Geumsoo Kim

Lack of methionine sulfoxide reductase A in mice increases sensitivity to oxidative stress but does not diminish life span.

Methionine sulfoxide reductase A (MsrA) repairs oxidized methionine residues within proteins and may also function as a general antioxidant. Previous reports have suggested that modulation of MsrA in mice and mammalian cell culture can affect the accumulation of oxidized proteins and may regulate resistance to oxidative stress. Thus, under the oxidative stress theory of aging, these results would predict that MsrA regulates the aging process in mammals. We show here that MsrA−/− mice are more susceptible to oxidative stress induced by paraquat. Skin‐derived fibroblasts do not express MsrA, but fibroblasts cultured from MsrA−/− mice were, nevertheless, also more susceptible to killing by various oxidative stresses. In contrast to previous reports, we find no evidence for neuromuscular dysfunction in MsrA−/− mice in either young adult or in older animals. Most important, we found no difference between MsrA−/− and control mice in either their median or maximum life span. Thus, our results show that MsrA regulates sensitivity to oxidative stress in mice but has no effect on aging, as determined by life span.—Salmon, A. B., Pérez, V. I., Bokov, A., Jernigan, A., Kim, G., Zhao, H., Levine, R. L., Richardson, A. Lack of methionine sulfoxide reductase A in mice increases sensitivity to oxidative stress but does not diminish life span. FASEB J. 23, 3601–3608 (2009). www.fasebj.org

Carbonic anhydrase III is not required in the mouse for normal growth, development, and life span.

ABSTRACT Carbonic anhydrase III is a cytosolic protein which is particularly abundant in skeletal muscle, adipocytes, and liver. The specific activity of this isozyme is quite low, suggesting that its physiological function is not that of hydrating carbon dioxide. To understand the cellular roles of carbonic anhydrase III, we inactivated the Car3 gene. Mice lacking carbonic anhydrase III were viable and fertile and had normal life spans. Carbonic anhydrase III has also been implicated in the response to oxidative stress. We found that mice lacking the protein had the same response to a hyperoxic challenge as did their wild-type siblings. No anatomic alterations were noted in the mice lacking carbonic anhydrase III. They had normal amounts and distribution of fat, despite the fact that carbonic anhydrase III constitutes about 30% of the soluble protein in adipocytes. We conclude that carbonic anhydrase III is dispensable for mice living under standard laboratory husbandry conditions.

Carbonic anhydrase III regulates peroxisome proliferator-activated receptor-γ2.

Carbonic anhydrase III (CAIII) is an isoenzyme of the CA family. Because of its low specific anhydrase activity, physiological functions in addition to hydrating CO(2) have been proposed. CAIII expression is highly induced in adipogenesis and CAIII is the most abundant protein in adipose tissues. The function of CAIII in both preadipocytes and adipocytes is however unknown. In the present study we demonstrate that adipogenesis is greatly increased in mouse embryonic fibroblasts (MEFs) from CAIII knockout (KO) mice, as demonstrated by a greater than 10-fold increase in the induction of fatty acid-binding protein-4 (FABP4) and increased triglyceride formation in CAIII(-/-) MEFs compared with CAIII(+/+) cells. To address the underlying mechanism, we investigated the expression of the two adipogenic key regulators, peroxisome proliferator-activated receptor-γ2 (PPARγ2) and CCAAT/enhancer binding protein-α. We found a considerable (approximately 1000-fold) increase in the PPARγ2 expression in the CAIII(-/-) MEFs. Furthermore, RNAi-mediated knockdown of endogenous CAIII in NIH 3T3-L1 preadipocytes resulted in a significant increase in the induction of PPARγ2 and FABP4. When both CAIII and PPARγ2 were knocked down, FABP4 was not induced. We conclude that down-regulation of CAIII in preadipocytes enhances adipogenesis and that CAIII is a regulator of adipogenic differentiation which acts at the level of PPARγ2 gene expression.

Stereospecific oxidation of calmodulin by methionine sulfoxide reductase A.

Methionine sulfoxide reductase A has long been known to reduce S-methionine sulfoxide, both as a free amino acid and within proteins. Recently the enzyme was shown to be bidirectional, capable of oxidizing free methionine and methionine in proteins to S-methionine sulfoxide. A feasible mechanism for controlling the directionality has been proposed, raising the possibility that reversible oxidation and reduction of methionine residues within proteins is a redox-based mechanism for cellular regulation. We undertook studies aimed at identifying proteins that are subject to site-specific, stereospecific oxidation and reduction of methionine residues. We found that calmodulin, which has nine methionine residues, is such a substrate for methionine sulfoxide reductase A. When calmodulin is in its calcium-bound form, Met77 is oxidized to S-methionine sulfoxide by methionine sulfoxide reductase A. When methionine sulfoxide reductase A operates in the reducing direction, the oxidized calmodulin is fully reduced back to its native form. We conclude that reversible covalent modification of Met77 may regulate the interaction of calmodulin with one or more of its many targets.

Transgenic Mice Overexpressing Methionine Sulfoxide Reductase A: Characterization of Embryonic Fibroblasts

Methionine residues in protein can be oxidized by reactive oxygen species to generate methionine sulfoxide. Aerobic organisms have methionine sulfoxide reductases capable of reducing methionine sulfoxide back to methionine. Methionine sulfoxide reductase A acts on the S-epimer of methionine sulfoxide, and it is known that altering its cellular level by genetic ablation or overexpression has notable effects on resistance to oxidative stress and on life span in species from microorganisms to animals. In mammals, the enzyme is present in both the cytosol and the mitochondria, and this study was undertaken to assess the contribution of each subcellular compartments reductase activity to resistance against oxidative stresses. Nontransgenic mouse embryonic fibroblasts lack methionine sulfoxide reductase A activity, providing a convenient cell type to determine the effects of expression of the enzyme in each compartment. We created transgenic mice with methionine sulfoxide reductase A targeted to the cytosol, mitochondria, or both and studied embryonic fibroblasts derived from each line. Unexpectedly, none of the transgenic cells gained resistance to a variety of oxidative stresses even though the expressed enzymes were catalytically active when assayed in vitro. Noting that activity in vivo requires thioredoxin and thioredoxin reductase, we determined the levels of these proteins in the fibroblasts and found that they were very low in both the nontransgenic and the transgenic cells. We conclude that overexpression of methionine sulfoxide reductase A did not confer resistance to oxidative stress because the cells lacked other proteins required to constitute a functional methionine sulfoxide reduction system.

Nucleotide sequence and structure of the mouse carbonic anhydrase III gene.

At least 14 distinct isozymes of carbonic anhydrase have been identified in mammals. These enzymes catalyze the hydration of carbon dioxide and are essential for regulation of cellular pH and carbon dioxide transport. Carbonic anhydrase III is highly expressed in certain tissues, including muscle and fat where it constitutes up to 25% of the soluble protein. We cloned a cDNA encoding mouse carbonic anhydrase III. This cDNA contains 1653 bp, consisting of 79 bp in the 5 UTR, a 780 bp open reading frame, and 794 bp of the 3 UTR, including two potential polyadenylation signals. Fluorescent in situ hybridization confirmed the existence of a single copy of the gene on chromosome 3. We then isolated the genomic DNA for mouse carbonic anhydrase III and analyzed its structure. The gene consists of seven exons and six introns which span 10.5 kb. The 5 flanking region of the genomic DNA is notable for a pyrimidine rich region consisting of two dinucleotide repeats containing 23 and 20 TC pairs separated by the same 15 bp spacer.

Effects of transgenic methionine sulfoxide reductase A (MsrA) expression on lifespan and age-dependent changes in metabolic function in mice

Mechanisms that preserve and maintain the cellular proteome are associated with long life and healthy aging. Oxidative damage is a significant contributor to perturbation of proteostasis and is dealt with by the cell through regulation of antioxidants, protein degradation, and repair of oxidized amino acids. Methionine sulfoxide reductase A (MsrA) repairs oxidation of free- and protein-bound methionine residues through enzymatic reduction and is found in both the cytosol and the mitochondria. Previous studies in Drosophila have shown that increasing expression of MsrA can extend longevity. Here we test the effects of increasing MsrA on longevity and healthy aging in two transgenic mouse models. We show that elevated expression of MsrA targeted specifically to the cytosol reduces the rate of age-related death in female mice when assessed by Gompertz analysis. However, neither cytosolic nor mitochondrial MsrA overexpression extends lifespan when measured by log-rank analysis. In mice with MsrA overexpression targeted to the mitochondria, we see evidence for improved insulin sensitivity in aged female mice. With these and our previous data, we conclude that the increasing MsrA expression in mice has differential effects on aging and healthy aging that are dependent on the target of its subcellular localization.

Methionine in Proteins: It’s Not Just for Protein Initiation Anymore

Methionine in proteins is often thought to be a generic hydrophobic residue, functionally replaceable with another hydrophobic residue such as valine or leucine. This is not the case, and the reason is that methionine contains sulfur that confers special properties on methionine. The sulfur can be oxidized, converting methionine to methionine sulfoxide, and ubiquitous methionine sulfoxide reductases can reduce the sulfoxide back to methionine. This redox cycle enables methionine residues to provide a catalytically efficient antioxidant defense by reacting with oxidizing species. The cycle also constitutes a reversible post-translational covalent modification analogous to phosphorylation. As with phosphorylation, enzymatically-mediated oxidation and reduction of specific methionine residues functions as a regulatory process in the cell. Methionine residues also form bonds with aromatic residues that contribute significantly to protein stability. Given these important functions, alteration of the methionine–methionine sulfoxide balance in proteins has been correlated with disease processes, including cardiovascular and neurodegenerative diseases. Methionine isn’t just for protein initiation.