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Fatty-acylation in RAW264.7 - Bioorthogonal Chemical Reporters for Monitoring Unsaturated Fatty-Acylated Proteins

ABSTRACT: Dietary unsaturated fatty acids beneficially affect human health, in part by modulating the immune system, but the mechanism is not completely understood. Given that unsaturated fatty acids have been shown to be covalently incorporated into a small subset of proteins, we designed three alkyne-tagged chemical reporters of unsaturated fatty acids, alk-16:1, alk-17:1 and alk-18:1, to explore the generality and diversity of this protein modification. Following cell lysis, proteins labelled with the reporters could be captured by azido-functionalized reagents via CuAAC for fluorescence detection or enrichment for proteomics analysis. These reporters label many proteins in mammalian cells and can be incorporated site-specifically, notably on Cys residues. Quantitative proteomics analysis (n= 4 biological replicates) of LPS/IFN-gamma stimulated RAW264.7 labelled with oleic acid (control), alk-16 (palmitic acid chemical reporter), alk-16:1, alk-17:1 and alk-18:1, revealed that unsaturated fatty acids modify similar protein targets to saturated fatty acids, including several immune proteins. Interestingly, some proteins can be differentially labeled with unsaturated and saturated fatty acid.

INSTRUMENT(S): LTQ Orbitrap XL

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Macrophage Cell Line, Macrophage

DISEASE(S): Leukemia

SUBMITTER: Emmanuelle Thinon

LAB HEAD: Howard C. Hang

PROVIDER: PXD003545 | Pride | 2018-10-26

REPOSITORIES: Pride

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Bioorthogonal Chemical Reporters for Monitoring Unsaturated Fatty-Acylated Proteins.

Thinon Emmanuelle E Percher Avital A Hang Howard C HC

Chembiochem : a European journal of chemical biology 20160729 19

Dietary unsaturated fatty acids, such as oleic acid, have been shown to be covalently incorporated into a small subset of proteins, but the generality and diversity of this protein modification has not been studied. We synthesized unsaturated fatty-acid chemical reporters and determined their protein targets in mammalian cells. The reporters can induce the formation of lipid droplets and be incorporated site-specifically onto known fatty-acylated proteins and label many proteins in mammalian cel ...[more]

PMID: 27350074

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Project description:Anaplastic Large Cell Lymphomas (ALCL) represent a subset of lymphomas in which the Anaplastic Lymphoma Kinase (ALK) gene is frequently fused to the NPM gene. We previously demonstrated that the constitutive phosphorylation of ALK chimeric proteins is sufficient to induce cellular transformation in vitro and in vivo, and that ALK activity is strictly required for the survival of ALK positive ALCL cells. To elucidate the signaling pathways required for ALK-mediated transformation and tumor maintenance, we analyzed the transcriptomes of multiple ALK positive ALCL cell lines abrogating their ALK-mediated signaling by inducible ALK RNA interference (RNAi) or with potent and cell permeable ALK inhibitors. Transcripts derived from the gene expression profiling (GEP) analysis uncovered a reproducible signature, which included a novel group of ALK-regulated genes. Functional RNAi screening on a set of these ALK transcriptional targets revealed that the transcription factor C/EBPb and the anti-apoptotic protein BCL2A1 are absolutely necessary to induce cell transformation and/or to sustain the growth and survival of ALK positive ALCL cells. Thus, we proved that an experimentally controlled and functionally validated GEP analysis represents a powerful tool to identify novel pathogenetic networks and validate biologically suitable target genes for therapeutic interventions. Experiment Overall Design: This series of microarray experiments contains the gene expression profiles of Anaplastic Large Cell Lymphoma (ALCL) cell lines (TS [a subclone of Sup-M2] and Su-DHL1) engineered to express ALK-A5 shRNA under a doxycycline-inducible promoter or treated with cell permeable pyrrolocarbazole-derived ALK inhibitors. A mutated ALK-A5M shRNA was used as control. Briefly, cells were transduced with pLV-DsRed-tTRKRAB, expanded, and used for transduction with pLVTH-GFP-shRNA lentiviral particles. Cells were induced with doxycycline (1 microg/ml) for 12 hours, double GFP+ DsRed+ cells selected by fluorescence-activated cell sorting. Cells expressing GFP in the absence of the inducer were removed by a second flow cytometry sorting, and expanded. shRNA expression was induced by doxycycline treatment for 72 or 84 hours. Drug treatments (300 nM), were performed in TS cells with ALK inhibitors (A2 or A3), mock compound (A1), or control diluent for 6 hours. 5 micrograms of total RNA was processed and hybridized to the Affymetrix HG-U133A chip following the manufacturer's instructions.

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Dataset Information

Fatty-acylation in RAW264.7 - Bioorthogonal Chemical Reporters for Monitoring Unsaturated Fatty-Acylated Proteins

Publications

Bioorthogonal Chemical Reporters for Monitoring Unsaturated Fatty-Acylated Proteins.

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