Quantitative phosphoproteomics of protein isoforms involved in EGF signalling.

This thesis describes the development of a new, powerful concept and approach to study signalling pathways based on top-down quantitative phosphoproteomics. This approach, called the Phospho-SILAC strategy, combines a first step of in-house developed phosphoprotein enrichment using Immobilized Affinity Metal Chromatography (IMAC), and Stable Isotope Labelling in Cell Culture (SILAC) with labelled arginine containing six 13C atoms. NRK49F cells grown in medium containing 13C6 Arg (heavy arginine, hR) were stimulated for short times with EGF and the cell lysate obtained was mixed in a ratio 1:1 with the lysate from unlabelled cells, used as control. Phosphoproteins were then enriched from the protein mixture using IMAC and displayed on 2D gels, allowing multiple isoform separation. Individual post-translational changes in different proteins and in different isoforms of the same protein upon EGF stimulation are described. Major focus was given to Hsp8 isoforms, as they were found to be differentially involved in EGF signalling when tested with antibodies against MAPK/CDK and PCK substrates. A potential MAPK phosphorylation site on Hsp8 N-terminal sequence was identified. The Phospho-SILAC strategy was also applied to Rho and Rab GTPases and their corresponding GDI (guanidine nucleotide dissociation inhibitor) molecules. Phosphorylation might regulate the binding between GTPase-GDI. The results obtained also suggested that RhoGDI-1 activity could be regulated by arginine methylation. A potent new concept and methodology is outlined for wider investigations of molecular dynamics and for guiding the selection of candidate proteins and their modification for further studies

Lipid droplet analysis in caveolin-deficient adipocytes: alterations in surface phospholipid composition and maturation defects.

Caveolins form plasmalemnal invaginated caveolae. They also locate around intracellular lipid droplets but their role in this location remains unclear. By studying primary adipocytes that highly express caveolin-1, we characterized the impact of caveolin-1 deficiency on lipid droplet proteome and lipidome. We identified several missing proteins on the lipid droplet surface of caveolin-deficient adipocytes and showed that the caveolin-1 lipid droplet pool is organized as multi-protein complexes containing cavin-1, with similar dynamics as those found in caveolae. On the lipid side, caveolin deficiency did not qualitatively alter neutral lipids in lipid droplet, but significantly reduced the relative abundance of surface phospholipid species: phosphatidylserine and lysophospholipids. Caveolin-deficient adipocytes can form only small lipid droplets, suggesting that the caveolin-lipid droplet pool might be involved in lipid droplet size regulation. Accordingly, we show that caveolin-1 concentration on adipocyte lipid droplets positively correlated with lipid droplet size in obese rodent models and human adipocytes. Moreover, rescue experiments by caveolin- green fluorescent protein in caveolin-deficient cells exposed to fatty acid overload demonstrated that caveolin-coated lipid droplets were able to grow larger than caveolin-devoid lipid droplets. Altogether, these data demonstrate that the lipid droplet-caveolin pool impacts on phospholipid and protein surface composition of lipid droplets and suggest a functional role on lipid droplet expandability

Dataset of differential lipid raft and global proteomes of SILAC-labeled cystic fibrosis cells upon TNF -α stimulation

Cystic fibrosis (CF) is a genetic disease due to mutations in the cystic fibrosis transmembrane regulator (CFTR), F508del-CFTR being the most frequent. Lipid raft-like microdomains (LRM) are regions of the plasma membrane that present a high cholesterol content and are insoluble to non-ionic detergents. LRM are essential functional and structural platforms that play an important role in the inflammatory response. CFTR is a known modulator of inflammation in LRM. Here we provide mass spectrometry data on the global impact of CFTR mutation and TNF-a stimulation on the LRM proteome. We used the Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) approach to quantify and identify 332 proteins in LRM upon TNF-a stimulation in CF cells and 1381 for the global proteome. We report two detailed tables containing lists of proteins obtained by mass spectrometry and the immunofluorescence validation results for one of these proteins, the G-protein coupled receptor 5A. These results are associated with the article “Changes in lipid raft proteome upon TNF-α stimulation of cystic fibrosis cells” (Chhuon et al., in press [1])