While the roles of cysteine as an antioxidant and in cell signaling are widely appreciated, only recently has it been recognized that methionine, like cysteine, functions as an antioxidant and as a key component of a system for regulation of cellular metabolism. The efficiency of methionine as an antioxidant or as a component of signaling systems depends on its ready interconversion between the reduced form (methionine) and the oxidized form (methionine sulfoxide). Methionine sulfoxide reductase catalyzes the reduction of methionine sulfoxide back to methionine. The major focus of our research in the last year continues to be the identification of proteins that interact with methionine sulfoxide reductase A in vivo. Our concerted effort with a variety of techniques over several years had not yielded candidates. This year, among other approaches, we utilized a human protein array with almost 10,000 recombinant proteins to screen for interacting proteins. It identified one strong candidate, STARD3, which is a late endosomal/lysosomal protein. Using 3 other independent techniques, we confirmed that the interaction is real. STARD3 is an important protein in intracellular cholesterol transport, and elucidating the effect of interaction with methionine sulfoxide reductase is now a major specific aim. We have also created a knock-in mouse that expresses calmodulin-1 mutated from methionine to glutamine at position 77. This was done because methionine sulfoxide reductase A stereospeifically oxidizes methionine 77, and glutamine is an analogue of methionine sulfoxide. We completed a phenotypic characterization of the mutant mouse, especially its cardiovascular and neurobehavioral phenotypes. While no cardiovascular changes were identified, the mutant clearly differs from its wild-type control in being much more active and less fearful. A major limitation in the field is the lack of a specific, sensitive, convenient assay of methionine sulfoxide in proteins. To this end, we have collaborated with chemists in the Imaging Probe Development Center to carry out a detailed study of the Pummerer reaction as a means to developing such an assay. This is in progress. Another major focus is to elucidate the mechanism by which alpha-synuclein forms covalent oligomers. Our on-going collaboration with the laboratory of Ad Bax continues to increase our understanding of the novel derivatives that arise from the interaction of DOPAL with lysine residues in alpha-synuclein. Our current emphasis is to develop specific assays to detect and quantitate these novel derivatives in vivo.