scholarly journals Broad and thematic remodeling of the surface glycoproteome on isogenic cells transformed with driving proliferative oncogenes

2019 ◽  
Author(s):  
Kevin K. Leung ◽  
Gary M. Wilson ◽  
Lisa L. Kirkemo ◽  
Nicholas M. Riley ◽  
Joshua J. Coon ◽  
...  
Keyword(s):  
Cell Surface ◽  
Electron Transfer Dissociation ◽  
Biomarker Discovery ◽  
Mapk Pathway ◽  
Strong Dependence ◽  
Mapk Signaling ◽  
Surface Proteins ◽  
Breast Epithelial Cell ◽  
Epithelial Cell Line ◽  
A Cell

AbstractThe cell surface proteome, the surfaceome, is the interface for engaging the extracellular space in normal and cancer cells. Here we apply quantitative proteomics of N-linked glycoproteins to reveal how a collection of some 700 surface proteins is dramatically remodeled in an isogenic breast epithelial cell line stably expressing any of six of the most prominent proliferative oncogenes, including the receptor tyrosine kinases, EGFR and HER2, and downstream signaling partners such as KRAS, BRAF, MEK and AKT. We find that each oncogene has somewhat different surfaceomes but the functions of these proteins are harmonized by common biological themes including up-regulation of nutrient transporters, down-regulation of adhesion molecules and tumor suppressing phosphatases, and alteration in immune modulators. Addition of a potent MEK inhibitor that blocks MAPK signaling brings each oncogene-induced surfaceome back to a common state reflecting their strong dependence on the MAPK pathway to propagate signaling. Using a recently developed glyco-proteomics method of activated ion electron transfer dissociation (AI-ETD) we found massive oncogene-induced changes in 142 N-linked glycans and differential increases in complex hybrid glycans especially for KRAS and HER2 oncogenes. Overall, these studies provide a broad systems level view of how specific driver oncogenes remodel the surface glycoproteome in a cell autologous fashion, and suggest possible surface targets, and combinations thereof, for drug and biomarker discovery.Significant statementThe cell surface glycoproteome (surfaceome) mediates interactions between the cell and the extracellular environment, and is a major target for immunotherapy in cancer. Using state-of-the-art proteomics methods, we compared how six neighboring proliferative oncogenes cause large and bidirectional expression of some 700 surface proteins and the 142 different glycans that decorate them. While each oncogene induces large and somewhat unique glycoproteomes relative to non-transformed cells, we find common functional consequences that are massively reversed by small molecule inhibition of the MAPK pathway. This large-scale comparative study provides important insights for how oncogenes remodel isogenic cells in a cell autologous fashion, and suggest possible new opportunities for antibody drug discovery in more complex tumor settings.

scholarly journals Broad and thematic remodeling of the surfaceome and glycoproteome on isogenic cells transformed with driving proliferative oncogenes

2020 ◽  
Vol 117 (14) ◽  
pp. 7764-7775 ◽  
Author(s):  
Kevin K. Leung ◽  
Gary M. Wilson ◽  
Lisa L. Kirkemo ◽  
Nicholas M. Riley ◽  
Joshua J. Coon ◽  
...  
Keyword(s):  
Cell Surface ◽  
Tyrosine Kinases ◽  
Electron Transfer Dissociation ◽  
Biomarker Discovery ◽  
Mapk Pathway ◽  
Strong Dependence ◽  
Mapk Signaling ◽  
Surface Proteins ◽  
Breast Epithelial Cell ◽  
Epithelial Cell Line

The cell surface proteome, the surfaceome, is the interface for engaging the extracellular space in normal and cancer cells. Here we apply quantitative proteomics ofN-linked glycoproteins to reveal how a collection of some 700 surface proteins is dramatically remodeled in an isogenic breast epithelial cell line stably expressing any of six of the most prominent proliferative oncogenes, including the receptor tyrosine kinases, EGFR and HER2, and downstream signaling partners such as KRAS, BRAF, MEK, and AKT. We find that each oncogene has somewhat different surfaceomes, but the functions of these proteins are harmonized by common biological themes including up-regulation of nutrient transporters, down-regulation of adhesion molecules and tumor suppressing phosphatases, and alteration in immune modulators. Addition of a potent MEK inhibitor that blocks MAPK signaling brings each oncogene-induced surfaceome back to a common state reflecting the strong dependence of the oncogene on the MAPK pathway to propagate signaling. Cell surface protein capture is mediated by covalent tagging of surface glycans, yet current methods do not afford sequencing of intact glycopeptides. Thus, we complement the surfaceome data with whole cell glycoproteomics enabled by a recently developed technique called activated ion electron transfer dissociation (AI-ETD). We found massive oncogene-induced changes to the glycoproteome and differential increases in complex hybrid glycans, especially for KRAS and HER2 oncogenes. Overall, these studies provide a broad systems-level view of how specific driver oncogenes remodel the surfaceome and the glycoproteome in a cell autologous fashion, and suggest possible surface targets, and combinations thereof, for drug and biomarker discovery.

Delivery of newly synthesized Na(+)-K(+)-ATPase to the plasma membrane of A6 epithelia

1997 ◽  
Vol 272 (6) ◽  
pp. C1781-C1789 ◽  
Author(s):  
B. Coupaye-Gerard ◽  
J. B. Zuckerman ◽  
P. Duncan ◽  
A. Bortnik ◽  
D. I. Avery ◽  
...  
Keyword(s):  
Steady State ◽  
Cell Surface ◽  
Apical Cell ◽  
Surface Proteins ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Epithelial Cell Line ◽  
Steady State Distribution ◽  
State Distribution ◽  
Sorting Signals

Na(+)-K(+)-ATPase is localized to the basolateral cell surface of most epithelial cells. Conflicting results regarding the intracellular trafficking of Na(+)-K(+)-ATPase in Madin-Darby canine kidney cells have been reported, with delivery to both apical and basolateral membranes or exclusively to the basolateral cell surface. We examined the delivery and steady-state distribution of Na(+)-K(+)-ATPase in the amphibian epithelial cell line A6 using an antibody raised against Na(+)-K(+)-ATPase alpha-subunit and sulfo-N-hydroxysuccinimidobiotin to tag cell surface proteins. The steady-state distribution of the Na(+)-K(+)-ATPase was basolateral, as confirmed by immunocytochemistry. Delivery of newly synthesized Na(+)-K(+)-ATPase to the cell surface was examined using [35S]methionine and [35S]cysteine in a pulse-chase protocol. After a 20-min pulse, the alpha-subunit and core glycosylated beta-subunit were present at both apical and basolateral cell surfaces. The alpha-subunit and core glycosylated beta-subunit delivered to the apical cell surface were degraded within 2 h. Mature alpha/beta-heterodimer was found almost exclusively at the basolateral surface after a 1- to 24-h chase. These data suggest that immature Na(+)-K(+)-ATPase alpha-subunit and core glycosylated beta-subunits are not retained in the endoplasmic reticulum of A6 cells and apparently lack sorting signals. Mature Na(+)-K(+)-ATPase is targeted to the basolateral surface, suggesting that basolateral targeting of the protein is conformation dependent.

scholarly journals The in silico human surfaceome

2018 ◽  
Vol 115 (46) ◽  
pp. E10988-E10997 ◽  
Author(s):  
Damaris Bausch-Fluck ◽  
Ulrich Goldmann ◽  
Sebastian Müller ◽  
Marc van Oostrum ◽  
Maik Müller ◽  
...  
Keyword(s):  
Cell Surface ◽  
In Silico ◽  
Embryonic Stem ◽  
Surface Protein ◽  
Surface Proteins ◽  
Cell Surface Protein ◽  
Cell Surface Proteins ◽  
A Cell ◽  
Visualization Tools ◽  
Protein Classes

Cell-surface proteins are of great biomedical importance, as demonstrated by the fact that 66% of approved human drugs listed in the DrugBank database target a cell-surface protein. Despite this biomedical relevance, there has been no comprehensive assessment of the human surfaceome, and only a fraction of the predicted 5,000 human transmembrane proteins have been shown to be located at the plasma membrane. To enable analysis of the human surfaceome, we developed the surfaceome predictor SURFY, based on machine learning. As a training set, we used experimentally verified high-confidence cell-surface proteins from the Cell Surface Protein Atlas (CSPA) and trained a random forest classifier on 131 features per protein and, specifically, per topological domain. SURFY was used to predict a human surfaceome of 2,886 proteins with an accuracy of 93.5%, which shows excellent overlap with known cell-surface protein classes (i.e., receptors). In deposited mRNA data, we found that between 543 and 1,100 surfaceome genes were expressed in cancer cell lines and maximally 1,700 surfaceome genes were expressed in embryonic stem cells and derivative lines. Thus, the surfaceome diversity depends on cell type and appears to be more dynamic than the nonsurface proteome. To make the predicted surfaceome readily accessible to the research community, we provide visualization tools for intuitive interrogation (wlab.ethz.ch/surfaceome). The in silico surfaceome enables the filtering of data generated by multiomics screens and supports the elucidation of the surfaceome nanoscale organization.

scholarly journals Identification of RAS-Mitogen-Activated Protein Kinase Signaling Pathway Modulators in an ERF1 Redistribution® Screen

2006 ◽  
Vol 11 (4) ◽  
pp. 423-434 ◽  
Author(s):  
Charlotta Grånäs ◽  
Betina Kerstin Lundholt ◽  
Frosty Loechel ◽  
Hans-Christian Pedersen ◽  
Sara Petersen Bjørn ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Signaling Pathway ◽  
Kinase Inhibitors ◽  
Mapk Pathway ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Mitogen Activated Protein ◽  
A Cell ◽  
Protein Kinase Signaling

The RAS-mitogen-activated protein kinase (MAPK) signaling pathway has a central role in regulating the proliferation and survival of both normal and tumor cells. This pathway has been 1 focus area for the development of anticancer drugs, resulting in several compounds, primarily kinase inhibitors, in clinical testing. The authors have undertaken a cell-based, high-throughput screen using a novel ERF1 Redistribution® assay to identify compounds that modulate the signaling pathway. The hit compounds were subsequently tested for activity in a functional cell proliferation assay designed to selectively detect compounds inhibiting the proliferation of MAPK pathway-dependent cancer cells. The authors report the identification of 2 cell membrane-permeable compounds that exhibit activity in the ERF1 Redistribution® assay and selectively inhibit proliferation of MAPK pathway-dependent malignant melanoma cells at similar potencies (IC50 =< 5 μM). These compounds have drug-like structures and are negative in RAF, MEK, and ERK in vitro kinase assays. Drugs belonging to these compound classes may prove useful for treating cancers caused by excessive MAPK pathway signaling. The results also show that cell-based, high-content Redistribution® screens can detect compounds with different modes of action and reveal novel targets in a pathway known to be disease relevant.

scholarly journals A high-molecular-mass cell-surface protein from Lactobacillus reuteri 1063 adheres to mucus components The GenBank accession number for the sequence reported in this paper is AF120104.

Microbiology ◽  
2002 ◽  
Vol 148 (2) ◽  
pp. 433-442 ◽  
Author(s):  
Stefan Roos ◽  
Hans Jonsson
Keyword(s):  
Cell Surface ◽  
Lactobacillus Reuteri ◽  
Surface Protein ◽  
Surface Proteins ◽  
Cell Surface Protein ◽  
Intestinal Mucus ◽  
A Cell ◽  
Molecular Masses ◽  
Ph Range

A gene from Lactobacillus reuteri 1063 encoding a cell-surface protein, designated Mub, that adheres to mucus components in vitro has been cloned and sequenced. The deduced amino acid sequence of Mub (358 kDa) shows the presence of 14 approximately 200 aa repeats and features typical for other cell-surface proteins of Gram-positive bacteria. Fusion proteins consisting of different repeats of Mub and the maltose-binding protein (MBP) were produced. These proteins adhered to pig mucus components, with molecular masses ranging from <0·1 to >2 MDa, to pig gastric mucin and to hen intestinal mucus. The binding of Mub to mucus components occurred in the pH range 3–7·4, with maximum binding at pH 4–5 and could be partly inhibited by the glycoprotein fetuin. Affinity-purified antibodies against recombinant Mub were used in immunofluorescence microscopy to demonstrate the presence of Mub on the cell surface of strain 1063. By using the antibodies in a Western blot analysis, Mub could also be detected in the growth medium. The results implicate Mub as a cell-surface protein that is involved in Lactobacillus interactions with mucin and in colonization of the digestive tract.

scholarly journals Functional correlation between cell adhesive properties and some cell surface proteins.

1977 ◽  
Vol 75 (2) ◽  
pp. 464-474 ◽  
Author(s):  
M Takeichi
Keyword(s):  
Cell Surface ◽  
Chinese Hamster ◽  
Surface Protein ◽  
Surface Proteins ◽  
Adhesive Properties ◽  
Cell Surface Proteins ◽  
Morphological Studies ◽  
Chinese Hamster V79 Cells ◽  
A Cell ◽  
In Cells

The adhesive properties of Chinese hamster V79 cells were analyzed and characterized by various cell dissociation treatments. The comparisons of aggregability among cells dissociated with EDTA, trypsin + Ca2+, and trypsin + EDTA, revealed that these cells have two adhesion mechanisms, a Ca2+-independent and a Ca2+-dependent one. The former did not depend on temperature, whereas the latter occurred only at physiological temperatures. Both mechanisms were trypsin sensitive, but the Ca2+-dependent one was protected by Ca2+ against trypsinization. In morphological studies, the Ca2+-independent adhesion appeared to be a simple agglutination or flocculation of cells, whereas the Ca2+-dependent adhesion seemed to be more physiological, being accompanied by cell deformation resulting in the increase of contact area between adjacent cells. Lactoperoxidase-catalyzed iodination of cell surface proteins revealed that several proteins are more intensely labeled in cells with Ca2+-independent adhesiveness than in cells without that property. It was also found that a cell surface protein with a molecular weight of approximately 150,000 is present only in cells with Ca2+-dependent adhesiveness. The iodination and trypsinization of this protein were protected by Ca2+, suggesting its reactivity to Ca2+. Possible mechanisms for each adhesion property are discussed, taking into account the correlation of these proteins with cell adhesiveness.

scholarly journals Large remodeling of the Myc-induced cell surface proteome in B cells and prostate cells creates new opportunities for immunotherapy

2021 ◽  
Vol 118 (4) ◽  
pp. e2018861118
Author(s):  
Wentao Chen ◽  
Kurt Yun Mou ◽  
Paige Solomon ◽  
Rahul Aggarwal ◽  
Kevin K. Leung ◽  
...  
Keyword(s):  
Cell Surface ◽  
B Cell ◽  
Cell Lines ◽  
Surface Proteins ◽  
Cell Type ◽  
Hematological Cancers ◽  
A Cell ◽  
Surface Proteome ◽  
The Impact ◽  
Cell Surface Proteome

MYC is a powerful transcription factor overexpressed in many human cancers including B cell and prostate cancers. Antibody therapeutics are exciting opportunities to attack cancers but require knowledge of surface proteins that change due to oncogene expression. To identify how MYC overexpression remodels the cell surface proteome in a cell autologous fashion and in different cell types, we investigated the impact of MYC overexpression on 800 surface proteins in three isogenic model cell lines either of B cell or prostate cell origin engineered to have high or low MYC levels. We found that MYC overexpression resulted in dramatic remodeling (both up- and down-regulation) of the cell surfaceome in a cell type-dependent fashion. We found systematic and large increases in distinct sets of >80 transporters including nucleoside transporters and nutrient transporters making cells more sensitive to toxic nucleoside analogs like cytarabine, commonly used for treating hematological cancers. Paradoxically, MYC overexpression also increased expression of surface proteins driving cell turnover such as TNFRSF10B, also known as death receptor 5, and immune cell attacking signals such as the natural killer cell activating ligand NCR3LG1, also known as B7-H6. We generated recombinant antibodies to these two targets and verified their up-regulation in MYC overexpression cell lines and showed they were sensitive to bispecific T cell engagers (BiTEs). Our studies demonstrate how MYC overexpression leads to dramatic bidirectional remodeling of the surfaceome in a cell type-dependent but functionally convergent fashion and identify surface targets or combinations thereof as possible candidates for cytotoxic metabolite or immunotherapy.

scholarly journals Cell-specific Bioorthogonal Tagging of Glycoproteins in Co-culture

2021 ◽  
Author(s):  
Anna Cioce ◽  
Beatriz Calle ◽  
Andrea Marchesi ◽  
Ganka Bineva-Todd ◽  
Helen Flynn ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Cell Surface ◽  
Cell Line ◽  
Specific Protein ◽  
Surface Proteins ◽  
Cancer Development ◽  
Biological Processes ◽  
Culture Studies ◽  
Cell Surface Proteins ◽  
A Cell

Interactions between cells fundamentally impact biological processes. In cancer development, such interactions define key stages of disease that cannot be adequately recapitulated in cell monoculture. Complex co-culture studies have been key to unraveling the complexity of these processes, usually by sorting cells and transcriptome or bulk proteome analyses. However, these methods invariably lead to sample loss and do not capture aberrant glycosylation as an important corollary of cancer formation. Here, we report the development of Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped with a biosynthetic AND gate that uses bioorthogonally tagged sugars to generate glycosylation precursors. The cellular glycosylation machinery then introduces bioorthogonal tags into glycoproteins exclusively in cell lines expressing the enzymes of the biosynthetic AND gate. Modification with clickable reporter moieties allows for imaging or enrichment with mass spectrometry-proteomics in a cell-specific fashion. Making use of glycans as a property of most cell surface proteins, we use BOCTAG as an efficient means for cell-specific protein tracing.

scholarly journals Spatiotemporal Control of Pathway Sensors and Cross-Pathway Feedback Regulate a Cell Differentiation MAPK Pathway in Yeast

2020 ◽  
Author(s):  
Aditi Prabhakar ◽  
Beatriz Gonzalez ◽  
Heather Dionne ◽  
Sukanya Basu ◽  
Paul J. Cullen
Keyword(s):  
Cell Differentiation ◽  
Mapk Pathway ◽  
Expression Profiles ◽  
Control Cell ◽  
Mapk Signaling ◽  
Hog Pathway ◽  
Temporal Regulation ◽  
Mapk Pathways ◽  
Bud Neck ◽  
A Cell

ABSTRACTMitogen-Activated Protein Kinase (MAPK) pathways control cell differentiation and the response to stress. MAPK pathways can share components with other pathways yet induce specific responses through mechanisms that remain unclear. In Saccharomyces cerevisiae, the MAPK pathway that controls filamentous growth (fMAPK) shares components with the MAPK pathway that regulates the response to osmotic stress (HOG). By exploring temporal regulation of MAPK signaling, we show here that the two pathways exhibited different patterns of activity throughout the cell cycle. The different patterns resulted from different expression profiles of genes encoding the mucin sensors (MSB2 for fMAPK and HKR1 for HOG). We also show that positive feedback through the fMAPK pathway stimulated the HOG pathway, presumably to modulate fMAPK pathway activity. By exploring spatial regulation of MAPK signaling, we found that the shared tetraspan protein, Sho1p, which has a dynamic localization pattern, induced the fMAPK pathway at the mother-bud neck. A Sho1p-interacting protein, Hof1p, which also localizes to the mother-bud neck and regulates cytokinesis, also regulated the fMAPK pathway. Therefore, spatial and temporal regulation of pathway sensors, and cross-pathway feedback, regulate a MAPK pathway that controls a cell differentiation response in yeast.

scholarly journals Exploring the surfaceome of Ewing sarcoma identifies a new and unique therapeutic target

2016 ◽  
Vol 113 (13) ◽  
pp. 3603-3608 ◽  
Author(s):  
Jennifer Town ◽  
Helio Pais ◽  
Sally Harrison ◽  
Lucy F. Stead ◽  
Carole Bataille ◽  
...  
Keyword(s):  
Cell Surface ◽  
Ewing Sarcoma ◽  
Somatic Tissue ◽  
Surface Proteins ◽  
Tumor Type ◽  
Rna Seq ◽  
Genes Encoding ◽  
A Cell ◽  
Surface Proteome ◽  
Cell Surface Proteome

The cell surface proteome of tumors mediates the interface between the transformed cells and the general microenvironment, including interactions with stromal cells in the tumor niche and immune cells such as T cells. In addition, the cell surface proteome of individual cancers defines biomarkers for that tumor type and potential proteins that can be the target of antibody-mediated therapy. We have used next-generation deep RNA sequencing (RNA-seq) coupled to an in-house database of genes encoding cell surface proteins (herein referred to as the surfaceome) as a tool to define a cell surface proteome of Ewing sarcoma compared with progenitor mesenchymal stem cells. This subtractive RNA-seq analysis revealed a specific surfaceome of Ewing and showed unexpectedly that the leucine-rich repeat and Ig domain protein 1 (LINGO1) is expressed in over 90% of Ewing sarcoma tumors, but not expressed in any other somatic tissue apart from the brain. We found that the LINGO1 protein acts as a gateway protein internalizing into the tumor cells when engaged by antibody and can carry antibody conjugated with drugs to kill Ewing sarcoma cells. Therefore, LINGO1 is a new, unique, and specific biomarker and drug target for the treatment of Ewing sarcoma.

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