Mitochondrial Thiol Regulation and Programmed Cell Death in Yeast

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Proteins are key molecules that control most biological processes. Each protein is made-up of a linear polymer of amino acids which adopt a unique three-dimensional structure. It is becoming increasingly recognized that the cysteine amino acid can play a key role in regulating the activity of many different proteins. This is because cysteine amino acids contain a sulphur atom in a thiol group which accounts for its high reactivity in oxidation and reduction reactions. This means that the redox state of cysteine residues in many different proteins, such as enzymes and transcription factors, can profoundly influence their activity. Not surprisingly therefore, alterations in the cellular redox balance are implicated in many disease processes. This work programme will investigate how cells maintain the redox state of mitochondrial proteins. Understanding mitochondrial redox regulation is particularly important since there are many established links between mitochondrial dysfunction and disease. The focus of this project will be on the glutathione/glutaredoxin and thioredoxin systems which constitute the main cellular redox systems. Our preliminary data, described in the application, show that the redox state of the mitochondrial thioredoxin is important for growth and its oxidation induces a form of programmed cell death (PCD). PCD is a normal process in the development of healthy organisms where cells die in a controlled, regulated fashion. It is a form of cell suicide which can be induced in response to a variety of stimuli including stress. It is important because cell death processes have been implicated in numerous disease processes and many medical treatments and interventions act through PCD. We will use the yeast model system to systematically examine and define the cellular concentrations and redox state of the mitochondrial redox regulatory systems. Importantly, we will examine these systems under conditions that induce PCD to determine the regulatory role of the mitochondrial thioredoxin in this process. Genetic approaches will be used to understand how the mitochondrial thioredoxin regulates cell death. A key technique will be to mutate the cysteine residues in the mitochondrial thioredoxin to directly examine their role in regulating PCD, measured using various known markers which act in the cell death pathway. Mitochondria are the main source of reactive oxygen species in most organisms and this is thought to represent a key signal that activates PCD. Thiol groups in proteins are known to be particularly sensitive to oxidation and so the final part of the project we will test the hypothesis that the mitochondrial thioredoxin system acts as a sensor of reactive oxygen species to activate PCD.