggest a role for Notch pathway activation by either Notch1 or Notch2 in supporting embryonic-stage HSC expansion subsequent to initial Notch1-mediated HSC specification. Disclosures Moon: Fate Therapeutics: co-founder Other. Rafii:Angiocrine Biosciences: Founder Other.

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Dll1/Notch Interaction Contributes to a Decreased Sensitivity of Myeloma Cells to Bortezomib

Blood ◽

10.1182/blood.v120.21.1840.1840 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1840-1840

Author(s):

Dehui Xu ◽

Jingsong Hu ◽

Elke De Bruyne ◽

Eline Menu ◽

Rik Schots ◽

...

Keyword(s):

Bone Marrow ◽

Drug Resistance ◽

Notch Signaling ◽

Stromal Cells ◽

Notch Pathway ◽

Surface Expression ◽

Myeloma Cells ◽

Notch Receptors ◽

Cyp1a1 Expression ◽

Notch Activation

Abstract Abstract 1840 One of the greatest challenges in multiple myeloma (MM) treatment is to overcome drug resistance. More and more evidence showed that not only the MM tumor cells should be targeted but also the bone marrow (BM) micro-environment. Interactions of MM cells with the BM micro-environment have a pivotal role in MM cell proliferation, survival, migration, angiogenesis as well as drug resistance. Many pathways are involved including the conserved Notch signaling pathway. The interaction of Notch receptors and ligands between adjacent cells induces proteolytic cleavage and release of the intracellular domain of the Notch receptor, also called Notch intracellular domains (NICD). NICD will then enter the nucleus and modify the expression of downstream target genes. Notch receptors are expressed by MM cells and Notch ligand Dll1 is present on bone marrow (BM) stromal cells. We investigated whether Notch activation in myeloma cells by the interaction with Dll1 on stromal cells contributes to bortezomib resistance. We analyzed Notch1 and Notch2 surface expression by flow cytometry on MM cells after Dll1 interaction using a stromal cell line modified to overexpress Dll1. Notch1 surface expression was not disturbed on mouse 5T33MMvt and human MMS1 and LP-1 cells while Notch2 expression on MM cells was significantly decreased after Dll1 interaction for 2 days. Next, we investigated NICD1 and NICD2 expression by western blot after Dll1/Notch interaction. NICD1 did not change in murine 5T33MMvt and human LP-1 and MMS-1 cells, while NICD2 is increased after Dll1 interaction. These results suggest that Dll1 can activate Notch signaling likely through the Notch2 receptor. We investigated whether Dll1/Notch activation could contribute to MM bortezomib resistance. MM cells were cocultured on immobilized recombinant Dll1 ligand and treated with 5 nM bortezomib for 48h. Compared to control, MM cells cocultured with Dll1 ligand were less sensitive to bortezomib. Furthermore, blocking the Notch pathway by DAPT (a gamma secretase inhibitor, GSI) could reverse this effect and increased the sensitivity to bortezomib. To delineate the molecular mechanism of Dll1-induced bortezomib resistance, we performed a drug resistance and metabolism gene array and found that CYP1A1 was significantly upregulated by Dll1/Notch interaction. CYP1A1 is a member of the cytochrome P450 family and regulates drug metabolism. We further demonstrated that inhibiting CYP1A1 by either α-Naphthoflavone (inhibitor) or CYP1A1-siRNA increases the sensitivity of MM cells to bortezomib, suggesting that CYP1A1 is involved in bortezomib resistance. As also previously demonstrated, CD138- 5T33MM cells are less sensitive to bortezomib than CD138+ 5T33MM cells. We analyzed CYP1A1 expression and activity and observed a higher CYP1A1 amount in CD138- cells compared to CD138+ MM cells. The higher CYP1A1 expression in CD138- cells might be a possible mechanism for their decreased bortezomib sensitivity compared to CD138+ cells. In addition, an in vivo experiment was performed. Combination treatment of DAPT with bortezomib was able to increase bortezomib sensitivity and prolonged overall survival in the 5T33MM mouse model. In conclusion, our results suggest that Dll1/Notch activation contributes to bortezomib resistance by upregulating CYP1A1, a molecule involved in drug metabolism. Our data provide a potential strategy to overcome bortezomib resistance by combination with a Notch pathway inhibitor. Disclosures: No relevant conflicts of interest to declare.

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Collective border cell migration requires the zinc transporter Catsup to limit endoplasmic reticulum stress

10.1101/2021.04.04.438395 ◽

2021 ◽

Author(s):

XIAORAN GUO ◽

Wei Dai ◽

Denise Montell

Keyword(s):

Endoplasmic Reticulum ◽

Cell Migration ◽

Er Stress ◽

Molecular Mechanisms ◽

Transmembrane Protein ◽

Zinc Transporter ◽

Collective Cell Migration ◽

Border Cells ◽

Border Cell ◽

Border Cell Migration

Collective cell migration is critical for normal development, wound healing, and in tumor progression and metastasis. Border cells in the Drosophila ovary provide a genetically tractable model to identify molecular mechanisms that drive this important cell behavior. In an unbiased screen for defects in border cell migration in mosaic clones, we identified a mutation in the catsup gene. Catsup, the Drosophila ortholog of Zip7, is a large, multifunctional, transmembrane protein of the endoplasmic reticulum (ER), which has been reported to negatively regulate catecholamine biosynthesis, to regulate Notch signaling, to function as a zinc transporter, and to limit ER stress. Here we report that catsup knockdown caused ER stress in border cells and that ectopic induction of ER stress was sufficient to block migration. Notch and EGFR trafficking were also disrupted. Wild type Catsup rescued the migration defect but point mutations known to disrupt the zinc ion transport of Zip7 did not. We conclude that migrating cells are particularly susceptible to defects in zinc transport and ER homeostasis.

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Long-range Notch-mediated tissue patterning requires actomyosin contractility

10.1101/259341 ◽

2018 ◽

Author(s):

Ginger L Hunter ◽

Li He ◽

Norbert Perrimon ◽

Guillaume Charras ◽

Edward Giniger ◽

...

Keyword(s):

Notch Signaling ◽

Myosin Ii ◽

Receptor Activation ◽

Notch Receptor ◽

Mechanical Forces ◽

Developmental Context ◽

Actomyosin Contractility ◽

Muscle Myosin ◽

Lateral Cell ◽

Notch Activation

AbstractDynamic, actin-based protrusions function in cell-cell signaling in a variety of systems. In the dorsal thorax of the developing fly, basal protrusions enable non-neighboring epithelial cells to touch, extending the range over which Notch-mediated lateral inhibition can occur during bristle patterning. Given that actin-based cell protrusions can exert mechanical forces on their environment and Notch receptor activation is mechanically sensitive, how might cytoskeletal contractility contribute to Notch signaling? We identify a pool of basal non-muscle myosin II (myosin II) that regulates protrusion dynamics, promotes Notch signaling, and is required in signal sending and receiving cells for Notch-dependent patterning. We show that interactions between protrusions are extensive and subject to actomyosin contractility. The effects of reducing myosin II activity are more pronounced for protrusion-mediated signaling than for signaling at lateral cell contacts. Together, these results reveal a role for actomyosin contractility in Notch activation, signaling, and patterning in a developmental context.

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