scholarly journals Phosphoproteomic analysis sheds light on intracellular signaling cascades triggered by Formyl-Peptide Receptor 2

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Fabio Cattaneo ◽  
Rosita Russo ◽  
Martina Castaldo ◽  
Angela Chambery ◽  
Cristiana Zollo ◽  
...  
Keyword(s):  
Intracellular Signaling ◽  
Drug Targets ◽  
Large Scale ◽  
Cellular Proliferation ◽  
Biological Effects ◽  
Formyl Peptide Receptor ◽  
Ros Generation ◽  
Systematic Analysis ◽  
Phosphorylated Proteins ◽  
Formyl Peptide

AbstractFormyl peptide receptors (FPRs) belong to the family of seven transmembrane Gi-protein coupled receptors (GPCR). FPR2 is considered the most promiscuous member of this family since it recognizes a wide variety of ligands. It plays a crucial role in several physio-pathological processes and different studies highlighted the correlation between its expression and the higher propensity to invasion and metastasis of some cancers. FPR2 stimulation by its synthetic agonist WKYMVm triggers multiple phosphorylations of intracellular signaling molecules, such as ERKs, PKC, PKB, p38MAPK, PI3K, PLC, and of non-signaling proteins, such as p47phox and p67phox which are involved in NADPH oxidase-dependent ROS generation. Biological effects of FPR2 stimulation include intracellular Ca2+ mobilization, cellular proliferation and migration, and wound healing. A systematic analysis of the phosphoproteome in FPR2-stimulated cells has not been yet reported. Herein, we describe a large-scale phosphoproteomic study in WKYMVm-stimulated CaLu-6 cells. By using high resolution MS/MS we identified 290 differentially phosphorylated proteins and 53 unique phosphopeptides mapping on 40 proteins. Phosphorylations on five selected phospho-proteins were further validated by western blotting, confirming their dependence on FPR2 stimulation. Interconnection between some of the signalling readout identified was also evaluated. Furthermore, we show that FPR2 stimulation with two anti-inflammatory agonists induces the phosphorylation of selected differentially phosphorylated proteins, suggesting their role in the resolution of inflammation. These data provide a promising resource for further studies on new signaling networks triggered by FPR2 and on novel molecular drug targets for human diseases.

scholarly journals Effects of Formyl Peptide Receptor Agonists Ac9-12 and WKYMV in In Vivo and In Vitro Acute Inflammatory Experimental Models

Cells ◽  
2022 ◽  
Vol 11 (2) ◽  
pp. 228
Author(s):  
Izabella Lice ◽  
José Marcos Sanches ◽  
Rebeca D. Correia-Silva ◽  
Mab P. Corrêa ◽  
Marcelo Y. Icimoto ◽  
...  
Keyword(s):  
Therapeutic Potential ◽  
Biological Effects ◽  
Experimental Models ◽  
Formyl Peptide Receptor ◽  
Saline Control ◽  
Acute Peritonitis ◽  
Formyl Peptide ◽  
Leukocyte Influx

Formyl peptide receptors (Fprs) are a G-protein-coupled receptor family mainly expressed on leukocytes. The activation of Fpr1 and Fpr2 triggers a cascade of signaling events, leading to leukocyte migration, cytokine release, and increased phagocytosis. In this study, we evaluate the effects of the Fpr1 and Fpr2 agonists Ac9-12 and WKYMV, respectively, in carrageenan-induced acute peritonitis and LPS-stimulated macrophages. Peritonitis was induced in male C57BL/6 mice through the intraperitoneal injection of 1 mL of 3% carrageenan solution or saline (control). Pre-treatments with Ac9-12 and WKYMV reduced leukocyte influx to the peritoneal cavity, particularly neutrophils and monocytes, and the release of IL-1β. The addition of the Fpr2 antagonist WRW4 reversed only the anti-inflammatory actions of WKYMV. In vitro, the administration of Boc2 and WRW4 reversed the effects of Ac9-12 and WKYMV, respectively, in the production of IL-6 by LPS-stimulated macrophages. These biological effects of peptides were differently regulated by ERK and p38 signaling pathways. Lipidomic analysis evidenced that Ac9-12 and WKYMV altered the intracellular lipid profile of LPS-stimulated macrophages, revealing an increased concentration of several glycerophospholipids, suggesting regulation of inflammatory pathways triggered by LPS. Overall, our data indicate the therapeutic potential of Ac9-12 and WKYMV via Fpr1 or Fpr2-activation in the inflammatory response and macrophage activation.

scholarly journals Interrogation of kinase genetic interactions provides a global view of PAK1-mediated signal transduction pathways

2020 ◽  
Vol 295 (50) ◽  
pp. 16906-16919
Author(s):  
Jae-Hong Kim ◽  
Yeojin Seo ◽  
Myungjin Jo ◽  
Hyejin Jeon ◽  
Young-Seop Kim ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Cell Migration ◽  
Intracellular Signaling ◽  
Mammalian Cells ◽  
Drug Targets ◽  
Large Scale ◽  
Genetic Interaction ◽  
Genetic Interactions ◽  
Signal Transduction Pathways ◽  
Global View

Kinases are critical components of intracellular signaling pathways and have been extensively investigated with regard to their roles in cancer. p21-activated kinase-1 (PAK1) is a serine/threonine kinase that has been previously implicated in numerous biological processes, such as cell migration, cell cycle progression, cell motility, invasion, and angiogenesis, in glioma and other cancers. However, the signaling network linked to PAK1 is not fully defined. We previously reported a large-scale yeast genetic interaction screen using toxicity as a readout to identify candidate PAK1 genetic interactions. En masse transformation of the PAK1 gene into 4,653 homozygous diploid Saccharomyces cerevisiae yeast deletion mutants identified ∼400 candidates that suppressed yeast toxicity. Here we selected 19 candidate PAK1 genetic interactions that had human orthologs and were expressed in glioma for further examination in mammalian cells, brain slice cultures, and orthotopic glioma models. RNAi and pharmacological inhibition of potential PAK1 interactors confirmed that DPP4, KIF11, mTOR, PKM2, SGPP1, TTK, and YWHAE regulate PAK1-induced cell migration and revealed the importance of genes related to the mitotic spindle, proteolysis, autophagy, and metabolism in PAK1-mediated glioma cell migration, drug resistance, and proliferation. AKT1 was further identified as a downstream mediator of the PAK1-TTK genetic interaction. Taken together, these data provide a global view of PAK1-mediated signal transduction pathways and point to potential new drug targets for glioma therapy.

scholarly journals Yeast-Based Genetic Interaction Analysis of Human Kinome

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1156
Author(s):  
Jae-Hong Kim ◽  
Yeojin Seo ◽  
Myungjin Jo ◽  
Hyejin Jeon ◽  
Won-Ha Lee ◽  
...  
Keyword(s):  
Cell Death ◽  
Intracellular Signaling ◽  
Drug Targets ◽  
Large Scale ◽  
Genetic Interaction ◽  
Interaction Analysis ◽  
Inflammatory Diseases ◽  
Molecular Transport ◽  
Multiple Drug ◽  
Network Analyses

Kinases are critical intracellular signaling proteins. To better understand kinase-mediated signal transduction, a large-scale human–yeast genetic interaction screen was performed. Among 597 human kinase genes tested, 28 displayed strong toxicity in yeast when overexpressed. En masse transformation of these toxic kinase genes into 4653 homozygous diploid yeast deletion mutants followed by barcode sequencing identified yeast toxicity modifiers and thus their human orthologs. Subsequent network analyses and functional grouping revealed that the 28 kinases and their 676 interaction partners (corresponding to a total of 969 genetic interactions) are enriched in cell death and survival (34%), small-molecule biochemistry (18%) and molecular transport (11%), among others. In the subnetwork analyses, a few kinases were commonly associated with glioma, cell migration and cell death/survival. Our analysis enabled the creation of a first draft of the kinase genetic interactome network and identified multiple drug targets for inflammatory diseases and cancer, in which deregulated kinase signaling plays a pathogenic role.

scholarly journals Effects of Fifty-Hertz Electromagnetic Fields on Granulocytic Differentiation of ATRA-Treated Acute Promyelocytic Leukemia NB4 Cells

2018 ◽  
Vol 46 (1) ◽  
pp. 389-400 ◽  
Author(s):  
Alfredo Errico Provenzano ◽  
Stefano Amatori ◽  
Maria Gemma Nasoni ◽  
Giuseppe Persico ◽  
Sergio Russo ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Acute Promyelocytic Leukemia ◽  
Electromagnetic Fields ◽  
Cellular Proliferation ◽  
Biological Effects ◽  
Promyelocytic Leukemia ◽  
Ros Generation ◽  
Granulocytic Differentiation ◽  
Nb4 Cells ◽  
Oxidative Balance

Background/Aims: Life on Earth is constantly exposed to electromagnetic fields (EMFs) and the effects induced by EMFs on biological systems have been extensively studied producing different and sometimes contradictory results. Extremely low-frequency electromagnetic fields (ELF-EMFs) have shown to play a role in regulating cell proliferation and differentiation, although how EMFs influence these processes remains unclear. Human acute promyelocytic leukemia (APL) cells are characterized by the arrest of differentiation at the promyelocytic stage due to epigenetic perturbations induced by PML/RARα fusion protein (Promyelocytic Leukemia protein - PML/Retinoic Acid Receptor alpha - RARα). Therapeutic administration of all-trans retinoic acid (ATRA) re-establishes the leukemogenic mechanism re-inducing the normal differentiation processes. Methods: We studied the effects of ELF-EMFs (50 Hz, 2 mT) on the ATRA-mediated granulocytic differentiation process of APL NB4 cells (a cell line established from the bone marrow of a patient affected by the acute promyelocytic leukemia) by monitoring cellular proliferation and morphology, nitrob lue tetrazolium (NBT) reduction and the expression of differentiation surface markers. Finally, we investigated mechanisms focusing on reactive oxygen species (ROS) generation and related molecular pathways. Results: ELF-EMF exposure decreases cellular proliferation potential and helps ATRA-treated NB4 cells to mature. Furthermore, the analysis of ROS production and the consequent extracellular signal regulated kinases (ERK1/2) phosphorylation suggest that a changed intracellular oxidative balance may influence the biological effects of ELF-EMFs. Conclusions: These results indicate that the exposure to ELF-EMF promotes ATRA-induced granulocytic differentiation of APL cells.

scholarly journals Synthesis, Biological Evaluation, and Molecular Modeling of Aza-Crown Ethers

Molecules ◽  
2021 ◽  
Vol 26 (8) ◽  
pp. 2225
Author(s):  
Stepan S. Basok ◽  
Igor A. Schepetkin ◽  
Andrei I. Khlebnikov ◽  
Anatoliy F. Lutsyuk ◽  
Tatiana I. Kirichenko ◽  
...  
Keyword(s):  
Crown Ethers ◽  
Density Functional ◽  
Biological Effects ◽  
Biological Evaluation ◽  
Cation Binding ◽  
Formyl Peptide Receptor ◽  
Human Neutrophils ◽  
Formyl Peptide ◽  
Diaza Crown Ethers ◽  
Aza Crown Ethers

Synthetic and natural ionophores have been developed to catalyze ion transport and have been shown to exhibit a variety of biological effects. We synthesized 24 aza- and diaza-crown ethers containing adamantyl, adamantylalkyl, aminomethylbenzoyl, and ε-aminocaproyl substituents and analyzed their biological effects in vitro. Ten of the compounds (8, 10–17, and 21) increased intracellular calcium ([Ca2+]i) in human neutrophils, with the most potent being compound 15 (N,N’-bis[2-(1-adamantyl)acetyl]-4,10-diaza-15-crown-5), suggesting that these compounds could alter normal neutrophil [Ca2+]i flux. Indeed, a number of these compounds (i.e., 8, 10–17, and 21) inhibited [Ca2+]i flux in human neutrophils activated by N-formyl peptide (fMLF). Some of these compounds also inhibited chemotactic peptide-induced [Ca2+]i flux in HL60 cells transfected with N-formyl peptide receptor 1 or 2 (FPR1 or FPR2). In addition, several of the active compounds inhibited neutrophil reactive oxygen species production induced by phorbol 12-myristate 13-acetate (PMA) and neutrophil chemotaxis toward fMLF, as both of these processes are highly dependent on regulated [Ca2+]i flux. Quantum chemical calculations were performed on five structure-related diaza-crown ethers and their complexes with Ca2+, Na+, and K+ to obtain a set of molecular electronic properties and to correlate these properties with biological activity. According to density-functional theory (DFT) modeling, Ca2+ ions were more effectively bound by these compounds versus Na+ and K+. The DFT-optimized structures of the ligand-Ca2+ complexes and quantitative structure-activity relationship (QSAR) analysis showed that the carbonyl oxygen atoms of the N,N’-diacylated diaza-crown ethers participated in cation binding and could play an important role in Ca2+ transfer. Thus, our modeling experiments provide a molecular basis to explain at least part of the ionophore mechanism of biological action of aza-crown ethers.

scholarly journals Single Cell Label-Free Probing of Chromatin Dynamics During B Lymphocyte Maturation

2021 ◽  
Vol 9 ◽  
Author(s):  
Rikke Morrish ◽  
Kevin Ho Wai Yim ◽  
Stefano Pagliara ◽  
Francesca Palombo ◽  
Richard Chahwan ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Fate ◽  
B Lymphocytes ◽  
Intracellular Signaling ◽  
Large Scale ◽  
B Lymphocyte ◽  
Biological Effects ◽  
Raman Microscopy ◽  
Phenotypic Characterization ◽  
Label Free

Large-scale intracellular signaling during developmental growth or in response to environmental alterations are largely orchestrated by chromatin within the cell nuclei. Chemical and conformational modifications of the chromatin architecture are critical steps in the regulation of differential gene expression and ultimately cell fate determination. Therefore, establishing chemical properties of the nucleus could provide key markers for phenotypic characterization of cellular processes on a scale of individual cells. Raman microscopy is a sensitive technique that is capable of probing single cell chemical composition—and sub-cellular regions—in a label-free optical manner. As such, it has great potential in both clinical and basic research. However, perceived limitations of Raman spectroscopy such as low signal intensity and the difficulty in linking alterations in vibrational signals directly with ensuing biological effects have hampered advances in the field. Here we use immune B lymphocyte development as a model to assess chromatin and transcriptional changes using confocal Raman microscopy in combination with microfluidic devices and correlative transcriptomics, thereby linking changes in chemical and structural properties to biological outcomes. Live B lymphocytes were assessed before and after maturation. Multivariate analysis was applied to distinguish cellular components within each cell. The spectral differences between non-activated and activated B lymphocytes were then identified, and their correlation with known intracellular biological changes were assessed in comparison to conventional RNA-seq analysis. Our data shows that spectral analysis provides a powerful tool to study gene activation that can complement conventional molecular biology techniques and opens the way for mapping the dynamics in the biochemical makeup of individual cells.

scholarly journals NOX2-Dependent Reactive Oxygen Species Regulate Formyl-Peptide Receptor 1-Mediated TrkA Transactivation in SH-SY5Y Cells

2019 ◽  
Vol 2019 ◽  
pp. 1-17 ◽  
Author(s):  
Martina Castaldo ◽  
Cristiana Zollo ◽  
Gabriella Esposito ◽  
Rosario Ammendola ◽  
Fabio Cattaneo
Keyword(s):  
Reactive Oxygen Species ◽  
Critical Role ◽  
Reactive Oxygen ◽  
Cellular Migration ◽  
Formyl Peptide Receptor ◽  
Neurotrophin Receptor ◽  
Ros Generation ◽  
Oxygen Species ◽  
Peptide Receptor ◽  
Formyl Peptide

Several enzymes are capable of producing reactive oxygen species (ROS), but only NADPH oxidases (NOX) generate ROS as their primary and sole function. In the central nervous system, NOX2 is the major source of ROS, which play important roles in signalling and functions. NOX2 activation requires p47phox phosphorylation and membrane translocation of cytosolic subunits. We demonstrate that SH-SY5Y cells express p47phox and that the stimulation of Formyl-Peptide Receptor 1 (FPR1) by N-fMLP induces p47phox phosphorylation and NOX-dependent superoxide generation. FPR1 is a member of the G protein-coupled receptor (GPCR) family and is able to transphosphorylate several tyrosine kinase receptors (RTKs). This mechanism requires ROS as signalling intermediates and is necessary to share information within the cell. We show that N-fMLP stimulation induces the phosphorylation of cytosolic Y490, Y751, and Y785 residues of the neurotrophin receptor TrkA. These phosphotyrosines provide docking sites for signalling molecules which, in turn, activate Ras/MAPK, PI3K/Akt, and PLC-γ1/PKC intracellular cascades. N-fMLP-induced ROS generation plays a critical role in FPR1-mediated TrkA transactivation. In fact, the blockade of NOX2 functions prevents Y490, Y751, and Y785 phosphorylation, as well as the triggering of downstream signalling cascades. Moreover, we observed that FPR1 stimulation by N-fMLP also improves proliferation, cellular migration, and neurite outgrowth of SH-SY5Y cells.

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