Methionine sulfoxide reductases and cholesterol transporter STARD3 constitute an efficient system for detoxification of cholesterol hydroperoxides.

Methionine sulfoxide reductases (MSRs) are key enzymes in the cellular oxidative defense system. Reactive oxygen species oxidize methionine residues to methionine sulfoxide, and the methionine sulfoxide reductases catalyze their reduction back to methionine. We previously identified the cholesterol transport protein STARD3 as an in vivo binding partner of MSRA (methionine sulfoxide reductase A), an enzyme that reduces methionine-S-sulfoxide back to methionine. We hypothesized that STARD3 would also bind the cytotoxic cholesterol hydroperoxides and that its two methionine residues, Met307 and Met427, could be oxidized, thus detoxifying cholesterol hydroperoxide. We now show that in addition to binding MSRA, STARD3 binds all three MSRB (methionine sulfoxide reductase B), enzymes that reduce methionine-R-sulfoxide back to methionine. Using pure 5, 6, and 7 positional isomers of cholesterol hydroperoxide, we found that both Met307 and Met427 on STARD3 are oxidized by 6α-hydroperoxy-3ß-hydroxycholest-4-ene (cholesterol-6α-hydroperoxide) and 7α-hydroperoxy-3ß-hydroxycholest-5-ene (cholesterol-7α-hydroperoxide). MSRs reduce the methionine sulfoxide back to methionine, restoring the ability of STARD3 to bind cholesterol. Thus, the cyclic oxidation and reduction of methionine residues in STARD3 provides a catalytically efficient mechanism to detoxify cholesterol hydroperoxide during cholesterol transport, protecting membrane contact sites and the entire cell against the toxicity of cholesterol hydroperoxide.

Lactate oxidation in Paracoccus denitrificans.

Paracoccus denitrificans has a classical cytochrome-dependent electron transport chain and two alternative oxidases. The classical transport chain is very similar to that in eukaryotic mitochondria. Thus, P. denitrificans can serve as a model of the mammalian mitochondrion that may be more tractable in elucidating mechanisms of regulation of energy production than are mitochondria. In a previous publication we reported detailed studies on respiration in P. denitrificans grown aerobically on glucose or malate. We noted that P. denitrificans has large stores of lactate under various growth conditions. This is surprising because P. denitrificans lacks an NAD+-dependent lactate dehydrogenase. The aim of this study was to investigate the mechanisms of lactate oxidation in P. denitrificans. We found that the bacterium grows well on either d-lactate or l-lactate. Growth on lactate supported a rate of maximum respiration that was equal to that of cells grown on glucose or malate. We report proteomic, metabolomic, and biochemical studies that establish that the metabolism of lactate by P. denitrificans is mediated by two non-NAD+-dependent lactate dehydrogenases. One prefers d-lactate over l-lactate (D-iLDH) and the other prefers l-lactate (L-iLDH). We cloned and produced the D-iLDH and characterized it. The Km for d-lactate was 34 µM, and for l-lactate it was 3.7 mM. Pyruvate was not a substrate, rendering the reaction unidirectional with lactate being converted to pyruvate for entry into the TCA cycle. The intracellular lactate was ∼14 mM such that both isomers could be metabolized by the enzyme. The enzyme has 1 FAD per molecule and utilizes a quinone rather than NAD + as an electron acceptor. D-iLDH provides a direct entry of lactate reducing equivalents into the cytochrome chain, potentially explaining the high respiratory capacity of P. denitrificans in the presence of lactate.

Oxidative stress-induced autonomous activation of the calcium/calmodulin-dependent kinase II involves disulfide formation in the regulatory domain.

Calcium/calmodulin-dependent protein kinase II δ (CaMKIIδ) has a pivotal role in cardiac signaling. Constitutive and deleterious CaMKII "autonomous" activation is induced by oxidative stress, and the previously reported mechanism involves oxidation of methionine residues in the regulatory domain. Here, we demonstrate that covalent oxidation leads to a disulfide bond with Cys273 in the regulatory domain causing autonomous activity. Autonomous activation was induced by treating CaMKII with diamide or histamine chloramine, two thiol-oxidizing agents. Autonomy was reversed when the protein was incubated with DTT or thioredoxin to reduce disulfide bonds. Tryptic mapping of the activated CaMKII revealed formation of a disulfide between Cys273 and Cys290 in the regulatory domain. We determined the apparent pKa of those Cys and found that Cys273 had a low pKa while that of Cys290 was elevated. The low pKa of Cys273 facilitates oxidation of its thiol to the sulfenic acid at physiological pH. The reactive sulfenic acid then attacks the thiol of Cys290 to form the disulfide. The previously reported CaMKII mutant in which methionine residues 281 and 282 were mutated to valine (MMVV) protects mice and flies from cardiac decompensation induced by oxidative stress. Our initial hypothesis was that the MMVV mutant underwent a conformational change that prevented disulfide formation and autonomous activation. However, we found that the thiol-oxidizing agents induced autonomy in the MMVV mutant and that the mutant undergoes rapid degradation by the cell, potentially preventing accumulation of the injurious autonomous form. Together, our results highlight additional mechanistic details of CaMKII autonomous activation.

Repurposing the Pummerer Rearrangement: Determination of Methionine Sulfoxides in Peptides.

The reversible oxidation of methionine residues in proteins has emerged as a biologically important post-translational modification. However, detection and quantitation of methionine sulfoxide in proteins is difficult. Our aim is to develop a method for specifically derivatizing methionine sulfoxide residues. We report a Pummerer rearrangement of methionine sulfoxide treated sequentially with trimethylsilyl chloride and then 2-mercaptoimidazole or pyridine-2-thiol to produce a dithioacetal product. This derivative is stable to standard mass spectrometry conditions, and its formation identified oxidized methionine residues. The scope and requirements of dithioacetal formation are reported for methionine sulfoxide and model substrates. The reaction intermediates have been investigated by computational techniques and by 13 C NMR spectroscopy. These provide evidence for an α-chlorinated intermediate. The derivatization allows for detection and quantitation of methionine sulfoxide in proteins by mass spectrometry and potentially by immunochemical methods.

Myristoylated methionine sulfoxide reductase A is a late endosomal protein.

Methionine residues in proteins provide antioxidant defense by reacting with oxidizing species, which oxidize methionine to methionine sulfoxide. Reduction of the sulfoxide back to methionine is catalyzed by methionine sulfoxide reductases, essential for protection against oxidative stress. The nonmyristoylated form of methionine sulfoxide reductase A (MSRA) is present in mitochondria, whereas the myristoylated form has been previously reported to be cytosolic. Despite the importance of MSRA in antioxidant defense, its in vivo binding partners and substrates have not been identified. Starting with a protein array, and followed by immunoprecipitation experiments, colocalization studies, and subcellular fractionation, we identified the late endosomal protein, StAR-related lipid transfer domain-containing 3 (STARD3), as a binding partner of myristoylated MSRA, but not of nonmyristoylated MSRA. STARD3 is known to have both membrane-binding and cytosolic domains that are important in STARD3-mediated transport of cholesterol from the endoplasmic reticulum to the endosome. We found that the STARD3 cytosolic domain localizes MSRA to the late endosome. We propose that the previous conclusion that myristoylated MSRA is strictly a cytosolic protein is artifactual and likely due to vigorous overexpression of MSRA. We conclude that myristoylated MSRA is a late endosomal protein that may play a role in lipid metabolism or may protect endosomal proteins from oxidative damage.

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.

Isoindole Linkages Provide a Pathway for DOPAL-Mediated Cross-Linking of α-Synuclein.

3,4-Dihydroxyphenylacetaldehyde (DOPAL) is a toxic and reactive product of dopamine catabolism. In the catecholaldehyde hypothesis for Parkinson's disease, it is a critical driver of the selective loss of dopaminergic neurons that characterizes the disease. DOPAL also cross-links α-synuclein, the main component of Lewy bodies, which are a pathological hallmark of the disease. We previously described the initial adduct formed in reactions between DOPAL and α-synuclein, a dicatechol pyrrole lysine (DCPL). Here, we examine the chemical basis for DOPAL-based cross-linking. We find that autoxidation of DCPL's catechol rings spurs its decomposition, yielding an intermediate dicatechol isoindole lysine (DCIL) product formed by an intramolecular reaction of the two catechol rings to give an unstable tetracyclic structure. DCIL then reacts with a second DCIL to give a dimeric, di-DCIL. This product is formed by an intermolecular carbon-carbon bond between the isoindole rings of the two DCILs that generates two structurally nonequivalent and separable atropisomers. Using α-synuclein, we demonstrate that the DOPAL-catalyzed formation of oligomers can be separated into two steps. The initial adduct formation occurs robustly within an hour, with DCPL as the main product, and the second step cross-links α-synuclein molecules. Exploiting this two-stage reaction, we use an isotopic labeling approach to show the predominant cross-linking mechanism is an interadduct reaction. Finally, we confirm that a mass consistent with a di-DCIL linkage can be observed in dimeric α-synuclein by mass spectrometry. Our work elucidates previously unknown pathways of catechol-based oxidative protein damage and will facilitate efforts to detect DOPAL-based cross-links in disease-state neurons.

Superoxide is the critical driver of DOPAL autoxidation, lysyl adduct formation, and crosslinking of α-synuclein.

Parkinson's disease has long been associated with redox imbalance and oxidative stress in dopaminergic neurons. The catecholaldehyde hypothesis proposes that 3,4-dihydroxyphenylacetaldehyde (DOPAL), an obligate product of dopamine catabolism, is a central nexus in a network of pathways leading to disease-state neurodegeneration, owing to its toxicity and potent ability to oligomerize α-synuclein, the main component of protein aggregates in Lewy bodies. In this work we examine the connection between reactive oxygen species and DOPAL autoxidation. We show that superoxide propagates a chain reaction oxidation, and that this reaction is dramatically inhibited by superoxide dismutase. Moreover, superoxide dismutase prevents DOPAL from forming dicatechol pyrrole adducts with lysine and from covalently crosslinking α-synuclein. Given that superoxide is a major radical byproduct of impaired cellular respiration, our results provide a possible mechanistic link between mitochondrial dysfunction and synuclein aggregation in dopaminergic neurons.

Lecithin:Cholesterol Acyltransferase Activation by Sulfhydryl-Reactive Small Molecules: Role of Cysteine-31.

Lecithin:cholesterol acyltransferase (LCAT) catalyzes plasma cholesteryl ester formation and is defective in familial lecithin:cholesterol acyltransferase deficiency (FLD), an autosomal recessive disorder characterized by low high-density lipoprotein, anemia, and renal disease. This study aimed to investigate the mechanism by which compound A [3-(5-(ethylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile], a small heterocyclic amine, activates LCAT. The effect of compound A on LCAT was tested in human plasma and with recombinant LCAT. Mass spectrometry and nuclear magnetic resonance were used to determine compound A adduct formation with LCAT. Molecular modeling was performed to gain insight into the effects of compound A on LCAT structure and activity. Compound A increased LCAT activity in a subset (three of nine) of LCAT mutations to levels comparable to FLD heterozygotes. The site-directed mutation LCAT-Cys31Gly prevented activation by compound A. Substitution of Cys31 with charged residues (Glu, Arg, and Lys) decreased LCAT activity, whereas bulky hydrophobic groups (Trp, Leu, Phe, and Met) increased activity up to 3-fold (P < 0.005). Mass spectrometry of a tryptic digestion of LCAT incubated with compound A revealed a +103.017 m/z adduct on Cys31, consistent with the addition of a single hydrophobic cyanopyrazine ring. Molecular modeling identified potential interactions of compound A near Cys31 and structural changes correlating with enhanced activity. Functional groups important for LCAT activation by compound A were identified by testing compound A derivatives. Finally, sulfhydryl-reactive ß-lactams were developed as a new class of LCAT activators. In conclusion, compound A activates LCAT, including some FLD mutations, by forming a hydrophobic adduct with Cys31, thus providing a mechanistic rationale for the design of future LCAT activators.

A Methionine Residue Promotes Hyperoxidation of the Catalytic Cysteine of Mouse Methionine Sulfoxide Reductase A.

Methionine sulfoxide reductase A (msrA) reduces methionine sulfoxide in proteins back to methionine. Its catalytic cysteine (Cys72-SH) has a low pKa that facilitates oxidation by methionine sulfoxide to cysteine sulfenic acid. If the catalytic cycle proceeds efficiently, the sulfenic acid is reduced back to cysteine at the expense of thioredoxin. However, the sulfenic acid is vulnerable to "irreversible" oxidation to cysteine sulfinic acid that inactivates msrA (hyperoxidation). We observed that human msrA is resistant to hyperoxidation while mouse msrA is readily hyperoxidized by micromolar concentrations of hydrogen peroxide. We investigated the basis of this difference in susceptibility to hyperoxidation and established that it is controlled by the presence or absence of a Met residue in the carboxyl-terminal domain of the enzyme, Met229. This residue is Val in human msrA, and when it was mutated to Met, human msrA became sensitive to hyperoxidation. Conversely, mouse msrA was rendered insensitive to hyperoxidation when Met229 was mutated to Val or one of five other residues. Positioning of the methionine at residue 229 is not critical, as hyperoxidation occurred as long as the methionine was located within the group of 14 carboxyl-terminal residues. The carboxyl domain of msrA is known to be flexible and to have access to the active site, and Met residues are known to form stable, noncovalent bonds with aromatic residues through interaction of the sulfur atom with the aromatic ring. We propose that Met229 forms such a bond with Trp74 at the active site, preventing formation of a protective sulfenylamide with Cys72 sulfenic acid. As a consequence, the sulfenic acid is available for facile, irreversible oxidation to cysteine sulfinic acid.

Nonenzymatic conversion of ADP-ribosylated arginines to ornithine alters the biological activities of human neutrophil peptide-1.

Activated neutrophils, recruited to the airway of diseased lung, release human neutrophil peptides (HNP1-4) that are cytotoxic to airway cells as well as microbes. Airway epithelial cells express arginine-specific ADP ribosyltransferase (ART)-1, a GPI-anchored ART that transfers ADP-ribose from NAD to arginines 14 and 24 of HNP-1. We previously reported that ADP-ribosyl-arginine is converted nonenzymatically to ornithine and that ADP-ribosylated HNP-1 and ADP-ribosyl-HNP-(ornithine) were isolated from bronchoalveolar lavage fluid of a patient with idiopathic pulmonary fibrosis, indicating that these reactions occur in vivo. To determine effects of HNP-ornithine on the airway, three analogs of HNP-1, HNP-(R14orn), HNP-(R24orn), and HNP-(R14,24orn), were tested for their activity against Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus; their cytotoxic effects on A549, NCI-H441, small airway epithelial-like cells, and normal human lung fibroblasts; and their ability to stimulate IL-8 and TGF-ß1 release from A549 cells, and to serve as ART1 substrates. HNP and the three analogs had similar effects on IL-8 and TGF-ß1 release from A549 cells and were all cytotoxic for small airway epithelial cells, NCI-H441, and normal human lung fibroblasts. HNP-(R14,24orn), when compared with HNP-1 and HNP-1 with a single ornithine substitution for arginine 14 or 24, exhibited reduced cytotoxicity, but it enhanced proliferation of A549 cells and had antibacterial activity. Thus, arginines 14 and 24, which can be ADP ribosylated by ART1, are critical to the regulation of the cytotoxic and antibacterial effects of HNP-1. The HNP analog, HNP-(R14,24orn), lacks the epithelial cell cytotoxicity of HNP-1, but partially retains its antibacterial activity and thus may have clinical applications in airway disease.

Regulation of the actin-activated MgATPase activity of Acanthamoeba myosin II by phosphorylation of serine 639 in motor domain loop 2.

It had been proposed previously that only filamentous forms of Acanthamoeba myosin II have actin-activated MgATPase activity and that this activity is inhibited by phosphorylation of up to four serine residues in a repeating sequence in the C-terminal nonhelical tailpiece of the two heavy chains. We have reinvestigated these issues using recombinant WT and mutant myosins. Contrary to the earlier proposal, we show that two nonfilamentous forms of Acanthamoeba myosin II, heavy meromyosin and myosin subfragment 1, have actin-activated MgATPase that is down-regulated by phosphorylation. By mass spectroscopy, we identified five serines in the heavy chains that can be phosphorylated by a partially purified kinase preparation in vitro and also are phosphorylated in endogenous myosin isolated from the amoebae: four serines in the nonhelical tailpiece and Ser639 in loop 2 of the motor domain. S639A mutants of both subfragment 1 and full-length myosin had actin-activated MgATPase that was not inhibited by phosphorylation of the serines in the nonhelical tailpiece or their mutation to glutamic acid or aspartic acid. Conversely, S639D mutants of both subfragment 1 and full-length myosin were inactive, irrespective of the phosphorylation state of the serines in the nonhelical tailpiece. To our knowledge, this is the first example of regulation of the actin-activated MgATPase activity of any myosin by modification of surface loop 2.

Toxic Dopamine Metabolite DOPAL Forms an Unexpected Dicatechol Pyrrole Adduct with Lysines of α-Synuclein.

Parkinson's disease has long been known to involve the loss of dopaminergic neurons in the substantia nigra and the coincidental appearance of Lewy bodies containing oligomerized forms of α-synuclein. The "catecholaldehyde hypothesis" posits a causal link between these two central pathologies mediated by 3,4-dihydroxyphenylacetaldehyde (DOPAL), the most toxic dopamine metabolite. Here we determine the structure of the dominant product in reactions between DOPAL and α-synuclein, a dicatechol pyrrole lysine adduct. This novel modification results from the addition of two DOPAL molecules to the Lys sidechain amine through their aldehyde moieties and the formation of a new carbon-carbon bond between their alkyl chains to generate a pyrrole ring. The product is detectable at low concentrations of DOPAL and its discovery should provide a valuable chemical basis for future studies of DOPAL-induced crosslinking of α-synuclein.

Methionine oxidation and reduction in proteins.

BACKGROUND: Cysteine and methionine are the two sulfur containing amino acids in proteins. While the roles of protein-bound cysteinyl residues as endogenous antioxidants are well appreciated, those of methionine remain largely unexplored. SCOPE: We summarize the key roles of methionine residues in proteins. MAJOR CONCLUSION: Recent studies establish that cysteine and methionine have remarkably similar functions. GENERAL SIGNIFICANCE: Both cysteine and methionine serve as important cellular antioxidants, stabilize the structure of proteins, and can act as regulatory switches through reversible oxidation and reduction. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.

Methionine sulfoxide reductase A is a stereospecific methionine oxidase.

Methionine sulfoxide reductase A (MsrA) catalyzes the reduction of methionine sulfoxide to methionine and is specific for the S epimer of methionine sulfoxide. The enzyme participates in defense against oxidative stresses by reducing methionine sulfoxide residues in proteins back to methionine. Because oxidation of methionine residues is reversible, this covalent modification could also function as a mechanism for cellular regulation, provided there exists a stereospecific methionine oxidase. We show that MsrA itself is a stereospecific methionine oxidase, producing S-methionine sulfoxide as its product. MsrA catalyzes its own autooxidation as well as oxidation of free methionine and methionine residues in peptides and proteins. When functioning as a reductase, MsrA fully reverses the oxidations which it catalyzes.

Characterization and solution structure of mouse myristoylated methionine sulfoxide reductase A.

Methionine sulfoxide reductase A is an essential enzyme in the antioxidant system which scavenges reactive oxygen species through cyclic oxidation and reduction of methionine and methionine sulfoxide. The cytosolic form of the enzyme is myristoylated, but it is not known to translocate to membranes, and the function of myristoylation is not established. We compared the biochemical and biophysical properties of myristoylated and nonmyristoylated mouse methionine sulfoxide reductase A. These were almost identical for both forms of the enzyme, except that the myristoylated form reduced methionine sulfoxide in protein much faster than the nonmyristoylated form. We determined the solution structure of the myristoylated protein and found that the myristoyl group lies in a relatively surface exposed "myristoyl nest." We propose that this structure functions to enhance protein-protein interaction.

A low pKa cysteine at the active site of mouse methionine sulfoxide reductase A.

Methionine sulfoxide reductase A is an essential enzyme in the antioxidant system which scavenges reactive oxygen species through cyclic oxidation and reduction of methionine and methionine sulfoxide. Recently it has also been shown to catalyze the reverse reaction, oxidizing methionine residues to methionine sulfoxide. A cysteine at the active site of the enzyme is essential for both reductase and oxidase activities. This cysteine has been reported to have a pK(a) of 9.5 in the absence of substrate, decreasing to 5.7 upon binding of substrate. Using three independent methods, we show that the pK(a) of the active site cysteine of mouse methionine sulfoxide reductase is 7.2 even in the absence of substrate. The primary mechanism by which the pK(a) is lowered is hydrogen bonding of the active site Cys-72 to protonated Glu-115. The low pK(a) renders the active site cysteine susceptible to oxidation to sulfenic acid by micromolar concentrations of hydrogen peroxide. This characteristic supports a role for methionine sulfoxide reductase in redox signaling.

Site-specific interaction between α-synuclein and membranes probed by NMR-observed methionine oxidation rates.

α-Synuclein (αS) is an intrinsically disordered protein that is water-soluble but also can bind negatively charged lipid membranes while adopting an α-helical conformation. Membrane affinity is increased by post-translational N-terminal acetylation, a common modification in all eukaryotic cells. In the presence of lipid vesicles containing a small fraction of peroxidized lipids, the N-terminal Met residues in αS (Met1 and Met5) rapidly oxidize while reducing the toxic lipid hydroperoxide to a nonreactive lipid hydroxide, whereas C-terminal Met residues remain unaffected. Met oxidation can be probed conveniently and quantitatively by NMR spectroscopy. The results show that oxidation of Met1 reduces the rate of oxidation of Met5 and vice versa as a result of decreased membrane affinity of the partially oxidized protein. The effect of Met oxidation on the αS-membrane affinity extends over large distances, as in the V49M mutant, oxidation of Met1 and Met5 strongly impacts the oxidation rate of Met49 and vice versa. When not bound to membrane, oxidized Met1 and Met5 of αS are excellent substrates for methionine sulfoxide reductase (Msr), thereby providing an efficient vehicle for water-soluble Msr enzymes to protect the membrane against oxidative damage.

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.

Ndufaf2, a protein in mitochondrial complex I, interacts in vivo with methionine sulfoxide reductases.

BACKGROUND: Methionine sulfoxide reductases are found in all aerobic organisms. They function in antioxidant defense, cellular regulation by reversible oxidation of methionine in proteins, and in protein structure. However, very few in vivo binding partners or substrates of the reductases have been identified. METHODS: We implemented a proximity labeling method, TurboID, to covalently link mitochondrial methionine sulfoxide reductase A (MSRA) to its binding partners in HEK293 cells. Proteomic analyses were performed to identify putative binding partners. RESULTS: We show that human Ndufaf2, also called mimitin, is a binding partner of MSRA as well as all 3 MSRBs. We found that both methionine residues in Ndufaf2 were susceptible to oxidation by hydrogen peroxide and that the methionine sulfoxide reductases can reduce these methionine sulfoxide residues back to methionine. CONCLUSION: Methionine sulfoxide reductases can reduce methionine sulfoxide back to methionine in Ndufaf2. In addition to a repair function, it also creates a mechanism that could regulate cellular processes by modulation of methionine oxidation in Ndufaf2.