How does the cytosol reduce non-native disulfides formed in the endoplasmic reticulum?

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 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.

This project will identify the key components required to ensure the correct folding and assembly of proteins within the secretory pathway. The work capitalises on recent discoveries from my group which address a topical aspect of the protein folding process, namely the removal of incorrect disulfide bonds from proteins entering the secretory pathway. This process is absolutely required for cells to be able to fold secreted proteins correctly and to remove misfolded proteins to alleviate cell stress. Breakdown of functional protein secretion and degradation leads to disease pathologies caused by a lack of function, or cell death resulting from an aggravated stress response. Hence, the efficient folding and secretion of proteins from mammalian cells is critically important for normal cell physiology.
The main aim of this project will be to identify the components required for coupling the reductive pathway in the cytosol to the resolution of non-native disulfides within the ER. We will take a biochemical approach to trap mixed disulfide intermediates between disulfide exchange proteins. In this way we will identify the missing membrane component that transfers disulfides from the cytosol to the ER. Also, we will determine the role of the thioredoxin-based reductive pathway in important cellular processes such as non-native disulfide formation, ER-associated degradation, induction of the unfolded protein response, and the maintenance of ER glutathione homeostasis.

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 the cell responds to oxidative stress conditions. In addition, companies interested in biopharmaceutical production may benefit from the knowledge gained from this project enabling them to rationally 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 make available any cell-lines, DNA constructs or antibodies created during the course of this project. Knowing how the cell balances the opposing reactions of reduction and formation of disulfides, will allow users to consider how the process may be manipulated during disease progression and aging. The discovery of Ero1 and its requirement for oxygen as an electron acceptor has led to an appreciation of the role of the ER in generating reactive oxygen species. A link between glucose metabolism and redox balance in the ER suggested in this work provides greater understanding of the consequence of glucose starvation on cellular physiology in the context of protein folding and secretion. In summary, the understanding of a fundamental cellular process may have wide benefits for not only greater understanding but also direct applications for users.