BACKGROUND-AIM D6 is an atypical chemokine receptor acting as a decoy and scavenger for inflammatory CC chemokines expressed in lymphatic endothelial cells. It is predominantly localized in endocytic vesicles and upon stimulation by the ligand is internalized via clathrin-coated pits. Once internalized, the ligand dissociates from the receptor and is targeted to degradation while the receptor is recycled back to the cell membrane. In order to characterize the phosphoproteome of HEK 293 cell line expressing D6 receptor, a SILAC-based proteomic approach in combination with protocols for the enrichment of phosphorylated proteins and high-resolution mass spectrometry, was applied. METHODS HEK 293 cells not expressing (CTR) and expressing D6 receptor were labelled with different isotopes. Cells that express D6 receptor were not stimulated (T0) or stimulated for 3 minutes (T3) and 30 minutes (T30) with chemokines. Three biological replicates were prepared for each condition. Three comparative experiments were performed: CTR vs T0, T3 vs T0 and T30 vs T0.Cells were lysed and combined prior to enzymatic digestion. Proteolytic digests were fractionated by HILIC and further enriched on Titansphere Phos-TiO resin. The phosphopeptides were subsequently separated by HPLC coupled online to an LTQ-Orbitrap Velos mass spectrometer operated using a TOP-20 data-dependent method including multistage activation. RESULTS Data analysis using MaxQuant (main search error 6 ppm, FDR 0.01% for peptide, protein and modification site identification) allowed to identify 3184, 3197 and 4183 phosphosites in CTR vs T0, T3 vs T0 and T0 vs T30 vs T0 experiments, respectively. Differential expression was calculated using the Perseus software. According to this analysis, 134/61, 157/93 and 607/136 phosphosites are up/down-regutaled in T0 vs CTR, T3 vs T0 and T30 vs T0, respectively. CONCLUSIONS The phosphoproteome profile analysis allowed for the first time to unravel how D6 receptor triggers the signal transduction in HEK 293 cell line.
Phosphoproteome characterization of HEK 293 cell line expressing the atypical chemokine D6 receptor / S. Nonnis, E. Borroni, E. Maffioli, F. Santagata, A. Negri, M. Locati, G. Tedeschi. ((Intervento presentato al 9. convegno Proteomics: Back to the Future tenutosi a Milano nel 2015.
|Titolo:||Phosphoproteome characterization of HEK 293 cell line expressing the atypical chemokine D6 receptor|
NONNIS, SIMONA (Primo)
BORRONI, ELENA MONICA (Secondo)
LOCATI, MASSIMO (Penultimo)
TEDESCHI, GABRIELLA (Ultimo)
|Data di pubblicazione:||23-ott-2015|
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
|Citazione:||Phosphoproteome characterization of HEK 293 cell line expressing the atypical chemokine D6 receptor / S. Nonnis, E. Borroni, E. Maffioli, F. Santagata, A. Negri, M. Locati, G. Tedeschi. ((Intervento presentato al 9. convegno Proteomics: Back to the Future tenutosi a Milano nel 2015.|
|Appare nelle tipologie:||14 - Intervento a convegno non pubblicato|