ERp27 is a 27.7 kDa redox-inactive member of the protein disulphide isomerase (PDI) family. It was found to interact with another PDI member, the well-known thiol oxidoreductase ERp57 (58 kDa) in vitro. Although it is known that ERp57 interacts with ERp27 in vitro this interaction was not investigated in living cells. In this research project we applied in vitro and in cellulo approaches to investigate the same interaction of ERp57 and ERp27 then to compare it to the interaction of ERp57/calnexin (CNX)/calreticulin (CRT) complex to determine if the ERp57 interaction with ERp27 competes with the ERp57/CNX/CRT complex. Additionally, we investigated the physiological role of ERp27. Protein expressions and purifications were carried out by the Nickel agarose affinity chromatography to obtain sufficient amount of proteins for analysis. Additionally, proteins were purified by gel filtration-chromatography. The interaction between purified ERp27 and ERp57 was determined using isothermal titration calorimetry (ITC) and by chemical cross-linking. The ITC results confirmed the interaction between ERp57 and the lectin CRT. However, we could not detect an interaction between ERp57 and ERp27 possibly due to low protein concentrations. Moreover, the in vitro cross-linking results were in agreement with the previous research and verified the binding of ERp57 with ERp27. However, in cellulo chemical cross-linking suggested that the same interaction does not occur in living cells. Nevertheless, this investigation revealed that ERp27 binds to other proteins in cellulo. Mass spectrometry results have identified protein candidates that interact with ERp27 in living cells which are the PDI homologous P5 and the ER oxidoreductin Ero1. These results provide new insights of the role of ERp27 and provide suggestions for further research.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:739215 |
| Date | January 2017 |
| Creators | Haji, Esraa |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/8793/ |
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