The mechanism of endoplasmic reticulum oxidoreductase 1 α (Ero1α) inactivation

Shepherd, Colin (2012) The mechanism of endoplasmic reticulum oxidoreductase 1 α (Ero1α) inactivation. PhD thesis, University of Glasgow.

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Printed Thesis Information: https://eleanor.lib.gla.ac.uk/record=b2996886

Abstract

Ero1α is a resident ER oxidase and is an important member of the oxidative protein folding machinery. It generates disulphide bonds de novo and donates them to protein disulphide isomerase (PDI), which in turn oxidises nascent substrate proteins within the ER. Ero1 activity must be tightly regulated for two key reasons: (i) it must maintain the balance of oxidised PDI to ensure oxidative protein folding can occur, but cannot be so active that the ER becomes hyperoxidising and dysfunctional, and (ii) Ero1 activity must be regulated to prevent the accumulation of hydrogen peroxide, a reactive oxygen species (ROS), within the ER. The regulation of Ero1α comes principally from 3 intramolecular disulphide bonds which are reduced by substrate upon activation, and re-oxidised upon inactivation by an unknown mechanism. Using an SDS-PAGE based assay we tested three hypotheses: that sulphenylation by Ero1α-produced hydrogen peroxide could induce re-oxidation; that an internal disulphide exchange mechanism could generate and distribute disulphide bonds within Ero1α; and that ER oxidoreductases could act to inactivate Ero1α. Having successfully expressed, purified and characterised a recombinant version of Ero1α, this was tested in a number of assays to address the above hypotheses.
In vitro findings show that Ero1α is specifically and rapidly oxidised by ERp46 and PDI. Sulphenylation and internal disulphide exchange-mediated oxidation of Ero1α provided a comparatively slow and incomplete method of re-oxidation. In vivo results suggest that ERp46 and PDI may have implications in Ero1α activity regulation. Overexpression of several ER oxidoreductases had no effect on Ero1α re-oxidation after DTT challenge, whereas Ero1α oxidation was impaired slightly in PDI- ERp46 double knockdown cells. Depletion of PDI from cells results in the DTT-resistance of Ero1α, suggesting that Ero1α, PDI and glutathione are involved in an intricate mechanism of sensing and reacting to ER redox conditions.
Two key ER oxidoreductases, PDI and ERp57, are oxidised in semi-permeabilised cells. Oxidation coincides with permeabilisation of the plasma membrane and the removal of cytosolic glutathione, directly implicating glutathione in the maintainence of the redox states of ER oxidoreductases. Oxidation during the permeabilisation of cells is an enzymatic process which is mediated in part by Ero1α. Semi-permeabilised cells harbour a more oxidising environment than do microsomes.
This study contributes significantly to the research field by complementing several previously reported findings, as well as providing a novel investigation into the molecular regulation of Ero1α and its relationship with PDI and glutathione in the cell.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: protein folding, disulphide bonds, oxidase, PDI, glutathione,
Subjects: Q Science > QH Natural history > QH301 Biology
Q Science > QH Natural history > QH345 Biochemistry
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Supervisor's Name: Bulleid, Prof. Neil J.
Date of Award: 2012
Depositing User: Mr Colin Shepherd
Unique ID: glathesis:2012-4647
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 21 Oct 2013 14:01
Last Modified: 21 Oct 2013 14:03
URI: http://theses.gla.ac.uk/id/eprint/4647

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