One characteristic of the aging process is the accumulation of damaged proteins, lipids, and nucleic acids that in turns cause cellular mal-function and death. Oxidation of methionine to methionine sulfoxide (MetO) is among the first events that may cause permanent protein damage if not properly reversed by the methionine sulfoxide reductase (Msr) system (consisting of MsrA and MsrB enzymes). A compromised performance of the Msr system may result in the accumulation of MetO-containing proteins (MetO-proteins) thereby enhancing the development of disease-state condition, such as in Alzheimer's. Therefore, determining the identity of the targeted proteins prone to methionine oxidation, in vivo, will greatly advance the possibilities in preventing cellular mal-function associated with these damaged proteins. Yet, no simple and reliable assay is available for an efficient identification of methionine-oxidized proteins in vivo; therefore halting the progress in developing new strategies in keeping the normal function of pivotal proteins. In this project, novel antibodies will be developed to specifically bind to MetO-protein. These antibodies will facilitate the identification of oxidized proteins in biological extracts. Preliminary results indicate that injecting oxidized recombinant Zea mays methionine-rich protein (containing 25% methionine residues) into a rabbit resulted in antibodies that can specifically recognize MetO residues in: pure non-homologues proteins, protein extracts of mouse tissues, proteins of yeast and human B-lymphocytes cells exposed to oxidative stress. To better facilitate the identification of cellular MetO-proteins, the use of anti-MetO antibodies in an affinity column and western blot analysis (coupled to biochemical and electrophoresis separations) are proposed. These methods are expected to enrich and purify MetO- proteins from cellular extracts that will be then identified by mass-spectrometry techniques. Yeast and mouse null mutants of the MsrA gene have shown to accumulate MetO-proteins as a consequence of oxidative stress and prolonged selenium deficiency through aging, respectively. Consequently, protein extracts of these organisms' cells/tissues (subjected to these conditions) will be used as a source for the isolation of the MetO-containing proteins. Identification of MetO- proteins in vivo may enhance the development of new preventive treatments for oxidative-stress and aging-related diseases by protecting the identified targeted proteins from oxidative damage. ? ? ?
