EAGER: Reactive Sulfide Species: Ubiquitous and Primordial Signaling Molecules

活性氧（ROS）、超氧化物和过氧化氢（H2O2）长期以来与生物系统中的氧化应激、细胞内信号传导和有害效应有关。然而，我们所知道的导致生命的大部分进化和物种间的机制保护发生在缺氧和硫化物环境中。有证据表明，早在大气中氧气出现之前，“抗氧化途径”就已经形成了。此外，许多测量ROS的方法对反应性硫化物（RSS）更敏感，这些硫化物在化学上比ROS更活跃，在生物学上也比ROS更通用。该研究提出了一个新的假设，即RSS、硫化氢（H2S）和多硫化物（H2Sn, n=2-7）参与细胞内信号传导，在许多情况下，RSS而不是ROS是真正的内源性信号，可能有助于氧化应激。也有人提出活性氮（RNS）、一氧化氮（NO-）和亚硝酸盐（NO2-）是原始的氧受体，它们在细胞内信号传导中与活性氮（RSS）相互作用。这项研究将为ROS介导细胞内信号传导和氧化应激的长期和根深蒂固的范式提供另一种选择。它将表明，活性氧本身是一种高度反应性和多用途的信号分子，许多被充分研究的活性氧的生理和病理作用可归因于活性氧。这项研究可能会开辟细胞内信号传导和氧化还原稳态的新领域，其中许多将适用于跨生物学学科，甚至可能具有相当大的治疗价值。拟进行的研究包括：研究RSS的氧化还原化学性质；确定RSS对细胞氧化还原信号的作用；并通过一些分析程序测试H2S2是否会被误认为H2O2。目标1将使用roGFP监测RSS的氧化还原活性，以及它如何受到游离金属、抗氧化酶（如超氧化物歧化酶、过氧化氢酶等）和血红素蛋白（血红蛋白、血红蛋白、肌红蛋白）的影响。这些研究将在常氧、缺氧和生理pO2、生理pH范围内以及存在和不存在原始电子受体RNS的情况下进行。采用敏感的H2S、O2和NO电流电极监测特定的RSS反应以及ROS、RSS和RNS之间的反应，建立化学计量关系。目的2将通过竞争性抑制剂和siRNA的抑制来研究与RSS生物合成相关的酶的具体作用。目的2还将研究外源性RSS和线粒体靶向H2S释放药物对细胞区室氧化还原状态的影响。目的3将测试“特异性”ROS检测是否可以区分ROS和RSS。PI的实验室长期以来一直支持高中、本科和研究生的培训，并特别为少数民族和代表性不足的群体营造了一个学习环境。这个项目将为继续进行培训提供资源。研究结果将通过在科学会议上的报告和同行评议的期刊出版物进行分发。

Reactive oxygen species (ROS), superoxide and hydrogen peroxide (H2O2) have long been associated with oxidative stress, intracellular signaling, and deleterious effects in biological systems. However, a large part of the evolution that led to life as we know it and the conservation of mechanisms across species occurred in anoxic and sulfidic environments. Evidence suggests that 'antioxidant pathways' were developed long before the appearance of atmospheric oxygen. Furthermore, many methods for measuring ROS are more sensitive to reactive sulfide species (RSS), which are chemically more reactive and biologically more versatile signaling molecules than ROS. The proposed research examines a novel hypothesis that RSS, hydrogen sulfide (H2S) and polysulfides (H2Sn, n=2-7) are involved in intracellular signaling, and in many instances RSS not ROS are the true endogenous signals and may contribute to oxidative stress. It is also proposed reactive nitrogen species (RNS), nitric oxide (NO-) and nitrite (NO2-) were primordial oxygen acceptors and they interact with RSS in intracellular signaling. This research will offer an alternative to the long-standing and entrenched paradigm that ROS mediate intracellular signaling and oxidative stress. It will show that RSS are highly reactive and versatile signaling molecules in their own right and that many of the well-studied physiological and pathological effects of ROS can be attributed to RSS. The research could open up new fields of intracellular signaling and redox homeostasis, many of which will be applicable in across biology disciplines and could even have considerable therapeutic value. The proposed research will: examine the redox chemistry of RSS; determine the contribution of RSS to redox signaling in cells; and test whether H2S2 may be mistaken for H2O2 by some analytical procedures. Aim 1 will use roGFP to monitor redox activity of RSS and how this is affected by free metals, antioxidant enzymes (e.g., superoxide dismutase, catalase, etc) and heme proteins (hemin, hemoglobin, myoglobin). These studies will be done in normoxia, hypoxia and physiological pO2's, over a range of physiological pH, and in the presence and absence of primordial electron acceptors RNS. Sensitive H2S, O2 and NO amperometric electrodes will be employed to monitor specific RSS reactions and reactions between ROS, RSS and RNS to establish stoichiometric relationships. Aim 2 will examine the specific roles enzymes associated with RSS biosynthesis by inhibition with competitive inhibitors and siRNA. Aim 2 will also examine the effects of exogenous RSS and mitochondrial-targeted H2S releasing drugs on cellular compartmental redox state. Aim 3 will test whether 'specific' ROS assays can distinguish between ROS and RSS. The PI's laboratory has long supported high school, undergraduate, and graduate training and has especially fostered a learning environment for minorities and under-represented groups. This project will provide the resources for this training to continue. Results from the studies will be distributed through presentations at scientific meetings and through peer-reviewed journal publications.