Identification of covalent modifications regulating immune signaling complex composition and phenotype

Area of Science:

• Cellular Biology
• Molecular Biology
• Immunology

Background:

• Cell signaling relies on dynamic protein community rearrangements.
• Understanding post-transcriptional modifications and protein interactions is crucial for identifying signaling checkpoints.

Purpose of the Study:

• To develop an efficient method for identifying post-transcriptional modifications regulating signaling complex composition and functional phenotypes.
• To investigate the interplay of modifications, interactions, and phenotypes in immune signaling.

Main Methods:

• Developed Modifications, Interactions, and Phenotypes by Affinity Purification Mass Spectrometry (MIP-APMS).
• Streamlined cloning and transduction of tagged proteins into reporter cells.
• Utilized affinity chromatography and MS-based quantification for time-resolved analysis.

Main Results:

• Characterized over 50 novel modifications and hundreds of protein-protein interactions in 19 immune complexes.
• Validated interdependencies in Toll-like receptor 2 signaling, including TRAF2 ISGylation and phosphorylation, and ARHGEF18 interaction.
• Identified distinct mechanisms of action for p38 (MAPK14) inhibitors.

Conclusions:

• MIP-APMS offers a fast, cost-effective pipeline for interrogating protein communities.
• The method facilitates the discovery of novel molecular signaling checkpoints in diverse biological systems.
• Provides insights into complex cellular signaling pathways and therapeutic targets.