Oxidative Modification Of Proteins

National Heart, Lung, And Blood Institute

Investigators

Linked publications, trials & patents

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. We previously reported the unexpected finding that a mouse genetically engineered to lack all 4 reductases was actually more resistant to oxidative stresses than the wild-type. Multiple efforts to establish the mechanism have not yet done so. Thus, we investigated the potential role of the microbiome in these mice. We found that it does not mediate the resistance. We are now carrying out proteomic and metabolomic characterization of the wild-type and mutant mice. We also elected to create a more tractable model system with the mouse liver cell line, ALM12. An ischemia-reperfusion challenge protocol was established. We conducted a genome-wide CRISPR knockout (KO) screen in AML12 hepatocyte cells using a library targeting 19,674 genes with four single-guide RNAs (sgRNAs) per gene. Functional analysis revealed that inhibition of aminoacyl-tRNA biosynthesis, ferroptosis, and quinone biosynthesis were the top pathways protecting against ischemia-reperfusion injury. Consistent with the implication of ferroptosis, we found that cells were completely protected from death if treated with the iron chelator deferoxamine during the ischemia phase. As is also characteristic of ferroptotic death, there was no DNA damage during ischemia-reperfusion. We continue to investigate the roles of reversible oxidative modification of Calcium-Calmodulin Dependent Protein Kinase II delta in cardiac physiology and pathology. We showed that oxidation causes two cysteine residues in the regulatory domain to form a disulfide bond and induce autonomous activity in Calcium-Calmodulin Dependent Protein Kinase II delta. We have created a transgenic mouse in which the critical low pKa cysteine is mutated to serine to prevent disulfide formation. We hypothesized that the mutation would protect the heart from the deleterious effects of oxidative stress and ischemia-reperfusion. Consistent with the hypothesis, we found that the heart carrying the mutation was protected from ischemia reperfusion injury in the Langendorff model. 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 established that lactate is metabolized by 2 NAD-independent lactate dehydrogenases, one specific for D-lactate and one for L-lactate. Due to the closure of the Balaban Laboratory, this project will be terminated.