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

项目摘要 内质网中蛋白质折叠的破坏--“ER应激”--与许多不同的细胞因子相关。 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， 由于这种特殊的疾病负担，了解导致ER的因素很重要 代谢失调时的压力然而，代谢活动和ER稳态的途径是 耦合是知之甚少。 线粒体是代谢的中心，TCA循环是这一活动的中心，接受底物 从糖酵解和脂肪酸氧化为catalysts，产生还原当量的电子传递 和维持细胞氧化还原稳态，并提供建筑材料的还原 脂质、葡萄糖和氨基酸的生物合成。由于它是众多流程的中心， TCA循环可能影响许多不同细胞途径，甚至那些没有明显直接联系的途径。 这包括ER蛋白质加工，其对氧化还原状态、氨基酸可用性和代谢的变化敏感。 细胞脂质含量 在母提案中，我们提供了一个以前未知的TCA之间的功能关系的证据， 代谢活性细胞中的周期活性和ER稳态，包括肝细胞、肌细胞和 脂肪细胞，这取决于生产NADPH的TCA循环和氧化还原调节谷胱甘肽。这 该提案旨在确定将TCA依赖性NADPH生产与 线粒体在内质网中的稳态。为此，我们提出了三个具体目标：（1）确定如何 NADPH的产生和区室化将营养流与内质网应激联系起来;（2）确定 线粒体和胞质谷胱甘肽氧化还原促进ER氧化;和（3）确定TCA活性和 谷胱甘肽氧化还原改变ER功能。我们将通过基因和 药理学工具来操纵TCA循环活动;尖端的生物传感器来监测细胞的变化， 氧化还原状态; ER-线粒体接触的操纵和分析;以及 操作和评估ER功能。这项工作的结果将是对如何进行机械性理解 代谢活动改变ER功能从而导致疾病。 这个补充申请要求资金，以支持沉浸式的夏季研究经验， 去年在Rutkowski实验室兼职工作的本科生。

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.