Research in the Section on Enzymes in the Laboratory of Biochemistry, NHLBI, is directed toward elucidation of basic mechanisms involved in the production of cellular damage during exposure to oxidative stress and the contributions of such damage to aging and disease. To this end, our current research involves studies in the following areas of exploration: (a) Metal-catalyzed oxidation of proteins in aging and disease. Previous studies in this laboratory led to the discovery that proteins are highly susceptible to metal-catalyzed oxidation and that this oxidation leads to conversion of the side chains of some amino acid residues to carbonyl derivatives. Based on this finding, the carbonyl content of protein has become a widely used marker of oxidative stress-mediated cellular damage and has led to the demonstration that the accumulation of oxidized protein is associated with aging and a number of age-related diseases. Taking advantage of sophisticated mass spectroscopic and high pressure liquid chromatographic technologies, we have now developed procedures for the identification and assay of those protein carbonyl derivatives known to be formed by metal-catalyzed reactions. Results of these studies demonstrate that oxidation of lysine, arginine, and proline residues of proteins account for at least 50% of the protein carbonyl groups in liver from old rats. (b) Antioxidant role of methionine residues of proteins. Surface-exposed methionine residues of proteins are highly susceptible to oxidation by almost every kind of reactive oxygen species (ROS). But, unlike other kinds of protein oxidation (except the oxidation of cysteine residues), the oxidation of methionine residues of proteins can be repaired by the action of methionine sulfoxide reductase that catalyzes the thioredoxin-dependent reduction of methionine sulfoxide back to methionine. Because the overall oxidation-reduction of methionine residues of proteins leads to conversion of various forms of ROS to unreactive products, we proposed that the cyclic oxidation/reduction of methionine residues of proteins may constitute an important antioxidant mechanism of cellular defense. To test this hypothesis, a mutant strain of mice was developed that lacked the dominant form of methionine sulfoxide reductase. Compared to the wild-type parental strain, the mutant exhibits enhanced sensitivity to oxidative stress (exposure to 100% oxygen), has a shorter life span under both normal and hyperoxic conditions, develops an atypical (tip-toe) walking behavior after six months of age, accumulated higher levels of oxidized protein (carbonyl derivatives) under oxidative stress, and exhibits abnormal patterns of expression of thioredoxin reductase under conditions of oxidative stress. Thus, it appears that methionine sulfoxide reductase may play an important role in aging and neurological disorders. (c) Oxidation of the prion protein. Studies on the oxidation of Syrian hamster SHa(29-231) prion protein were initiated because this protein binds copper with high affinity and could therefore be highly susceptible to metal-catalyzed oxidation. Indeed, exposure of the prion protein to the ascorbate/oxygen/copper mixed function oxidation system led to rapid oxidation of the protein and to its aggregation, similar to that observed during conversion of the prion protein to its pathogenic counterpart. Because the prion protein contains numerous surface-exposed methionine residues, structural changes associated with the oxidation of these residues is also under investigation. It was established that exposure of the protein to hydrogen peroxide in the absence of copper leads to rapid oxidation of methionine residues 109 and 112, which are known to be essential for the properties of the toxic peptide, the fibrillogenic prion fragment PrP 106-126. Several other residues, including Met 129, were also oxidized. In contrast to the metal-catalyzed oxidation, this oxidation did not result in aggregation. (d) Regulation of methionine sulfoxide reductase transcription. Results of studies described in the above section and results of earlier studies in this laboratory with yeast and bacteria demonstrate that methionine sulfoxide reductase serves an important biological function as an antioxidant under conditions of oxidative stress. To identify which proteins are involved in the regulation of methionine sulfoxide reductase gene (msrA) transcription, nuclear proteins were isolated from both wild-type and null mutant strains of yeast and their ability to bind msrA promoter DNA was determined by electrophoretic mobility shift assays. By using this technique, several proteins that are candidates for a role in msrA transcription have been detected. Some of these have been cloned and antibodies are being prepared to confirm their binding specificities. Further studies are needed to establish their roles, if any, in msrA transcription. (e) Oxidation of methionine residues by hypochlorous acid. The biosynthesis of hyporchlorous acid by neutrophils and macrophages represents a major mechanism for antibacterial action in mammals. Hypochlorous acid is also able to oxidize methionine residues of proteins. Results of preliminary studies indicate that oxidation of free methionine by hypochlorous acid proceeds by an oxygen-independent mechanism in which chloramine derivatives are intermediates. However, if the alpha-amino group of methionine is acylated, as occurs in proteins, then the oxidation proceeds by a mechanism that does not involve a chloramine intermediate. Further studies are designed to determine whether the oxidation of methionine residues in proteins involves direct transfer of oxygen from hypochlorous acid to form methionine sulfoxide or if it involves interactions with water or molecular oxygen. (f) Role of reactive oxygen species in apoptosis. The activation of one or more proteases (caspases) is fundamental to the elimination of damaged (non-functional) cells in animal tissues by a process referred to as apoptosis. We demonstrated previously that activation of caspase-3 like activity of HeLa cells is induced by hydrogen peroxide and that this induction is inhibited by a general caspase inhibitor and also a caspase-3 specific inhibitor. Results of current investigations indicate that the caspase-3 activation does not involve either the caspase-9 (mitochondrial-dependent) or the caspase-8 (death receptor-dependent) mechanism, but may involve an actin-dependent focal adhesion process. Further studies are designed to confirm this possibility. (g) Manganese-induced apoptosis. We reported earlier that at high concentrations manganese (Mn) induces apoptosis by a non-mitochondrial-mediated mechanism. In continuing studies, we have demonstrated that Mn-induced activation of caspase-3 like activity in 3T3 cells is suppressed by calpain inhibitors I, II, and by the p38 inhibitor, SB 202190. However, after activation has occurred, these inhibitors have no effect on caspase-3 like activity. Further studies are directed toward explanation of the linkages between p38, calpain, and caspase-12 in Mn-induced apoptosis.