Modulation of the NOTCH1 Pathway by LUNATIC FRINGE Is Dominant over That of MANIC or RADICAL FRINGE

Fringes are glycosyltransferases that transfer a GlcNAc to O-fucose residues on Epidermal Growth Factor-like (EGF) repeats. Three Fringes exist in mammals: LUNATIC FRINGE (LFNG), MANIC FRINGE (MFNG), and RADICAL FRINGE (RFNG). Fringe modification of O-fucose on EGF repeats in the NOTCH1 (N1) extracellular domain modulates the activation of N1 signaling. Not all O-fucose residues of N1 are modified by all Fringes; some are modified by one or two Fringes and others not modified at all. The distinct effects on N1 activity depend on which Fringe is expressed in a cell. However, little data is available on the effect that more than one Fringe has on the modification of O-fucose residues and the resulting downstream consequence on Notch activation. Using mass spectral glycoproteomic site mapping and cell-based N1 signaling assays, we compared the effect of co-expression of N1 with one or more Fringes on modification of O-fucose and activation of N1 in three cell lines. Individual expression of each Fringe with N1 in the three cell lines revealed differences in modulation of the Notch pathway dependent on the presence of endogenous Fringes. Despite these cell-based differences, co-expression of several Fringes with N1 demonstrated a dominant effect of LFNG over MFNG or RFNG. MFNG and RFNG appeared to be co-dominant but strongly dependent on the ligands used to activate N1 and on the endogenous expression of Fringes. These results show a hierarchy of Fringe activity and indicate that the effect of MFNG and/or RFNG could be small in the presence of LFNG.

Download Full-text

Notch Activation Induces Endothelial Cell Cycle Arrest and Participates in Contact Inhibition: Role of p21Cip1 Repression

Molecular and Cellular Biology ◽

10.1128/mcb.24.20.8813-8822.2004 ◽

2004 ◽

Vol 24 (20) ◽

pp. 8813-8822 ◽

Cited By ~ 145

Author(s):

Michela Noseda ◽

Linda Chang ◽

Graeme McLean ◽

Jonathan E. Grim ◽

Bruce E. Clurman ◽

...

Keyword(s):

Cell Cycle ◽

Endothelial Cells ◽

Vascular Function ◽

Nuclear Translocation ◽

Notch Pathway ◽

Contact Inhibition ◽

Cyclin D ◽

A Cell ◽

Rb Phosphorylation ◽

Notch Activation

ABSTRACT Although previous studies demonstrate that appropriate Notch signaling is required during angiogenesis and in vascular homeostasis, the mechanisms by which Notch regulates vascular function remain to be elucidated. Here, we show that activation of the Notch pathway by the ligand Jagged1 reduces the proliferation of endothelial cells. Notch activation inhibits proliferation of endothelial cells in a cell-autonomous manner by inhibiting phosphorylation of the retinoblastoma protein (Rb). During cell cycle entry, p21Cip1 is upregulated in endothelial cells. Activated Notch inhibits mitogen-induced upregulation of p21Cip1 and delays cyclin D-cdk4-mediated Rb phosphorylation. Notch-dependent repression of p21Cip1 prevents nuclear localization of cyclin D and cdk4. The necessity of p21Cip1 for nuclear translocation of cyclin D-cdk4 and S-phase entry in endothelial cells was demonstrated by targeted downregulation of p21Cip1 by using RNA interference. We further demonstrate that when endothelial cells reach confluence, Notch is activated and p21Cip1 is downregulated. Inhibition of the Notch pathway at confluence prevents p21Cip1 downregulation and induces Rb phosphorylation. We suggest that Notch activation contributes to contact inhibition of endothelial cells, in part through repression of p21Cip1 expression.

Download Full-text

Lysosome Targeting Chimeras (LYTACs) for the Degradation of Secreted and Membrane Proteins

10.26434/chemrxiv.7927061.v1 ◽

2019 ◽

Cited By ~ 2

Author(s):

Steven Banik ◽

Kayvon Pedram ◽

Simon Wisnovsky ◽

Nicholas Riley ◽

Carolyn Bertozzi

Keyword(s):

Epidermal Growth Factor Receptor ◽

Membrane Proteins ◽

Growth Factor Receptor ◽

Basic Research ◽

Biochemical Pathway ◽

Programmed Death Ligand 1 ◽

Programmed Death ◽

A Cell ◽

Epidermal Growth ◽

Powerful Strategy

<p>Targeted protein degradation is a powerful strategy to address the canonically undruggable proteome. However, current technologies are limited to targets with cytosolically-accessible and ligandable domains. Here, we designed and synthesized conjugates capable of binding both a cell surface lysosome targeting receptor and the extracellular domain of a target protein. These lysosome targeting chimeras (LYTACs) consist of an antibody fused to agonist glycopeptide ligands for the cation-independent mannose-6-phosphate receptor (CI-M6PR). LYTACs enabled a CRISPRi knockdown screen revealing the biochemical pathway for CI-M6PR-mediated cargo internalization. We demonstrated that LYTACs mediate efficient degradation of Apolipoprotein-E4, epidermal growth factor receptor (EGFR), CD71, and programmed death-ligand 1 (PD-L1). LYTACs represent a modular strategy for directing secreted and membrane proteins for degradation in the context of both basic research and therapy. <b></b></p>

Download Full-text

Computational Evaluation and In Vitro Validation of New Epidermal Growth Factor Receptor Inhibitors

Current Topics in Medicinal Chemistry ◽

10.2174/1568026620666200603122726 ◽

2020 ◽

Vol 20 (18) ◽

pp. 1628-1639

Author(s):

Sergi Gómez-Ganau ◽

Josefa Castillo ◽

Andrés Cervantes ◽

Jesus Vicente de Julián-Ortiz ◽

Rafael Gozalbes

Keyword(s):

Cell Proliferation ◽

Epidermal Growth Factor Receptor ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Binding Affinity ◽

Cell Lines ◽

Growth Factor Receptor ◽

Significant Activity ◽

Epidermal Growth

Background: The Epidermal Growth Factor Receptor (EGFR) is a transmembrane protein that acts as a receptor of extracellular protein ligands of the epidermal growth factor (EGF/ErbB) family. It has been shown that EGFR is overexpressed by many tumours and correlates with poor prognosis. Therefore, EGFR can be considered as a very interesting therapeutic target for the treatment of a large variety of cancers such as lung, ovarian, endometrial, gastric, bladder and breast cancers, cervical adenocarcinoma, malignant melanoma and glioblastoma. Methods: We have followed a structure-based virtual screening (SBVS) procedure with a library composed of several commercial collections of chemicals (615,462 compounds in total) and the 3D structure of EGFR obtained from the Protein Data Bank (PDB code: 1M17). The docking results from this campaign were then ranked according to the theoretical binding affinity of these molecules to EGFR, and compared with the binding affinity of erlotinib, a well-known EGFR inhibitor. A total of 23 top-rated commercial compounds displaying potential binding affinities similar or even better than erlotinib were selected for experimental evaluation. In vitro assays in different cell lines were performed. A preliminary test was carried out with a simple and standard quick cell proliferation assay kit, and six compounds showed significant activity when compared to positive control. Then, viability and cell proliferation of these compounds were further tested using a protocol based on propidium iodide (PI) and flow cytometry in HCT116, Caco-2 and H358 cell lines. Results: The whole six compounds displayed good effects when compared with erlotinib at 30 μM. When reducing the concentration to 10μM, the activity of the 6 compounds depends on the cell line used: the six compounds showed inhibitory activity with HCT116, two compounds showed inhibition with Caco-2, and three compounds showed inhibitory effects with H358. At 2 μM, one compound showed inhibiting effects close to those from erlotinib. Conclusion: Therefore, these compounds could be considered as potential primary hits, acting as promising starting points to expand the therapeutic options against a wide range of cancers.

Download Full-text

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.

Download Full-text

Aspects of the biology of regeneration and repair in the human gastrointestinal tract

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.1998.0257 ◽

1998 ◽

Vol 353 (1370) ◽

pp. 925-933 ◽

Cited By ~ 45

Author(s):

Nicholas A. Wright

Keyword(s):

Stem Cells ◽

Gastric Gland ◽

Cell Lineage ◽

Mucosal Ulceration ◽

Cell Lineages ◽

Trefoil Peptides ◽

Pyloric Mucosa ◽

A Cell ◽

Epidermal Growth ◽

Altered Gene

The main pathways of epithelial differentiation in the intestine, Paneth, mucous, endocrine and columnar cell lineages are well recognized. However, in abnormal circumstances, for example in mucosal ulceration, a cell lineage with features distinct from these emerges, which has often been dismissed in the past as ‘pyloric’ metaplasia, because of its morphological resemblance to the pyloric mucosa in the stomach. However, we can conclude that this cell lineage has a defined phenotype unique in gastrointestinal epithelia, has a histogenesis that resembles that of Brunner's glands, but acquires a proliferative organization similar to that of the gastric gland. It expresses several peptides of particular interest, including epidermal growth factor, the trefoil peptides TFF1, TFF2, TFF3, lysozyme and PSTI. The presence of this lineage also appears to cause altered gene expression in adjacent indigenous cell lineages. We propose that this cell lineage is induced in gastrointestinal stem cells as a result of chronic mucosal ulceration, and plays an important part in ulcer healing; it should therefore be added to the repertoire of gastrointestinal stem cells.

Download Full-text

lncLocator 2.0: a cell-line-specific subcellular localization predictor for long non-coding RNAs with interpretable deep learning

Bioinformatics ◽

10.1093/bioinformatics/btab127 ◽

2021 ◽

Author(s):

Yang Lin ◽

Xiaoyong Pan ◽

Hong-Bin Shen

Keyword(s):

Subcellular Localization ◽

Cell Line ◽

Cell Lines ◽

Short Term Memory ◽

Computational Method ◽

Language Models ◽

Supplementary Information ◽

Deep Model ◽

A Cell ◽

Non Coding Rnas

Abstract Motivation Long non-coding RNAs (lncRNAs) are generally expressed in a tissue-specific way, and subcellular localizations of lncRNAs depend on the tissues or cell lines that they are expressed. Previous computational methods for predicting subcellular localizations of lncRNAs do not take this characteristic into account, they train a unified machine learning model for pooled lncRNAs from all available cell lines. It is of importance to develop a cell-line-specific computational method to predict lncRNA locations in different cell lines. Results In this study, we present an updated cell-line-specific predictor lncLocator 2.0, which trains an end-to-end deep model per cell line, for predicting lncRNA subcellular localization from sequences.We first construct benchmark datasets of lncRNA subcellular localizations for 15 cell lines. Then we learn word embeddings using natural language models, and these learned embeddings are fed into convolutional neural network, long short-term memory and multilayer perceptron to classify subcellular localizations. lncLocator 2.0 achieves varying effectiveness for different cell lines and demonstrates the necessity of training cell-line-specific models. Furthermore, we adopt Integrated Gradients to explain the proposed model in lncLocator 2.0, and find some potential patterns that determine the subcellular localizations of lncRNAs, suggesting that the subcellular localization of lncRNAs is linked to some specific nucleotides. Availability The lncLocator 2.0 is available at www.csbio.sjtu.edu.cn/bioinf/lncLocator2 and the source code can be found at https://github.com/Yang-J-LIN/lncLocator2. Supplementary information Supplementary data are available at Bioinformatics online.

Download Full-text

Quantitative cell fusion: the fusion sensitivity (FS) potential

Journal of Cell Science ◽

10.1242/jcs.49.1.87 ◽

1981 ◽

Vol 49 (1) ◽

pp. 87-97

Author(s):

D. Rohme

Keyword(s):

Cell Line ◽

Cell Fusion ◽

Cell Lines ◽

Dose Response ◽

Sendai Virus ◽

Biological Significance ◽

Diploid Cell ◽

Mammalian Cell Lines ◽

Virus Dose ◽

A Cell

The dose response of Sendai virus-induced cell fusion was studied in 10 mammalian cell lines, comprising 5 continuous and 5 diploid cell lines originating from 5 species. The extent of fusion was calculated using a parameter directly proportional to the number of fusion events (t-parameter). At lower levels of fusion the dose response was found to be based on the same simple kinetic rules in all cell lines and was defined by the formula: t = FS. FAU/(I + FS. FAU), where FS (fusion sensitivity) is a cell-specific constant of the fusion rate and FAU (fusion activity units) is the virus dose. The FS potential of a cell line was determined as the linear regression coefficient of the fusion index (t/(I - t)) on the virus dose. At higher levels of fusion, when the fusion extent reached cell-line-specific maximal levels, the dose response was not as uniform. In general, and particularly in the cases of the diploid cell lines, these maximal levels were directly proportional to the FS potentials. Thus, it was concluded that the FS potential is the basic quantitative feature, which expresses the cellular fusion efficiency. The fact that FS varied extensively between cell lines, but at the same time apparently followed certain patterns (being higher in continuous compared to diploid cell lines and being related to the species of origin of the cells), emphasizes it biological significance as well as its possible usefulness in studies of the efficiency of various molecular interactions in the cell membrane/cytoskeleton system.

Download Full-text

Introns excised from immunoglobulin pre-mRNAs exist as discrete species

Molecular and Cellular Biology ◽

10.1128/mcb.4.10.2017-2022.1984 ◽

1984 ◽

Vol 4 (10) ◽

pp. 2017-2022

Author(s):

C Coleclough ◽

D Wood

Keyword(s):

Cell Lines ◽

Resolving Power ◽

Linear Molecule ◽

Immunoglobulin Kappa ◽

Polyadenylic Acid ◽

Nonionic Detergent ◽

A Cell ◽

Active Locus ◽

B Lineage ◽

High Level

We have discovered a new class of transcripts of immunoglobulin kappa genes in RNA from B-lineage cells. These transcripts have the properties predicted of free introns excised from kappa mRNA precursors. RNA extracted from populations of normal mouse spleen cells polyclonally activated with B-cell mitogens contains four such transcripts; their electrophoretic mobilities correspond to the distances between the intron-exon boundary of the C kappa region and the four useable J kappa elements, and their relative abundance reflects the relative usage of those J segments. Analysis of RNA from monoclonal kappa-expressing cell lines reveals that one active locus produces one free intron, its size determined by which J element is used in that locus. Apart from their distinctive size, free introns are identified by their lack of polyadenylic acid and their ability to hybridize to cloned probes containing intron sequences, but not to the adjacent V or C exonic sequences. They have a characteristic subcellular distribution, being extractable from nuclei by treatment with nonionic detergent; nuclei thus treated retain most of the primary mRNA precursors, but few of the free introns. A high level of kappa gene expression is not a prerequisite of a cell containing detectable free kappa introns; the lymphoma 38c has only 5% or less of the amount of kappa mRNA that the plasmacytoma MCP-11 contains, yet the ratio of free intron to mRNA precursor is about the same in both cell lines. When analyzed by electrophoretic separation of sufficient resolving power, the free introns due to a single kappa locus resolve into two discrete species. We consider that this most likely reflects the existence of two conformers of the intron, one presumably a covalently intact circle and the other linear molecule.

Download Full-text