Periodic Reporting for period 1 - Cytokineproteomics (Investigating inflammatory signaling by combining phospho- and ubiquitin proteomics with CRISPR/Cas9 technology)
Período documentado: 2017-03-01 hasta 2019-02-28
Signal transduction of cytokine signalling pathways occur through posttranslational modifications (PTMs) of signalling components. To investigate signaling crosstalk it is important to capture and analyse those PTMs in an unbiased, system-based manner. Mass spectrometry-based proteomics has matured remarkably in the last years and now provides a comprehensive discovery tool to address diverse biological questions. To analyse signaling events we enrich for posttranslational modification, which are subsequently analysed in a highly quantitative fashion. Thereby we aim to identify crucial signaling events for cytokine signaling in isolation and in context of other cytokines. These findings will give more insights into cytokine signaling and might provide more suitable drug targets. Targeting downstream signaling events instead of inhibiting cytokine pathways upstream (e.g. TNF inhibition) could be a potential approach to reduce treatment toxicity.
We conducted further analyses of the TNF phosphoproteome in myeoloid cells and investigated its kinetics, from very early time points of one minute to one hour of stimulation. Thereby we required the generation of a deep library acquired by DDA, which allowed identification of DIA acquired MS2 spectra. We detected up to 20,000 phosphopeptides per sample and identified a range of phosphorylations on E3 ligases, kinases and transcription factor that are significantly regulated by TNF in a time dependent manner. Several of these candidates are critical for TNF signaling or have been implicated in other innate immune signaling pathways.
Also RIPK3, a kinase essential for TNF-induced necroptosis has been found regulated in response to TNF. We investigated the impact of RIPK3 on the phosphoproteome upon TNF stimulation and induction of TNF-induced apoptosis and necroptosis. Therefore we firstly generated U937 cells deficient for RIPK3 using the CRISPR/Cas9 system. These cells were stimulated accordingly and phosphopeptides were enriched. The analysis revealed RIPK3 dependent phosphorylations of several proteins.
We also analysed the phosphoproteome of myeloid cells undergoing TNF-induced apoptosis and necroptosis for 15 minutes and 3 hours. At the early 15 min time point was no significant difference between the phosphoproteomes of TNF and TNF-induced apoptosis and necroptosis. After 3 hours of stimulation TNF-induced apoptosis induced phosphorylation on many proteins involved in the DNA damage response as a result of caspase activity at this later time point. This was not detected in TNF-induced necroptosis when caspases are inhibited. Consistently, we detected upregulation of phosphorylations in apoptosis compared to necroptosis and control on three sites of the caspase inhibitor XIAP, a key player in TNF signalling.