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Gatekeepers of Mitochondrial NAD+

线粒体 NAD 的看门人

基本信息

批准号：
10301237
负责人：
Xiaolu Ang Cambronne
金额：
$ 7.93万
依托单位：
UNIVERSITY OF TEXAS AT AUSTIN
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-30 至 2022-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10301237
关键词：
Address Affect Age Back Binding Proteins Bioenergetics Biosensor Cardiovascular Diseases Cell Nucleus Cell Respiration Cells Chromatin Citric Acid Cycle Cultured Cells Cytoplasm Data Detection Development Disease Dyes Etiology Fluorescence Gatekeeping Gene Expression Generations Genetic Health Homeostasis Human Pathology In Vitro Infection Intervention Lead Learning Link Malignant Neoplasms Masks Measurement Measures Metabolic Metabolic Pathway Metabolic syndrome Metabolism Methods Mitochondria Mitochondrial Matrix Modification Molecular Monitor Mutagenesis Myopathy Nerve Degeneration Neurologic Nicotinamide adenine dinucleotide Noise Nuclear Onset of illness Organelles Oxides Pathology Physiological Positioning Attribute Regulation Reporter Reporting Role Series Signal Transduction Structure System Technology Testing Time Tissues Variant Whole Organism base cell type design experimental study improved in vivo insight interest novel strategies response sensor temporal measurement

项目摘要

Project summary We are working on new in vivo methods to study the intermediary metabolite nicotinamide adenine dinucleotide (NAD+). NAD+ is critical for cellular metabolism and health, and its decreased steady-state levels have been linked to human pathologies such as neurodegeneration, cardiovascular disease, metabolic syndrome, and cancer. NAD+ concentrations are highly compartmentalized by cell type, subcellular localization, and its protein- bound or free fractions. A lack of methods that can monitor free NAD+ in cells with spatial and temporal information has hindered our learning about the relevant pools, threshold concentrations, and fluctuations that NAD+ may undergo leading to disease onset. This precludes our ability to identify treatments or approaches to intervene before NAD+ levels are misregulated. In particular, there are no genetically-encoded methods for in vivo mitochondrial NAD+ measurements. Free mitochondrial NAD+ is required for cellular respiration, and diminished levels due to genetics, age, or infection can promote reductive metabolism. Thus, to address this gap we will improve the brightness (Aim 1) of a variant of the current NAD+ sensor, which is able to monitor free mitochondrial NAD+ in cells. We will also generate a series of constructs that will be used to test a new approach for improving the dynamic range of fluorescent readout (Aim 2). Together, data from these the aims will identify a feasible method to robustly obtain free mitochondrial NAD+ measurements in vivo.

项目总结我们正在研究体内研究中间代谢物烟酰胺腺嘌呤二核苷酸的新方法。 (NAD+)。NAD+对细胞新陈代谢和健康至关重要，其降低的稳态水平一直是与神经退行性变、心血管疾病、代谢综合征和癌症。NAD+浓度由细胞类型、亚细胞定位及其蛋白高度划分- 束缚分数或自由分数。缺乏能够监测细胞内游离NAD+的方法信息阻碍了我们对相关池、阈值浓度和波动的了解 NAD+可能导致疾病的发生。这就排除了我们识别治疗方法或方法的能力在NAD+水平被错误调控之前进行干预。具体地说，目前还没有遗传编码的方法活体线粒体NAD+测量。自由线粒体NAD+是细胞呼吸所必需的，由于遗传、年龄或感染导致的水平降低可以促进还原代谢。因此，为了解决这一差距，我们将改进当前NAD+传感器的一个变体的亮度(目标1)，该传感器能够免费监控细胞内线粒体NAD+。我们还将生成一系列用于测试新方法的构造用于提高荧光读数的动态范围(目标2)。总而言之，来自这些目标的数据将确定一种可靠地获取体内线粒体NAD+游离值的可行方法。