Gunasekara, Sanjika Dias (2006) Characterising the expression and interactions of the Endoplasmic Reticulum Oxidoreductase Erol β. Doctoral thesis, Durham University.
Proteins destined for the secretory pathway and endoplasmic reticulum (ER) resident proteins are targeted to the ER through an N-terminal signal sequence. Proteins then follow a post-translational maturation process to fold into functional proteins. For most of these proteins the formation of disulfide bonds (S-S) at the correct position is essential for structure and function. In eukaryotes, protein oxidation in the ER is catalysed by Endoplasmic reticulum oxidoreductases (EROs) which donate disulfide bonds to (and accept electrons from) Protein Disulfide Isomerase (PDI). In yeast, Erol p is essential for viability and protein secretion. Two ER resident homologues have been identified in the human genome, Erolα and Erol β, which complement the yeast erol-1 temperature sensitive mutation. The main focus of this project was Erol β. Under steady state conditions in transfected cells, Ero 1 β was captured in higher molecular weight complexes in the presence and in the absence of an alkylating agent. Ero-PDI covalent interactions are alkylation dependent, but alkylation independent Ero Iß complexes were found in transfected cells and endogenous tissue. In Erolα the CXXCXXC C-terminal motif is important for protein folding, structural integrity and complex formation with the PDI. In Ero Iß, the AXXCXXC (C390A) mutation disrupts covalent PDI interactions but does not interfere with homodimer formation. The CXXCXXA (C396A) mutation caused disruption of Ero Iß homodimer formation. Modelling the dimer onto the Erol p crystal structure suggested that this 396 cysteine is indirectly disrupting dimmer formation by most likely displacing the cofactor FAD. Two FAD binding domain mutants, initially discovered in yeast, were constructed in Ero Iß and were found to cause instability of Ero Iß through misoxidation during temperature and reducing stress conditions. Ero Iß expression patterns were studied using immunohistochemistry on human stomach and pancreas tissues. The data indicated that Ero Iß is constitutively strongly expressed in enzyme producing chief cells and hormone producing pancreatic islet cells. An interesting result from immunohistochemistry stainings of healthy, Barrett's and oesophageal tumour tissue showed that the ER chaperone protein ERp57 is up-regulated only ill dysplastic and oesophageal tumour tissue. These initial findings suggest ERp57 has potential as a marker in oesophageal tumour diagnosis.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Faculty and Department:||Faculty of Science > Biological and Biomedical Sciences, School of|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||09 Sep 2011 09:51|