Functional roles of oxidative modifications of S100A8 and S100A9

Abstract

The S100 proteins are Ca2+ binding proteins belonging to the EF-hand superfamily with diverse intracellular and extracellular functions. Three members (S100A8, S100A9 and S100A12) are upregulated in patients with acute and chronic inflammatory disorders, hence are known as inflammation-associated S100s. S100A8 and S100A9 are co-expressed constitutively in neutrophils, and induced in other cells by inflammatory mediators or oxidative stress. These proteins form homo- and hetero-complexes and have distinct intracellular and extracellular activities depending on their oligomeric forms. S100A8 and S100A9 are highly sensitive to oxidation by hypochlorite and peroxides and may have a protective role in oxidative defense by scavenging oxidants. Studies in this thesis investigated the physiological and molecular effects of S100A8 in yeast Saccharomyces cerevisiae during oxidative stress to elucidate its function in mammalian cells. Several differentially expressed genes involved in anti-oxidative defense and lipid metabolism were upregulated in microarray of S100A8-expressing yeast cells treated with hydrogen peroxide. PLB1, encoding phospholipase B1 that hydrolyzes phospholipids to generate lipid precursors, was upregulated in S100A8-expressing HeLa cells following hydrogen peroxide treatment. S100A8 also localized to peroxisomes and/or lipid bodies in yeast, and closely associated with these structures in some mammalian cells. Collectively, these studies suggest possible roles for S100A8 in anti-oxidative defense and lipid metabolism. Oxidation of S100A8 and S100A9 generates various reversible and irreversible modifications that may regulate their activities. Studies in this thesis characterized two novel modifications of S100A8 and S100A9, S-nitrosylation and S-glutathionylation, and their functional effects. S-nitrosylated S100A8 (S100A8-SNO) was detected by mass spectrometry in neutrophils treated with nitric oxide donors. S100A8-SNO reduced mast cell degranulation and mast cell-mediated leukocyte adhesion and extravasation in the rat mesenteric microcirculation, suggesting novel anti-inflammatory functions that are regulated by S-nitrosylation. S-glutathionylated S100A9 (S100A9-SSG) was generated in neutrophils activated by phorbol myristate acetate, and S100A9-SSG decreased neutrophil adhesion to fibronectin in complex with S100A8, suggesting that it may suppress excessive leukocyte transmigration. Studies in this thesis have elucidated several possible mechanisms whereby S100A8 contributes to anti-oxidative defense, and emphasized that specific oxidative modifications of S100A8 and S100A9 are functionally important.