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New approaches to studying redox metabolism using time-resolved NAD(P)H fluorescence and anisotropy

利用时间分辨 NAD(P)H 荧光和各向异性研究氧化还原代谢的新方法

基本信息

批准号：
BB/P018726/1
负责人：
Angus Bain
金额：
$ 73.16万
依托单位：
University College London
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2017
资助国家：
英国
起止时间：
2017 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FP018726%2F1
关键词：
New approaches studying redox metabolism

项目摘要

Living cells require the constant input of energy to maintain their characteristic order. This is provided by the chemical reactions of metabolism. A vast number of these reactions are "redox" (reduction-oxidation) reactions, in which electrons are transferred from one molecule to another. During the breakdown of nutrient molecules to extract energy from food, bonds are broken and electrons are passed to a carrier molecule known as NADH. Removal of electrons is referred to as oxidation, and their addition is known as reduction. The metabolism of food therefore involves the oxidation of sugars, proteins and fats and the simultaneous reduction of NADH.The energy passed to NADH during its reduction is converted into a useable form, the cell's "energy currency" adenosine triphosphate (ATP), inside the mitochondria. This is achieved by using the electrons carried by NADH to reduce oxygen to water. This is the fate of almost 100% of the oxygen consumed by the body, and the energy released is stored as ATP. Defects in this process cause the production of reactive oxygen species (ROS). These are highly damaging molecules, and so the cell possesses specific defence mechanisms against their deleterious effects. These defences are maintained by another electron carrier molecule known as NADPH.The antioxidant systems supported by NADPH act to neutralise the harmful ROS produced during the energy generating processes regulated by NADH. These reactions are collectively known as redox metabolism. Imbalance between the two processes is a known factor in the development of a wide range of diseases, including cancer, diabetes and neurodegenerative disorders. In order to make progress in understanding how these diseases occur and testing potential treatments, it is crucial that biomedical researchers are provided with highly accurate tools to investigate redox metabolism in cells and tissues.As a collection of scientists whose expertise includes the use of lasers to study the dynamic behaviour of molecules and the application of these methods to investigate metabolic processes in living tissues, we propose to develop the next generation of approaches to studying the role of redox metabolism in health and disease. We will exploit the intrinsic fluorescence of NADH and NADPH to construct our new experimental technique. While the two molecules emit light of the same colour, the two independent sets of enzymes that they bind to in order to perform their distinct roles will cause contrasting effects on other characteristics of their fluorescence.We will first prepare solutions of NADH and NADPH bound to their respective enzymes to investigate the resulting properties of their fluorescence. The distinct binding sites to which these molecules attach may cause contrasting effects on the time taken for fluorescence to emerge following absorption of a laser pulse, the so-called fluorescence lifetime. Different forces acting on the molecules in the binding site will also cause differences in the freedom of their motion, which can be detected by measuring the rate of change of the polarisation of the light emitted with respect to the light absorbed, the so-called time-resolved fluorescence anisotropy.Following characterisation of the differences in NADH and NADPH fluorescence when bound to their separate sets of enzymes, we will construct a microscope in which these properties can be detected inside living tissues. Inside the complex environment of the cell, the fluorescence signals would be expected to arise from a mixture of NADH and NADPH, both bound to their enzymes and free. We will develop approaches to extract information from these signals, allowing the function of the separate redox pathways that they are involved in to be investigated. As redox metabolism is being found to play a role in an ever growing range of processes, these new approaches will play a key role in enhancing our fundamental understanding of biology.

活细胞需要持续不断的能量输入来维持其特有的秩序。这是由新陈代谢的化学反应提供的。这些反应中的大量是“氧化还原”（还原-氧化）反应，其中电子从一个分子转移到另一个分子。在营养分子分解以从食物中提取能量的过程中，键被打破，电子被传递到称为NADH的载体分子。电子的去除被称为氧化，它们的加入被称为还原。因此，食物的新陈代谢包括糖、蛋白质和脂肪的氧化，以及同时还原的NADH。还原过程中传递给NADH的能量在线粒体内转化为可用的形式，即细胞的“能量货币”三磷酸腺苷（ATP）。这是通过使用NADH携带的电子将氧气还原为水来实现的。这是几乎100%的氧气被人体消耗的命运，释放的能量被储存为ATP。该过程中的缺陷会导致活性氧物质（ROS）的产生。这些都是高度破坏性的分子，因此细胞具有针对其有害作用的特定防御机制。这些防御是由另一种电子载体分子NADPH维持的。NADPH支持的抗氧化系统可以中和由NADH调节的能量产生过程中产生的有害ROS。这些反应统称为氧化还原代谢。这两个过程之间的不平衡是一个已知的因素，在发展中的一系列疾病，包括癌症，糖尿病和神经退行性疾病。为了在了解这些疾病的发生机制和测试潜在治疗方法方面取得进展，至关重要的是为生物医学研究人员提供高度精确的工具来研究细胞和组织中的氧化还原代谢。作为一个科学家的集合，他们的专业知识包括使用激光研究分子的动态行为，以及将这些方法应用于研究活组织中的代谢过程，我们建议开发下一代的方法来研究氧化还原代谢在健康和疾病中的作用。我们将利用NADH和NADPH的内源荧光来构建我们的新实验技术。虽然这两种分子发出相同颜色的光，但它们所结合的两组独立的酶为了发挥各自不同的作用，会对它们的荧光的其他特性产生对比效应。我们将首先制备与各自酶结合的NADH和NADPH溶液，以研究它们的荧光特性。这些分子附着的不同结合位点可能会对吸收激光脉冲后荧光出现所需的时间产生对比效应，即所谓的荧光寿命。作用在结合位点中的分子上的不同力也会导致它们运动自由度的差异，这可以通过测量发射光相对于吸收光的偏振变化率来检测，即所谓的时间分辨荧光各向异性。我们将建造一个显微镜，在显微镜中，这些特性可以在活组织内被检测到。在细胞的复杂环境中，荧光信号预计将来自NADH和NADPH的混合物，两者都与它们的酶结合并游离。我们将开发从这些信号中提取信息的方法，从而研究它们所参与的单独氧化还原途径的功能。随着氧化还原代谢在越来越多的过程中发挥作用，这些新方法将在增强我们对生物学的基本理解方面发挥关键作用。