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

脱氢抗坏血酸和（磷酸）脂质氢过氧化物的生物成像：荧光蛋白生物传感器的开发

• 批准号: BB/P026656/1
• 负责人: Phillip Mullineaux
• 金额: $ 19.31万
• 依托单位: University of Essex
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FP026656%2F1
• 关键词: Bioimaging; dehydroascorbate; phospho; lipid; hydroperoxides

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.

我们呼吸的氧气（O2）是由植物光合作用产生的。然而，所有产生和/或消耗O2（通过呼吸）的细胞都面临着不可避免的危险，即活性氧（ROS）的产生。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O2的不可避免的后果。ROS积累与衰老、神经变性、癌症风险增加和与抵抗感染相关的炎症反应有关。通常作为氧化损伤的指标测量的重要ROS是脂质（或磷脂）氢过氧化物，其特别有效，因为它们可以溶解在细胞膜中并损伤细胞膜。植物还产生脂质过氧化氢，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中重要的风味成分，如番茄和面粉中的异味（例如）。然而，进化往往会把潜在的破坏性变成有用的东西。脂质氢过氧化物的情况就是如此，它们是一些重要细胞信号分子的前体，例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，以响应促进脂质过氧化氢积累的条件，从而导致打开防御机制，最大限度地减少进一步的氧化损伤。维生素C（抗坏血酸盐），我们需要在我们的饮食是一种有效的抗氧化剂由植物（也是许多哺乳动物，但不是人类）。植物和动物需要抗氧化剂，以最大限度地减少ROS的积累，从而防止上述由它们引起的许多问题。植物和动物中的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，作为植物生长调节剂和在动物中用于胶原蛋白的合成中是重要的。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，但目前还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，如果我们能够准确测量它们在活细胞中的水平，那么关于这些分子的许多未回答的问题就可以得到解决。我们的目标是制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方法来精确定位和测量抗坏血酸盐和脂质过氧化氢。我们建议这样做，在体外和体内的所谓的“氧化还原继电器”荧光蛋白生物传感器的建设和测试。我们希望分别从大米和萝卜中合成结合抗坏血酸和脂质过氧化氢的酶，并将它们与一种经过极大修饰的水母荧光蛋白roGFP 2结合。当传感器酶与其伴侣化合物反应时，它被氧化（漂白），它将其氧化传递给其roGFP 2伴侣，这改变了其荧光特性。这种荧光可以在表达这些传感器的细胞中使用专门的显微镜可视化。这将意味着我们可以获得前所未有的信息水平，关于植物和动物细胞在应对许多不同情况和挑战时所具有的抗坏血酸和脂质过氧化物在细胞中的位置，时间和数量。