The study of post-translational modifications (PTMs) in the modulation of cellular function has revealed the complexity of signaling networks and the existence of interactions or cross-talk between PTMs in the same or different proteins. Different combinations of PTMs can yield different functional outcomes, including conformational changes, different activities or the interaction with different molecules. Previous studies have reported separately that phosphorylation and ubiquitination participate in T-cell receptor (TCR) signaling. However, the combined analysis of multiple PTMs and their potential cross-talk in T cells has not been addressed so far. In this study, we determined the phosphorylation, ubiquitination and acetylation dynamics during physiological activation in primary human T cells to elucidate post-translational regulatory events involved in TCR signaling. Mononuclear cells were obtained from concentrated buffy coats using a Ficoll gradient. T cells were purified by negative selection and activated using anti-CD3/CD28 antibodies during 15 or 120 minutes in four biological replicates. Proteins were extracted and digested using LFASP (Casanovas, Anal.Chem. 2017) and subjected to serial PTM enrichment. Briefly, peptides containing ubiquitination sites were purified using an anti-K-GG antibody, the unbound peptides were incubated with anti-K-Ac antibodies for the isolation of acetylated peptides, and the anti-K-Ac-unbound peptides were used for phosphopeptide enrichment. Peptide extracts were analyzed by LC-MS/MS in an Orbitrap-XL instrument coupled to an Agilent 1200 nanoflow HPLC. Database search was done with Proteome Discoverer 1.4 and the label-free quantitative analysis using Progenesis and DanteR. We quantified 4415 phosphorylated, 2772 ubiquitinated and 1895 acetylated peptides, in primary human T cells. Activation of primary T cells with anti-CD3/CD28 antibodies induced the ubiquitination of several proteins involved in the initial stages of signal transduction, including ZAP70, CD3¿, CD3¿, CD28 and PI3K. In particular, the ubiquitination of ZAP70 in Lys-217 upon activation had been already reported by other authors. Also, the detection of a regulated ubiquitination site in PI3K would be in agreement with studies indicating that the recruitment of p85 to CD28 and to the TCR is regulated by ubiquitination. In our search for potential cross-talk phenomena, we quantified more than 100 sites of direct negative regulation (amino acids that can present different modification) and 52 peptides with double modification in these cells. Several regulated multi-modified peptides are derived from proteins involved in the T cell activation pathway, including nine phosphorylated and ubiquitinated peptides in different subunits of the TCR complex. This is a unique data on the extent and dynamics of PTMs and their cross-talk in primary human T cells upon activation.

Trabajo presentado en el BD Clinical Proteomics: towards personalized medicine and health, celebrado en Barcelona los días 7 y 8 de noviembre de 2018

Peer reviewed