Interrogating and manipulating mitochondrial ROS, energetics and proteomics

描述(由申请人提供)：我们提出了对挑战06-AG-104的回应，其中包括三种新工具和技术的开发，这些工具和技术可以改变或询问体内人类线粒体的功能。然后，我们将把这些工具应用到老化心脏和肌肉的研究中，以证明它们可以使人们对线粒体ROS、线粒体能量学以及线粒体蛋白质组成和周转在年龄相关性骨骼肌减少和心脏病中的作用有一个全面的了解。在目标1中，我们将通过使用新型线粒体靶向抗氧化剂和保护肽来操纵体内线粒体的结构和活性。这在人类衰老中很重要，因为来自我们和其他实验室的数据表明，线粒体和线粒体ROS在与年龄相关的器官功能下降中发挥着关键作用。在目标2中，我们将应用新的磁共振和光学光谱技术来同时测量O2和ATP的通量，以创建一种完全非侵入性的、更多功能的线粒体功能测量方法。在Aim 3中，我们将使用新的蛋白质组学方法来表征体内衰老组织和抗氧化肽保护组织中线粒体功能变化的结构基础。这将包括使用体内重同位素标记来测量全球线粒体蛋白质丰度以及线粒体蛋白质组在蛋白质合成和周转率方面的广泛差异。在每个目标中，我们都将应用和验证使用两种组织开发的工具，对于这两种组织，有大量证据表明线粒体和线粒体ROS在衰老中发挥关键作用：心脏和骨骼肌。我们将检查健康的年轻和老年肌肉，以及血管紧张素II应激的年轻心脏和AZT治疗应激的年轻肌肉。这三种方法的结合使用使我们能够在监控和测量老化组织中线粒体的功能和功能障碍的同时操纵人类线粒体的结构和活性。因此，我们可以对线粒体、ROS、能量组学和蛋白质组学在衰老和疾病中的体内相互关系进行综合评估。 与公共卫生相关：本应对挑战06-AG-104整合了三个响应元素，将联合使用以提供对线粒体、ROS、能量学和蛋白质组学在衰老和疾病中的体内相互关系的综合评估：1)我们将利用新型线粒体靶向抗氧化剂和保护肽，在体内操纵线粒体的结构和活性；2)我们将使用新型磁共振和光学光谱分析工具，创建体内线粒体能量学的完全非侵入性、更多功能的测量方法；3)我们将使用新型蛋白质组学工具，研究线粒体功能变化的结构基础。这些方法的结合使用使我们能够在监控和测量老化组织中线粒体的功能和功能障碍的同时操纵人类线粒体的结构和活动。

DESCRIPTION (provided by applicant): We propose a response to Challenge 06-AG-104 that incorporates development of three novel tools and technologies that can alter or interrogate human mitochondrial function in vivo. We will then apply these tools to the study of aging heart and muscle in order to demonstrate that they can enable an integrated understanding of the role of mitochondrial ROS, mitochondrial energetics and changes in mitochondrial protein composition and turnover in age-related sarcopenia and heart disease. In Aim 1 we will manipulate in vivo mitochondrial structure and activity by the use of novel mitochondrial targeted antioxidant and protective peptides. This is important in human aging because data from our and other laboratories indicate that mitochondria and mitochondrial ROS play a critical role in age-related declines in organ function. In Aim 2 we will apply novel magnetic resonance and optical spectroscopy techniques to measure O2 and ATP fluxes simultaneously to create a totally non-invasive and more versatile measure of mitochondrial function. In Aim 3 w e will use novel proteomic methodologies to characterize the structural basis of changes in mitochondrial function in aging tissues in vivo and tissues protected by antioxidant peptides. This will include measurement of global mitochondrial protein abundance and mitochondrial proteome-wide differences in protein syntheses and turnover rates using in vivo heavy isotope labeling. In each of these aims we will apply and validate the tools developed using two tissues for which there is abundant evidence for the critical role of mitochondria and, mitochondrial ROS in aging: heart and skeletal muscle. We will examine healthy young and old muscle, plus young hearts stressed with angiotensin II and young muscle stressed by AZT treatment. The combined use of these three methods allows us to manipulate human mitochondrial structure and activity at the same time as we monitor and measure mitochondrial function and dysfunction in aging tissues. Thus, we can obtain an integrated assessment of the in vivo inter-relationships of mitochondrial, ROS, energetics and proteomics in aging and disease. PUBLIC HEALTH RELEVANCE: This response to Challenge 06-AG-104 incorporates three responsive elements that will be used together in concert to provide an integrated assessment of the in vivo inter-relationships of mitochondrial, ROS, energetics and proteomics in aging and disease: 1) we will manipulate in vivo mitochondrial structure and activity by the use of novel mitochondrial targeted antioxidant and protective peptides; 2) we will use novel magnetic resonance and optical spectroscopy tools to create totally non-invasive and more versatile measures of mitochondrial energetics in vivo, and 3) we will use novel proteomic tools to characterize the structural basis of changes in mitochondrial function. The combined use of these methods allows us to manipulate human mitochondrial structure and activity at the same time as we monitor and measure mitochondrial function and dysfunction in aging tissues.