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

点击翻译按钮获取中文摘要

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.