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

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.

We will design and build novel genetically encoded fluorescent protein biosensors for imaging the levels, at tissue and sub-cellular resolution, of ascorbate and (phospho)lipid hydroperoxides ((PH)LOOHs) in vivo in real time. Both biosensors will be based on "proximity redox relay" probes. These probe types have been used to image hydrogen peroxide and oxidised glutathione dynamics in yeast, animal and plant cells.
For the ascorbate biosensor, we will use a rice dehydroascorbate reductase (DHAR) for which a structure has just been published. Recombinant DHAR will be tethered to the redox sensitive roGFP2. This will create a "redox relay" from dehydroascorbate via DHAR to roGFP2. To get this to work, we anticipate having to solve several problems which require a multi-disciplinary collaboration between protein structural biochemists, cell biologist imaging experts, and plant and animal molecular biologists. We will use structure-guided design to modify DHAR and rapidly test variants of the enzyme for its ability to interact with roGFP2 in vitro. This will optimise electron transfer between roGFP2 and DHAR in response to the presence of dehydro- (oxidised) ascorbate. Based on the best in vitro design, we will build chimeric genes to express in animal and plant cells targeted to mitochondria, chloroplasts and the cytosol to show they encode a functional biosensor.
A similar approach will be used to design a biosensor for (PH)LOOHs. For this probe, we will tether to roGFP2 a radish (PH)LOOH glutathione peroxidase (PHGPX). TRX is the preferred electron donor for plant GPXs, so the challenge is to eliminate its interaction with the enzyme. This is so roGFP2 becomes the preferred reductant source, even if less efficient than TRX. Again, we will use structure-guided design to optimise an interaction between roGFP2 and PHGPX and remove TRX binding. To do this, we will determine the structure of a PHGPX-TRX complex to provide the necessary data.

Although this resource is intended to underpin fundamental life science research (see Academic Beneficiaries), we envisage that it will impact on agriculture, horticulture and parts of the food supply chain. In the long run we envisage that these and other biosensors could spin out technologies that contribute to more efficient production systems. For example, our Pathways to Impact envisages using these biosensors to develop "sentinel" plants to report, using remote sensing, very precisely the condition of a crop. An extension of this idea is that it could lead to technology that would allow, easily and cheaply, the early detection of metabolic processes that would herald future deterioration in stored live crop products. The availability of sentinel plants and sentinel harvested products could spawn new cheap technology such as the development of hand-held electronic devices to detect sensor fluorescence and give accurate readouts of specific parameters, such as the amount of lipid hydroperoxides or oxidised ascorbate.
Equally, this type of biosensor technology could impact on the biotechnology and life science industries in several ways. For example, more accurate, sensitive and comprehensive means of measuring both a key vitamin (ascorbic acid) and the involvement of lipid peroxidation in triggering or suppressing disease states though fundamental processes such as apoptosis, could open the way to an even better understanding of the link between diet, health and factors such as aging. In addition, we envisage exploiting specific biosensors, such as for lipid peroxidation, to develop screens for novel therapeutics that would ultimately treat human diseases such as cancer.
The potential for commercial exploitation of our research and the development of impact will be assessed by experts from the University's Research and Enterprise Office (REO). We shall meet them regularly. Communications outside the scientific community will be directed in a coordinated manner by the University's Communications Office.
Industry needs skilled personnel. A key element in the knowledge transfer process is the training of researchers at all levels. The proposed research will enable training or further development in synthetic biology, cell biology, imaging and image processing, structural biology, recombinant protein technology and project management.
An important group of beneficiaries is the General Public and in particular students. We plan to engage University of Essex students to develop a set of images and videos, which would be particularly useful to our outreach work with local schools. At Essex, "Science in Society" schemes organise talks to schools, colleges and the public during frequent Open Days to highlight the range of research activities in the biological sciences. Such project related materials should prove to be highly informative at these and the many external events we attend.
Finally, this project maps very well onto the aims of the bioimaging TRDF and this will be its major impact. This is because it will be an early stage investigation of new tools and technologies. This proof-of-concept pilot project is expressly about technology development and will clearly deliver an "improved technological approach" that enables "measurements inside cellular compartments", allowing us "to study (ascorbate and lipid hydroperoxide) dynamics, further enhancing resolution, sensitivity and precision". While there is an element of risk in this project it is contained and a mitigation plan is presented and a successful outcome would by highly rewarding and have a substantial impact on the UK and International Life Science community.