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

线粒体是细胞的发电站，从主要燃料分子的分解中释放能量，即碳水化合物，脂肪和蛋白质作为“高能”化学ATP。ATP是所有生物体的通用“能量货币”，它是为与生命本身相关的所有正常身体活动提供动力所必需的。线粒体也是细胞内呼吸（氧消耗）的主要位点，其中活性氧物质（ROS）（本质上是氧的部分还原形式，所谓的超氧阴离子、羟基自由基和过氧化氢）也作为呼吸的天然存在的有毒副产物连续产生，其对组织具有潜在的非常大的损害。因此，生物体已经进化出一个基于酶的抗氧化防御系统的综合网络，以确保快速清除这些潜在的有害物质。然而，当ROS的产生不平衡时，可能会发生氧化应激，这可能会暂时压倒细胞的抗氧化防御。由于这是一个潜在的致命事件，细胞必须做出适当的反应，尽可能减少不可逆损伤的程度，并确保其最终存活。最近对一组新出现的抗氧化酶，过氧化物酶（Prxs）的研究表明，它们作为细胞内过氧化氢浓度的关键调节剂，具有双重作用，在组织保护和过氧化氢介导的细胞信号转导反应，以提高ROS水平。在我们的实验室中，我们最近重建了主要的人类线粒体peroxiredoxin途径在体外，并阐明了一些新的特性，通过生产和纯化其3组成酶在细菌中允许详细调查其在抗氧化防御中的作用。还确定了其主要成分PrxIII的独特3D结构，PrxIII在其伴侣蛋白硫氧还蛋白和硫氧还蛋白还原酶的帮助下直接将过氧化氢还原为水。PrxIII具有一个显著的亚基组织，包括两个机械互锁的环，每个环由12个相同的蛋白质亚基组装而成，这是所谓的蛋白质链烷的仅有的两个已知例子之一。此外，PrxIII可以在不同条件下以各种组装状态（低聚状态）存在。这些包括基本的二聚体单元（2个亚基），单个十二聚体环，2-环索烃和含有多个环的长的规则的丝状结构。我们现在希望调查的中央生物学的重要性，这一关键的抗氧化防御系统在更详细的和相关性的存在下，多种结构形式的Prx III的调节途径的活性作为氧化应激是牵连作为一个主要的致病因素，在一系列的人类疾病，包括心血管疾病，癌症，糖尿病和神经退行性疾病。我们的研究还将侧重于PrxIII途径如何与参与降解主要燃料分子的线粒体多酶复合物相互作用并保护一组重要的线粒体多酶复合物;确定在氧化应激条件下易受损伤的其他关键线粒体蛋白质;并阐明PrxIII体内功能缺陷如何影响线粒体完整性，能量产生和细胞活力。

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.