Determining the substrate specificity of ER oxidoreductases

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 address the general question of how the cell ensures that proteins are made correctly and adopt the correct shape. 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. A family of enzymes that are located within the cell are responsible for ensuring that some proteins are made correctly and adopt the correct shape. However little is known about which proteins these enzymes are able to help or indeed what their exact function is. We will use a newly developed technique to identify the substrate proteins for each of these enzymes and use this information to determine precisely their function during the folding of proteins within the cell. The information gained from this work will help with our attempts to understand what the cell needs to produce proteins. Understanding the requirements for protein folding in more depth will aid us to make rational decisions when we try to produce proteins in a recombinant form for either structural biology studies or for medical uses.

The ER provides an environment that allows the oxidative folding and post-translational modification of proteins entering the secretory pathway. The compartmentalisation of the ER away from the cytosol allows the correct redox conditions to be established that enable a distinct set of folding catalysts to facilitate the formation and isomerisation of disulphide bonds. A growing family of ER oxidoreductases is thought to be responsible for catalysing the formation; isomerisation and reduction of these disulphide bonds. However, we know little about the function of each of these oxidoreductases, which proteins are substrate for these enzymes or how their activity is regulated. The objective of this research proposal is to determine the substrate specificity of several of the ER oxidoreductases. To address this objective we propose to take an unbiased proteomics approach to determine the range of substrates that each oxidoreductase interacts with. To facilitate this analysis we will take advantage of the fact that during the formation and reduction of disulphide bonds a mixed disulphide must form between the protein substrate and the enzyme involved in catalysis. Such a proteomics approach has the advantage of not relying on any prior knowledge of potential substrates and enables the substrate specificity of each oxidoreductase to be defined within the same cellular environment. Such an approach has already been successful in determining the range of substrates interacting with ERp57 in cell grown in culture. Once the protein substrates have been identified we will then characterise the role of the individual ER oxidoreductase during disulphide bond formation, folding, assembly or transport of the substrate protein. We will achieve this aim by establishing folding and transport assays for selected substrate proteins. The results from this work will, for the first time, allow us to determine the precise requirements for the folding, assembly and secretion of individual proteins and potentially allow us to engineer cell-lines for more efficient production of recombinant proteins.