Oxidative Modification Of Proteins

National Heart, Lung, And Blood Institute

Investigators

Linked publications & trials

Abstract

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. In order to elucidate the mechanism of protection against oxidative stress in mice lacking methionine sulfoxide reductases, we elected to create a more tractable model system with the mouse liver cell line, ALM12. An ischemia-reperfusion challenge protocol was established, along with chemical challenges including hydrogen peroxide and paraquat. We have systematically knocked out the 4 methionine sulfoxide reductases, attempting to produce a quadruple knockout. Triple knockouts are viable and do not show resistance to oxidative stress. However, many attempts to make the quadruple knockout have failed to yield viable cells. We conclude that the quadruple knockout in liver cells is lethal, unlike the situation in the intact mouse. We speculate that the difference is a consequence of inter-organ communication and cooperation in the intact animal. We continue our attempts to identify proteins that interact with the reductases. Utilizing a protein array and probing with methionine sulfoxide reductase A, we previously identified one high confidence candidate, STARD3. We have now extended these studies and demonstrated that all 4 methionine sulfoxide reductases bind to STARD3. Cholesterol is bound by and transported to and from organelles by STARD3, and not surprisingly, it also binds cholesterol hydroperoxide, a highly toxic oxidizing agent. We showed that both methionines in STARD3 are oxidized after binding of the hydroperoxide, and that the reductases reduce the resulting methionine sulfoxide back to methionine. The process is catalytic, so that STARD3 and the reductases form an efficient system for detoxification of cholesterol hydroperoxide. We continue to collaborate with the Anderson laboratory at Johns Hopkins to investigate the roles of reversible oxidative modification of CAMKII delta (Calcium-Calmodulin Dependent Protein Kinase II delta) in cardiac physiology and pathology. We extended our studies to revisit the reported mechanism of oxidation, namely the conversion of methionine residues in the regulatory domain to methionine sulfoxide. We found that the proposed mechanism was incorrect. The methionines are not oxidized. Rather, two cysteine residues in the regulatory domain form a disulfide bond and induce autonomous activity in CAMKII delta. We have created a transgenic mouse in which the critical low pKa cysteine is mutated to serine to prevent disulfide formation. We are currently assessing whether this transgenic mouse is protected against deleterious effects of chronic oxidation of the kinase. Our collaborative study with the Balaban laboratory focused on understanding regulation of oxygen consumption and ATP production in Paracoccus denitrificans continues. The bacterium lacks a classical lactate dehydrogenase, yet we found that it vigorously carries out oxidative respiration when grown on lactate as a sole carbon and energy source. We hypothesized that a ubiquinone-dependent lactate dehydrogenase was responsible for ability of the bacterium to utilize lactate. However, when that enzyme was knocked out, the bacterium could still be grown on either D or L lactate. Our efforts to elucidate the metabolic pathway utilized for lactate oxidation are ongoing.