Mitochondrial Thiol Regulation and Programmed Cell Death in Yeast

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.

All organisms contain complex regulatory machinery to maintain the redox status of -SH groups in proteins and low molecular weight sulphydryls. Glutaredoxins and thioredoxins are key oxidoreductases that constitute the main cellular redox systems. Whilst there has been extensive research on the cytoplasmic systems, little is known regarding the mitochondrial systems. This work programme will define the thiol regulatory systems which maintain the mitochondrial thioredoxin system and determine the role of mitochondrial thioredoxin (Trx3) in programmed cell death (PCD). The starting point for this project will be to determine the absolute cellular concentrations of the mitochondrial redox proteins. The GSSG/2GSH redox couple will be measured as a general indicator of the mitochondrial redox state and the redox state of Trx3 will be measured directly. These data will be used to establish how the mitochondrial thiol regulatory network is altered during conditions which induce PCD. We will test the hypothesis that oxidation of Trx3 plays a key role in regulating PCD by examining the requirement for Trx3 and its cysteine residues. The central question to be addressed is whether oxidation of Trx3 is a common marker of PCD and the availability of trx3 cysteine mutants will allow us to directly confirm that oxidation of Trx3 is mediating any effects. The mechanism of Trx3-induced PCD will be addressed by examining mitochondrial events which have been linked to apoptosis including changes in the mitochondrial membrane potential, respiratory activity and cytochrome c release. A particular focus will be to identify potential interactions with the yeast Yca1 metacaspase and Aif1 Apoptosis-inducing factor and other redox-dependent interactions which might be important for promoting PCD. We have preliminary data that the mitochondrial 1-Cys peroxiredoxin (Prx1) interacts with Trx3 and the final part of the project will examine the role of Prx1 in regulating Trx3-mediated PCD.

Who will benefit from this research?
Academic researchers with wide-ranging interests in cell biology, biochemistry and molecular biology will benefit from the methodology and data generated from this research project. The research will be of interest to researchers in medical biosciences since disrupting redox control is implicated in numerous disease processes and ageing. Additionally, data generated from this research will be of benefit to industrial researchers who are interested in how cells respond to stress conditions and the use of yeasts in biotechnological applications.

How will they benefit from this research?
This research will increase our understanding of sulphydryl homeostasis during oxidative stress conditions. Redox changes accompany developmental changes in the life cycle of multicellular organisms including proliferation, differentiation, senescence and cell death. Our yeast studies will provide a framework for researchers in medical and disease-related fields to understand these changes. An added benefit is that studies using the yeast model system provide an alternative to animal-based studies. The yeast system offers significant advantages in terms of its tractability to genetic and biochemical manipulation. This will potentially benefit researchers in the study of diverse diseases and pathologies including cancer, viral infections and neurodegenerative disorders. Living with environmental change is a priority area for the BBSRC. Our research will uncover mechanisms used by cells to cope with changes in oxidative stress. This research will provide information and tools to increase the knowledge needed to enable organisms to build resilience and adapt to environmental change. The project may have significant industrial implications since yeast fermentations are extensively used in baking and brewing as well as for the production of biofuels and biophamaceuticals. Industrial yeast strains are subjected to various stress conditions during the course of manufacturing processes. Our findings will increase understanding of the yeast oxidative stress response which may be extended to industrial usage of yeast in commercial settings. Commercial yeasts need to be active in a variety of environments (e.g. doughs of different sugar contents) and following a range of treatments that expose them to stress (e.g. drying). Our studies will examine the changes in cytoplasmic and mitochondrial redox homeostasis which yeast cells are exposed to during changing growth and stress conditions. Researchers in these industries will benefit from conceptual advances generated during this work.

What will be done to ensure that they benefit from this research?
Results will be disseminated through research seminars, presentations at conferences and publications in scientific journals. Funding is requested to attend national and international research conferences to allow the researchers to publicize this research. Resources generated from this project are likely to include yeast strains, plasmids and antibodies and will be made available to the scientific community upon request. Detailed protocols and primary data will be made freely available to academic collaborators. Manchester University has a good track record of encouraging public engagement. This includes regular open days to inform school children and the public about University research and tours of the research facilities at Manchester. This will allow the researchers to share their research findings with the wider public and to raise awareness of the importance of basic research. Manchester University maintains excellent links with the business sector which will allow us to exploit any potential for collaboration with industry. This is managed by the faculty Business Development Team which provides support and information for staff wishing to develop relationships with business.