ProteinS-Glutathionylation Mediates Macrophage Responses to Metabolic Cues from the Extracellular Environment

Abstract

Aims: Protein S-glutathionylation, the formation of a mixed disulfide between glutathione and protein thiols, is an oxidative modification that has emerged as a new signaling paradigm, potentially linking oxidative stress to chronic inflammation associated with heart disease, diabetes, cancer, lung disease, and aging. Using a novel, highly sensitive, and selective proteomic approach to identify S-glutathionylated proteins, we tested the hypothesis that monocytes and macrophages sense changes in their microenvironment and respond to metabolic stress by altering their protein thiol S-glutathionylation status. Results: We identified over 130 S-glutathionylated proteins, which were associated with a variety of cellular functions, including metabolism, transcription and translation, protein folding, free radical scavenging, cell motility, and cell death. Over 90% of S-glutathionylated proteins identified in metabolically stressed THP-1 monocytes were also found in hydrogen peroxide (H 2 O 2 )-treated cells, suggesting that H 2 O 2 mediates metabolic stress-induced protein S-glutathionylation in monocytes and macrophages. We validated our findings in mouse peritoneal macrophages isolated from both healthy and dyslipidemic atherosclerotic mice and found that 52% of the S-glutathionylated proteins found in THP-1 monocytes were also identified in vivo. Changes in macrophage protein S-glutathionylation induced by dyslipidemia were sexually dimorphic. Innovation: We provide a novel mechanistic link between metabolic (and thiol oxidative) stress, macrophage dysfunction, and chronic inflammatory diseases associated with metabolic disorders. Conclusion: Our data support the concept that changes in the extracellular metabolic microenvironment induce S-glutathionylation of proteins central to macrophage metabolism and a wide array of cellular signaling pathways and functions, which in turn initiate and promote functional and phenotypic changes in macrophages. Antioxid. Redox Signal. 25, 836-851.

Protein Thiol Redox Signaling in Monocytes and Macrophages

“…More recently, our group and others have used proteomic approaches to characterize protein-S-glutathionylation in monocytes and macrophages (148,187,209). These proteomic approaches rely on trapping protein thiols in their native redox state by the addition of a thiol-alkylating agent such as N-ethyl maleimide (NEM) (34).…”

“…In turn, GSSG is reduced back to two molecules of GSH by glutathione reductase (GR), which uses electrons from NADPH. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars proteomics approach to identify S-glutathionylated proteins in both the THP-1 monocyte cell line and purified mouse peritoneal macrophages under baseline conditions as well as under conditions of metabolic stress induced by hyperglycemia and high low-density lipoprotein levels (HG+LDL) (209). In this study, we selectively deglutathionylated NEM-treated monocyte or macrophage lysates with recombinant Grx and labeled the reduced thiol residues with iodoacetamide conjugated to desthiobiotin.…”

“…In this study, we selectively deglutathionylated NEM-treated monocyte or macrophage lysates with recombinant Grx and labeled the reduced thiol residues with iodoacetamide conjugated to desthiobiotin. These labeled proteins were then affinity purified and examined by LC-MS/MS (209). We identified over 100 proteins that were S-glutathionylated under physiological conditions in monocytes or macrophages, many of which showed changes in their S-glutathionylation status in response to metabolic stress (209).…”

“…These labeled proteins were then affinity purified and examined by LC-MS/MS (209). We identified over 100 proteins that were S-glutathionylated under physiological conditions in monocytes or macrophages, many of which showed changes in their S-glutathionylation status in response to metabolic stress (209).…”

“…Our group has recently shown that the a subunit of ATP synthase undergoes S-glutathionylation in THP-1 monocytes cultured under physiological conditions (5 mM glucose) as well as THP-1 cells cultured under metabolic stress (25 mM glucose +100 lg/ml LDL) (209). (63).…”