Mitochondrial protein thiol modifications during oxidative stress

The focus of this thesis is to investigate the interactions of mitochondrial protein thiols with ROS and examine the oxidation of these thiols in response to oxidative stress. Among these protein thiol modifications, an area of great interest is the interactions of mitochondrial protein thiols with glutathione, therefore possible connections between the modifications and cell death were investigated. I explored the possibility that staurosporine (STS) induces apoptosis via the mitochondrial pathway, causing early changes in the mitochondrial membrane potential (Δψ), by changing the thiol redox status of the cell. The hypothesis tested was that there may be a common link between oxidative stress and thiol changes of mitochondrial proteins and oxidation of the cellular glutathione pool which then initiates the critical mitochondrial events that lead to apoptosis. STS caused apoptosis after 2-4 hours of treatment and caused a decrease in total glutathione measured in cells, with the depletion of cellular glutathione occurring after the induction of apoptosis. There were no changes in glutathione (GSH)/glutathione disulphide (GSSG) redox state up to one hour of STS treatment, and thus no association of cellular GSH oxidation with the early mitochondrial Δψ changes observed. However, the GSH pool was significantly oxidised after 2 hours. Even so, no significant changes in protein glutathionylation by STS were observed at 2 hours. I next explored the possibility that protein thiol modifications might respond to oxidative stress for the purpose of redox signalling, whereby redox-sensitive modifications might contribute to biological regulation. Therefore, I went on to quantify total and exposed protein thiols in subcellular fractions including mitochondria. This analysis showed that 74% of total liver cell lysate protein thiols are exposed compared to 57% of mitochondrial proteins, 67% of cytosolic proteins and 62% of soluble proteins. The amount of exposed thiols as a percentage of total thiols in each of the four fractions did not differ when comparing rat liver and rat heart tissue. I then further characterised the distribution of protein thiols in the mitochondria and found that approximately two-thirds of protein thiols present in the mitochondrial membrane fraction were exposed compared to 78% in the mitochondrial matrix fraction. In addition, by using two membrane-impermeant thiol-alkylating agents, I was able to show that approximately a third of all the exposed protein thiols in the mitochondrial membrane fraction were either on the outer membrane, facing outward into the cytosol or facing inward into the intermembrane space, within the intermembrane space itself or on the outer leaflet of the inner membrane. The possibility that protein thiols might have a protective role acting as a redox buffer was also explored, and loss of exposed protein thiols during oxidative stress was investigated.EThOS - Electronic Theses Online ServiceGBUnited Kingdo