Metabolic rewiring coupled to the production of reactive oxygen and nitrogen species (RONS)

代谢重新布线与活性氧和氮 (RONS) 的产生相结合

• 批准号: 10672344
• 负责人: Jing Fan
• 金额: $ 42.63万
• 依托单位: MORGRIDGE INSTITUTE FOR RESEARCH, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10672344
• 关键词: Acetyl Coenzyme A; Automobile Driving; Biochemical; Blood Vessels; Cardiovascular Diseases; Cell physiology; Cells; Coenzyme A; Coupled; Cysteine; Disease; Endothelial Cells; Enzymes; Foundations; Free Radicals; Glycolysis; Histone Acetylation; Immune; Immune response; Inflammation; Inflammatory; Isotopes; Ketoglutarate Dehydrogenase Complex; Knowledge; Link; Macrophage; Mediating; Metabolic; Metabolism; Mitochondria; Modification; Molecular; NADP; NADPH Oxidase; Nerve Degeneration; Neurons; Neurophysiology - biologic function; Nitric Oxide Synthase; Nitrogen; Oxidation-Reduction; Oxygen; Pathologic; Pentosephosphate Pathway; Periodicity; Physiological Processes; Play; Production; Proteins; Reaction; Reactive Nitrogen Species; Reactive Oxygen Species; Recovery; Regulation; Role; Signal Transduction; Sulfhydryl Compounds; Time; Tissues; arm; electron donor; enzyme substrate; genetic manipulation; glucose metabolism; improved; neutrophil; novel; oxidation; pyruvate dehydrogenase

Project Summary/Abstract Free radicals are more than toxic by-products of metabolism. Many cells produce reactive oxygen species (ROS) and reactive nitrogen species (RNS) using specialized enzymes, NADPH oxidases (NOXs) and nitric oxide synthases (NOSs) respectively, for specific functions. For instance, innate immune cells produce RONS in large amounts during inflammation, and endothelial cells and neurons can produce them for signaling. RONS production is an important part of many physiological processes as well as numerous diseases, including inflammatory disorders, cardiovascular diseases, neurodegeneration, etc. We propose to investigate how NOX- and NOS-dependent RONS production is coupled to substantial remodeling of cellular metabolism. Specifically, we will address two key questions: (1) How is cellular metabolism remodeled to enable RONS production? Both NOXs and NOSs require NADPH as the electron donor to drive the reactions. Thus, activation of NOX or NOS greatly increases cellular NADPH demand and can significantly lower NADPH/NADP+ redox ratio. We recently discovered that in activated neutrophils, NOX activation is quantitatively coupled to the metabolic shift from glycolysis to cyclic pentose phosphate pathway (a unique mode for glucose metabolism with ultra-high NADPH yield) to meet this demand. We propose to investigate the molecular mechanism allowing neutrophils to complete such substantial metabolic switch in a very short time, which is essential for them to immediately mount the first line of innate immune defense. We will next reveal the metabolic strategies other cells use to power up RONS production using approaches including isotopic-tracing based metabolic flux analysis and genetic manipulation. (2) How does the increase in RNS regulates cellular metabolism? It is well known that RONS can impact cell functions through protein cysteine oxidation or nitrosylation. Intriguingly, we recently found that the inducible RNS production in activated macrophages drives dynamic metabolic remodeling via a novel mechanism: RNS specifically and efficiently inactivate two crucial mitochondrial enzymes, pyruvate dehydrogenase and oxoglutarate dehydrogenase, via covalent modifications of the catalytic lipoic arm in their E2 subunits. We seek to understand of the biochemical mechanisms for such RNS-driven regulation in-depth, specifically, (i) elucidate the role of coenzyme A, the thiol-containing substrate for these enzymes, in delivering NO- mediated modifications specifically onto the catalytic lipoic moiety; (ii) examine the mechanistic link between such lipoic modifications with the cysteine nitrosylation on their E3 subunit; (iii) examine the reversibility of such RNS- mediated modifications of lipoic arm and the mechanism driving the recovery. We will further investigate how this mechanism regulates other lipoic arm dependent- enzymes, and elucidate the downstream effects of such regulation in cellular physiology in various RNS-producing cells (e.g. regulation of pyruvate dehydrogenase by NO can modulate histone acetylation and downstream cell functions by changing acetyl-coA availability).

项目概要/摘要 自由基不仅仅是新陈代谢的有毒副产品。许多细胞产生活性氧 (ROS) 和活性氮 (RNS)，使用专门的酶、NADPH 氧化酶 (NOX) 和一氧化氮 合酶（NOS）分别用于特定功能。例如，先天免疫细胞大量产生 RONS 炎症期间的量，内皮细胞和神经元可以产生它们用于信号传导。罗恩斯 生产是许多生理过程以及许多疾病的重要组成部分，包括 炎症性疾病、心血管疾病、神经退行性疾病等。我们建议研究 NOX- NOS 依赖性 RONS 的产生与细胞代谢的实质性重塑相关。具体来说， 我们将解决两个关键问题：（1）如何重塑细胞代谢以实现 RONS 的产生？两个都 NOX 和 NOS 需要 NADPH 作为电子供体来驱动反应。因此，NOX或NOS的激活 大大增加细胞 NADPH 需求，并能显着降低 NADPH/NADP+ 氧化还原比。我们最近 发现在活化的中性粒细胞中，NOX 活化与从 糖酵解至环戊糖磷酸途径（超高 NADPH 葡萄糖代谢的独特模式 产量）来满足这一需求。我们建议研究使中性粒细胞完成的分子机制 在很短的时间内发生如此巨大的代谢转变，这对于他们立即启动第一个过程至关重要 先天免疫防御线。接下来我们将揭示其他细胞用来增强 RONS 的代谢策略 使用包括基于同位素示踪的代谢通量分析和遗传操作的方法进行生产。 （2）RNS的增加如何调节细胞代谢？众所周知，RONS 可以影响细胞 通过蛋白质半胱氨酸氧化或亚硝基化发挥作用。有趣的是，我们最近发现诱导 活化巨噬细胞中 RNS 的产生通过一种新机制驱动动态代谢重塑：RNS 特异性且有效地灭活两种关键的线粒体酶：丙酮酸脱氢酶和 氧化戊二酸脱氢酶，通过其 E2 亚基中的催化硫辛臂的共价修饰。我们寻求 深入了解这种 RNS 驱动调节的生化机制，具体来说，(i) 阐明 辅酶 A（这些酶的含硫醇底物）在传递 NO 介导的过程中的作用 专门针对催化硫辛部分进行修饰； (ii) 检查此类硫辛酸之间的机制联系 E3 亚基上的半胱氨酸亚硝基化修饰； (iii) 检查这种 RNS 的可逆性- 硫辛臂介导的修饰和驱动恢复的机制。我们将进一步研究如何 该机制调节其他硫辛臂依赖性酶，并阐明此类酶的下游效应 各种产生 RNS 的细胞中细胞生理学的调节（例如通过调节丙酮酸脱氢酶） NO 可以通过改变乙酰辅酶 A 的可用性来调节组蛋白乙酰化和下游细胞功能。