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Development of methods to monitor maturation and trafficking of Carboxypeptidase Y (CPY) and its G255R mutant (CPY*)

TANNA, YESHURUN,AMARASINGHAM (2022) Development of methods to monitor maturation and trafficking of Carboxypeptidase Y (CPY) and its G255R mutant (CPY*). Masters thesis, Durham University.

PDF (Thesis (MSc)) - Accepted Version


Protein folding is a vital biological process which underpins many cellular functions in both eukaryotic and prokaryotic cells. This mechanism is a prime example of macromolecular self-assembly which leads to important biological function, such as molecular trafficking to specific cellular parts and cellular differentiation. However, whilst for the majority of cases proteins fold into their correct 3D structure with long-term stability, there is a propensity for proteins to misfold due to insufficient molecular interactions between the amino acids within the polypeptide chain. Once formed, these misfolded proteins have the potential to aggregate and cause pathological or even neurological diseases. Thus, it is of importance to probe the mechanism(s) of protein misfolding to uncover its molecular origins.
The yeast species known as baker’s yeast (S. Cerevisiae) is a model organism to probe this mechanism, and carboxypeptidase Y (CPY) has been proposed as a suitable model protein, due to its high abundance within the yeast endoplasmic reticulum (ER), to understand this process. Literature has shown that CPY is a widely used model protein in understanding protein sorting events within the ER of S. Cerevisiae. The advantages, and subsequent choice of this protein, are based upon its structure and role. It plays a part in the C-terminal chemistry of polypeptides, and thus may inform on mis-interactions which contribute to misfolding. Furthermore, CPY trafficking from the ER to the Golgi to the vacuole has provided information on sorting signal events which are like mammalian cellular signals, and thus share similar features with other organisms. It is also a preferable model protein due to its unique catalytic triad (active site). Although classified as a serine protease, it has a much greater pH and temperature range than other proteases, and can thus maintain high activity across environmental changes. Its mutated analogue, carboxypeptidase Y* (CPY*), has also been chosen as a model protein to compare its molecular sorting mechanism with CPY. It is characterised by a glycine-arginine mutation at the 255 amino acid position.
The purpose of this project is to uncover the molecular mechanisms of misfolding, namely, post trafficking of CPY & CPY*, whether these misfolded proteins renature and continue trafficking or whether they are degraded by cellular machinery. Alternatively, whether there is evidence of competition between these processes. This would also shed light on the kinetics of these processes and the likelihood of clearance of these misfolded proteins from the ER. To probe these processes, the maturation of pre-cursor forms of both CPY and CPY* have been studied, as they undergo cellular trafficking across the secretory pathway from the ER to the Golgi to the vacuole.
The initial experiments have been used to test whether CPY/CPY* can be detected in a western blot through SDS-PAGE gels, and whether CPY/CPY* can be detected in an immunoprecipitate. The final experiment was used to assess whether the dose-dependent cell-cycle regulator 2 (DCR2) plays a role in ER-induced stress, by specifically affecting CPY* degradation. All such experiments employ classical molecular biology techniques. These findings could shed light on whether degradation, by means of disulphide bond breaking and thus slower migration on SDS-PAGE gels, or renaturation, by means of cellular mechanisms, is the dominant mechanism within the ER.

Item Type:Thesis (Masters)
Award:Master of Science
Faculty and Department:Faculty of Science > Biological and Biomedical Sciences, School of
Thesis Date:2022
Copyright:Copyright of this thesis is held by the author
Deposited On:03 Nov 2022 10:27

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