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

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.

Methionine sulfoxide reductase A and a dietary supplement S-methyl-L-cysteine prevent Parkinson's-like symptoms.

Parkinson's disease (PD), a common neurodegenerative disease, is caused by loss of dopaminergic neurons in the substantia nigra. Although the underlying cause of the neuronal loss is unknown, oxidative stress is thought to play a major role in the pathogenesis of PD. The amino acid methionine is readily oxidized to methionine sulfoxide, and its reduction is catalyzed by a family of enzymes called methionine sulfoxide reductases (MSRs). The reversible oxidation-reduction cycle of methionine involving MSRs has been postulated to act as a catalytic antioxidant system protecting cells from oxidative damage. Here, we show that one member of the MSR family, MSRA, inhibits development of the locomotor and circadian rhythm defects caused by ectopic expression of human alpha-synuclein in the Drosophila nervous system. Furthermore, we demonstrate that one way to enhance the MSRA antioxidant system is dietary supplementation with S-methyl-L-cysteine (SMLC), found abundantly in garlic, cabbage, and turnips. SMLC, a substrate in the catalytic antioxidant system mediated by MSRA, prevents the alpha-synuclein-induced abnormalities. Therefore, interventions focusing on the enzymatic reduction of oxidized methionine catalyzed by MSRA represent a new prevention and therapeutic approach for PD and potentially for other neurodegenerative diseases involving oxidative stress.

Identification of a new functional splice variant of the enzyme methionine sulphoxide reductase A (MSRA) expressed in rat vascular smooth muscle cells.

Reactive oxygen species contribute to ageing of the vascular system and development of cardiovascular disease. Methionine-S-sulphoxide, an oxidized form of methionine, is repaired by the enzyme methionine sulphoxide reductase A (MSRA). The enzyme, targeted to mitochondria or the cytosol by alternative splicing, is vital for oxidative stress resistance. This study was designed to examine the endogenous expression and intracellular localization of MSRA in rat aortic vascular smooth muscle cells (VSMCs). We detected robust MSRA immunoreactivity exclusively in mitochondria. Sequence analysis of msrA transcripts revealed the presence of a novel mitochondrial splice variant, msrA2a, in cultured rat VSMCs as well as in aortic tissue preparations. The enzymatic activity of a recombinant MSRA2a protein was confirmed by the reduction of methionine sulphoxide in a model substrate peptide. We conclude that multiple MSRA variants participate in the repair of oxidized proteins in VSMC mitochondria, but that other protective mechanisms may exist in the cytoplasmic compartment.

Three methionine residues located within the regulator of conductance for K+ (RCK) domains confer oxidative sensitivity to large-conductance Ca2+-activated K+ channels.

Methionine-directed oxidation of the human Slo1 potassium channel (hSlo1) shifts the half-activation voltage by -30 mV and markedly slows channel deactivation at low concentrations of intracellular Ca2+ ([Ca2+]i). We demonstrate here that the contemporaneous mutation of M536, M712 and M739 to leucine renders the channel functionally insensitive to methionine oxidation caused by the oxidant chloramine-T (Ch-T) without altering other functional characteristics. Coexpression with the auxiliary beta1 subunit fails to restore the full oxidative sensitivity to this triple mutant channel. The Ch-T effect is mediated specifically by M536, M712 and M739 because even small changes in this residue combination interfere with the ability to remove the oxidant sensitivity following mutation. Replacement of M712 or M739, but not M536, with the hydrophilic residue glutamate largely mimics oxidation of the channel and essentially removes the Ch-T sensitivity, suggesting that M712 and M739 may be part of a hydrophobic pocket disrupted by oxidation of non-polar methionine to the more hydrophilic methionine sulfoxide. The increase in wild-type hSlo1 open probability caused by methionine oxidation disappears at high [Ca2+]i and biophysical modelling of the Ch-T effect on steady-state activation implicates a decrease in the allosteric coupling between Ca2+ binding and the pore. The dramatic increase in open probability at low [Ca2+]i especially within the physiological voltage range suggests that oxidation of M536, M712 or M739 may enhance the Slo1 BK activity during conditions of oxidative stress, such as those associated with ischaemia-reperfusion and neurodegenerative disease, or in response to metabolic cues.