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Analysis of SLP-76 interactomes of human primary CD4+ T cells in presence or absence of Dasatinib treatmentAnalysis of SLP-76 interactomes of human primary CD4+ T cells in presence or absence of Dasatinib treatment

ABSTRACT: Using a CRISPR/Cas9-based approach, we engineered human primary CD4+ T cells in which the bait protein SLP76 was tagged with an affinity Twin-Strep-tag (OST). Through affinity purification coupled with quantitative mass spectrometry, this system allows determining the composition and dynamics of the SLP76 signalosome following T cell activation, and it can be used to assess the mechanisms of action of drugs targeting human T cell activation. Dasatinib is an inhibitor that blocks the adenosine triphosphate binding sites of LCK. Here, we assessed the effect of the Dasatinib treatment on the SLP76 signalosome in CD4+ human T cells. Affinity purification of the OST tagged protein was performed using Streptactin beads, from T cells left non-stimulated, or stimulated for 30s or 120s with anti-CD3 and anti-CD28 antibodies. Prior to stimulation, they were preincubated for 45 min at 37°C with either Dasatinib (100 nM) or vehicle alone (DMSO). Each AP-MS purification was 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=4 for all conditions.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Primary Cell, T Cell

SUBMITTER: Anne Gonzalez de Peredo

LAB HEAD: Odile Schiltz

PROVIDER: PXD028939 | Pride | 2022-01-28

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.

			Action	DRS
	Experimental_design_SLP76_Dasatinib.txt	Txt
	MaxQuant_files.zip	Other
	QEMYC210903_02.raw	Raw
	QEMYC210903_04.raw	Raw
	QEMYC210903_06.raw	Raw

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Analysis of SLP-76 interactomes of human primary CD4+ T cells in presence or absence of Dasatinib treatmentAnalysis of SLP-76 interactomes of human primary CD4+ T cells in presence or absence of Dasatinib treatment

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