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

许多化学信使触发细胞对细胞内外的环境变化作出反应。一个重要的信使是过氧化氢，它对细胞既有害又有益。多年来，我们的团队一直在研究一种叫做过氧化物酶的酶，它能将过氧化氢转化为水。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，特别是酵母中的细胞色素c过氧化物酶（CcP）。尽管如此，我们意识到，除非我们在细胞环境中检测它们，否则我们无法完全了解这些酶的生物学功能。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没有预料到的。重要的是，我们发现CcP不是通过直接分解过氧化氢来保护细胞，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，过氧化氢酶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揭示CcP是如何传递这个信号的。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用化学方法修饰CcP和其他信号蛋白来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，我们可以通过基因操纵酵母细胞来去除、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，以产生不具有过氧化物酶功能的CcP突变体，从而将CcP的过氧化物酶活性与其信号作用分离开来。酵母中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与CcP在细胞信号传导和防御中的协调作用。人类细胞具有与酵母相似的信号通路，因此所获得的知识将加速我们对过氧化氢信号在健康和疾病中的分子机制的理解。

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.