Protection of vascular smooth muscle cells by over-expressed methionine sulphoxide reductase A: Role of intracellular localization and substrate availability

Abstract

Methionine sulphoxide reductase A (MSRA) that reduces methionine-S-sulphoxide back to methionine constitutes a catalytic antioxidant mechanism to prevent oxidative damage at multiple sub-cellular loci. This study examined the relative importance of protection of the cytoplasm and mitochondria by MSRA using A-10 vascular smooth muscle cells, a cell type that requires a low level of reactive oxygen species (ROS) for normal function but is readily damaged by higher concentrations of ROS. Adenoviral over-expression of human MSRA variants, targeted to either mitochondria or the cytoplasm, did not change basal viability of non-stressed cells. Oxidative stress caused by treatment with the methionine-preferring oxidizing reagent chloramine-T decreased cell viability in a concentration-dependent manner. Cytoplasmic MSRA preserved cell viability more effectively than mitochondrial MSRA and co-application of S-methyl-L-cysteine, an amino acid that acts as a substrate for MSRA when oxidized, further increased the extent of protection. This suggests an important role for an MSRA catalytic antioxidant cycle for protection of the cytoplasmic compartment against oxidative damage.

Functions and evolution of selenoprotein methionine sulfoxide reductases

“…It should be noted, however, that the alternatively spliced MsrA form was not detected in examined mouse tissues, including brain, kidney, and liver, by western blot assays, and only a weak signal was detected at the mRNA level. Studies showed that other alternative forms of MsrA mRNA including an additional exon 2 could be detected in rat aortic vascular smooth muscle cells and aortic tissue preparations [53] and this form was localized exclusively to mitochondria [53, 54]. …”

Impact of Hydrogen Peroxide on the Activity, Structure, and Conformational Stability of the Oxidized Protein Repair Enzyme Methionine Sulfoxide Reductase A

“…5 Indeed, MsrAoverexpressing cells have an increased resistance to oxidative stress, a characteristic that has been associated to an improved detoxication of ROS. 6,7,22 For example, SV40-WI38 fibroblasts overexpressing MsrA, which were found to be more resistant to H 2 O 2 treatment, were exhibiting a lower content of ROS after treatment with H 2 O 2 , resulting in an almost total protection of intracellular proteins against irreversible oxidative modification. 6 In a previous study, we have shown that both cytosolic and mitochondrial isoforms of MsrA purified from rat liver exhibited oxidative modification of up to three of their five cysteine residues.…”

Deletion of Methionine Sulfoxide Reductase A Does Not Affect Atherothrombosis but Promotes Neointimal Hyperplasia and Extracellular Signal-Regulated Kinase 1/2 Signaling

“…57, 58 Of note, the rate of proliferation in the fibroblast studies was mainly examined by MTT assay, which depends on the cellular metabolic activity due to NAD(P)H flux. Since MsrA is expressed in both cytoplasm and the mitochondria in VSMC, 59 it may affect NAD(P)H flux, possibly independently from its role in proliferation. In fact, a proteomic analysis of 293T cells after MsrA knockdown revealed a subunit of NADH dehydrogenase 1β as target of methionine oxidation.…”