Structural Alternation in Heat Shock Proteins of Activated Macrophages

Wenhao Zhang,Wenxuan Hou,Jing Liu,Zongyuan Liu,Zhaoyun Zong,Ying Wei,Yuling Chen,Songbiao Zhu,Huaijin Zhang,Haiteng Deng

doi:10.3390/cells10123507

Abstract

The inflammatory response of macrophages is an orderly and complex process under strict regulation accompanied by drastic changes in morphology and functions. It is predicted that proteins will undergo structural changes during these finely regulated processes. However, changes in structural proteome in macrophages during the inflammatory response remain poorly characterized. 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 the Gene Ontology (GO) biological process enrichment, we discovered that proteins with altered structures were enriched into protein folding-related terms, 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 stability of HSP60 was enhanced in activated macrophages and formed an 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 during macrophage activation.

Highlights

IntroductionMacrophages can be activated by a wide range of cytokines and microbial ligands via the
Macrophages play a major role in the host defense and inflammatory response.Macrophages can be activated by a wide range of cytokines and microbial ligands via theToll-like receptor (TLR) signaling pathway [1,2,3]
When RAW264.7 macrophages are stimulated by lipopolysaccharide (LPS), the TLR4 signaling pathway is activated, which in turn activates the transcription factor nuclear factor κ B (NF- κB) and secretes different cytokines, such as tumor necrosis factor-α, IL1-β, and IL-6

Summary

Introduction

Macrophages can be activated by a wide range of cytokines and microbial ligands via the. Toll-like receptor (TLR) signaling pathway [1,2,3]. When RAW264.7 macrophages are stimulated by lipopolysaccharide (LPS), the TLR4 signaling pathway is activated, which in turn activates the transcription factor nuclear factor κ B (NF- κB) and secretes different cytokines, such as tumor necrosis factor-α, IL1-β, and IL-6. The process of macrophage activation has been well defined, the proteome-wide structural changes during macrophage activation have not been well characterized. Limited proteolysis mass spectrometry (LiP-MS) is a powerful approach to characterize global structural changes [7,8]. An in-depth characterization of proteome-wide structural changes during macrophage activation will help us to have a deeper understanding of this process

Methods

Results

Conclusion