Scientific coordinator of the project: Anne Gonzalez de Peredo
Taking advantage of the great improvements in large-scale proteomic analysis of complex samples, we use state-of-the-art mass spectrometry techniques to study the molecular mechanisms of the inflammatory and immune responses in specific cellular systems, such as endothelial cells and different types of immune cells.
Major research axis:
- Proteomic analysis of the inflammatory response of endothelial cells
- Functional mechanisms of interleukin-33, a cytokine playing a major role in innate immunity and allergy
- Fine molecular characterization of the T Cell Receptor (TCR) pathway of T lymphocytes
- In-depth proteomic analysis of regulatory T cells (Tregs)
Proteomic analysis of the inflammatory response of endothelial cells
In collaboration with the team of J-P Girard at IPBS, we use large-scale proteomic analysis associated with label-free quantification to study the pathways and processes activated in endothelial cells (ECs) under inflammatory conditions. Using such methods, we could extensively map the ECs proteome, and provided the most complete proteomic characterization to date of the human EC response to several inflammatory cytokines, such as TNFα, IFNg and IL1β.
Recently we used such a global proteomic approach to analyze IL-33 function in endothelial cells. IL-33 is a nuclear cytokine from the IL-1 family that plays important roles in health and disease. We compared the extracellular and intracellular roles of IL-33 in primary human endothelial cells, and found that exogenous extracellular IL-33 acts as a cytokine inducing expression of a distinct set of proteins associated with inflammatory responses in endothelial cells, but not as a nuclear factor.
Proteomic analysis of the inflammatory response
of endothelial cells upon TNFa/IFNg stimulation
Functional mechanisms of interleukin-33, a cytokine playing a major role in innate immunity and allergy
Interleukin-33 (IL-33) is a nuclear cytokine from the IL-1 family with critical roles in tissue homeostasis and repair, type 2 immunity, viral infection, inflammation and allergy. It binds to the ST2 receptor expressed on cells of the innate and adaptive immune system. Major target cells of IL-33 include group 2 innate lymphoid cells (ILC2s), mast cells, and certain subsets of regulatory T cells. IL-33 plays a role in many important diseases. Particularly, several genome-wide association identified the genes of IL-33 and ST2 as major susceptibility loci for human asthma.
In collaboration with the team of J-P Girard at IPBS, we use mass spectrometry techniques to better characterize the mechanisms of IL-33 action.
Mechanisms of IL-33 activation by proteolytic maturation
IL-33 contains a N-terminal chromatin-binding domain: it is mainly sequestered in the nucleus of epithelial end endothelial cells from barrier tissues, and is released upon cellular damage or necrose. On the other hand, its C-terminal part is composed of a bioactive IL1-like cytokine domain. The group of J-P Girard demonstrated that IL-33 is not regulated as IL-1b through maturation by caspase-1 (which rather inactivates IL-33 by cleaving inside the IL1-like domain), but through proteolytic maturation by inflammatory proteases.
We use mass spectrometry analysis to identify highly active mature forms of the protein resulting from processing by activator proteases, and map the cleavage sites in the central domain of IL-33.
Detection of Neo-Nterminal peptides in maturated forms of IL-33 through quantitative comparison with peptides from full-length protein
Unbiased global proteomic studies to characterize IL-33 action
Extracellular IL-33 is known to activate a growing number of target cells (including group 2 innate lymphoid cells, mast cells, regulatory T cells, and endothelial cells). We use large-scale proteomics to characterize the extracellular and intracellular role of IL-33 on these cells.
- Lefrancais, E., Roga, S., Gautier, V., Gonzalez-de-Peredo, A., Monsarrat, B., Girard, J. P., and Cayrol, C. (2012) IL-33 is processed into mature bioactive forms by neutrophil elastase and cathepsin G. PNAS 109, 1673-1678.
- Gautier et al (2016) Extracellular IL-33 cytokine, but not endogenous nuclear IL-33, regulates protein expression in endothelial cells. Sci Rep 6, 34255.
Proteomic analysis of primary endothelial cells response to the stimulation with extracellular IL-33
Characterization of the T Cell Receptor (TCR) pathway in primary T lymphocytes
One of our research areas is to decipher, at the cellular and molecular levels, the functional mechanisms of T lymphocytes using proteomic approaches. In collaboration with teams bringing complementary expertise in immunopathology and T cell signaling (B. Malissen, CIML, Marseille; R. Lesourne, CPTP, Toulouse), we are involved in the detailed characterization of the T Cell Receptor (TCR) pathway, by analyzing the signalosome of several key components of this complex system using AP-MS strategies.
In collaboration with R. Roncagalli and B. Malissen (CIML, Marseille), we use KI mouse model expressing different bait proteins of the TCR pathway with a C-terminal One-Strep-tag to enrich endogenous signaling complexes at different time points following TCR activation. Time resolved proteomics allows to obtain dynamics information and to gain insight into the mechanisms of action of several proteins and the internal organization of the interactomes.
Using this methodology, we characterized the interactome of PAG (Phosphoprotein associated with glycosphingolipid-enriched microdomains), a ubiquitously expressed transmembrane adaptor protein present in lipid rafts and involved in the regulation of TCR signaling.
Conversely to the previously proposed model, we showed that PAG is quickly phosphorylated and binds CSK and other inhibitory molecules upon T cell activation.
Analysis of PAG interactome
Binding kinetics of proteins interacting with PAG
Fine characterization of phosphorylation kinetics of PAG tyrosines
We also applied a similar approach to analyze the differences between the molecular networks formed around the E3 ubiquitin–protein ligases CBL and CBLB, two negative regulators of the TCR.
We monitored the dynamics of the CBL and CBLB signaling complexes that assemble in T cells following TCR stimulation, and exploit correlations in protein association with CBL and CBLB as a function of time for predicting the occurrence of direct physical association between them.
By combining co-recruitment analysis with biochemical analysis, we demonstrated that the CD5 transmembrane receptor constitutes a key scaffold for CBL- and CBLBmediated ubiquitylation following TCR engagement.
Analysis of Cbl and Cbl interactomes
Analysis of Themis1 interactome
MS-based analysis of Themis1 interactome in thymocytes
- Immunopurification of endogenous Themis1
- Control: same immunoprecipitation in Themis KO thymocytes
- Label-free quantitative comparison of immunopurified samples from WT and Themis1-/- mice
Evaluation of absolute protein partners abundance
- Reginald et al (2015) Revisiting the Timing of Action of the PAG Adaptor Using Quantitative Proteomics Analysis of Primary T Cells. Journal of immunology 195, 5472-5481.
- Voisinne et al (2016) Co-recruitment analysis of the CBL and CBLB signalosomes in primary T cells identifies CD5 as a key regulator of TCR-induced ubiquitylation. Mol Syst Biol 12, 876.
- Zvezdova et al (2016) Themis1 enhances T cell receptor signaling during thymocyte development by promoting Vav1 activity and Grb2 stability. Sci Signal 9, ra51.
In-depth proteomic analysis of regulatory T cells (Tregs)
Regulatory T cells (Treg) represent a minor sub-population of T lymphocytes which is crucial for the maintenance of immune homeostasis. In collaboration with A. Saoudi (CPTP, Toulouse), we used large-scale quantitative mass spectrometry to define a specific proteomic “signature” of Treg.
Treg and conventional T lymphocyte (Tconv) sub-populations were sorted by flow cytometry and subjected to global proteomic analysis by single-run nanoLC-MS/MS on a fast-sequencing Q-Exactive mass spectrometer. Besides “historical” proteins that characterize Treg, our study identified numerous new proteins that are up- or down-regulated in Treg versus Tconv.
We focused on Themis1, a protein particularly under-represented in Treg, and recently described as being involved in the pathogenesis of immune diseases. Using a transgenic mouse model over-expressing Themis1, we provided in vivo and in vitro evidence of its importance for Treg suppressive functions, in an animal model of inflammatory bowel disease and in co-culture assays.
- Duguet F, Locard-Paulet M et al (2017) Proteomic analysis of regulatory T cells reveals the importance of Themis1 in the control of their suppressive function. Mol Cell Proteomics. 2017