Alternative oxidases in animals

地球上生命的货币是能量。生物体利用电子传递系统（ETS）将各种分子转化为三磷酸腺苷（ATP）以进行生长和繁殖。在大多数ETS中，细胞色素c氧化酶是最终的电子受体，但许多生物体也含有第二种终端氧化酶，称为替代氧化酶（AOX）。AOX的生理作用相当神秘，因为从生物能量学的角度来看，它的存在似乎是违反直觉的，但AOX mRNA表达和AOX蛋白水平在应激时增加。这些观察结果表明，AOX在ETS中具有灵活性，并使生物体能够应对压力环境。最初被认为不存在于动物体内的AOX，于2005年在动物体内被发现。从那时起，这一新的研究领域得到了探索，但我们的知识仍然存在重大差距。我提议解决构成我的研究计划基础的三个主要问题。(1)动物体内的AOX是如何被翻译后调控的？很少有研究关注动物体内的天然AOX蛋白。AOX蛋白存在于几种动物物种中，但我们对动物中该蛋白活性的翻译后调控缺乏了解。我们将在加利福尼亚虎脚类桡足动物中研究这一过程，因为我的实验室花了几年时间开发工具来研究这种生物中的AOX。我们将使用高分辨率呼吸测定法来鉴定AOX的激活剂和抑制剂，以及潜在的呼吸底物对该途径容量的影响。(2) AOX在动物体内的生理作用是什么？植物中的AOX通过允许ETS的灵活性，通过初级代谢有效调节ATP需求和碳通量，对逆境耐受性有重要贡献。也许AOX在动物身上也起着类似的生理作用。我们将控制加州虎爪鼠体内AOX蛋白的水平，然后将这些动物暴露在高温胁迫下，看看AOX是否在这些条件下对它们的生存起作用。(3) AOX在动物中的分类分布如何？这是我研究计划的一个持续目标，因为除非我们能确定哪些动物含有AOX，否则不可能研究AOX。这项工作是通过结合生物信息学分析来鉴定含有AOX的动物，然后通过分子生物学实验来确认这些物种中AOX mRNA的表达和AOX蛋白的存在。更好地了解AOX的分类分布将有助于我们了解该酶的起源和进化历史。从我们的研究中获得的知识可能会导致治疗人类线粒体疾病和由寄生动物引起的人类感染。使用抑制剂靶向AOX可能使我们能够杀死威胁加拿大农业和水产养殖的寄生虫。

The currency of life on Earth is energy. Living organisms use electron transport systems (ETS) to convert various molecules into adenosine triphosphate (ATP) for growth and reproduction. Cytochrome c oxidase is the final electron acceptor in most ETS, but many organisms also contain a second terminal oxidase called alternative oxidase (AOX). The physiological role for AOX is rather mysterious, as its presence seems counterintuitive from a bioenergetics perspective, but AOX mRNA expression and AOX protein levels increase during stress. These observations suggest that AOX confers flexibility in the ETS and allows organisms to deal with stressful environments. AOX, initially thought to be absent from animals, was discovered in animals in 2005. This new field of research has been explored since then, but there are still significant gaps in our knowledge. I propose to address three main questions that form the basis of my research program. (1) How is AOX post-translationally regulated in animals? Very few studies have looked at a natural AOX protein in animals. The AOX protein is present in several animal species, but our knowledge of the post-translational regulation of the protein's activity in animals is lacking. We will study this process in the copepod Tigriopus californicus, as my lab has spent several years developing tools to study AOX in this organism. We will use high-resolution respirometry to identify the activators and inhibitors of AOX, as well as the impact of potential respiratory substrates on the capacity of this pathway. (2) What is the physiological role(s) of AOX in animals? AOX in plants contributes significantly to stress tolerance by allowing flexibility in the ETS for effective regulation of ATP demand and carbon flux through primary metabolism. Perhaps AOX plays a similar physiological role in animals. We will manipulate the levels of AOX protein in Tigriopus californicus and then will expose these animals to high temperature stress to see if AOX plays a role in their survival under these conditions. (3) What is the taxonomic distribution of AOX in animals? This is a continuing goal of my research program, as it is impossible to study AOX unless we can identify which animals contain it. This work is done by using a combination of bioinformatics analyses to identify animals that contain AOX, followed by molecular biology experiments to confirm the expression of AOX mRNA and the presence of AOX protein in these species. A better understanding of the taxonomic distribution of AOX will contribute to our knowledge on the origin and evolutionary history of the enzyme. The knowledge gained from our research may lead to treatments for human mitochondrial diseases and human infections caused by parasitic animals. Targeting AOX using inhibitors may allow us to kill parasites that threaten Canadian agriculture and aquaculture.