Defining the Connections between Respiratory Chain Structure and Oxidative Stress

描述（由申请人提供）：线粒体电子传递链（ETC）是所有真核细胞生存的核心，驱动ATP的合成，为细胞生物能量提供燃料。尽管ETC具有基本的作用，但在不同的生物体中，ETC的结构存在显著差异。与高等动物相对简单的ETC特征不同，植物具有复杂的分支呼吸链，其中包含II型NAD(P)H脱氢酶（ND）和替代氧化酶（AOX），它们为电子流动提供了替代途径。最近的几项研究表明，这些替代呼吸酶可以最大限度地减少ETC的“电子泄漏”，减少有害活性氧（ROS）的产生。活性氧的产生和由此产生的氧化应激具有重要的生物医学意义，因为氧化损伤似乎在衰老以及从阿尔茨海默氏症到糖尿病等多种病理中起着重要作用。值得注意的是，与动物衰老相关的进行性氧化损伤在植物中不存在，并且ND在动物系统（果蝇）中的表达减少了线粒体ROS的产生并延长了寿命。因此，本提案的首要目标是确定ETC结构与ROS产生/进行性氧化损伤之间的关系。为此，我们计划利用诱导型RNA干扰载体对模式植物拟南芥（Arabidopsis thaliana）中的AOX基因家族、NDinternal基因家族和NDexternal基因家族进行实验修饰。由此产生的转基因植物（独立的aox沉默，ndin沉默和ndout沉默系）将允许调节抑制不同的替代呼吸途径，创造植物型和哺乳动物型呼吸链结构之间的中间产物。为了将这些独特的呼吸结构与细胞生理学的可量化影响联系起来，我们计划测量转基因系中ROS的产生、氧化损伤以及细胞抗氧化池的大小和氧化状态。此外，我们将研究转基因系转录组的全局变化，以表征呼吸链结构的改变如何影响ros相关的信号通路。该提案扩展并建立在目前NIH score资助的研究基础上，该研究侧重于拟南芥的发展，作为研究基本细胞氧化还原生物学和氧化损伤的模型系统。总的来说，拟议的项目将对我们对线粒体相关ROS产生的基本理解产生重大影响，这一过程对于细胞生物学的基础领域和人类健康的维持都是至关重要的。

DESCRIPTION (provided by applicant): The mitochondrial electron transport chain (ETC) is central to the survival of all eukaryotic cells, driving the synthesis of ATP that fuels cellular bioenergetics. Despite its fundamental role, significant variation exists in the structure of the ETC in different organisms. Unlike the relatively simple ETC characteristic of higher animals, plants possess a complex, branched respiratory chain containing type II NAD(P)H dehydrogenases (ND) and alternative oxidases (AOX), which provide alternative pathways for electron flow. Several recent studies have suggested that these alternative respiratory enzymes may minimize "electron leakage" from the ETC, diminishing the production of damaging reactive oxygen species (ROS). ROS production and resulting oxidative stress are of significant biomedical interest, since oxidative damage appears to play a significant role in aging as well as a diverse array of pathologies, from Alzheimer's to diabetes. Notably, the progressive oxidative damage associated with aging in animals is absent in plants, and the expression of an ND in an animal system (Drosophila) decreases mitochondrial ROS production and increases lifespan. Thus, the overarching goal of this proposal is to define the relationship between ETC structure and ROS production/progressive oxidative damage. Toward this end, we plan to experimentally modify the plant ETC by using an inducible RNA interference vector to silence the AOX gene family, the NDinternal gene family, and the NDexternal gene family in the model plant species Arabidopsis thaliana. The resulting transgenic plants (independent AOX-silenced, NDin-silenced, and NDout-silenced lines) will allow the regulated suppression of distinct alternative respiratory pathways, creating intermediates between plant-type and mammalian-type respiratory chain configurations. To link these unique respiratory structures to quantifiable effects on cell physiology, we plan to measure ROS production, oxidative damage, and the size and oxidation state of cellular antioxidant pools in the transgenic lines. In addition, we will examine global changes in the transcriptomes of the transgenic lines in order to characterize how altered respiratory chain structure affects ROS-associated signaling pathways. This proposal expands and builds upon current NIH SCORE-funded research focused on the development of Arabidopsis as a model system to study basic cellular redox biology and oxidative damage. Overall, the proposed project will have a major impact on our fundamental understanding of mitochondrial- associated ROS production, a process which is central to both the basic field of cell biology and the maintenance of human health. PUBLIC HEALTH RELEVANCE: A variety of diseases, from Alzheimer's to diabetes, are related to the oxidative stress that arises from disruptions in the cell's delicate "electron economy". The proposed project will elucidate how the structure of a cell's mitochondrial electron transport chain affects the production of reactive oxygen species and oxidative stress, using the plant Arabidopsis thaliana as an experimental model.