Abstract
Gamma interferon-inducible thiol reductase (GILT) is an enzyme involved in the initial steps of antigen processing and presentation. Recently we have shown that GILT is also expressed in mouse T cells, where it exerts an inhibitory role on T cell activation. In this study, we identified mitochondrial manganese superoxide dismutase (SOD2) as one of the key intermediaries affected by GILT expression in fibroblasts. Expression and activity of SOD2 is reduced in the absence of GILT because of reduced SOD2 protein stability. The forced increase in SOD2 expression in the absence of GILT restores fibroblast proliferation to wild-type levels. Thus, GILT appears to have a fundamental role in cellular proliferation mediated through its influence on SOD2 protein activity and expression.
Highlights
Enzymes of the thiol reductase family carry out reduction, oxidation, and isomerization of protein disulfide bonds in cytosol [1, 2], mitochondria [3], endoplasmic reticulum [2], and lysosomes.2 The majority of these enzymes are functional at neutral or slightly alkaline conditions [4], they have similar three-dimensional structures, and all feature a conservative active site loop containing two cysteines in the sequence -CGPC- [5]
Using GILTϪ/Ϫ mice as a model, we have shown that Gamma interferon-inducible thiol reductase (GILT) catalyzes initial unfolding of antigenic protein and facilitates protein/peptide binding to MHC class II molecules [10]
We examined whether GILT might affect cellular proliferation in general
Summary
Primary Cells, and Cell Lines—C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME). GILTϪ/Ϫ and WT SV40 large T antigen immortalized mouse fibroblast cell line was generated from GILTϪ/Ϫ and WT mice in Dr Peter Creswell’s laboratory at Yale University. TaqMan probe for SOD2 was designed to span the junction between two exons to avoid amplification of the genomic DNA This was not possible for 18 S rRNA, which has no introns. Superoxide Dismutase Activity Assay—2 ϫ 107 GILTϪ/Ϫ and WT mouse fibroblast cells were homogenized in 1 ml of cold buffer (20 mM HEPES, 1 mM EGTA, 210 mM mannitol, and 70 mM sucrose), pH 7.2, using a Dounce homogenizer. The SOD activity assay was performed as suggested by the supplier (Cayman Chemical) This protocol is based on the tetrazolium salt for the detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. GILTϪ/Ϫ and WT mouse fibroblast cell lines were starved overnight in phenol red-free medium with 1% FBS. Cells were washed again in PBS, resuspended in cold PBS, 0.5% bovine serum albumin buffer, and immediately analyzed by flow cytometry
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