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Proteomic characterization of signal transduction by P2X7 in regulatory T cells (609.8)
Author(s) -
Tedeschi Gabriella,
Maffioli Elisa,
Negri Armando,
Romagnani Andrea,
Grassi Fabio
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.609.8
Subject(s) - purinergic receptor , stable isotope labeling by amino acids in cell culture , chemistry , microbiology and biotechnology , signal transduction , receptor , biochemistry , biology , gene , proteomics
ATP released from CD4 + helper T cells upon TCR stimulation contributes to the activation of MAPK signalling through purinergic P2X receptors. T regs produce lower amounts of ATP than conventional CD4 + T cells, although the gene encoding P2X7 receptor is comprised in T regs signature genes. Activation of purinergic P2X7 receptor by ATP mediates T regs conversion to pro‐inflammatory T cells, thus promoting autoimmunity. We identified and characterized the phosphoproteome triggered by BzATP as P2X7 agonist both in stimulated and non‐stimulated T regs employing a SILAC‐based proteomic approach. C57BL/6 wild‐type (WT) and p2rx7 ‐/‐ T regs labelled with different isotopes (R0K0 or R10K8, respectively) in SILAC media were stimulated with BzATP. After T regs stimulation, cells were lysed and combined prior to enzymatic digestion. Following fractionation by HILIC and further enrichement on a Titansphere Phos‐TiO resin, the phosphopeptides were analyzed by an LTQ‐Orbitrap velos. For identification and quantification of phosphopeptides, MaxQuant 1.3.0.5 was used