Metal-Catalyzed Oxidation Of Proteins

National Heart, Lung, And Blood Institute

Investigators

Linked publications, trials & patents

Abstract

For our first specific aim, we have created a series of transgenic mice which either overexpress methionine sulfoxide reductase in specific cellular locations or which lack the enzyme. We have established cell cultures of embryonic fibroblasts from these animals and have challenged them with a series of oxidative stresses and compared their response to that of wild-type fibroblasts. In two collaborative studies with other NIH investigators, we are studying the susceptibility of the transgenic heart to ischemia and reperfusion and of the eye to light-induced oxidative damage. In a separate series of experiments, we directly tested the hypothesis that methionine in proteins functions as a protective antioxidant. We engineered E. coli with decreased content of methionine residues in its proteins and subjected the bacteria to a variety of oxidative stresses. We found that these methionine-deficient cells were much more readily killed by the stresses than their wild-type counterparts.[unreadable] [unreadable] Work on the second specific aim has 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 are employing genetic and biochemical techniques to identify and characterize the proteolytic system responsible for the non-proteasomal turnover.[unreadable] [unreadable] Multiple attempts to demonstrate a specific interaction of mutant superoxide dismutases with the 20S proteasome from rat and cow failed to find such an interaction. We then turned to the much simpler archaebacteria proteosomes, which have only a single alpha and beta subunit rather than the seven present in the mammalian enzyme. This system allowed us to establish the mechanism by which the proteasome selectively degrades the mutant superoxide dismutases. The discrimination is not a property of the proteasome, as we had previously thought. Rather, it is a function of the inherent propensity of the mutants to unfold. The unfolded protein then enters the proteasome through the open gate of the alpha subunits.