Structure-Function Analysis of a Key Mitochondrial PrxIII Antioxidant Defence Pathway: Roles in Antioxidant Defence & Chaperone-Mediated Protection

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Mitochondria are the powerhouses of cells liberating energy from the breakdown of the major fuel molecules, namely carbohydrates, fats and proteins as the 'high-energy' chemical ATP. ATP is the universal 'energy currency' of all living organisms where it is required for powering all the normal bodily activities associated with life itself. Mitochondria are also the principal sites of intracellular respiration (oxygen consumption) where reactive oxygen species (ROS), in essence partially-reduced forms of oxygen, so-called superoxide anions, hydroxyl radicals and hydrogen peroxide, are also produced continuously as naturally-occurring, toxic by-products of respiration that are potentially very damaging to tissues. As a result, organisms have evolved an integrated network of enzyme-based, antioxidant defence systems to ensure the rapid removal of these potentially harmful species. However, oxidative stress can occur when there is an imbalance in the production of ROS that can temporarily overwhelm cellular antioxidant defences. As this is a potentially lethal event, cells must respond appropriately to minimise the extent of irreversible damage wherever possible and ensure their ultimate survival. Recent studies on a newly-emerging group of antioxidant enzymes, the peroxiredoxins (Prxs) have revealed that they serve as critical regulators of intracellular hydrogen peroxide concentrations with dual roles in tissue protection and hydrogen peroxide-mediated cell signalling responses to elevated ROS levels. In our laboratory, we have recently reconstituted the main human mitochondrial peroxiredoxin pathway in vitro and elucidated some its novel properties by producing and purifying its its 3 constituent enzymes in bacteria permitting detailed investigation of its role in antioxidant defence. A unique 3D structure has also been determined for its principal component, PrxIII that directly reduces hydrogen peroxide to water with the aid of its partner proteins, thioredoxin and thioredoxin reductase. PrxIII has a remarkable subunit organisation comprising two mechanically-interlocked rings, each assembled from 12 identical protein subunits, one of only two known examples of a so-called protein catenane. Moreover, PrxIII can exist in various states of assembly (oligomeric states) under different conditions. These include basic dimeric units (2 subunits), single dodecameric rings, 2-ring catenanes and long, regular filamentous structures containing multiple rings. We now wish to investigate the central biological importance of this key antioxidant defence system in greater detail and the relevance of the presence of multiple structural forms of Prx III in the regulation of pathway activity as oxidative stress is implicated as a major causative factor in a range of human diseases including cardiovascular disease, cancer, diabetes and neurodegenerative disorders. Our research will also focus on how the PrxIII pathway interacts with and protects a vital group of mitochondrial multienzyme complexes involved in degrading the major fuel molecules; on identifying other key mitochondrial proteins that are susceptible to damage under conditions of oxidative stress; and on elucidating how defective functioning of PrxIII in vivo affects mitochondrial integrity, energy production and cell viability.