Proteome-Wide Acetylome Profiling Suggests Extensive Aspirin-Driven Remodeling of Networks Relevant to THP-1...

Acetylsalicylic Acid (aspirin or ASA) is known to exhibit immunomodulatory effects not only through Cyclooxygenase (COX) inhibition but also through direct protein acetylation. However, its impact on innate immune cell differentiation remains unclear.

To fill this gap, the study used quantitative acetyl-proteomics to track changes in the lysine acetylome during THP-1 monocyte differentiation into macrophages following ASA preconditioning.

Our results showed that preconditioning of THP-1 macrophages with 300 μg/ml ASA for 3 hours before differentiation induced persistent acetylation changes. We identified 5,199 differentially acetylated sites across 2,678 proteins, with 2,595 sites upregulated and 2,604 downregulated. The sequence analysis revealed a strong preference of ASA for acidic residues like E_K motifs and hydrophobic regions. The subcellular localization analysis showed notable enrichment in the nucleus (1,166 proteins), cytoplasm (850 proteins), and mitochondria (405 proteins), and frequently contained functional domains like PWWP, SET, RhoGEF, and RNA recognition motifs.

The Gene Ontology analysis linked these proteins to cellular metabolism, regulation, and stimulus response, while our KEGG analysis connected them to neurodegeneration, infection, and metabolic pathways. Furthermore, the protein-protein interaction networks further showed coordinated changes in ribosomal, signaling, and chromatin complexes.

The findings show that ASA preconditioning leaves a lasting acetylome signature during macrophage differentiation reprogramming regulatory networks relevant to macrophage differentiation and functional networks via motif-directed acetylation. The results provide a plausible COX-independent model in which structural motif-targeted acetylation may underlie ASA’s immunomodulatory role.