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Durham e-Theses
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ER quality control in male fertility

ARTHURS, HAYLEY,MELANIE (2018) ER quality control in male fertility. Masters thesis, Durham University.



Protein disulphide isomerase is a member of the thioredoxin superfamily, resides in the endoplasmic reticulum (ER) and catalyses the formation of disulphide bonds within or between polypeptides, enabling protein quality control. PDILT is a member of the PDI family that specifically resides in the testes and is expressed under developmental control. Recent work has found that, unlike PDI (which possesses the classical CXXC sequence), PDILT has an SXXC domain. This suggests that PDILT does not function fully as a disulphide oxidoreductase but may work as a chaperone. We wished to investigate PDILT and compare it with other PDI proteins, to study the evolution of the PDILT gene and to decipher its protein-protein interactions.
Using sequence analysis of PDILT using the BLAST program, the PDILT protein was compared with other PDI proteins in homosapiens and in other species. We then probed further to investigate the expression and interaction of PDILT with other proteins. A transfected cell line model to investigate wt and mutant PDILT was established, and mouse testis tissue lysate was also used to study PDILT interactions. Using immunofluorescence, western blot analysis, and immunoprecipitation techniques, it was established that PDILT forms intermolecular-disulphide complexes and interacts with other chaperones such as CALR3 and CLGN.
Sequence analysis demonstrated conserved amino acids in the b and b’ domains that reflect the specialised roles of the a-a’-b-b’ domains of PDILT. The cysteine residues responsible for inter-molecular disulphide bond formation were identified and found to be important in the interaction of PDILT with CLGN. Lastly, a novel function of the unique tail domain of PDILT was found. The work in this thesis suggests that the tail may be important for the integrity and half-life of the PDILT protein. These studies will help to elucidate the role of PDILT in governing male fertility.

Item Type:Thesis (Masters)
Award:Master of Science
Faculty and Department:Faculty of Science > Biological and Biomedical Sciences, School of
Thesis Date:2018
Copyright:Copyright of this thesis is held by the author
Deposited On:25 Jan 2018 10:08

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