Characterization of Inflammatory and Immune Processes

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.

Characterization of the TCR signalosome using transgenic mouse models

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.

 

AP-MS workflow for analysis of the TCR signalosome

 

 

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

 

Identification of new interactors

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

 


Characterization of the Themis1 interactome
In collaboration with R. Lesourne (CPTP, Toulouse), we characterized the interactome of Themis1 in primary mouse thymocytes, through immunopurification of endogenous Themis1. Quantitative proteomics identified SHP-1/PTN6 and Vav1 as predominant interacting partners of Themis1. Further biological studies identified a primary role for Themis1 in regulating Grb2 stability and Vav1 effector function. Conversely to the previously proposed model of Themis1 acting as an attenuator of TCR signaling, these studies suggested that Themis1 might rather activate TCR signaling.

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
References

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.