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Title: Identification and characterisation of the missing components of the ER reductive pathway
Author: Cao, Xiaofei
ISNI:       0000 0004 9355 9320
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2020
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Efficient folding of proteins frequently requires the introduction of disulfide bonds. The insertion of disulfide bonds could result in native disulfides, which can be found in the correctly folded and functional protein structure, and non-native disulfides which are not present in the native protein. The resolution of non-native disulfides is absolutely required for cells to fold secreted proteins correctly and to remove misfolded proteins to alleviate cell stress. The pathways for disulfide formation are well characterised. However, our understanding of the reduction of non-native disulfides is still limited. How the reducing equivalents are transferred across the ER membrane and whether there are additional components needed in the reduction pathway either in cytosol or ER lumen are the questions we need to address. Microsomes including redox-sensitive green fluorescence protein (roGFP) were used to address which membrane protein was involved in the ER reductive pathway. We demonstrated that a membrane protein is required for the reduction of microsomal proteins by the membrane impermeable reducing agent Tris(2-carboxyethyl)phosphine (TCEP) or a reconstituted thioredoxin/thioredoxin reductase (Trx/TrxR) pathway. Previous research in our group suggests that ERp57 has a specific role in the isomerisation of non-native disulfide bonds in specific glycoprotein. We optimised the determination of redox state of ERp57 in semi-permeabilised (SP) cells. It was found that the reduction of ERp57 in SP cells required reducing equivalents generated by the Trx/TrxR pathway and the reduction was dependent on catalytic cysteines in the Trx motif. It was also shown by proteinase experiments that different membrane proteins are required for the transduction of electrons from TCEP and the Trx/TrxR pathway. In addition, we took advantage of an in vitro translational system supplemented with SP cells. A post-translationally folding assay was developed to evaluate the ability of the cytosolic components to resolve non-native disulfides in nascent polypeptides proteins in the ER. We showed that the minimal components in the cytosolic Trx/TrxR pathway are sufficient for disulfide rearrangement in the ER and that the reduction process is Trx - dependent.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Q Science (General)