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Analysis of the interactome of CD6 in CRISPR/CAS9 modified mouse CD4+ T cells

ABSTRACT: Using a CRISPR/Cas9-based approach we engineer primary CD4+ T cells in which the CD6 protein was tagged with an affinity Twin-Strep-tag (OST) with the purpose of determining by quantitative mass spectrometry the composition and dynamics of the signalosome assembling around the tagged protein prior to and following T cell activation. Affinity purification of the OST tagged protein was performed using Streptactin beads, from T cells that were non-stimulated, or stimulated for 30s or 120s with anti-CD3 and anti-CD4 antibodies, or 300s with pervanadate. Each AP-MS purification is associated with a corresponding control (purification from non edited WT CD4+ T cells, cultured and stimulated in the same conditions). The number of replicate biological experiments was n=5 (cells non-stimulated or stimulated for 30s with anti-CD3/CD4), n=2 (cells stimulated for120s with antiCD3/CD4), or n=3 (cells stimulated 300s with pervanadate), and each sample was analyzed in duplicate or triplicate by LC-MS, resulting in 78 raw files.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Spleen, T Cell, Lymph Node

SUBMITTER: Anne Gonzalez de Peredo

LAB HEAD: Odile Burlet-Schiltz

PROVIDER: PXD018527 | Pride | 2020-09-29

REPOSITORIES: Pride

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Project description:T-cell receptor (TCR) signaling is essential for the function of T cells and negatively regulated by the E3 ubiquitin-protein ligases CBL and CBLB. Here we combine mouse genetics and affinity purification coupled to quantitative mass spectrometry to monitor the dynamics of the CBL and CBLB signaling complexes that assemble in normal T cells over 600 seconds of TCR stimulation. We identify many previously known CBL and CBLB interacting partners, as well as a majority of proteins that have not yet been implicated in those signaling complexes. We exploit correlations in protein association with CBL and CBLB as a function of time of TCR stimulation for predicting the occurrence of direct physical association between them. The dataset is divided in two distinct subsets corresponding to the Cbl and to the Cblb interactomes. Each of them contains mass spectrometry results from the analysis of 10 different conditions of AP-MS purifications (based on affinity purification on Streptactin beads of One-Strep-tagged proteins) starting from CD4+ T cells which were either non stimulated or stimulated with anti-CD3 and anti-CD4 antibodies as follows: CBL interactome: - CBL-OST transgenic mice, CD4+ T cells non stimulated (noted Cbl_0) - CBL-OST transgenic mice, CD4+ T cells stimulated 30s (noted Cbl_30) - CBL-OST transgenic mice, CD4+ T cells stimulated 120s (noted Cbl_120) - CBL-OST transgenic mice, CD4+ T cells stimulated 300s (noted Cbl_300) - CBL-OST transgenic mice, CD4+ T cells stimulated 600s (noted Cbl_600) - WT mice, CD4+ T cells non stimulated (noted WT_0) - WT mice, CD4+ T cells stimulated 30s (noted WT_30) - WT mice, CD4+ T cells stimulated 120s (noted WT_120) - WT mice, CD4+ T cells stimulated 300s (noted WT_300) - WT mice, CD4+ T cells stimulated 600s (noted WT_600) For the CBL interactome, 3 biological replicates were prepared for these 10 different conditions (noted Ech1, Ech2, Ech3), yielding 30 analyzed samples. Three technical nanoLC-MS runs were acquired for each sample (noted R1, R2, R3), leading to 90 nanoLC-MS raw files. CBL interactome: - CBLB-OST transgenic mice, CD4+ T cells non stimulated (noted Cblb_0) - CBLB-OST transgenic mice, CD4+ T cells stimulated 30s (noted Cblb_30) - CBLB-OST transgenic mice, CD4+ T cells stimulated 120s (noted Cblb_120) - CBLB-OST transgenic mice, CD4+ T cells stimulated 300s (noted Cblb_300) - CBL-OST transgenic mice, CD4+ T cells stimulated 600s (noted Cblb_600) - WT mice, CD4+ T cells non stimulated (noted WT_0) - WT mice, CD4+ T cells stimulated 30s (noted WT_30) - WT mice, CD4+ T cells stimulated 120s (noted WT_120) - WT mice, CD4+ T cells stimulated 300s (noted WT_300) - WT mice, CD4+ T cells stimulated 600s (noted WT_600) For the CBLB interactome, 4 biological replicates were prepared for these 10 different conditions (noted S1, S2, S3, S4), yielding 40 analyzed samples. For series S1 to S3, 2 technical nanoLC-MS runs were acquired for each sample (noted R1, R2) and for serie S4, 3 technical nanoLC-MS runs were acquired for each sample (noted R1, R2, R3), leading in total to 90 nanoLC-MS raw files.

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Analysis of the interactome of CD6 in CRISPR/CAS9 modified mouse CD4+ T cells

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