Dataset Information

LC-MS/MS identification of Mycolactone protein targets

ABSTRACT: Mycolactone is a diffusible macrolide produced by the skin pathogen Mycobacterium ulcerans that suppresses the development of protective immunity against Buruli ulcer disease. In this proteomics study we aimed to identify proteins of which the expression levels changed upon mycolactone treatment. Jurkat T cells were labeled light or heavy by stable isotope labeling by amino acids in cell culture (SILAC), then treated with mycolactone (light) or vehicle (heavy) for 1h prior to activation with PMA/IO for 6h. After mixing light- and heavy-labeled cultures, cells were lysed and extracted proteins were digested with trypsin. The resulting peptide mixture was analyzed by LC-MS/MS and proteins were identified by the MaxQuant software and quantified based on the intensity of the light and heavy signals in the MS spectra of their peptides. The analysis was repeated with reversed labeling, leading to a total of 4,636 proteins that were quantified in both analyses. Interestingly, 52 proteins were down-regulated in mycolactone-treated cells (mycolactone/control ratio < 1.4), while only two proteins were up-regulated (mycolactone/control ratio > 1.4). Gene ontology analysis further revealed that the down-regulated proteins were significantly enriched in proteins located in the plasma membrane and endoplasmic reticulum and we could show that this enrichment was not due to a bias in protein extraction, since the distribution of all identified proteins across different subcellular compartments was similar to those of all human proteins. Analysis of SP-PIR Family-Domains keywords in the UniProt database confirmed this observation and showed an additional enrichment in glycoproteins, proteins with an immunoglobulin domain and proteins involved in immune responses. Furthermore, we observed that the down-regulated proteins were significantly enriched in single-pass type I/II membrane proteins. In contrast, the fraction of multi-pass membrane proteins was comparable among the down-regulated and all identified proteins, suggesting that multi-pass membrane proteins may at least partially resist mycolactone-mediated down-regulation.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): T Cell, Cell Culture, T-lymphocyte Cell Line, Jurkat Cell

DISEASE(S): Buruli Ulcer Disease

SUBMITTER: Francis Impens

LAB HEAD: Caroline Demangel

PROVIDER: PXD002971 | Pride | 2016-11-04

REPOSITORIES: Pride

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Publications

Proteomics Reveals Scope of Mycolactone-mediated Sec61 Blockade and Distinctive Stress Signature.

Morel Jean-David JD Paatero Anja O AO Wei Jiajie J Yewdell Jonathan W JW Guenin-Macé Laure L Van Haver Delphi D Impens Francis F Pietrosemoli Natalia N Paavilainen Ville O VO Demangel Caroline C

Molecular & cellular proteomics : MCP 20180618 9

Mycolactone is a bacteria-derived macrolide that blocks the biogenesis of a large array of secretory and integral transmembrane proteins (TMP) through potent inhibition of the Sec61 translocon. Here, we used quantitative proteomics to delineate the direct and indirect effects of mycolactone-mediated Sec61 blockade in living cells. In T lymphocytes, dendritic cells and sensory neurons, Sec61 substrates downregulated by mycolactone were in order of incidence: secretory proteins (with a signal pept ...[more]

PMID: 29915147

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Project description:Snail1 is a transcriptional repressor required for a correct embryonic development. In cancer, Snail1 promotes the epithelial to mesenchymal transition in tumorigenic epithelial cells. In this work, we have analyzed the control of Snail1 in the differentiation of the 3T3-L1 cell line derived from murine embryo cells. The activation by snail of 3T3-L1 induced typical markers of cancer-activated fibroblasts as S100A4 or CD44. We generated 3T3-L1 cells stably over expressing Snail1 (3T3L1/Snail1) and control (3T3-L1/control) cells. We used SILAC quantitative approach to identify and characterize protein alterations induced by Snail1. Cells were fractionated in 5 subcellular fractions. The nuclear fraction of the cells was separated by 10% SDS-PAGE. Gels with forward and reverse experiments were stained with Coomassie Blue and cut into 18 slices prior to reduction, alkylation and digestion with trypsin. Tryptic peptides were scanned and fragmented with a linear ion trap-Orbitrap Velos (ThermoScientific). We identified a total of 3108 proteins, with 2572 quantified proteins, and 565 proteins modulated >1.5-fold by Snail1 overexpression. Among them, we found interesting up-regulated proteins associated to early differentiation of adipogenesis (C/EBPβ) and down-regulated proteins implicated in the final stages of differentiation to adipocytes (Fatty acid-binding protein or Fatty acid synthase). We also observed as down-regulated proteins important mediators of PPARγ pathway. We also observed downregulation of proteins implicated in mTOR, SRC and JAK/STAT pathway. We validated these proteomics data by western blot and qPCR in 3T3-L1 cells and other types of fibroblasts with capable to differentiate to terminal mesenchymal phenotypes, as well as in mesenchymal stem cells (MSC). This work provided insight into novel proteins with potential roles in the regulation of differentiation of the 3T3-L1 and MSCs as Nr2F6, ASC-1, Prrx1 or Cbx6. These candidates are down regulated due to the overexpression of Snail1 in 3T3-L1 cells. We next investigated the potential binding of Snail1 to promoter of these candidates. In silico analysis with MatInspector program revealed various putative E-box consensus motifs for Snail1. We performed ChIP and Luciferase assay to validate Snail1 binds to different E-box motifs of our candidates. Additionally, we analyzed the ability to prevent the differentiation to adipocytes of the 3T3-L1 cells using siRNAs. This work provided insight into novel proteins with potential roles in the regulation of differentiation to adipocytes of the 3T3-L1 and mMSC cells as Nr2F6, ASC-1, Prrx1 or Cbx6 controlled by Snail1.

Dataset Information

LC-MS/MS identification of Mycolactone protein targets

Publications

Proteomics Reveals Scope of Mycolactone-mediated Sec61 Blockade and Distinctive Stress Signature.

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