Biological chemistry of heme-mediated peroxide signalling and heme trafficking

血红素介导的过氧化物信号传导和血红素运输的生物化学

• 批准号: RGPIN-2018-05032
• 负责人: English, Ann
• 金额: $ 5.76万
• 依托单位: Concordia University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=748454
• 关键词: Biological; chemistry; heme; mediated; peroxide

Heme in hemoglobin transports oxygen but when it associates with other proteins, it performs different functions. For example, we recently demonstrated that the heme in cytochrome c peroxidase (Ccp1) signals when levels of hydrogen peroxide (a reactive oxygen species) in yeast mitochondria become potentially harmful. This occurs as cells deplete the glucose in their medium and switch from fermentation to respiration, which produces hydrogen peroxide as a by-product. Serendipitously, we noticed that heme-free Ccp1 exits respiring mitochondria, which at the same time accelerate their decomposition of hydrogen peroxide with oxygen generation. This is indicative of catalase activity and we showed that the heme from Ccp1 is transferred to catalase, an enzyme that uses heme to rapidly decompose hydrogen peroxide. We also showed using mass spectrometry that hydrogen peroxide weakens heme binding to Ccp1 by oxidizing the protein but that the heme remains undamaged.Such peroxide-induced heme mobilization has not been reported before so we aim to find out in atomic detail how this occurs in Ccp1. We will prepare mutant forms of Ccp1 to test possible mechanisms as well as model the reaction using computational methods to identify species that might not be stable enough to be detected experimentally. In characterizing the oxidation of Ccp1, we noted that its heme is wired' to many surface residues. We think that these residues locate binding sites for antioxidants present in the cell that protect Ccp1 or for compounds that Ccp1 converts to bioactive metabolites. We will examine the roles of these wires in detail and we will test if specific wires can be turned off by mutating residues along them. Heme is made in mitochondria but how it is transported to the many heme-requiring proteins in the cell remains a mystery. Inspired by our discovery that Ccp1's heme is transferred to catalase, we used a proteomics approach to discover other proteins involved in heme trafficking to or from Ccp1. We identified several proteins of interest, and we will explore heme transfer between them using sophisticated microscopy techniques. A hallmark of our research program is our use of yeast as a living test tube to guide our experiments and to discover new chemistry. Importantly, yeast and human cells share common signalling pathways so we will create key knowledge on hydrogen peroxide signalling and heme trafficking in health and disease that will contribute to drug discovery.

血红蛋白中的血红素运输氧气，但当它与其他蛋白质结合时，它发挥不同的功能。例如，我们最近证明，当酵母线粒体中的过氧化氢(一种活性氧物种)水平变得潜在有害时，细胞色素c过氧化物酶(Ccp1)中的血红素发出信号。这发生在细胞耗尽其培养液中的葡萄糖并从发酵转换到呼吸作用时，呼吸作用产生副产品过氧化氢。我们偶然地注意到，不含血红素的Ccp1存在呼吸线粒体，线粒体同时加速了过氧化氢的分解和氧气的产生。这表明了过氧化氢酶的活性，我们发现Ccp1中的血红素转移到了过氧化氢酶上，过氧化氢酶是一种使用血红素快速分解过氧化氢的酶。我们还用质谱仪表明，过氧化氢通过氧化蛋白质来削弱血红素与Ccp1的结合，但血红素保持不变。这种由过氧化氢诱导的血红素动员以前从未报道过，所以我们的目标是从原子细节上找出这是如何发生在Ccp1中的。我们将准备Ccp1的突变形式来测试可能的机制，并使用计算方法对反应进行建模，以识别可能不够稳定而无法通过实验检测到的物种。在表征Ccp1的氧化时，我们注意到它的血红素与许多表面残基相连。我们认为这些残基定位于细胞中保护Ccp1的抗氧化剂的结合部位，或者是Ccp1转化为生物活性代谢物的化合物的结合部位。我们将详细研究这些导线的作用，并测试是否可以通过沿它们突变残基来关闭特定的导线。血红素是在线粒体中形成的，但它是如何运输到细胞中许多需要血红素的蛋白质中仍然是一个谜。受我们发现CCP1‘S血红素被转移到过氧化氢酶的启发，我们使用蛋白质组学的方法来发现与CCP1之间的血红素运输有关的其他蛋白质。我们鉴定了几种感兴趣的蛋白质，并将使用复杂的显微镜技术探索它们之间的血红素转移。我们研究计划的一个特点是我们使用酵母作为活的试管来指导我们的实验和发现新的化学物质。重要的是，酵母和人类细胞共享共同的信号通路，因此我们将创建有关过氧化氢信号和血红素贩运在健康和疾病方面的关键知识，这将有助于药物发现。