Ehrlichia SLiM ligand mimetic activates Notch signaling in human monocytes

Ehrlichia chaffeensis evades innate host defenses by reprogramming the mononuclear phagocyte through mechanisms that involve exploitation of multiple evolutionarily conserved cellular signaling pathways including Notch. This immune evasion strategy is directed in part by tandem repeat protein (TRP) effectors. Specifically, the TRP120 effector activates and regulates Notch signaling through interactions with the Notch receptor and the negative regulator, F-Box and WD repeat domain-containing 7 (FBW7). However, the specific molecular interactions and motifs required for E. chaffeensis TRP120-Notch receptor interaction and activation have not been defined. To investigate the molecular basis of TRP120 Notch activation, we compared TRP120 with endogenous canonical/non-canonical Notch ligands and identified a short region of sequence homology within the tandem repeat (TR) domain. TRP120 was predicted to share biological function with Notch ligands, and a function-associated sequence in the TR domain was identified. To investigate TRP120-Notch receptor interactions, colocalization between TRP120 and endogenous Notch-1 was observed. Moreover, direct interactions between full length TRP120, the TRP120 TR domain containing the putative Notch ligand sequence, and the Notch receptor LBR were demonstrated. To molecularly define the TRP120 Notch activation motif, peptide mapping was used to identify an 11-amino acid short linear motif (SLiM) located within the TRP120 TR that activated Notch signaling and downstream gene expression. Peptide mutants of the Notch SLiM or anti-Notch SLiM antibody reduced or eliminated Notch activation and NICD nuclear translocation. This investigation reveals a novel molecularly defined pathogen encoded Notch SLiM mimetic that activates Notch signaling consistent with endogenous ligands.

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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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TAMI-11. IMPACT OF oHSV ACTIVATED NOTCH SIGNALING IN TUMOR MICROENVIRONMENT, AND ITS IMPACT ON ANTI-TUMOR IMMUNITY

Neuro-Oncology ◽

10.1093/neuonc/noaa215.900 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii215-ii215

Author(s):

Yoshihiro Otani ◽

Ji Young Yoo ◽

Samantha Chao ◽

Toshihiko Shimizu ◽

Cole Lewis ◽

...

Keyword(s):

Tumor Microenvironment ◽

Notch Signaling ◽

Tumor Immunity ◽

Cell Communication ◽

Notch Signaling Pathway ◽

Therapeutic Benefit ◽

Notch Receptor ◽

Negative Regulator ◽

Gamma Secretase ◽

The Impact

Abstract NOTCH signaling is a method of cell-cell communication where membrane bound NOTCH ligands on signal-sending cells can bind to and initiate cleavage of the NOTCH receptor, releasing NICD which can initiate signal transduction in adjacent “signal-receiving” cells. We have recently shown that oHSV treatment of GBM cells induces NICD cleavage and NOTCH activation in adjacent uninfected glioma cells. RNA sequencing of GBM cells post-infection also uncovered Gene Ontology NOTCH signaling pathway to be significantly upregulated. This activation was induced by viral miRNA-H16, which represses FIH-1 expression. FIH-1 was found to be a negative regulator of Mib1, a ubiquitin ligase, which activates NOTCH ligand-mediated activation of adjacent signal-receiving cells bearing the NOTCH receptor (Otani et al Clin. Can. Res. 2020). Here we have investigated the impact of oHSV-induced NOTCH signaling on the tumor microenvironment. Treatment of brain tumors in immune competent mice with oHSV and NOTCH blocking gamma secretase inhibitor (GSI) induced an anti-tumor memory immune response. Long term survivors in mice treated with the combination also completely rejected subsequent tumor re-challenge in the other hemisphere. UMAP of flow cytometry of tumor-bearing hemispheres and functional analysis of isolated cellular fractions from treated mice showed a significant influx of MDSC cells after oHSV treatment that was rescued in mice treated with oHSV and GSI. Ongoing mechanistic studies are uncovering a significant induction of NOTCH in tumor associated macrophages that aids in recruitment of MDSC cells. Overall these studies have uncovered a significant impact of oHSV therapy on GBM tumor microenvironment and presents opportunities for combination therapies that can help improve therapeutic benefit and anti-tumor immunity.

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NUMB enhances Notch signaling by repressing ubiquitination of NOTCH1 intracellular domain

Journal of Molecular Cell Biology ◽

10.1093/jmcb/mjz088 ◽

2019 ◽

Vol 12 (5) ◽

pp. 345-358 ◽

Cited By ~ 2

Author(s):

Zhiyuan Luo ◽

Lili Mu ◽

Yue Zheng ◽

Wenchen Shen ◽

Jiali Li ◽

...

Keyword(s):

Notch Signaling ◽

Posttranslational Modifications ◽

Transcriptional Activation ◽

Neural Progenitor Cells ◽

Nuclear Translocation ◽

Notch Receptor ◽

Deubiquitinating Enzyme ◽

Intracellular Domain ◽

Ubiquitin Proteasome ◽

Notch1 Receptor

Abstract The release and nuclear translocation of the intracellular domain of Notch receptor (NICD) is the prerequisite for Notch signaling-mediated transcriptional activation. NICD is subjected to various posttranslational modifications including ubiquitination. Here, we surprisingly found that NUMB proteins stabilize the intracellular domain of NOTCH1 receptor (N1ICD) by regulating the ubiquitin–proteasome machinery, which is independent of NUMB’s role in modulating endocytosis. BAP1, a deubiquitinating enzyme (DUB), was further identified as a positive N1ICD regulator, and NUMB facilitates the association between N1ICD and BAP1 to stabilize N1ICD. Intriguingly, BAP1 stabilizes N1ICD independent of its DUB activity but relying on the BRCA1-inhibiting function. BAP1 strengthens Notch signaling and maintains stem-like properties of cortical neural progenitor cells. Thus, NUMB enhances Notch signaling by regulating the ubiquitinating activity of the BAP1–BRCA1 complex.

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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 ◽

10.1074/jbc.m112.366807 ◽

2012 ◽

Vol 287 (35) ◽

pp. 29429-29441 ◽

Cited By ~ 42

Author(s):

Julien Moretti ◽

Patricia Chastagner ◽

Chih-Chao Liang ◽

Martin A. Cohn ◽

Alain Israël ◽

...

Keyword(s):

Notch Signaling ◽

Receptor Trafficking ◽

Notch Receptor ◽

Negative Regulator ◽

Specific Protease ◽

Ubiquitin Specific Protease

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Protein O-fucosyltransferase 1 (Pofut1) regulates lymphoid and myeloid homeostasis through modulation of Notch receptor ligand interactions

Blood ◽

10.1182/blood-2010-12-326074 ◽

2011 ◽

Vol 117 (21) ◽

pp. 5652-5662 ◽

Cited By ~ 68

Author(s):

David Yao ◽

Yuanshuai Huang ◽

Xiaoran Huang ◽

Weihuan Wang ◽

Quanjian Yan ◽

...

Keyword(s):

Notch Signaling ◽

Notch Receptor ◽

Cell Surface Expression ◽

Developmental Defects ◽

Marginal Zone B Cells ◽

Notch Ligand ◽

Notch Receptors ◽

Ligand Interactions ◽

Notch Ligands ◽

T Lymphopoiesis

Abstract Notch signaling is essential for lymphocyte development and is also implicated in myelopoiesis. Notch receptors are modified by O-fucosylation catalyzed by protein O-fucosyltransferase 1 (Pofut1). Fringe enzymes add N-acetylglucosamine to O-fucose and modify Notch signaling by altering the sensitivity of Notch receptors to Notch ligands. To address physiologic functions in hematopoiesis of Notch modified by O-fucose glycans, we examined mice with inducible inactivation of Pofut1 using Mx-Cre. These mice exhibited a reduction in T lymphopoiesis and in the production of marginal-zone B cells, in addition to myeloid hyperplasia. Restoration of Notch1 signaling rescued T lymphopoiesis and the marrow myeloid hyperplasia. After marrow transfer, both cell-autonomous and environmental cues were found to contribute to lymphoid developmental defects and myeloid hyperplasia in Pofut1-deleted mice. Although Pofut1 deficiency slightly decreased cell surface expression of Notch1 and Notch2, it completely abrogated the binding of Notch receptors with Delta-like Notch ligands and suppressed downstream Notch target activation, indicating that O-fucose glycans are critical for efficient Notch-ligand binding that transduce Notch signals. The combined data support a key role for the O-fucose glycans generated by Pofut1 in Notch regulation of hematopoietic homeostasis through modulation of Notch-ligand interactions.

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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 ◽

10.1093/genetics/152.2.567 ◽

1999 ◽

Vol 152 (2) ◽

pp. 567-576 ◽

Cited By ~ 13

Author(s):

M Cornell ◽

D A P Evans ◽

R Mann ◽

M Fostier ◽

M Flasza ◽

...

Keyword(s):

Notch Signaling ◽

Signaling Pathway ◽

Cell Fate ◽

Ubiquitin Ligase ◽

Notch Pathway ◽

Notch Receptor ◽

Negative Regulator ◽

Loss Of Function ◽

Wing Vein ◽

Cell Fate Decisions

Abstract 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 drives development of Barrett’s metaplasia from Dclk1-positive epithelial tuft cells in the murine gastric mucosa

Scientific Reports ◽

10.1038/s41598-021-84011-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Bettina Kunze ◽

Moritz Middelhoff ◽

H. Carlo Maurer ◽

Tatiana Agibalova ◽

Akanksha Anand ◽

...

Keyword(s):

Notch Signaling ◽

Secretory Cell ◽

Notch Receptor ◽

Neoplastic Progression ◽

Epithelial Tumor ◽

Genetic Ablation ◽

Stem Cell Maintenance ◽

Cellular Origin ◽

Tuft Cells ◽

Notch Activation

AbstractBarrett’s esophagus (BE) is a precursor to esophageal adenocarcinoma (EAC), but its cellular origin and mechanism of neoplastic progression remain unresolved. Notch signaling, which plays a key role in regulating intestinal stem cell maintenance, has been implicated in a number of cancers. The kinase Dclk1 labels epithelial post-mitotic tuft cells at the squamo-columnar junction (SCJ), and has also been proposed to contribute to epithelial tumor growth. Here, we find that genetic activation of intracellular Notch signaling in epithelial Dclk1-positive tuft cells resulted in the accelerated development of metaplasia and dysplasia in a mouse model of BE (pL2.Dclk1.N2IC mice). In contrast, genetic ablation of Notch receptor 2 in Dclk1-positive cells delayed BE progression (pL2.Dclk1.N2fl mice), and led to increased secretory cell differentiation. The accelerated BE progression in pL2.Dclk1.N2IC mice correlated with changes to the transcriptomic landscape, most notably for the activation of oncogenic, proliferative pathways in BE tissues, in contrast to upregulated Wnt signalling in pL2.Dclk1.N2fl mice. Collectively, our data show that Notch activation in Dclk1-positive tuft cells in the gastric cardia can contribute to BE development.

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Mature human eosinophils express functional Notch ligands mediating eosinophil autocrine regulation

Blood ◽

10.1182/blood-2008-05-155937 ◽

2009 ◽

Vol 113 (13) ◽

pp. 3092-3101 ◽

Cited By ~ 30

Author(s):

Amy L. Radke ◽

Lauren E. Reynolds ◽

Rossana C. N. Melo ◽

Ann M. Dvorak ◽

Peter F. Weller ◽

...

Keyword(s):

Notch Signaling ◽

Neutralizing Antibodies ◽

Receptor Activation ◽

Notch Receptor ◽

Gm Csf ◽

Tissue Eosinophilia ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Blood Eosinophils ◽

Notch Ligands

Abstract Eosinophil chemotaxis and survival within tissues are key components in the development of tissue eosinophilia and subsequent effector responses. In this study, we demonstrate a novel mechanism of eosinophil autoregulation affecting migration and survival mediated through Notch signaling. We show for the first time that human blood eosinophils express Notch receptors and Notch ligands, expressions of which are influenced by the presence of eosinophil-activating granulocyte-macrophage colony-stimulating factor (GM-CSF). Evidence of Notch receptor activation and subsequent transcription of the Notch-responsive gene HES1 were observed in GM-CSF–stimulated eosinophils, confirming functionality of eosinophil-expressed Notch-signaling components. Moreover, by inhibiting Notch signaling with γ-secretase inhibitors or Notch receptor–specific neutralizing antibodies, we demonstrate that autocrine Notch signaling enhances stimulus-mediated actin rearrangement and eosinophil chemokinesis, and impairs eosinophil viability. Taken together, these data suggest autocrine Notch signaling, enhanced in response to tissue- or inflammatory-derived signals, influences eosinophil activity and longevity, which may ultimately contribute to the development of tissue eosinophilia and exacerbation or remediation of eosinophil effector functions.

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PKCθ links proximal T cell and Notch signaling through localized regulation of the actin cytoskeleton

eLife ◽

10.7554/elife.20003 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 9

Author(s):

Graham J Britton ◽

Rachel Ambler ◽

Danielle J Clark ◽

Elaine V Hill ◽

Helen M Tunbridge ◽

...

Keyword(s):

T Cell ◽

Notch Signaling ◽

Cell Receptor ◽

Negative Regulator ◽

T Cell Signaling ◽

Costimulatory Receptor ◽

Th17 Differentiation ◽

Antigen Presenting ◽

Cell Interface ◽

Notch Activation

Notch is a critical regulator of T cell differentiation and is activated through proteolytic cleavage in response to ligand engagement. Using murine myelin-reactive CD4 T cells, we demonstrate that proximal T cell signaling modulates Notch activation by a spatiotemporally constrained mechanism. The protein kinase PKCθ is a critical mediator of signaling by the T cell antigen receptor and the principal costimulatory receptor CD28. PKCθ selectively inactivates the negative regulator of F-actin generation, Coronin 1A, at the center of the T cell interface with the antigen presenting cell (APC). This allows for effective generation of the large actin-based lamellum required for recruitment of the Notch-processing membrane metalloproteinase ADAM10. Such enhancement of Notch activation is critical for efficient T cell proliferation and Th17 differentiation. We reveal a novel mechanism that, through modulation of the cytoskeleton, controls Notch activation at the T cell:APC interface thereby linking T cell receptor and Notch signaling pathways.

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four-jointed interacts with dachs, abelson and enabled and feeds back onto the Notch pathway to affect growth and segmentation in the Drosophila leg

Development ◽

10.1242/dev.128.18.3533 ◽

2001 ◽

Vol 128 (18) ◽

pp. 3533-3542

Author(s):

Gerri R. Buckles ◽

Cordelia Rauskolb ◽

John Lee Villano ◽

Flora N. Katz

Keyword(s):

Notch Signaling ◽

Molecular Basis ◽

Feedback Loop ◽

Regional Growth ◽

Notch Pathway ◽

Notch Signaling Pathway ◽

Normal Pattern ◽

Notch Ligands ◽

Notch Activation

The molecular basis of segmentation and regional growth during morphogenesis of Drosophila legs is poorly understood. We show that four-jointed is not only required for these processes, but also can direct ectopic growth and joint initiation when its normal pattern of expression is disturbed. These effects are non-autonomous, consistent with our demonstration of both transmembrane and secreted forms of the protein in vivo. The similarities between four-jointed and Notch phenotypes led us to further investigate the relationships between these pathways. Surprisingly, we find that although four-jointed expression is regulated downstream of Notch activation, four-jointed can induce expression of the Notch ligands, Serrate and Delta, and may thereby participate in a feedback loop with the Notch signaling pathway. We also show that four-jointed interacts with abelson, enabled and dachs, which leads us to suggest that one target of four-jointed signaling is the actin cytoskeleton. Thus, four-jointed may bridge the gap between the signals that direct morphogenesis and those that carry it out.

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