The quarternary structure of complex IV of the electron transport chain of Drosophila melanogaster is a key study of Prof. Bill Ballard.
How Do Cells Regulate Redox Environment at the Cellular and Organellar Level?
Altered redox homeostasis is associated with many diseases including, diabetes and more generally metabolic syndrome, neurodegenerative disorders including Alzheimer’s, Parkinson’s diseases and has been implicated as an important factor regulating cell growth, senescence and ageing. In humans altered redox is associated with age-related changes at the cellular, tissue and organ level, and with age-related disease states. For example the redox environment of blood plasma becomes progressively more pro-oxidising with age. In addition to age-related changes in cellular redox, cells of aerobic organisms are continually exposed to reactive oxygen species (ROS) as part of normal metabolism. These ROS are toxic, affect redox balance, and have been implicated in many diseases including cancer, cardiovascular disease, arthritis and ageing. Organisms have evolved a complex array of defences comprised of low-molecular weight (non-enzymatic) and enzymatic antioxidants. These defences include redox buffers such as the tripeptide GSH (γ-glutamylcysteinylglycine) that are responsible for maintaining redox homeostasis. Oxidative stress results from redox imbalance e.g. when cellular defence mechanisms are unable to adequately adapt to an oxidant challenge leading to oxidative damage to cellular constituents.
Maintenance of a stable redox environment is critical for appropriate functioning of cellular processes and cell survival. The ability of a cell to maintain its redox environment in an optimal range is as important as its ability to maintain pH homeostasis, since many reactions are dependent on redox and cysteine residues in proteins are sensitive to oxidation. Cells and tissues need to maintain an overall reducing environment. Numerous species contribute to buffering the redox environment of cells including oxidised and reduced: NADP(H), thioredoxin, ascorbic acid, and thiol-containing molecules including glutathione (GSH), cysteine and protein thiols.
Appropriate functioning of a broad range of cellular processes is facilitated by maintenance of a highly reducing environment in most cellular organelles/compartments. Glutathione (GSH) is generally regarded as the primary redox buffering system in most cellular compartments due to its low redox potential and relatively high abundance. The redox environment in distinct cellular compartments may also differ due to differences in localisation of redox-regulating enzymes, movement of small molecules between compartments and/or the biochemical processes conducted in each. In eukaryotic cells there are different cell compartments (cytosol, mitochondria, nucleus, peroxisome, endoplasmic reticulum) and some of these have dramatically different redox states, consistent with the different processes that occur in them. Traditionally most measurements of cell redox couples have been performed on whole cell homogenates. This mixes compartments and can be misleading and very inaccurate. This has important implications, not only for regulation of cellular redox, but also for pathological conditions in which redox is perturbed.
The development of redox-sensitive green fluorescent protein (roGFP) has provided a significant leap forward in terms of assessing cellular redox state in a dynamic ‘real-time’ and non-invasive manner. roGFP probes can also be targeting to specific organelles or compartments. This project aims to exploit roGFP, to indentify the gentic and environmental factors affecting redox homeostasis in cells and cellular compartments. The project will exploit the power and versatility of the model eukaryote organism Saccharomyces cerevisiae (baker’s yeast). The approach will involve genetically engineering roGFP to target the probe to specific compartments including the mitochondrial matrix, endoplasmic reticulum, peroxisome and nucleus. The redox environment of the cytosol and/or organelle will then be assessed by measuring the response of the probe in wild-type and mutant cells of the genome-wide yeast deletion collection. The yeast deletion collection (~4600 mutants) is a very powerful tool that can be exploited to assess the effect of changes in cellular processes or metabolic pathways on a given trait, in this case compartmental redox, on a genome-wide scale. For a limited number of mutants, hand selected based on their putative role in redox, or mutants discovered through genome-wide screening as ‘mutants of interest’, the dynamic (short-term) response to changes in redox will also be studied more intensely. For these selected mutants a range of growth/stress conditions including exposure of cells oxidants, selected drugs and nutrients. This more focused approach will be used to establish the exact role of a gene(s) in regulation of cellular and/or organellar redox. The methodology used will include both high (robotic) and low-throughput screening, flow cytometry and imaging of cells using fluorescence microscopy. The methodology employed will include a broad range of recombinant DNA, microbiological, biochemical and cellular biology techniques.
The project will yield interesting data that will help us understand how redox environment is established and maintained at the cellular and organellar level. The data generated may have important implications for understanding the role of altered redox in disease processes.
References:
- The integration of glutathione homeostasis and redox signaling. Meyer AJ. J Plant Physiol. 165 (13):1390-403.
- Real-time imaging of the intracellular glutathione redox potential. Gutscher M, et al. Nat Methods. 5 (6):553-9.
BABS personnel that are responsible for this project