EAGER: Reactive Sulfide Species: Ubiquitous and Primordial Signaling Molecules

EAGER：反应性硫化物种类：普遍存在的原始信号分子

• 批准号: 1446310
• 负责人: Kenneth Olson
• 金额: $ 30万
• 依托单位: Indiana University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1446310&HistoricalAwards=false
• 关键词: EAGER; Reactive; Sulfide; Species; Ubiquitous

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.

长期以来，活性氧(ROS)、超氧阴离子和过氧化氢(H_2O_2)与生物系统中的氧化应激、细胞内信号转导和有害效应密切相关。然而，我们所知的导致生命的进化的很大一部分以及跨物种机制的保护发生在缺氧和硫化环境中。有证据表明，“抗氧化途径”早在大气中氧气出现之前就已经形成了。此外，许多测量ROS的方法对活性硫化物物种(RSS)更敏感，RSS在化学上比ROS更具活性，在生物学上比ROS具有更多功能的信号分子。这项研究验证了一种新的假说，即RSS、硫化氢(H_2S)和多硫化物(H_2S，n=2-7)参与了细胞内信号传递，在许多情况下，RSS而不是ROS是真正的内源信号，可能参与氧化应激。也有人认为，活性氮(RNS)、一氧化氮(NO-)和亚硝酸盐(NO2-)是原始的氧受体，它们在细胞内信号转导中与RSS相互作用。这项研究将提供一种替代长期存在的和根深蒂固的范例，即ROS介导细胞内信号和氧化应激。这将表明RSS本身就是高度反应性和多功能的信号分子，许多被广泛研究的ROS的生理和病理效应都可以归因于RSS。这项研究可能会开辟细胞内信号和氧化还原动态平衡的新领域，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。这项拟议的研究将：检查RSS的氧化还原化学；确定RSS对细胞中氧化还原信号的贡献；并测试一些分析方法是否会将H2S2误认为H2O2。目标1将使用roGFP来监测RSS的氧化还原活性，以及自由金属、抗氧化酶(如超氧化物歧化酶、过氧化氢酶等)和血红素蛋白(血红素、血红蛋白、肌红蛋白)如何影响RSS的氧化还原活性。这些研究将在常氧、低氧和生理性PO2‘S中进行，在一定的生理pH范围内，在存在和不存在原始电子受体RNS的情况下进行。敏感的H_2S、O_2和NO安培电极将被用来监测特定的RSS反应以及ROS、RSS和RNS之间的反应，以建立化学计量关系。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还将检查外源RSS和线粒体靶向的硫化氢释放药物对细胞间隔室氧化还原状态的影响。目标3将测试“特定的”ROS分析是否能区分ROS和RSS。长期以来，国际学生联合会的实验室一直支持高中、本科生和研究生的培训，特别是为少数族裔和代表性不足的群体营造了一个学习环境。该项目将为继续开展这项培训提供资源。研究结果将通过在科学会议上的陈述和同行评议的期刊出版物分发。