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The propagation and diversification of signals downstream of the T cell receptor (TCR) involve several adaptor proteins that control the assembly of multimolecular signaling complexes (signalosomes). The global characterization of changes in protein-protein interactions (PPI) following genetic perturbations is critical to understand the resulting phenotypes. Here, by combining genome editing techniques in T cells and interactomics studies based on affinity purification coupled to mass spectrometry (AP-MS) analysis, we determined and quantified the molecular reorganization of the SLP76 interactome resulting from the ablation of each of the three GRB2-family adaptors. Our data showed that the absence of GADS or GRB2 induces a major remodeling of the PPI network associated with SLP76 following TCR engagement. Unexpectedly, this PPI network rewiring minimally affects proximal molecular events of the TCR signaling pathway. Nevertheless, during prolonged TCR stimulation, GRB2- and GADS-deficient cells displayed a reduced level of activation and cytokine secretion capacity. Using the canonical SLP76 signalosome, this analysis highlights the plasticity of PPI networks and their reorganization following specific genetic perturbations.

Figure S1. Related to Fig. 2: Heatmap of the H3K27me3 level in a new cohort of 78 NPM1mut CN-AML patients. Figure S2. Related to Fig. 2: Analysis of H3K27me3 HIST1 status in CD34low and CD34high sorted blasts. Figure S3 Related to Fig. 4. Representative Integrative Genomics Viewer (IGV) tracks of H3K27me3 signal obtained from ChIP-chip data published in Tiberi et al., 2015 Figure S4. Related to Fig. 5: Histone protein extraction in NPM1mut patients. Figure S5. Related to Fig. 5:Total protein abundance of each histone types determined by IBAQ label-free quantification method. Figure S6 related to Fig. 6. Effect of H1-3 KD on histone H1 subtype mRNA and protein expression. Figure S7 related to Fig. 6. Effect of H1d KD on CD11b expression in shRNA1 (clones KD#2 and KD#3) and in shRNA2 conditions. Figure S7 related to Figure 6. Effect of H1d KD on CD11b expression in shRNA1 (clones KD#2 and KD#3) and in shRNA2 conditions

Table S1. Clinical and molecular characteristics in the GOELAMS Cohort (n = 46) according to H3K27me3 HIST1 status. Table S2. Multivariate analyses in the validation CN-AML cohort (n = 46). Table S3. Univariate and Multivariate Analyses for 3-HIST1-mRNA signature in TCGA and Metzeler cohorts. Table S4. Clinical characteristics of NPM1mut patients selected for transcriptomic analysis.

Introduction treatments targeting the Human Epidermal Growth Factor Receptor 2 (HER2/ERBB2) have improved the natural history of HER2-positive breast cancer. However, except HER2 protein expression and gene amplification, there is no predictive biomarker to guide the HER2-targeted therapies. We developed Parallel reaction monitoring (PRM) a powerful approach, to quantify and evaluate key proteins involved in the HER2 pathway and/or anti-HER2 treatment sensitivity. Results in BCLs, PRM measurements correlated with western blot immunocytochemistry and transcriptomic data. At baseline, higher expression of HER2, EGFR, PTEN and HER3 but lower expression of phospho-HER2 correlated with trastuzumab sensitivity. Under trastuzumab, PRM demonstrated a decrease in HER2 and an increase in phospho-HER2, which correlated with drug sensitivity. The opposite was observed under lapatinib. HER2 quantification was also correlated with immunohistochemistry in PDXs and clinical breast cancer samples. Discussion in conclusion, PRM-based assay, developed to quantify proteins of the HER2 pathway in breast cancer samples revealed a large magnitude of expression, which may have relevance in terms of treatment sensitivity. Materials and Methods we first evaluated PRM in term of sensitivity, linearity and reproducibility. PRM was then applied to breast cancer cell lines (BCLs) including BCLs exposed to anti-HER2 agents, patient-derived xenografts (PDXs) and frozen breast cancer samples.

Additional file 7. Table summary of patients selected for the absolute quantification of the two isoforms of tAP-F13A1 by LC-PRM.

Additional file 6. Coefficients of Variation (CVs) of the ratios light/heavy.

Additional file 1. Workflow of the study from the biomarker discovery step to the validation of tAP-F13A1 by LC-PRM.

Additional file 5. Representative mass spectra of the AP-F13A1 activation peptide.

Figure S2. Baseline biomarker values according to medical history (de novo versus radio-induced angiosarcoma). (PPTX 290 kb)

Additional file 3. MS/MS Spectra from the Mascot DAT file imported in Skyline software as MS/MS library.

Additional file 4. MS/MS Filteringâ transition setting tabs in Skyline software.

Figure S1. Location map of the tumors in each treatment arm (A/B). (PPTX 117 kb)

Figure S3. Forest plot: evaluation of the predictive value of clinical factors and biomarkers for the treatment effect in terms of PFS. Biomarkers were categorized as binary variables using the observed median value as the cut-off to illustrate the results. (PPTX 415 kb)

Additional file 2. Table of synthetic light and heavy stables isotope for tAP-F13A1 during inclusion mass screening and PRM-based assays.

Table S1. All Grade ≥ 3 drug-related adverse events. (DOCX 13 kb)

Protein footprinting is a new methodology that is based on probing, typically with the use of mass spectrometry, of reactivity of different aminoacid residues to a modifying reagent. Data thus obtained allow one to make inferences about protein conformations and their intermolecular interactions. Most of the protein footprinting studies so far have been performed on individual proteins in vitro. We explore whether a similar approach is possible with the proteins inside of living cells, employing dimethylsulfate (DMS), a reagent widely used for the in vivo footprinting of nucleic acids. DMS can induce methylation of the lysine, histidine and glutamate residues on proteins. Using models of the histone H2B/H2AZ heterodimer assembled in vitro and from chromatin treated in vivo, we show that the methylation by deuterated DMS allows one to distinguish the accessibility of a particular residue in and out of the protein's environmental/structural context. The detection of changes in protein conformations or their interactions in vivo can provide a new approach to the identification of proteins involved in various intracellular pathways and help in the search for perspective drug targets and biomarkers of diseases., Comment: 28 pages, 5 figures