Regulation of ER homeostasis by TCA cycle activity: mechanisms and consequences

TCA 循环活动调节 ER 稳态：机制和后果

• 批准号: 10799333
• 负责人: David Thomas Rutkowski
• 金额: $ 3.45万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10799333
• 关键词: Acetyl Coenzyme A; Adipocytes; Adult; Affect; Amino Acids; Atherosclerosis; Biosensor; Catabolism; Cells; Citric Acid Cycle; Client; Communication; Coupled; Cytosol; Data; Data Collection; Diabetes Mellitus; Disease; Electron Transport; Endoplasmic Reticulum; Enzymes; Funding; Genetic; Glucose; Glutathione; Glutathione Disulfide; Glutathione Reductase; Glycolysis; Goals; Hepatocyte; Homeostasis; Human; Isocitrate Dehydrogenase; Knowledge; Lead; Link; Lipids; Liver diseases; Maintenance; Mediating; Metabolic; Metabolic Diseases; Metabolism; Mitochondria; Modeling; Molecular Biology; Molecular Chaperones; Monitor; Muscle Cells; NADP; Nutrient; Nutritional; Obesity; Outcome; Oxidation-Reduction; Parents; Pathway interactions; Process; Production; Proteins; Pyruvate; Quality Control; Reader; Reduced Glutathione; Regulation; Request for Applications; Signal Transduction; Steatohepatitis; Stress; Sulfhydryl Compounds; Testing; Time; Work; building materials; burden of illness; cell type; cofactor; comorbidity; design; endoplasmic reticulum stress; fatty acid oxidation; genetic manipulation; improved; insight; knock-down; lipid biosynthesis; malic enzyme; novel; oxidation; pharmacologic; protein folding; public health relevance; pyruvate carrier; response; tool

Project Summary Disruption of protein folding in the endoplasmic reticulum—“ER stress”—is associated with many different metabolic diseases, particularly those associated with obesity that affect between 22 and 30 percent of adults in the U.S. Because of this exceptional disease burden, it is important to understand the factors that cause ER stress during metabolic dysregulation. Yet the pathways by which metabolic activity and ER homeostasis are coupled are poorly understood. Mitochondria are central to metabolism, and the TCA cycle is the hub of this activity, accepting substrates from glycolysis and fatty acid oxidation for catabolism, generating reducing equivalents for electron transport and for the maintenance of cellular redox homeostasis, and providing building materials for the reductive biosynthesis of lipids, glucose, and amino acids. Because of its centrality to so many processes, flux through the TCA cycle is likely to affect many diverse cellular pathways, even those with no obvious direct connection. This includes ER protein processing, which is sensitive to changes in redox state, amino acid availability, and cellular lipid content. In the parent proposal, we provide evidence for a previously unknown functional relationship between TCA cycle activity and ER homeostasis in metabolically active cells, including hepatocytes, myocytes, and adipocytes, that depends on production of NADPH by the TCA cycle and redox regulation of glutathione. This proposal is designed to identify the basic mechanisms linking TCA-dependent NADPH production in the mitochondria to homeostasis in the ER. Toward that end, we propose three specific aims: (1) Determine how NADPH production and compartmentalization link nutrient flow to ER stress; (2) Determine how changes to mitochondrial and cytosolic glutathione redox promote ER oxidation; and (3) Determine how TCA activity and glutathione redox alter ER function. We will achieve these aims using a combination of genetic and pharmacological tools to manipulate TCA cycle activity; cutting-edge biosensors to monitor changes in cellular redox status; manipulation and analysis of ER-mitochondrial contacts; and molecular biology approaches to manipulate and assess ER functionality. The outcome of this work will be a mechanistic understanding of how metabolic activity alters ER function to contribute to disease. This supplemental application requests funds for the purchase of a multi-functional plate reader, to end our reliance on widely distributed and functionally limited machines for data collection.

项目摘要 内质网中蛋白质折叠的破坏 - “ ER应激”与许多不同的 代谢疾病，特别是与肥胖有关的疾病，影响22％至30％的成年人 在美国，由于这种特殊的疾病伯恩，重要的是要了解导致ER的因素 代谢失调期间的压力。然而，代谢活动和ER稳态的途径是 耦合知之甚少。 线粒体是代谢的核心，TCA周期是该活动的枢纽，接受底物 从分解代谢的糖酵解和脂肪酸氧化中，产生了电子传输的等效物 并维持细胞氧化还原稳态，并为减少的建筑材料提供建筑材料 脂质，葡萄糖和氨基酸的生物合成。由于它的中心地位如此多，因此磁通 TCA周期可能会影响许多不同的细胞途径，即使是没有明显直接连接的细胞途径。 这包括ER蛋白质加工，该加工对氧化还原态的变化，氨基酸的可用性和 细胞脂质含量。 在父母的建议中，我们提供了以前未知的TCA功能关系的证据 代谢活性细胞中的循环活性和ER稳态，包括肝细胞，肌细胞和 脂肪细胞，取决于TCA周期和谷胱甘肽的氧化还原调节的NADPH的产生。这 建议旨在确定与TCA依赖性NADPH生产相关的基本机制 线粒体在急诊室中稳态。为此，我们提出了三个具体目标：（1）确定如何 NADPH的生产和分区化将养分流与ER应力联系起来； （2）确定如何变化 线粒体和胞质谷胱甘肽氧化还原促进氧化； （3）确定TCA活动和 谷胱甘肽氧化还原ER功能。我们将使用遗传和 操纵TCA周期活动的药理工具；尖端的生物传感器以监测细胞的变化 氧化还原状态；操纵和分析ER-线粒体接触；和分子生物学方法 操纵和评估ER功能。这项工作的结果将是对如何理解 代谢活性改变了ER的功能，从而导致疾病。 该补充申请要求购买资金以购买多功能板读取器，以结束我们的 依赖广泛分布的数据收集机器。