Shedding light on oxidative stress: Identifying factors modulating the redox balance in the endoplasmic reticulum of Caenorhabditis elegans.

Reactive oxygen species (ROS), oxidative stress and the reduction-oxidation (redox) balance play key roles in many different processes in living organisms. The importance of these are reflected in the number of diseases and conditions where they play a major role, including, inflammation (including arthritis), in cancer, heart disease and stroke, and in the ageing process. In addition to involvement in disease, redox and ROS play essential roles in key normal cellular functions. The cellular compartment where proteins are folded and assembled prior to secretion is called the endoplasmic reticulum (ER). A critical step in ER protein folding is the formation of disulphide bonds, an event that is highly sensitive to the redox environment. If the ER is too reducing then disulphides will not form yet if it is too oxidising then non-native forms become very stable leading to misfolding, an event that may lead to disease. Many of the factors involved in controlling this delicate balance to allow correct disulphide formation and to prevent oxidative stress in the ER remain to be identified. In this project we will combine our diverse but complementary expertise in genetics, biochemistry and cell biology to fully dissect how the redox balance is maintained in the ER of a small model organism called Caenorhabditis elegans. We have developed a strain of C. elegans that emits a specific light signal that is dependent on the redox environment in the ER and we propose to use this as an experimental tool to uncover the genes involved in redox balance in this organelle. These experiments will be used to inform and direct experiments in mammalian cells and will have a direct bearing on human cell biology, human disease states, ageing and the biotechnological production of proteins.

The endoplasmic reticulum (ER) is the subcellular organelle where proteins destined for secretion are folded, modified and assembled prior to their secretion. This process includes the formation of disulphide bonds, an event that is highly sensitive to the redox balance. If the ER is too reducing then disulphides cannot efficiently form yet if it is too oxidising non-native disulphides are stabilised leading to misfolding. Numerous proteins are involved in maintaining the redox homeostasis in the ER to allow disulphide formation and to prevent oxidative stress in the organelle. This project combines expertise in genetics of a nematode model system as well as mammalian cell biology to fully dissect the key players contributing to maintaining the redox balance in the ER. The project will generate Caenorhabditis elegans mutants selecting for redox stress to identify proteins involved in preventing ER stress. In addition live organism imaging will be used to assess redox stress using a redox-sensitive variant of GFP (roGFP). The ultimate goal of this study is the use of the model nematode system to inform and direct experiments in mammalian cells.

This project will, for the first time, allow the evaluation of the redox balance at the physiological whole-body context of a multicellular animal. Our approach will also allow detailed cell, tissue and organ level examination of redox. The collaboration will make a direct connection between work carried out in a model organism and studies carried out in mammalian systems. It is well established that C. elegans represents an excellent, genetically amenable animal model and work in this simple worm has led to the award of three separate Nobel prizes in the last decade. The development of sensitive GFP reporter molecules represented a major advance in cell biology that was recognized by the award of a joint Nobel Prize in 2008 for work on C. elegans. Likewise the development of calcium sensitive variants of GFP has allowed the direct monitoring of intracellular changes in live cells.
We are proposing to combine our expertise in genetics, cell biology and biochemistry by applying this model system to help understand important questions in human cell biology, namely the redox balance and protein folding process in the endoplasmic reticulum. Oxidative folding, isomerisation and reduction all occur in the ER and the fine control of this redox balance is critical since its disruption will lead to ER stress and ultimately protein misfolding and aggregation. In addition to playing a key role in the ageing process, ER stress can lead to a wide range of diseases such as diabetes, inflammation and several neurodegenerative disorders such as Alzheimer's and Parkinson's disease. The exact mechanism for the maintenance of redox balance in the ER remains a major unanswered question in cell biology.