Structural Alternation in Heat Shock Proteins of Activated Macrophages

The inflammatory response of macrophages is an orderly and complex process under strict reg-ulation accompanied by drastic changes in morphology and functions. It is predicted that pro-teins will undergo structural changes during these finely regulated processes. However, changes in structural proteome in macrophages during the inflammatory response remain poorly char-acterized. In the present study, we applied limited proteolysis coupled mass spectrometry (LiP-MS) to identify proteome-wide structural changes in lipopolysaccharide (LPS)-activated macrophages. We identified 386 structure-specific proteolytic fingerprints from 230 proteins. Using GO molecular function enrichment, we discovered that proteins with altered structures were enriched into unfolded protein binding and protein folding chaperone, in which HSP60 was ranked as the most changed protein. We verified the structural changes in HSP60 by using cellular thermal shift assay (CETSA) and native CETSA. Our results showed that the thermal sta-bility of HSP60 was enhanced in activated macrophages and formed a HSP10-less complex. In conclusion, we demonstrate that in situ structural systems biology is an effective method to characterize proteomic structural changes and reveal that the structures of chaperone proteins vary significantly in macrophage activation.