Determining the substrate specificity of ER oxidoreductases
Lead Research Organisation:
University of Manchester
Department Name: Life Sciences
Abstract
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.
Technical Summary
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.
Organisations
People |
ORCID iD |
Neil Bulleid (Principal Investigator) |
Publications
Berry J
(2009)
ERp57 is involved in the oxidative folding of the low-density lipoprotein receptor in the endoplasmic reticulum
in Bioscience Horizons
Chalmers F
(2017)
Inhibition of IRE1a-mediated XBP1 mRNA cleavage by XBP1 reveals a novel regulatory process during the unfolded protein response
in Wellcome Open Research
Chalmers F
(2017)
Inhibition of IRE1a-mediated XBP1 mRNA cleavage by XBP1 reveals a novel regulatory process during the unfolded protein response.
in Wellcome open research
Chambers JE
(2008)
Formation of a major histocompatibility complex class I tapasin disulfide indicates a change in spatial organization of the peptide-loading complex during assembly.
in The Journal of biological chemistry
Jessop CE
(2009)
Substrate specificity of the oxidoreductase ERp57 is determined primarily by its interaction with calnexin and calreticulin.
in The Journal of biological chemistry
Jessop CE
(2009)
Protein disulphide isomerase family members show distinct substrate specificity: P5 is targeted to BiP client proteins.
in Journal of cell science
Jessop CE
(2007)
ERp57 is essential for efficient folding of glycoproteins sharing common structural domains.
in The EMBO journal
Oka OB
(2019)
ERp18 regulates activation of ATF6a during unfolded protein response.
in The EMBO journal
Pisoni GB
(2015)
Division of labor among oxidoreductases: TMX1 preferentially acts on transmembrane polypeptides.
in Molecular biology of the cell
Description | Identification of substrates for folding enzymes present in the secretory pathway |
Exploitation Route | Design of novel cell lines for biopharmaceutical production. |
Sectors | Pharmaceuticals and Medical Biotechnology |
Description | Provided data for the identification of enzymes involved in removal of non-native disulfides. Used to demonstrate a link between the unfolded protein response and disulfide reduction. |
First Year Of Impact | 2012 |
Sector | Manufacturing, including Industrial Biotechology |
Description | Determining the reductive pathway in the endoplasmic reticulum of mammalian cells |
Amount | £384,687 (GBP) |
Funding ID | BB/L00593X/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 03/2014 |
End | 06/2017 |