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

项目摘要 内质网蛋白质折叠的破坏--“内质网应激”--与许多不同的 代谢性疾病，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负担，了解导致ER的因素很重要 代谢失调期间的应激。然而，代谢活动和内质网动态平衡的途径 人们对此知之甚少。 线粒体是新陈代谢的中心，而TCA循环是这一活动的中枢，接受底物 从糖酵解和脂肪酸氧化分解代谢，生成电子传递的还原当量 并用于维持细胞氧化还原动态平衡，并为还原剂提供建材 脂肪、葡萄糖和氨基酸的生物合成。由于它对如此多的过程处于中心地位，因此通过 TCA循环可能会影响许多不同的细胞途径，即使是那些没有明显直接联系的途径。 这包括内质网蛋白质的处理，它对氧化还原状态、氨基酸可用性和 细胞内脂肪含量。 在父提案中，我们提供了TCA之间以前未知的功能关系的证据 代谢活跃的细胞，包括肝细胞、心肌细胞和 脂肪细胞，这依赖于TCA循环和谷胱甘肽的氧化还原调节产生NADPH。这 该提案旨在确定TCA依赖的NADPH生产在 线粒体到内质网内的动态平衡。为此，我们提出了三个具体目标：(1)确定如何 NADPH的产生和区划将营养流与内质网应激联系起来；(2)确定如何改变 线粒体和胞质谷胱甘肽氧化还原促进内质网氧化；以及(3)确定TCA活性和 谷胱甘肽氧化还原改变内质网功能。我们将利用基因和基因的组合来实现这些目标 操纵TCA循环活动的药理学工具；监测细胞变化的尖端生物传感器 氧化还原状态；内质网-线粒体接触的操作和分析；以及分子生物学方法 操作和评估ER功能。这项工作的结果将是机械地理解如何 代谢活动改变内质网功能，从而导致疾病。 此补充应用程序请求资金来支持身临其境的夏季研究体验 去年在鲁特科夫斯基实验室兼职的本科生。

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 to support an immersive summer research experience for an undergraduate who has worked part-time in the Rutkowski lab over the last year.