Date of Award

1997

Degree Name

Biomedical Sciences

College

Joan C. Edwards School of Medicine

Type of Degree

Ph.D.

Document Type

Dissertation

First Advisor

Todd Green

Second Advisor

Carl Gruetter

Third Advisor

Michael Moore

Fourth Advisor

Richard Niles

Fifth Advisor

Vernon Reichenbecher

Sixth Advisor

Leonard J. Deutsch

Abstract

The mitochondrion imports and processes the vast majority of the proteins which comprise its structural elements and metabolic pathways. Studies in the last decade have been successful at defining the mechanism and protein machinery responsible for recognizing, importing, and processing cytosolic precursor proteins into the mature mitochondrial proteins. Little is known, however, about physiological factors which might influence or compromise mitochondrial protein processing. The objective of this work was to develop a novel approach to study mitochondrial import and assembly of precursor proteins as a process within the context of the cellular environment. To these ends we employed the baculovirus expression system to overexpress the prototypical precursor protein, human manganese-dependent superoxide dismutase (hMn-SOD). It was found that hMn-SOD precursor was correctly processed and assembled by Spodoptera frugiperda insect cell mitochondria into fully active mature hMn-SOD. Using this model, in which the expression of a single precursor protein is greatly exaggerated, we were able to study factors which might influence mitochondrial import of proteins. Others have reported that under hyperoxic conditions a post-translational block of the functional expression of Mn-SOD occurs [Clerch et al. (1993) J. Clin Invest., 91:499-508]. This led us to hypothesize that this block of Mn-SOD expression could occur at the level of mitochondrial import. In particular, we reasoned that mitochondrial import of precursor proteins may represent a process which is susceptible to dysfunction; and which, if compromised, would lead to a post-translational block of the functional expression of mitochondrial protein. Consistent with our hypothesis it was found that oxidizing stress applied with hyperoxic culture conditions (>95% O2 atm.) or the redox cycling agent, paraquat, leads to a lesion of the import/processing of precursor hMn-SOD in the baculovirus model. The use of 1mM manganese chloride to activate the superoxide dismutase activity of the recombinant hMn-SOD was found to ablate the effects of hyperoxia and paraquat treatments, directly demonstrating the involvement of superoxide anion in the dysfunction of hMn-SOD processing. The oxidation of key sulfhydryl groups as a component of the mitochondrial processing lesion was implicated by virtue of the observation that the sulfhydryl reducing agent, dithiothreitol (DTT), was completely effective in preventing the block of hMn-SOD processing induced by paraquat. In addition, the recent suggestion that the peripheral benzodiazepine receptor (PBzR) is associated with the mitochondrial protein translocation machinery led us to explore the effects of the known binding ligands for this receptor. Strikingly, the peripheral benzodiazepine receptor agonists PK11194, Ro5-4864 and Protoporphyrin IX were all found to enhance mitochondrial processing of hMn-SOD precursor, suggesting a role for the PBzR in regulation of mitochondrial import of proteins. Collectively, our results suggest a possible physiological role of a redox-regulated mechanism for mitochondrial biogenesis which could explain, at least in part, the broad heterogeneity of mitochondrial phenotypes.

Subject(s)

Mitochondria.

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