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做出反应。一个重要的信使是过氧化氢，它对细胞既有毒性又有好处。多年来，我们团队研究了一种名为过氧化氢酶的酶，这种酶能将过氧化氢转化为水。我们从细胞中提取了过氧化物酶，发现它们具有许多有趣的性质，特别是从酵母中提取的细胞色素C过氧化物酶(CCP)。然而，我们意识到，除非我们在它们的细胞环境中检查它们，否则我们不能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不可预测的。重要的是，我们发现CCP不是通过像预期的那样直接分解过氧化氢来保护细胞，而是通过感知它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解开CCP是如何传递这一信号的。我们拥有先进的工具来观察活细胞内部，我们可以检测细胞如何通过化学修饰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合作的新型蛋白质，我们可以通过基因操作酵母细胞来移除、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，以产生一种不具有过氧化物酶功能的突变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与CCP在细胞信号传递和防御中的协同作用。人类细胞拥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在健康和疾病中信号传递的分子机制的理解。

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.