ALEXANDER, MICHAEL,HUGH (2018) A Study of the PDILT Protein and its Expression in Immortalised Cells and Tissues. Masters thesis, Durham University.
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Author-imposed embargo until 27 October 2021.
Protein Disulfide Isomerase-like protein of the Testis (PDILT) is an endoplasmic reticulum (ER) protein that is required for male fertility and is a member of the Protein Disulfide Isomerase (PDI) family. PDI has a well-established role in the formation of disulfide bonds in newly synthesised proteins, which is facilitated by thioredoxin active sites (a CXXC motif) in the a domains. PDILT, however, lacks these classic redox active sites and has an SXXS and a SXXC motif in the a and a’ domain respectively but nevertheless interacts with clients in the ER. PDILT also contains a unique C-terminal extension, the function of which is unknown.
In order to examine the role that the unique structural elements of PDILT contribute to client binding, several PDILT mutant constructs were transfected into HT1080 cells and subjected to a series of western blotting and immunofluorescence experiments. In this thesis, data is shown to support the assertion that the two solvent-exposed cysteines present in PDILT mediate all disulphide dependent interactions with client proteins. We also show that the deletion of the C-terminal extension caused a large increase in client interactions and thus we theorise that the tail of PDILT regulates PDILT/client protein interactions.
PDILT is specifically expressed in the post-meiotic spermatid cells of the testis, which limits the potential to study the expression and function of endogenous PDILT in cell lines. Thus, work in this thesis has also examined a variety of immortalised cell lines for PDILT expression, to develop better systems for the further study of testis specific chaperones. Along with PDILT, a range of immortalised cell lines were also examined for the derepression of other testis specific ER chaperone proteins. As a result of this investigation, this thesis also reports on the unique expression of calmegin in cell lines with different genotypes.
|Item Type:||Thesis (Masters)|
|Award:||Master of Science|
|Faculty and Department:||Faculty of Science > Biological and Biomedical Sciences, School of|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||01 Nov 2018 14:32|