Lemin, Andrew James (2007) Characterisation of the oxidoreductase Erol-Lß and the misfolding of the secretory pathway substrate HLA-B27. Unspecified thesis, Durham University.
The endoplasmic reticulum (ER) is the site of oxidative folding for proteins entering the secretory pathway. Here, nascent polypeptides acquire disulfide bonds, which confer both stability and functionality on secretory and ER-resident proteins. In eukaryotes, this process is catalysed by the disulfide oxidoreductase protein disulfide isomerase (PDI). Many mammalian homologs of PDI have been described including the pancreas-specific homolog PDIp. PDI is 'recharged' by the disulfide oxidoreductase Его. Accepted electrons are then passed to molecular oxygen via an Ero-bound flavin adenine dinucleotide (FAD) molecule. We provide data showing that human Erol-Lß protein is able to form disulfide-dependent homodimers in vivo. We also provide evidence that the yeast G252S and H254Y FAD- binding mutants exhibit reduced affinity for PDI. Since the Erol-La C391A mutant can rescue the erol-l temperature-sensitive mutant, Ero-PDI association and Ero-Ero dimensation may be significant in maintaining the oxidative protein folding pathway in the ER. Homodimerisation was not affected by FAD-binding mutants, suggesting that the erol-1 and erol-2 phenotypes cannot be accredited to the failure of Erol p to homodimerise. We also make the first steps towards characterising the interactions of PDIp with the human Erol-La and Erol-Lß proteins. In order to observe protein-protein interactions, we characterise a polyclonal anti֊Erol֊Lß antibody for intended use in immunoprecipitations and immunoblotting. Quality control measures are in place to ensure that only natively folded proteins are permitted to exit the secretory pathway. Chaperone molecules such as immunoglobulin- binding protein (BiP) retain unfolded or misfolded proteins in the ER， which are eventually retrotranslocated out of the ER and degraded. When misfolded proteins accumulate in the ER， however, the folding capacity of the ER may be exceeded. This triggers a cellular response pathway called the unfolded protein response, aimed at restoring homeostasis in the ER via transcription regulation and translational attenuation. We provide evidence that misfolding/misoxidation of the major histocompatability complex (MHC) class I heavy chain HLA-B27 in HeLa cells causes the UPR to be triggered. Possession of the HLA-B27 allele in lymphoblastoid cell lines results in some UPR signalling. Interestingly, analysis of IREI-mediated XBPl splicing shows a distinct difference in sensitivity of the UPR to induction by the pharmacological agents dithiothreitol (DTT) and tunicamycin. Since possession of HLA-B27 is highly associated with development of the chronic inflammatory disease ankylosing spondylitis, induction of the UPR as a consequence of HLA-B27 misfolding may have implications in disease pathogenesis.
|Item Type:||Thesis (Unspecified)|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||09 Sep 2011 09:56|