Alfred Hansel

Mitochondrial targeting of the human peptide methionine sulfoxide reductase (MSRA), an enzyme-involved in the repair of oxidized proteins

Peptide methionine sulfoxide reductase (MSRA) catalyzes the reduction of methionine sulfoxide to methionine. This widely expressed enzyme constitutes an important repair mechanism for oxidatively damaged proteins, which accumulate during the manifestation of certain degenerative diseases and aging processes. In addition, it is discussed to be involved in regulatory processes. Here we address the question of how the enzymes diverse functions are reflected in its subcellular localization. Using fusions of the human version of MSRA with the enhanced green fluorescence protein expressed in various mammalian cell lines, we show a distinct localization at mitochondria. The N‐terminal 23 amino acid residues contain the signal for this mitochondrial targeting. Activity tests showed that they are not required for enzyme function. Mitochondrial localization of native MSRA in mouse and rat liver slices was verified with an MSRA‐specific antibody by using immunohistochemical methods. The protein was located in the mitochondrial matrix, as demonstrated by using pre‐embedding immunostaining and electron microscopy. Mitochondria are the major source of reactive oxygen species (ROS). Therefore, MSRA has to be considered an important means for the general reduction of ROS release from mitochondria.

Methionine Sulfoxide Reductase A and a Dietary Supplement S-Methyl-L-Cysteine Prevent Parkinson's-Like Symptoms

Parkinsons 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 α-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 α-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.

Molecular interaction of δ-conotoxins with voltage-gated sodium channels

Various neurotoxic peptides modulate voltage‐gated sodium (NaV) channels and thereby affect cellular excitability. δ‐Conotoxins from predatory cone snails slow down inactivation of NaV channels, but their interaction site and mechanism of channel modulation are unknown. Here, we show that δ‐conotoxin SVIE from Conus striatus interacts with a conserved hydrophobic triad (YFV) in the domain‐4 voltage sensor of NaV channels. This site overlaps with that of the scorpion α‐toxin Lqh‐2, but not with the α‐like toxin Lqh‐3 site. δ‐SVIE functionally competes with Lqh‐2, but exhibits strong cooperativity with Lqh‐3, presumably by synergistically trapping the voltage sensor in its “on” position.

Molecular cloning and functional expression of a human peptide methionine sulfoxide reductase (hMsrA)

Oxidation of methionine residues in proteins to methionine sulfoxide can be reversed by the enzyme peptide methionine sulfoxide reductase (MsrA, EC 1.8.4.6). We cloned the gene encoding a human homologue (hMsrA) of the enzyme, which has an 88% amino acid sequence identity to the bovine version (bMsrA). With dot blot analyses based on RNA from human tissues, expression of hMsrA was found in all tissues tested, with highest mRNA levels in adult kidney and cerebellum, followed by liver, heart ventricles, bone marrow and hippocampus. In fetal tissue, expression was highest in the liver. No expression of hmsrA was detected in leukemia and lymphoma cell lines. To test if hMsrA is functional in cells, we assayed its effect on the inactivation time course of the A‐type potassium channel ShC/B since this channel property strongly depends on the oxidative state of a methionine residue in the N‐terminal part of the polypeptide. Co‐expression of ShC/B and hMsrA in Xenopus oocytes significantly accelerated inactivation, showing that the cloned enzyme is functional in an in vivo assay system. Furthermore, the activity of a purified glutathione‐S‐transferase‐hMsrA fusion protein was demonstrated in vitro by measuring the reduction of [3H]N‐acetyl methionine sulfoxide.

Oxidation of multiple methionine residues impairs rapid sodium channel inactivation

Reactive oxygen species (ROS) readily oxidize the sulfur-containing amino acids cysteine and methionine (Met). The impact of Met oxidation on the fast inactivation of the skeletal muscle sodium channel NaV1.4 expressed in mammalian cells was studied by applying the Met-preferring oxidant chloramine-T or by irradiating the ROS-producing dye Lucifer Yellow in the patch pipettes. Both interventions dramatically slowed down inactivation of the sodium channels. Replacement of Met in the Ile–Phe–Met inactivation motif with Leu (M1305L) strongly attenuated the oxidizing effect on inactivation but did not eliminate it completely. Mutagenesis of Met1470 in the putative receptor of the inactivation lid also markedly diminished the oxidation sensitivity of the channel, while that of other conserved Met residues in intracellular linkers connecting the membrane-spanning segments (442, 1139, 1154, 1316, 1469) were of minor importance. The results of mutagenesis, assays of other NaV channel isoforms (NaV1.2, NaV1.5, NaV1.7), and the kinetics of the oxidation-induced removal of inactivation collectively indicate that multiple Met residues need to be oxidized to completely impair inactivation. This arrangement using multiple Met residues confers a finely graded oxidative modulation of NaV channels and allows organisms to adapt to a variety of oxidative stress conditions, such as ischemic reperfusion.

Activity, tissue distribution and site-directed mutagenesis of a human peptide methionine sulfoxide reductase of type B: hCBS1

Human CBS1 is a methionine sulfoxide reductase of type B (MSRB) as it specifically reduced Met‐R‐SO in peptides with dithiothreitol or the thioredoxin system as reductants. Mutation C169S in the active site completely abolished enzymatic activity, while mutation W110A only reduced activity and C105S had no effect. Like human MSRA, hCBS1 showed in vivo reducing activity coexpressed with the Drosophila ShC/B potassium channel in oocytes, by accelerating the overall inactivation time course. hCBS1‐encoding mRNA is most abundant in muscle tissues, especially in the heart and thereby shows an expression pattern different to the human MSRA.

The μO-conotoxin MrVIA inhibits voltage-gated sodium channels by associating with domain-3

Several families of peptide toxins from cone snails affect voltage‐gated sodium (NaV) channels: μ‐conotoxins block the pore, δ‐conotoxins inhibit channel inactivation, and μO‐conotoxins inhibit NaV channels by an unknown mechanism. The only currently known μO‐conotoxins MrVIA and MrVIB from Conus marmoreus were applied to cloned rat skeletal muscle (NaV1.4) and brain (NaV1.2) sodium channels in mammalian cells. A systematic domain‐swapping strategy identified the C‐terminal pore loop of domain‐3 as the major determinant for NaV1.4 being more potently blocked than NaV1.2 channels. μO‐conotoxins therefore show an interaction pattern with NaV channels that is clearly different from the related μ‐ and δ‐conotoxins, indicative of a distinct molecular mechanism of channel inhibition.

Differential sensitivity of sodium channels from the central and peripheral nervous system to the scorpion toxins Lqh-2 and Lqh-3.

The scorpion α‐toxins Lqh‐2 and Lqh‐3, isolated from the venom of the Israeli yellow scorpion Leiurus quinquestriatus hebraeus, were previously shown to be very potent in removing fast inactivation of rat skeletal muscle sodium channels ( Chen et al., 2000 ). Here, we show that tetrodotoxin‐sensitive neuronal channels NaV1.2 and NaV1.7, which are mainly expressed in mammalian central and peripheral nervous systems, respectively, are differentially sensitive to these two toxins. rNaV1.2 and hNaV1.7 channels were studied with patch‐clamp methods upon expression in mammalian cells. While Lqh‐3 was about 100‐times more potent in removing inactivation in hNaV1.7 channels compared with rNaV1.2, Lqh‐2 was about 20‐times more active in the other direction. Site‐directed mutagenesis showed that the differences in the putative binding sites for these toxins, the S3‐4 linkers of domain 4, are of major importance for Lqh‐3, but not for Lqh‐2.

A second human methionine sulfoxide reductase (hMSRB2) reducing methionine-R-sulfoxide displays a tissue expression pattern distinct from hMSRB1

Abstract Peptide methionine sulfoxide reductases are important enzymes in the defense against cellular oxidative stress as they reduce methionine sulfoxide, the product of methionine oxidation by physiologically relevant reactive oxygen species. Two distinct enzyme classes, MSRA and MSRB, have evolved for selectively reducing the two epimers, methionine-S-sulfoxide and methionine-Rsulfoxide. A new human MSR enzyme (hMSRB2) specifically reducing methionine-R-sulfoxide, which showed a conversion rate for peptide-bound methionine-S-sulfoxide similar to hMSRB1, was characterized with respect to its tissue expression. As previously found for hMSRB1, expression of hMSRB2 mRNA was weak in brain, but strong in heart and skeletal muscle. In contrast to hMSRB1, its expression was high in smooth muscle-containing organs (digestive system, bladder), lung and aorta, while hMSRB1 displayed a higher expression than hMSRB2 in liver and kidney.

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 cultured 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 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.