Functional characterisation of the Endoplasmic Reticulum protein (ERp27)

Haji, Esraa (2017) Functional characterisation of the Endoplasmic Reticulum protein (ERp27). PhD thesis, University of Glasgow.

Abstract

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.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: ERp27, PDI.
• Subjects: Q Science > QR Microbiology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Supervisor's Name: Bulleid, Professor Neil
• Date of Award: 2017
• Depositing User: Dr Esraa Haji
• Unique ID: glathesis:2017-8793
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 26 Feb 2018 16:53
• Last Modified: 25 Apr 2018 08:16
• URI: https://theses.gla.ac.uk/id/eprint/8793