Abstract
BackgroundMethionine (Met) residues in proteins can be readily oxidized by reactive oxygen species to Met sulfoxide (MetO). MetO is a promising physiological marker of oxidative stress and its inefficient repair by MetO reductases (Msrs) has been linked to neurodegeneration and aging. Conventional methods of assaying MetO formation and reduction rely on chromatographic or mass spectrometry procedures, but the use of Met-rich proteins (MRPs) may offer a more streamlined alternative.ResultsWe carried out a computational search of completely sequenced genomes for MRPs deficient in cysteine (Cys) residues and identified several proteins containing 20% or more Met residues. We used these MRPs to examine Met oxidation and MetO reduction by in-gel shift assays and immunoblot assays with antibodies generated against various oxidized MRPs. The oxidation of Cys-free MRPs by hydrogen peroxide could be conveniently monitored by SDS-PAGE and was specific for Met, as evidenced by quantitative reduction of these proteins with Msrs in DTT- and thioredoxin-dependent assays. We found that hypochlorite was especially efficient in oxidizing MRPs. Finally, we further developed a procedure wherein antibodies made against oxidized MRPs were isolated on affinity resins containing same or other oxidized or reduced MRPs. This procedure yielded reagents specific for MetO in these proteins, but proved to be ineffective in developing antibodies with broad MetO specificity.ConclusionOur data show that MRPs provide a convenient tool for characterization of Met oxidation, MetO reduction and Msr activities, and could be used for various aspects of redox biology involving reversible Met oxidation.
Highlights
Methionine (Met) residues in proteins can be readily oxidized by reactive oxygen species to Met sulfoxide (MetO)
ORFs encoded in completely sequenced genomes, revealed approximately 170 sequences with more than 20% Met content (10 fold higher Met content than expected by chance based on Met content of proteins in the NCBI database), which were present in organisms ranging from bacteria to mammals
We previously demonstrated that oxidation of Met residues in the first-generation, Cys-containing Met-rich protein (MRP) could be monitored by protein mobility shift using SDS-PAGE gels, and that MetO-containing MRPs migrated slower than the corresponding reduced proteins [18]
Summary
Methionine (Met) residues in proteins can be readily oxidized by reactive oxygen species to Met sulfoxide (MetO). Methionine oxidation results in the formation of a mixture of two diastereomeric forms of methionine sulfoxide (MetO), which can be reduced back to Met by methionine sulfoxide reductases (Msrs) A (MsrA) and B (MsrB). These enzymes play an important role in protection of proteins against oxidative stress and have been implicated in regulation of the aging process. Insufficient repair of oxidized Met by these enzymes may alter protein structure and cause protein aggregation and/or loss of function, accelerating age-associated diseases [1,2,3,4,5,6,7,8,9,10]. Identification of proteins most susceptible to Met oxidation may help better understand age-associated diseases and signaling pathways
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