For our first specific aim, we created a series of transgenic mice which either overexpress methionine sulfoxide reductase A in specific cellular locations or which lack the enzyme. We established cell cultures of embryonic fibroblasts from these animals and challenged them with a series of oxidative stresses and compared their response to that of wild-type fibroblasts. Notably, there was no difference in resistance among the wild-type, knockout, and overexpressing cell lines. We found that wild-type fibroblasts lack methionine sulfoxide reductase, so that they are phenotypically knockouts. That however, did not explain the failure of overexpression to confer protection against oxidative stress. The enzyme is part of a cycle which requires thioredoxin and thioredoxin reductase to be functional, and we thus investigated the expression of these latter proteins in the fibroblasts. We found that levels were very low in comparison to liver, providing an explanation of the failure of overexpression to confer protection. We are also investigating the hypothesis that methionine sulfoxide reductase is important in nutrition, functioning to salvage the metabolically """"""""expensive"""""""" amino acid methionine by reducing its unusable sulfoxide. Weanling animals with different genotypes are being raised on diets varying in methionine content, and their growth and biochemical parameters are being measured. In a collaborative study with other NHLBI investigators, we are studying the susceptibility of the transgenic heart to ischemia and reperfusion. At the biochemical level, we have discovered that the reductase is myristoylated and are carrying out a variety of studies to elucidate the function of this covalent modification. Thus far we have not documented a role in cellular translocation. We are investigating the effect of myristoylation on enzymatic parameters, protein-protein interaction, and structure. The latter is being determined by NMR. Work on the second specific aim progressed rapidly after the development of very sensitive assays for iron regulatory protein-2. We confirmed the published observation that iron-deficient cells suddenly exposed to high concentrations of iron rapidly degrade iron-regulatory protein-2 through the proteasome pathway. However, when examining the physiological regulation of cells not stressed by exposure to high concentrations of iron, we found that turnover of the protein is not mediated by the proteasome. We have shown that the proteolytic system responsible for the non-proteasomal turnover is calcium-dependent but is not a calpain. Microarray studies were conducted to identify protease candidates, but none were found. Similarly, a battery of protease inhibitors failed to block the non-proteasomal protease. We are now employing gene reporter techniques and affinity chromatography as approaches to identifying the novel protease.

Project Start
Project End
Budget Start
Budget End
Support Year
33
Fiscal Year
2010
Total Cost
$1,591,019
Indirect Cost
Name
National Heart, Lung, and Blood Institute
Department
Type
DUNS #
City
State
Country
Zip Code
Williamson, Kim C; Levine, Rodney L; Miller, Louis H (2018) Even malaria parasites watch their host's diet. Nat Microbiol 3:130-131
Werner-Allen, Jonathan W; Monti, Sarah; DuMond, Jenna F et al. (2018) Isoindole Linkages Provide a Pathway for DOPAL-Mediated Cross-Linking of ?-Synuclein. Biochemistry 57:1462-1474
Lim, Jung Mi; Lim, Jung Chae; Kim, Geumsoo et al. (2018) Myristoylated methionine sulfoxide reductase A is a late endosomal protein. J Biol Chem 293:7355-7366
Lim, Jung Mi; Kim, Geumsoo; Levine, Rodney L (2018) Methionine in Proteins: It's Not Just for Protein Initiation Anymore. Neurochem Res :
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Werner-Allen, Jon W; DuMond, Jenna F; Levine, Rodney L et al. (2016) Toxic Dopamine Metabolite DOPAL Forms an Unexpected Dicatechol Pyrrole Adduct with Lysines of ?-Synuclein. Angew Chem Int Ed Engl 55:7374-8
Salmon, Adam B; Kim, Geumsoo; Liu, Chengyu et al. (2016) Effects of transgenic methionine sulfoxide reductase A (MsrA) expression on lifespan and age-dependent changes in metabolic function in mice. Redox Biol 10:251-256
Kim, Geumsoo; Levine, Rodney L (2016) A Methionine Residue Promotes Hyperoxidation of the Catalytic Cysteine of Mouse Methionine Sulfoxide Reductase A. Biochemistry 55:3586-93

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