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. Progress this year has been severely limited because of the COVID-19 pandemic that required complete shutdown of the laboratory for 4 months, followed by a very limited reopening. Experiments with genetically modified animals are still suspended, and colonies are kept at a minimal population. This will continue until a much higher staffing level is permitted. Planned follow up experiments with mice on the protective effect of knocking out all 4 methionine sulfoxide reductases are thus still suspended. However, we have initiated a new tissue culture model of ischemia-reperfusion with the mouse liver cell line, ALM12. Several hours of anoxia followed by re-oxygenation induced 50% cell death. we are now utilizing CRISPR-cas9 to knockout the reductases. We continue our attempts to identify proteins that interact with the reductases. Utilizing a protein array and probing with methionine sulfoxide reductase A, we identified one high confidence candidate, STARD3. It is a late lysosomal protein located in the lysosomal membrane with a cystosolic domain. It is known to bind lipids and is believed to facilitate transfer of cholesterol from the late endosome to mitochondria and possibly other cellular locations. We confirmed that FLAG-tagged STARD3 and myristoylated msrA interact by overexpressing both in HEK293 cells, immunopreciptating with anti-FLAG, and immunodetecting with anti-msrA. We showed that almost all of the cellular reductase A is bound to the late endosome and is not a cytosolic protein as had been proposed from studies of cells overexpressing the enzyme. We are now focused on elucidating the role of the STARD3-MSRA protein-protein interaction. We continue to collaborate with the Anderson laboratory at Johns Hopkins to investigate the roles of reversible oxidative modification of CAMKII delta in cardiac physiology and pathology. A paper reporting that MICAL specifically oxidizes Met308 and that methionine sulfoxide B reverses oxidation has been published. Our collaborative study with the Balaban laboratory focused on understanding regulation of oxygen consumption and ATP production in Paracoccus denitrificans continues. The organism is difficult to manipulate genetically because of lack of classical targeting vectors and the organism's resistance to CRISPR-cas9. However, a brute force selection method succeeding in generating needed knockout strains and a knockin expressing a pH sensitive fluorimetric probe.