| The mitochondrial-localized manganese superoxide dismutase (MnSOD) is a critical component of the cellular defense system providing protection from harmful, and potentially toxic reactive oxygen species. My thesis has dealt with two facets of MnSOD biology, (1) the identification and characterization of an element within the MnSOD mRNA that is responsible for message turnover, and (2) the functional evaluation of both wildtype and a series of active site-mutants in response to pro-apototic stimuli. I have performed stimulus washout experiments designed to determine a 4 h half-life of the endogenous MnSOD mRNA. By utilizing a transient transfection system, I was able to localize the RNA stability element to approximately 282 nt of the coding region of the MnSOD mRNA. RNA electrophoretic mobility shift assays and northwestern blot analyses have demonstrated interactions between the RNA stability element and at least four cytosolic factors.; I have also demonstrated the ability of MnSOD to protect against a nephrotoxic dose of the chemotherapeutic agent cisplatin, CP. By showing that overexpression of a mitochondrial-targeted catalase enzyme does not protect against CP, I have demonstrated in vivo, that the mechanism of CP-mediated cytotoxicity is at the level of superoxide radical generation. Additionally, I have evaluated the in vivo consequences of active site mutations within MnSOD. These results have provided interesting insights into the mechanism of MnSOD-mediated cytoprotection. This work will not only add to the body of knowledge of the molecular regulation of MnSOD, but may provide key insights into the possibilities of targeted gene delivery to tissues in variety of pathophysiologies. |
