Mechanistic underpinnings of biological oxidative processes: Chemical, computational and genetic analysis of reactions between metalloproteins and redox signalling molecules

生物氧化过程的机制基础：金属蛋白和氧化还原信号分子之间反应的化学、计算和遗传分析

• 批准号: 1530-2013
• 负责人: English, Ann
• 金额: $ 3.93万
• 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: 2014
• 资助国家: 加拿大
• 起止时间: 2014-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=543978
• 关键词: Mechanistic; underpinnings; biological; oxidative; processes

Many chemical messengers trigger cells to respond to environmental changes, both inside and outside the cell. One important messenger is hydrogen peroxide, which has the power to be both toxic and beneficial to cells. For many years, our group studied enzymes called peroxidases that convert hydrogen peroxide to water. We extracted the peroxidases from cells and discovered that they possess many interesting properties, in particular cytochrome c peroxidase (CcP) from yeast. Nonetheless, we realized that we could not fully understand the biological function of these enzymes unless we examined them in their cellular environment. Thus, about six years ago we began investigating the role of CcP in yeast cells and most of the results we obtained so far were totally unpredicted. Importantly, we found that CcP protects cells not by directly decomposing hydrogen peroxide as expected but by sensing its presence and signalling to the cell to produce catalase, an enzyme that is a more efficient hydrogen peroxide scavenger. Now our aim is to unravel at the molecular level how CcP conveys this signal. We have the sophisticated tools to look inside live cells, we can detect how cells chemically modify CcP and other signalling proteins to alter their function or location, we can capture novel proteins that partner with CcP, and we can genetically manipulate yeast cells to remove, mutate or tag any protein of interest. In fact, we have already genetically altered yeast to produce a mutant CcP that does not function as a peroxidase in order to decouple CcP's peroxidase activity from its signalling roles. Yeast contains other enzymes that react with hydrogen peroxide and we will investigate at the molecular level their coordination with CcP in cell signalling and defense. Human cells possess similar signalling pathways as yeast so the knowledge gained will accelerate our understanding of the molecular mechanisms of hydrogen peroxide signalling in health and disease.

许多化学信使触发细胞对细胞内外的环境变化做出反应。一个重要的信使是过氧化氢，它具有对细胞既有毒又有益的能力。多年来，我们小组研究了将过氧化氢转化为水的过氧化物酶。我们从细胞中提取过氧化物酶，发现它们具有许多有趣的性质，特别是来自酵母的细胞色素c过氧化物酶（CcP）。尽管如此，我们意识到，我们不能完全理解这些酶的生物学功能，除非我们在细胞环境中检查它们。因此，大约六年前，我们开始研究CcP在酵母细胞中的作用，到目前为止，我们获得的大多数结果都是完全无法预测的。重要的是，我们发现CcP保护细胞的方式不是像预期的那样直接分解过氧化氢，而是通过感知过氧化氢的存在并向细胞发出信号来产生过氧化氢酶，这是一种更有效的过氧化氢清除剂。现在我们的目标是在分子水平上解开CcP如何传递这种信号。我们有复杂的工具来观察活细胞内部，我们可以检测细胞如何化学修饰CcP和其他信号蛋白以改变它们的功能或位置，我们可以捕获与CcP合作的新蛋白质，我们可以遗传操纵酵母细胞以去除，突变或标记任何感兴趣的蛋白质。事实上，我们已经对酵母进行了基因改造，使其产生一种不起过氧化物酶作用的突变型CcP，以使CcP的过氧化物酶活性与其信号作用脱钩。酵母含有与过氧化氢反应的其他酶，我们将在分子水平上研究它们在细胞信号传导和防御中与CcP的协调。人类细胞具有与酵母相似的信号传导途径，因此所获得的知识将加速我们对健康和疾病中过氧化氢信号传导的分子机制的理解。