Identification of covalent modifications regulating immune signaling complex composition and phenotype

Abstract

Cells signal through rearrangements of protein communities governed by covalent modifications and reversible interactions of distinct sets of proteins. A method that identifies those posttranscriptional modifications regulating signaling complex composition and functional phenotypes in one experimental setup would facilitate an efficient identification of novel molecular signaling checkpoints. Here, we devised modifications, interactions and phenotypes by affinity purification mass spectrometry (MIP-APMS), comprising the streamlined cloning and transduction of tagged proteins into functionalized reporter cells as well as affinity chromatography, followed by MS-based quantification. We report the time-resolved interplay of more than 50 previously undescribed modification and hundreds of protein-protein interactions of 19 immune protein complexes in monocytes. Validation of interdependencies between covalent, reversible, and functional protein complex regulations by knockout or site-specific mutation revealed ISGylation and phosphorylation of TRAF2 as well as ARHGEF18 interaction in Toll-like receptor 2 signaling. Moreover, we identify distinct mechanisms of action for small molecule inhibitors of p38 (MAPK14). Our method provides a fast and cost-effective pipeline for the molecular interrogation of protein communities in diverse biological systems and primary cells.

Transcriptomic Context of RUNX3 Expression in Monocytes: A Cross-Sectional Analysis

“…This process, mediated by small GTPases of the Rho family, stabilizes leukocyte adhesion and improves cell resistance to deformation. Several studies have established the role of actin cytoskeleton genes for cell dynamic response in mitogen-activated protein kinase (MAPK) signaling cascade with its downstream nuclear factor kappa B (NFκB) [ 32 , 33 , 34 ]. Because MAP3K7 is a central kinase in the pathway, the knockout of MAP3K7 and the interactor ARHGEF18 partially reduces NFκB activity in monocytes [ 33 ].…”

“…Several studies have established the role of actin cytoskeleton genes for cell dynamic response in mitogen-activated protein kinase (MAPK) signaling cascade with its downstream nuclear factor kappa B (NFκB) [ 32 , 33 , 34 ]. Because MAP3K7 is a central kinase in the pathway, the knockout of MAP3K7 and the interactor ARHGEF18 partially reduces NFκB activity in monocytes [ 33 ]. The NF-κB pathway promotes the expression of pro-inflammatory genes and to some extent induces expression of Runx3.…”

Cell-selective proteomics segregates pancreatic cancer subtypes by extracellular proteins in tumors and circulation

“…For stable MetRS* expression, PDAC cells were lentivirally transduced using a modified Precision LentiORF Collection (pLOC) library (GE Healthcare) plasmid (pLOC-CMV > MetRS*:IRES:TurboGFP:P2A:BlastR; enrichment method comparison experiment), generated as described previously 121 , or a pLV-EF1A > MetRS*:P2A:EGFP:T2A:Puro plasmid, constructed by VectorBuilder.…”

A proteomic perspective on TNF-mediated signalling and cell death

“…MS is also becoming more readily utilised in structural studies and has proven exceptionally successful in combination with various pulldown- and enrichment strategies to unravel enzyme-substrate relationships ( Figure 2 ). It is primarily used in the signalling field to investigate post-translational modifications like phosphorylation, ubiquitylation and less studied PTMs including acetylation, methylation, citrullination and other modifications like protein cleavage [ 7 , 35 , 40 ]. An enrichment strategy for caspase substrates recently identified the NEDD4-binding protein 1 (N4BP1) as a target of caspase-8 and suppressor of the cytokine response upon lipopolysaccharide (LPS) or TNF stimulation [ 41 ].…”