Bioimaging of dehydroascorbate and (phospho)lipid hydroperoxides: The development of fluorescent protein biosensors

我们呼吸的氧气（O2）是植物光合作用时产生的。然而，所有通过呼吸产生和/或消耗氧气的细胞都面临着一个不可避免的危险，那就是活性氧（ROS）的产生。活性氧是氧化学不可避免的结果，如果它们积累起来，它们会对细胞成分造成氧化损伤，并可能引发细胞死亡。氧化损伤是产生或消耗氧气不可避免的后果。活性氧积累与衰老、神经退化、癌症风险增加以及与防御感染相关的炎症反应有关。通常作为氧化损伤指标的重要ROS是脂质（或磷脂）氢过氧化物，它们特别有效，因为它们可以溶解并破坏细胞膜。植物也产生脂质氢过氧化物，这通常与光合作用的抑制、感染引起的组织损伤、食草动物的放牧和臭氧等大气污染物有关。脂质过氧化物也是一些成熟水果的重要风味成分，例如西红柿和面粉中的异味。然而，进化往往会将潜在的破坏性转化为有用的东西。脂质氢过氧化物就是这种情况，它是一些重要细胞信号分子的前体，如动物中的前列腺素和植物中的茉莉酸。这些分子刺激细胞信号传导，以应对促进脂质氢过氧化物积累的条件，从而开启防御机制，最大限度地减少进一步的氧化损伤。我们饮食中需要的维生素C（抗坏血酸）是一种强效抗氧化剂，由植物（以及许多哺乳动物，但不是人类）产生。植物和动物需要抗氧化剂，以尽量减少活性氧的积累，从而防止上述由活性氧引起的许多问题。在植物和动物中，抗坏血酸不仅可以防止ROS，还可以发挥许多其他作用。例如，它在植物细胞壁强化中很重要，作为植物生长调节剂，在动物中合成胶原蛋白。维生素C在蔬菜和水果中也会积累到很高的水平，尽管我们知道它对我们的饮食很重要，但我们还不清楚为什么植物的一些储存器官和水果中会积累这么多维生素C。对于抗坏血酸和脂质氢过氧化物，如果我们能够准确地测量它们在活细胞中的水平，那么关于这些分子的许多悬而未决的问题就可以得到解决。我们的目标是制造出能够做到这一点的传感器。我们的目标是为英国生物科学界提供一种低成本的方法来精确定位和测量抗坏血酸和脂质氢过氧化物。我们建议通过在体外和体内建立和测试所谓的“氧化还原继电器”荧光蛋白生物传感器来做到这一点。我们想要合成一种酶，分别从大米和萝卜中提取抗坏血酸和脂质氢过氧化物，并将它们与一种被称为roGFP2的经过极大修饰的水母荧光蛋白捆绑在一起。当传感器酶与其伙伴化合物发生反应时，它被氧化（漂白），它将其氧化传递给它的roGFP2伙伴，这改变了它的荧光特性。使用专门的显微镜，可以在表达这些传感器的细胞中看到这种荧光。这将意味着我们可以获得前所未有的信息，关于细胞中的位置，时间和抗坏血酸和脂质氢过氧化物的数量，植物和动物细胞对许多不同的情况和挑战作出反应。

The oxygen (O2) we breathe is produced by plants when they photosynthesise. However, all cells that produce and/or consume O2 (by respiration) face an unavoidable danger, which is the production of reactive oxygen species (ROS). ROS arise as an inevitable consequence of O2 chemistry and if they accumulate, they cause oxidative damage to cell components and can trigger the death of the cell. Oxidative damage is an inescapable consequence of producing or consuming O2. ROS accumulation is associated with aging, nerve degeneration, increased cancer risk and inflammation responses associated with defence against infection. Important ROS which are often measured as an indicator of oxidative damage are lipid (or phosopholipid) hydroperoxides, which are particularly potent because they can dissolve in and damage cell membranes. Plants also produce lipid hydroperoxides, which are often associated with the inhibition of photosynthesis, tissue damage due to infection, grazing by herbivores and atmospheric pollutants such as ozone. Lipid peroxides are also important as flavour components in some ripe fruits, such as tomatoes and off-flavours in flour (for example). Evolution, though, often turns the potentially damaging into something useful. This is the case for lipid hydroperoxides, which are the precursors for some important cell signalling molecules such as prostaglandins in animals and jasmonic acid in plants. These molecules stimulate cell signalling in response to conditions that promote accumulation of lipid hydroperoxides causing the switching on of defences that minimise further oxidative damage.Vitamin C (ascorbate) which we require in our diet is a potent antioxidant made by plants (and also many mammals, but not humans). Plants and animals need antioxidants in order to minimise the accumulation of ROS and thus prevent many of the problems caused by them that were described above. Ascorbate in both plants and animals not only protects against ROS, but play many other roles. For example, it is important in cell wall strengthening in plants, as a plant growth regulator and in animals for the synthesis of collagen. Vitamin C also accumulates to high levels in vegetables and fruits, although we know it is important for our diet, it is not clear why plants accumulate so much in some storage organs and fruits. For both ascorbate and lipid hydroperoxides, the many unanswered questions about these molecules could be addressed if we could accurately measure their levels in the living cell. We aim to build sensors that can do this. Our aim is to provide the UK bioscience community with a low cost means to precisely locate and measure ascorbate and lipid hydroperoxides. We propose to do this by building and testing in vitro and in vivo so-called "redox relay" fluorescent protein biosensors. We want to take synthetic versions of enzymes that bind ascorbate and lipid hydroperoxides, from rice and radishes respectively, and tether them to a greatly modified jellyfish fluorescent protein called roGFP2. When the sensor enzyme reacts with its partner compound it becomes oxidised (bleached), it passes on its oxidation to its roGFP2 partner, which changes its fluorescence characteristics. This fluorescence can be visualised in cells expressing these sensors using specialised microscopes. This will mean we can obtain unprecedented levels of information on the places in the cell, the time and the amount of ascorbate and lipid hydroperoxides that plant and animal cells have in response to many different situations and challenges.