scholarly journals Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Dongjin Jeong ◽  
Hye Sung Kim ◽  
Hye Young Kim ◽  
Min Jueng Kang ◽  
Hyeryeon Jung ◽  
...  
Keyword(s):  
Fas Ligand ◽  
Affinity Purification ◽  
Mass Spectrometry Analysis ◽  
Dependent Manner ◽  
Chemokine Production ◽  
Spectrometry Analysis ◽  
Soluble Fas ◽  
Soluble Fas Ligand ◽  
Deficient Mice

To date, no study has demonstrated that soluble Fas ligand (sFasL)-mediated inflammation is regulated via interaction with Fas in vivo. We found that FasL interacts specifically with tumor necrosis factor receptor superfamily (TNFRSF)10B, also known as death receptor (DR)5. Autoantibody-induced arthritis (AIA) was attenuated in FasL (Faslgld/gld)- and soluble FasL (FaslΔs/Δs)-deficient mice, but not in Fas (Faslpr/lpr and Fas–/–)- or membrane FasL (FaslΔm/Δm)-deficient mice, suggesting sFasL promotes inflammation by binding to a Fas-independent receptor. Affinity purification mass spectrometry analysis using human (h) fibroblast-like synovial cells (FLSCs) identified DR5 as one of several proteins that could be the elusive Fas-independent FasL receptor. Subsequent cellular and biochemical analyses revealed that DR5 interacted specifically with recombinant FasL–Fc protein, although the strength of this interaction was approximately 60-fold lower than the affinity between TRAIL and DR5. A microarray assay using joint tissues from mice with arthritis implied that the chemokine CX3CL1 may play an important downstream role of the interaction. The interaction enhanced Cx3cl1 transcription and increased sCX3CL1 production in FLSCs, possibly in an NF-κB-dependent manner. Moreover, the sFasL–DR5 interaction-mediated CX3CL1–CX3CR1 axis initiated and amplified inflammation by enhancing inflammatory cell influx and aggravating inflammation via secondary chemokine production. Blockade of FasL or CX3CR1 attenuated AIA. Therefore, the sFasL–DR5 interaction promotes inflammation and is a potential therapeutic target.

scholarly journals Functional Characterization of a Chimeric Soluble Fas Ligand Polymer with In Vivo Anti-Tumor Activity

PLoS ONE ◽  
2013 ◽  
Vol 8 (1) ◽  
pp. e54000 ◽  
Author(s):  
Sophie Daburon ◽  
Christel Devaud ◽  
Pierre Costet ◽  
Aurore Morello ◽  
Laure Garrigue-Antar ◽  
...  
Keyword(s):  
Fas Ligand ◽  
Functional Characterization ◽  
Soluble Fas ◽  
Soluble Fas Ligand ◽  
Anti Tumor Activity

scholarly journals Roles for thrombin and fibrin(ogen) in cytokine/chemokine production and macrophage adhesion in vivo

Blood ◽  
2002 ◽  
Vol 99 (3) ◽  
pp. 1053-1059 ◽  
Author(s):  
Frank M. Szaba ◽  
Stephen T. Smiley
Keyword(s):  
Inflammatory Responses ◽  
Direct Injection ◽  
Peritoneal Macrophages ◽  
Dependent Manner ◽  
Chemokine Production ◽  
Monocyte Chemoattractant Protein 1 ◽  
Macrophage Adhesion ◽  
Pleiotropic Functions ◽  
Deficient Mice

Abstract Extravascular coagulation leading to fibrin deposition accompanies many immune and inflammatory responses. Although recognized by pathologists for decades, and probably pathologic under certain conditions, the physiologic functions of extravascular coagulation remain to be fully defined. This study demonstrates that thrombin can activate macrophage adhesion and prompt interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) production in vivo. Peritoneal macrophages were elicited with thioglycollate (TG) and then activated in situ, either by intraperitoneal injection of lipopolysaccharide (LPS) or by injection of antigen into mice bearing antigen-primed T cells. Others previously established that such treatments stimulate macrophage adhesion to the mesothelial lining of the peritoneal cavity. The present study demonstrates that thrombin functions in this process, as macrophage adhesion was suppressed by Refludan, a highly specific thrombin antagonist, and induced by direct peritoneal administration of purified thrombin. Although recent studies established that protease activated receptor 1 (PAR-1) mediates some of thrombin's proinflammatory activities macrophage adhesion occurred normally in PAR-1–deficient mice. However, adhesion was suppressed in fibrin(ogen)-deficient mice, suggesting that fibrin formation stimulates macrophage adhesion in vivo. This study also suggests that fibrin regulates chemokine/cytokine production in vivo, as direct injection of thrombin stimulated peritoneal accumulation of IL-6 and MCP-1 in a fibrin(ogen)-dependent manner. Given that prior studies have clearly established inflammatory roles for PAR-1, thrombin probably has pleiotropic functions during inflammation, stimulating vasodilation and mast cell degranulation via PAR-1, and activating cytokine/chemokine production and macrophage adhesion via fibrin(ogen).

scholarly journals Cloning and purification of functionally active Fas ligand interfering protein (FIP) expressed in Escherichia coli.

2008 ◽  
Vol 55 (1) ◽  
pp. 51-56 ◽  
Author(s):  
Pawel Wisniewski ◽  
Adam Master ◽  
Bozena Kaminska
Keyword(s):  
Escherichia Coli ◽  
Metal Ion ◽  
Inclusion Bodies ◽  
Structural Changes ◽  
Fas Ligand ◽  
Biological Activities ◽  
Single Step ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Soluble Fas

This report presents purification and characterization of the extracellular domain of rat Fas protein, called FIP (FasL interfering protein), expressed as inclusion bodies in Escherichia coli. FIP was extracted from the inclusion bodies, solubilized with 8 M urea, purified by a single-step immobilized metal ion (Ni(2+)) affinity chromatography and refolded. SDS/PAGE and mass spectrometry analysis of the purified protein verified its purity. Fluorescence spectrum analysis showed that the refolding procedure caused structural changes which presumably might have led to oligomerization. The purified FIP has biological activities: it binds specifically soluble Fas ligand and protects human Jurkat lymphocytes against FasL-dependent apoptosis. This efficient procedure of FIP expression in E. coli and renaturation may be useful for production of therapeutically important proteins.

scholarly journals Locus-Conserved Circular RNA cZNF292 Controls Endothelial Cell Flow Responses

2021 ◽  
Author(s):  
Andreas W Heumüller ◽  
Alisha Nicole Jones ◽  
André Mourão ◽  
Marius Klangwart ◽  
Chenyue Shi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Laminar Flow ◽  
Affinity Purification ◽  
Mass Spectrometry Analysis ◽  
Circular Rnas ◽  
Cell Orientation ◽  
Spectrometry Analysis ◽  
Functional Conservation

Background : Circular RNAs (circRNAs) are generated by back-splicing of mostly mRNAs and are gaining increasing attention as a novel class of regulatory RNAs that control various cellular functions. However, their physiological roles and functional conservation in vivo are rarely addressed, given the inherent challenges of their genetic inactivation. Here we aimed to identify locus conserved circRNAs in mice and humans, which can be genetically deleted due to retained intronic elements not contained in the mRNA host gene to eventually address functional conservation. Methods: Mechanistically, we identified the protein syndesmos (SDOS) to specifically interact with cZNF292 in endothelial cells by RNA affinity purification and subsequent mass spectrometry analysis. Silencing of SDOS or its protein binding partner Syndecan-4, or mutation of the SDOS-cZNF292 binding site, prevented laminar flow-induced cytoskeletal reorganisation thereby recapitulating cZfp292 phenotypes. Results: Combining published endothelial RNA sequencing datasets with circRNAs of the circATLAS databank, we identified locus-conserved circRNA retaining intronic elements between mice and humans. CRISPR/Cas9 mediated genetic depletion of the top expressed circRNA cZfp292 resulted in an altered endothelial morphology and aberrant flow alignment in the aorta in vivo. Consistently, depletion of cZNF292 in endothelial cells in vitro abolished laminar flow-induced alterations in cell orientation, paxillin localisation and focal adhesion organisation. Conclusion: Together, our data reveal a hitherto unknown role of cZNF292/cZfp292 in endothelial flow responses, which influences endothelial shape.

scholarly journals N-Terminal Ubiquitination of Extracellular Signal-Regulated Kinase 3 and p21 Directs Their Degradation by the Proteasome

2004 ◽  
Vol 24 (14) ◽  
pp. 6140-6150 ◽  
Author(s):  
Philippe Coulombe ◽  
Geneviève Rodier ◽  
Eric Bonneil ◽  
Pierre Thibault ◽  
Sylvain Meloche
Keyword(s):  
Mitogen Activated Protein Kinase ◽  
Mass Spectrometry Analysis ◽  
Dependent Manner ◽  
Proliferating Cells ◽  
Intact Cells ◽  
Spectrometry Analysis ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

ABSTRACT Extracellular signal-regulated kinase 3 (ERK3) is an unstable mitogen-activated protein kinase homologue that is constitutively degraded by the ubiquitin-proteasome pathway in proliferating cells. Here we show that a lysineless mutant of ERK3 is still ubiquitinated in vivo and requires a functional ubiquitin conjugation pathway for its degradation. Addition of N-terminal sequence tags of increasing size stabilizes ERK3 by preventing its ubiquitination. Importantly, we identified a fusion peptide between the N-terminal methionine of ERK3 and the C-terminal glycine of ubiquitin in vivo by tandem mass spectrometry analysis. These findings demonstrate that ERK3 is conjugated to ubiquitin via its free NH2 terminus. We found that large N-terminal tags also stabilize the expression of the cell cycle inhibitor p21 but not that of substrates ubiquitinated on internal lysine residues. Consistent with this observation, lysineless p21 is ubiquitinated and degraded in a ubiquitin-dependent manner in intact cells. Our results suggests that N-terminal ubiquitination is a more prevalent modification than originally recognized.

scholarly journals N-Terminomics Strategies for Protease Substrates Profiling

Molecules ◽  
2021 ◽  
Vol 26 (15) ◽  
pp. 4699
Author(s):  
Mubashir Mintoo ◽  
Amritangshu Chakravarty ◽  
Ronak Tilvawala
Keyword(s):  
Mass Spectrometry ◽  
Protease Activity ◽  
Viral Infections ◽  
Mass Spectrometry Analysis ◽  
Post Translational Modification ◽  
Spectrometry Analysis ◽  
Physiological Conditions ◽  
Inflammatory Conditions ◽  
Biological Mixtures

Proteases play a central role in various biochemical pathways catalyzing and regulating key biological events. Proteases catalyze an irreversible post-translational modification called proteolysis by hydrolyzing peptide bonds in proteins. Given the destructive potential of proteolysis, protease activity is tightly regulated. Dysregulation of protease activity has been reported in numerous disease conditions, including cancers, neurodegenerative diseases, inflammatory conditions, cardiovascular diseases, and viral infections. The proteolytic profile of a cell, tissue, or organ is governed by protease activation, activity, and substrate specificity. Thus, identifying protease substrates and proteolytic events under physiological conditions can provide crucial information about how the change in protease regulation can alter the cellular proteolytic landscape. In recent years, mass spectrometry-based techniques called N-terminomics have become instrumental in identifying protease substrates from complex biological mixtures. N-terminomics employs the labeling and enrichment of native and neo-N-termini peptides, generated upon proteolysis followed by mass spectrometry analysis allowing protease substrate profiling directly from biological samples. In this review, we provide a brief overview of N-terminomics techniques, focusing on their strengths, weaknesses, limitations, and providing specific examples where they were successfully employed to identify protease substrates in vivo and under physiological conditions. In addition, we explore the current trends in the protease field and the potential for future developments.

Levels of soluble Fas ligand in myocarditis

1998 ◽  
Vol 82 (2) ◽  
pp. 246-248 ◽  
Author(s):  
Tetsuya Toyozaki ◽  
Michiaki Hiroe ◽  
Masato Tanaka ◽  
Shigekazu Nagata ◽  
Hidemi Ohwada ◽  
...  
Keyword(s):  
Fas Ligand ◽  
Soluble Fas ◽  
Soluble Fas Ligand

Soluble fas ligand levels during rejection after paediatric liver transplantation

1998 ◽  
Vol 28 ◽  
pp. 67
Author(s):  
N. Hadzic ◽  
M. Hussain ◽  
P. Cheeseman ◽  
R. Francavilla ◽  
G. Mieli-Vergani ◽  
...  
Keyword(s):  
Liver Transplantation ◽  
Fas Ligand ◽  
Soluble Fas ◽  
Soluble Fas Ligand ◽  
Paediatric Liver Transplantation

scholarly journals Phytotoxicity of Essential Oils on Selected Weeds: Potential Hazard on Food Crops

Plants ◽  
2018 ◽  
Vol 7 (4) ◽  
pp. 79 ◽  
Author(s):  
María Ibáñez ◽  
María Blázquez
Keyword(s):  
Seed Germination ◽  
Essential Oil ◽  
Essential Oils ◽  
Mass Spectrometry Analysis ◽  
Gas Chromatography Mass Spectrometry ◽  
Food Crops ◽  
Potential Hazard ◽  
Spectrometry Analysis

The chemical composition of winter savory, peppermint, and anise essential oils, and in vitro and in vivo phytotoxic activity against weeds (Portulaca oleracea, Lolium multiflorum, and Echinochloa crus-galli) and food crops (maize, rice, and tomato), have been studied. Sixty-four compounds accounting for between 97.67–99.66% of the total essential oils were identified by Gas Chromatography-Mass Spectrometry analysis. Winter savory with carvacrol (43.34%) and thymol (23.20%) as the main compounds produced a total inhibitory effect against the seed germination of tested weed. Menthol (48.23%), menthone (23.33%), and iso-menthone (16.33%) from peppermint only showed total seed germination inhibition on L. multiflorum, whereas no significant effects were observed with trans-anethole (99.46%) from anise at all concentrations (0.125–1 µL/mL). Low doses of peppermint essential oil could be used as a sustainable alternative to synthetic agrochemicals to control L. multiflorum. The results corroborate that in vivo assays with a commercial emulsifiable concentrate need higher doses of the essential oils to reproduce previous in vitro trials. The higher in vivo phytotoxicity of winter savory essential oil constitutes an eco-friendly and less pernicious alternative to weed control. It is possible to achieve a greater in vivo phytotoxicity if less active essential oil like peppermint is included with other active excipients.

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