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CCDC88B affinity proteomic analysis

ABSTRACT: The CCDC88B gene (11q13) is a risk factor for several chronic inflammatory diseases in humans, including inflammatory bowel disease (IBD), multiple sclerosis and psoriasis. A Ccdc88b mutation in mice conveys protection against pathological inflammation in experimental models of microbial (cerebral malaria) and autoimmune (experimental autoimmune encephalomyelitis) of neuroinflammation and colitis. At the cellular level, loss of Ccdc88b function is phenotypically expressed as a migratory defect in vivo, and a cell mobility defect in vitro in mutant dendritic cells and lymphocytes. At the molecular level, CCDC88B belongs to a family of protein scaffold associated with the cytoskeleton and that feature protein:protein interactions domains. We used a co-immunoprecipitation / mass spectrometry-based approach to identify physical interactions of CCDC88B from mouse thymocytes and BI-141 cells.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): T Cell, Thymus

DISEASE(S): Inflammation,Colitis,Thymus

SUBMITTER: John LaCava

LAB HEAD: John LaCava

PROVIDER: PXD023779 | Pride | 2024-01-10

REPOSITORIES: Pride

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Project description:CD5 is characterized as an inhibitory co-receptor with important regulatory role during T cell development. To study the molecular mechanism by which CD5 operates, we used quantitative mass spectrometry to analyze the components of the CD5 signaling machinery in primary T cells. In a first set of experiments, CD5-containing complexes were immunoprecipitated from thymocytes of wild-type (WT) C57BL/6 mice. Thymocytes were treated with pervanadate to induce widespread activation of protein tyrosine kinases. Corresponding samples prepared from thymocytes of Cd5−/− mice were used as controls, to discriminate CD5-binding molecules from the background of contaminant proteins. Eight biological replicates were prepared for both conditions, and samples were analyzed in duplicate LC-MS runs. This first set of experiment is presented in TableS1 of the paper, and is associated to the MaxQuant result file “Interactome WT” in the present PX dataset. We analyzed the CD5 interactome at different time of stimulation with pervanadate (1min and 10min) using thymocytes from wild-type (WT) C57BL/6 mice. Corresponding samples prepared from thymocytes of Cd5−/− mice were used as controls. Three biological replicates were prepared for both conditions (WT and KO) and each time point (1min and 10min). This set of experiments is presented in TableS2 of the paper and is associated to the MaxQuant result file “Interactome at 1min and 10min” in the present PX dataset. We checked that the activation of CD5 (phosphorylation sites) and the formation of the signaling complex was comparable in different stimulatory conditions (comparison of WT thymocytes stimulated with either pervanadate or anti-CD3+anti-CD4 antibodies). We also analyzed the binding of CD5 interactors in thymocytes from a c-Cbl-/- mouse model (comparison of WT and c-Cbl-/-thymocytes stimulated with pervanadate). These experiments and the associated experimental design of the comparisons are summarized in TableS3 of the paper, and are based on the MaxQuant result file “Comparison of different stimulatory conditions” in this dataset. To check the phosphorylation status of the CD5 tyrosine residues at different time of stimulation, we analyzed samples immunoprecipitated from thymocytes of wild-type (WT) C57BL/6 mice, following stimulation with anti-CD3+anti-CD4 antibodies for 1min, 3min and 10min. This set of experiment is presented in TableS4 of the paper and is associated to the MaxQuant result file “CD5 pY kinetics” in the present PX dataset. To determine the role of Y429 in the context of CD5 signaling, we expressed a wild-type (CD5tgWt) or a mutated form of CD5, containing a tyrosine to phenylalanine substitution at position 429 (CD5tgY429F) in transgenic mice. We analyzed the CD5 interactome by comparing samples immunoprecipitated from thymocytes of these 2 mice models (following pervanadate stimulation, 6 and 5 biological replicates respectively) with samples prepared in the same way from Cd5−/− mice as controls (6 biological replicates). The results are shown in TableS5 of the paper, and correspond to the MaxQuant result file “interactome CD5tgWt_CD5tgY429F”.

			Action	DRS
	LEGEND.rtf	Other
	MaxQuant_output.zip	Other
	ccdc11_160831095019.raw	Raw
	ccdc12_160831112257.raw	Raw
	ccdc15_160831043729.raw	Raw

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CCDC88B affinity proteomic analysis

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