Determining the reductive pathway in the endoplasmic reticulum of mammalian cells

The ability of cells to correctly fold and assemble proteins is the final stage in protein synthesis. Protein folding requires a subset of proteins able to either catalyse folding reactions or act as molecular chaperones preventing non-productive protein aggregation and cell stress. The inability of cells to carry out the folding process results in cell death and consequently some of the most catastrophic disease pathologies such as diabetes, Alzheimer's and Parkinson's.

For cells and tissues to remain healthy they must be able to make proteins and the proteins they make must be able to function correctly. The cell has complex machinery for ensuring that when new proteins are made they are functional and are transported to the correct location, be it within the cell or outside. This project will determine one crucial process that allows proteins to be made efficiently and be delivered outside the cell and, in particular, how this process breaks down during disease. The production and delivery of proteins can be summarised into two key stages: i) ensuring proteins are made correctly and adopt the correct shape, ii) transport of the proteins from the inside to the outside of the cell.

Proteins are made as a string of amino acids which coil-up or fold to adopt a characteristic shape or three-dimensional structure. Only one such shape is functional and the cell ensures that this shape is adopted by providing helper proteins or chaperones to aid this process. If cells are unable to correctly fold proteins then disease results. For the secreted proteins to function they need to be robust and to ensure this is the case they form links within the protein to tie the protein together. These links are called disulfide bonds. Without these bonds the proteins would not function and would not be secreted. We know very little about how the correct links are introduced or how incorrect linkages are removed. This project will investigate how the correct bonds are formed. Our group wants to understand in detail how cells provide the correct environment to allow proteins to fold and to form the correct disulfide bonds to ensure their stability. To understand how cells fold and assemble proteins we are studying this process in mammalian cells using a combination of cell biological and biochemical techniques.

While the pathway for protein thiol reduction in the cytosol and oxidation in the endoplasmic reticulum (ER) are well characterised in mammalian cells, our understanding of a reduction pathway for protein thiols within the ER is completely lacking. The importance of a reductive pathway can best be illustrated when cells are producing large amounts of disulfide-bonded proteins such as insulin in pancreatic beta cells or antibodies in plasma cells. The accumulation of non-native disulfide-bonded insulin following glucose stimulation can lead to loss of insulin secretion, oxidative stress and apoptosis, similar to that seen in type II diabetes. Similarly aggregation of immunoglobulin occurs when the accumulation of misfolded protein exceeds the cellular capacity to remove the aberrantly disulfide bonded protein from the ER. Without a reductive pathway the cell would not be able to reduce non-native or regulatory disulfides or to reverse the sulfenylation or nitrosylation of cysteines caused by hydrogen peroxide or nitric oxide respectively. The main aim of this project, therefore, will be to discover how the cell maintains an optimised redox balance in the ER to both allow correct disulfide formation and to recycle sulfenylated, nitrosylated or glutathionylated thiols. We will take a cell biological approach to identify factors that influence the redox status monitored in live cells using a novel redox-sensitive GFP. We will take also a biochemical approach and trap mixed disulfide intermediates between disulfide exchange proteins and putative reductases. We will also identify ER proteins that contain redox-sensitive cysteines residues as these proteins are likely to be substrates for the reductive pathway. Finally we will verify any potential components of the reductive pathway using several well established assays such as non-native disulfide formation, induction of the UPR, changes to thiol modification or the rate of recovery of ER proteins following oxidation.

The main users who would benefit from this work include researchers interested in investigating how the correct disulfides are formed in proteins, what regulates the redox conditions with the ER of mammalian cells and how protein thiol modifications are reversed. In addition, companies interested in biopharmaceutical production may benefit from the knowledge gained from this project enabling them rationally to engineer cells to maximise protein production from mammalian cells. Hence any discoveries made will have a very broad and significant impact of the research community.

The major benefit for users will be from the knowledge gained during the project. However, we will also make available the enhanced redox sensitive GFP that we have developed. This protein has a much higher fluorescent signal than the previous versions making possible the high throughput screen. Previous versions worked fine for microscopy applications but could not be used effectively in a plate reader format due to low signal to noise ratios.