Biosensor-Linked Immunosorbent Assay for the Quantification of Methionine Oxidation in Target Proteins.

Methionine oxidation is involved in regulating the protein activity and often leads to protein malfunction. However, tools for quantitative analyses of protein-specific methionine oxidation are currently unavailable. In this work, we developed a biological sensor that quantifies oxidized methionine in the form of methionine-R-sulfoxide in target proteins. The biosensor "tpMetROG" consists of methionine sulfoxide reductase B (MsrB), circularly permuted yellow fluorescent protein (cpYFP), thioredoxin, and protein G. Protein G binds to the constant region of antibodies against target proteins, specifically capturing them. Then, MsrB reduces the oxidized methionine in these proteins, leading to cpYFP fluorescence changes. We assessed this biosensor for quantitative analysis of methionine-R-sulfoxide in various proteins, such as calmodulin, IDLO, LegP, Sacde, and actin. We further developed an immunosorbent assay using the biosensor to quantify methionine oxidation in specific proteins such as calmodulin in animal tissues. The biosensor-linked immunosorbent assay proves to be an indispensable tool for detecting methionine oxidation in a protein-specific manner. This is a versatile tool for studying the redox biology of methionine oxidation in proteins.

Structural and kinetic insights into flavin-containing monooxygenase and calponin-homology domains in human MICAL3.

MICAL is an oxidoreductase that participates in cytoskeleton reorganization via actin disassembly in the presence of NADPH. Although three MICALs (MICAL1, MICAL2 and MICAL3) have been identified in mammals, only the structure of mouse MICAL1 has been reported. Here, the first crystal structure of human MICAL3, which contains the flavin-containing monooxygenase (FMO) and calponin-homology (CH) domains, is reported. MICAL3 has an FAD/NADP-binding Rossmann-fold domain for mono-oxygenase activity like MICAL1. The FMO and CH domains of both MICAL3 and MICAL1 are highly similar in structure, but superimposition of the two structures shows a different relative position of the CH domain in the asymmetric unit. Based on kinetic analyses, the catalytic efficiency of MICAL3 dramatically increased on adding F-actin only when the CH domain was available. However, this did not occur when two residues, Glu213 and Arg530, were mutated in the FMO and CH domains, respectively. Overall, MICAL3 is structurally highly similar to MICAL1, which suggests that they may adopt the same catalytic mechanism, but the difference in the relative position of the CH domain produces a difference in F-actin substrate specificity.

Expression of the methionine sulfoxide reductase lost during evolution extends Drosophila lifespan in a methionine-dependent manner.

Accumulation of oxidized amino acids, including methionine, has been implicated in aging. The ability to reduce one of the products of methionine oxidation, free methionine-R-sulfoxide (Met-R-SO), is widespread in microorganisms, but during evolution this function, conferred by the enzyme fRMsr, was lost in metazoa. We examined whether restoration of the fRMsr function in an animal can alleviate the consequences of methionine oxidation. Ectopic expression of yeast fRMsr supported the ability of Drosophila to catalyze free Met-R-SO reduction without affecting fecundity, food consumption, and response to starvation. fRMsr expression also increased resistance to oxidative stress. Moreover, it extended lifespan of flies in a methionine-dependent manner. Thus, expression of an oxidoreductase lost during evolution can enhance metabolic and redox functions and lead to an increase in lifespan in an animal model. More broadly, our study exposes the potential of a combination of genetic and nutritional strategies in lifespan control.