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，所有产生和/或消耗氧气(通过呼吸)的细胞都面临着一个不可避免的危险，那就是产生活性氧物种(ROS)。ROS的产生是O2化学的必然结果，如果它们积累，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的不可避免的后果。ROS的积累与衰老、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。重要的ROS经常被测量为氧化损伤的指示物是脂(或磷脂)氢过氧化氢，它特别有效，因为它们可以溶解在细胞膜中并破坏细胞膜。植物还会产生脂质过氧化氢，这往往与光合作用的抑制、感染造成的组织破坏、草食动物的放牧以及臭氧等大气污染物有关。过氧化脂质在一些成熟水果中也是重要的风味成分，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。这就是脂质过氧化氢的情况，它是一些重要的细胞信号分子的前体，如动物的前列腺素和植物的茉莉酸。这些分子刺激细胞信号，以响应促进脂质过氧化氢积累的条件，导致防御系统的启动，从而将进一步的氧化损伤降至最低。我们饮食中所需的维生素C(抗坏血酸)是一种由植物(以及许多哺乳动物，但不是人类)制造的有效抗氧化剂。植物和动物需要抗氧化剂，以最大限度地减少ROS的积累，从而防止由它们引起的上述许多问题。植物和动物体内的抗坏血酸不仅可以抵御ROS，还可以发挥许多其他作用。例如，在植物中，作为一种植物生长调节剂，在动物中合成胶原蛋白，它在加强细胞壁方面很重要。维生素C也在蔬菜和水果中积累到很高的水平，尽管我们知道它对我们的饮食很重要，但我们不清楚为什么植物在一些储存器官和水果中积累了这么多。对于抗坏血酸和脂质过氧化氢来说，如果我们能够准确地测量它们在活细胞中的水平，关于这些分子的许多悬而未决的问题就可以得到解决。我们的目标是建造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手段，以精确定位和测量抗坏血酸和脂质过氧化氢。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实现这一点。我们想要分别从大米和萝卜中提取结合抗坏血酸和过氧化脂质的酶的合成版本，并将它们与一种名为roGFP2的经过大幅修改的水母荧光蛋白捆绑在一起。当传感器酶与其伙伴化合物反应时，它被氧化(漂白)，它将其氧化传递给它的伙伴roGFP2，这改变了它的荧光特性。这种荧光可以用特殊的显微镜在表达这些传感器的细胞中可视化。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，植物和动物细胞在应对许多不同情况和挑战时所拥有的抗坏血酸和脂质过氧化氢的时间和数量。

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.