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.

项目概要 内质网中蛋白质折叠的破坏（“ER 应激”）与许多不同的因素有关。 代谢疾病，特别是与肥胖相关的疾病，影响 22% 至 30% 的成年人 在美国，由于这种特殊的疾病负担，了解导致 ER 的因素非常重要 代谢失调期间的压力。然而代谢活动和 ER 稳态的途径 耦合的理解甚少。 线粒体是新陈代谢的核心，TCA 循环是该活动的中心，接受底物 来自糖酵解和脂肪酸氧化的分解代谢，产生电子传输的还原当量 维持细胞氧化还原稳态，并为还原提供建筑材料 脂质、葡萄糖和氨基酸的生物合成。由于它在许多过程中处于中心地位，因此通过 TCA 循环可能会影响许多不同的细胞途径，甚至是那些没有明显直接联系的途径。 这包括 ER 蛋白质加工，它对氧化还原状态、氨基酸可用性和 细胞脂质含量。 在父提案中，我们提供了 TCA 之间先前未知的功能关系的证据 代谢活跃细胞（包括肝细胞、肌细胞和细胞）的循环活动和 ER 稳态 脂肪细胞，这取决于 TCA 循环产生 NADPH 和谷胱甘肽的氧化还原调节。这 该提案旨在确定 TCA 依赖性 NADPH 生产的基本机制 线粒体在 ER 中保持稳态。为此，我们提出三个具体目标：（1）确定如何 NADPH 的产生和区隔将营养流与 ER 应激联系起来； (2) 确定如何改变 线粒体和细胞质谷胱甘肽氧化还原促进内质网氧化； (3) 确定 TCA 活性和 谷胱甘肽氧化还原改变 ER 功能。我们将结合遗传和基因来实现这些目标 操纵 TCA 循环活性的药理学工具；监测细胞变化的尖端生物传感器 氧化还原状态； ER-线粒体接触的操作和分析；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 代谢活动改变内质网功能从而导致疾病。 此补充申请要求资金购买多功能读板器，以结束我们的 依赖广泛分布且功能有限的机器来收集数据。