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

项目总结/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） 和活性氮物质（RNS）使用专门的酶，NADPH氧化酶（NOX）和一氧化氮 具体功能分别为NOS。例如，先天免疫细胞产生大量的RONS， 在炎症期间，内皮细胞和神经元可以产生它们用于信号传导。Rons 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，我们建议调查如何氮氧化物- 并且NOS依赖性RONS的产生与细胞代谢的实质性重塑相关。具体地说， 我们将解决两个关键问题：（1）细胞代谢是如何被改造以使RONS产生的？两 NOX和NOS需要NADPH作为电子供体来驱动反应。因此，NOX或NOS的活化 大大增加了细胞对NADPH的需求，并可显著降低NADPH/NADP+氧化还原比。我们最近 发现，在活化的中性粒细胞中，NOX活化与代谢转变定量相关， 糖酵解至环戊糖磷酸途径（具有超高NADPH的葡萄糖代谢的独特模式 #21453;，以满足这一需求。我们建议调查的分子机制，使中性粒细胞完成 这种实质性的代谢转换在很短的时间内，这是必不可少的，他们立即安装第一个 先天免疫防御系统接下来我们将揭示其他细胞用于启动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起作用。有趣的是，我们最近发现， 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 特异性和有效地抑制两种关键的线粒体酶，丙酮酸脱氢酶和 酮戊二酸脱氢酶，通过共价修饰其E2亚基中的催化硫辛酸臂。我们寻求 为了深入了解这种RNS驱动调节的生化机制，具体而言，（i）阐明 辅酶A，这些酶的含巯基底物，在递送NO介导的 （ii）检查这种硫辛酸部分之间的机械连接; 在其E3亚基上用半胱氨酸亚硝基化修饰;（iii）检查此类RNS的可逆性- 介导的脂质臂修饰和驱动恢复的机制。我们将进一步调查 该机制调节其它类脂臂依赖性酶，并阐明这些酶的下游效应。 调节各种RNS-产生细胞中的细胞生理学（例如， 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）。

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).