Analytical & Biological Chemistry Research Facility - Masters by Research Theses
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Item Differential regulation of the phosphorylated and dephosphorylated forms of the ubiquitin-conjugating enzyme Ubc6e(University College Cork, 2024) Burns, Stephanie; Fleming, John V (Eoin)The ubiquitin proteasome system (UPS) is a key stress response employed by cells to remove damaged and misfolded proteins that accumulate in the endoplasmic reticulum (ER) during energy deprivation, hypoxia, or viral infection. Ubc6e is an ER-localised ubiquitin-conjugating enzyme that plays an important role in the proteasomal degradation of misfolded proteins and can be phosphorylated at serine residue 184. Recent studies have demonstrated that Ubc6e is co-localised with p62/sequestrosome, which is a cargo receptor that delivers ubiquitinated cargo to autophagosomes for degradation. To further investigate the possible role that Ubc6e may play in autophagy, we aimed to investigate any protein-protein interactions between p62 and Ubc6e and we also aimed to investigate the effect of autophagic-related proteins, p62 and ULK1 on Ubc6e. From our research, we saw that Ubc6e levels are reduced when co-expressed with proteins that promote autophagy. Specifically for the autophagy inducer ULK1, we saw differential regulation between the phosphorylated and dephosphorylated forms of Ubc6e, suggesting that the S184 phosphorylation may protect the protein from autophagic degradation. Degradation of the dephosphorylated form, on the other hand, did not depend on S184 phosphorylation, or the catalytic activity of Ubc6e. Neither was it dependent absolutely on ER localisation. Finally, although ULK1 co-expression did not appear to specifically alter the ER/ perinuclear localisation of Ubc6e, it was noted that amino-terminal tagging with GFP led to a ULK1-related localisation of Ubc6e to intracellular vesicles. Together our results suggest that Ubc6e does play a role in autophagy however it is not mediated by the direct interaction with p62.Item Enhancing the potential of hydrolases in enantioselective synthesis(University College Cork, 2024) Torley, Rachel; Maguire, Anita; Collins, Stuart; Synthesis and Solid State Pharmaceutical Centre; Thermo Fisher ScientificThis interdisciplinary research project focuses on the utilisation of a novel hydrolase of marine origin as a biocatalyst in asymmetric synthesis, bringing together synthetic organic chemistry and molecular biology. The primary objective of this research was to investigate the effect of histidine tags on the activity and selectivity of this biocatalyst. Chapter 1 summarises literature reports of the applications of biocatalysts in asymmetric processes and, specifically, the reported effects of histidine tags on enzyme activity. Chapter 2 describes the synthesis and use of ester substrates for biocatalysis screening against three variants of the novel marine esterase, esterase 26D. The enzyme variants included the wild-type, non-histidine tagged strain, and two histidine tagged enzymes (C-terminal and N-terminal tagged). Overall, the investigation showed that the histidine tag had no detectable effect on the selectivity or activity of the esterase 26D. An important outcome of the work was development of a standard procedure to be undertaken at the outset of future investigations of novel biocatalysts to definitively establish whether the histidine tag impacts on the enzyme activity. Chapter 3 details the expression of the novel esterase, esterase 26D, variants. The non-histidine tag variant was cloned into E. coli where it was subsequently expressed alongside the readily available histidine tagged counterparts. Additionally, variants of other enzymes, esterase 32C and 51C, were cloned and expressed to provide another set of enzymes that could be screened for activity in the future. Lastly, Chapter 4 contains the full experimental details and spectroscopic characterisation of the compounds synthesised in the work, the biocatalysis studies, along with the general methods employed for generating the desired enzymes.Item Rhodium catalysed 1,4-addition of N-Boc-indol-3-ylboronic acid to α,β-unsaturated carbonyls(University College Cork, 2024) O'Driscoll, Conor C.; McGlacken, Gerard; Irish Research Council for Science, Engineering and TechnologyIn recent years, work within the McGlacken group has focused on the asymmetric conjugate additions of indole boronic acids. The work presented in this thesis is concerned with the exploration of the asymmetric rhodium-catalysed conjugate addition of indol-3-ylboronic acids to α,β-unsaturated carbonyl compounds. Optimisation studies towards the asymmetric addition of N-Boc-indol-3-ylboronic acid to 2-cyclohexen-1-one are described herein. With the optimised reaction conditions, a variety of substrates was explored and the synthesis of a number of novel compounds is described. Modest yields of up to 39% were achieved, with excellent enantioselectivities of up to 96%.Item Design and use of novel metal catalysts in organic synthesis(University College Cork, 2023) Fitzgerald, Deirbhile; Maguire, Anita; Collins, Stuart; Irish Research CouncilThis project focuses on the design and synthesis of novel dirhodium carboxylate catalysts, and their precursor ligands, for use as enantioselective catalysts in transformations of α-diazocarbonyl compounds. Chapter One focuses on the literature background over the past thirty years in transition metal-catalysed asymmetric transformations of α-diazocarbonyl compounds. This overview aims to give an insight into the use of both copper and rhodium-based catalysts in intramolecular and intermolecular C-H insertion reactions, cyclopropanation and desymmetrisation reactions. The evolution of dirhodium carboxylate design since the 1980s is explored. This summary provides insight into their use as enantioselective catalysts in asymmetric transformations of α-diazocarbonyl compounds, thus providing context for this work. Chapter Two describes the design and synthesis of novel, enantiopure dirhodium carboxylate complexes, drawing inspiration from the rhodium (S)-mandelate skeleton. Initially, two enantiopure rhodium carboxylates were synthesised, incorporating carboxylic acid ligands bearing a fenchyl chiral auxiliary. Synthesis of carboxylic acid ligands bearing a bulky adamantyloxy group or a tert-butoxy group in place of the fenchyl substituent was investigated to simplify the stereochemical features, with just one stereogenic centre in each ligand. Two target carboxylic acids were obtained in racemic form, one bearing an adamantyloxy group and the other bearing a tert-butoxy group adjacent to the carboxylic acid moiety. Resolution of these carboxylic acids was explored through formation of diastereomeric salts or enzymatic kinetic resolution; while enantioenrichment was observed, further work is required to obtain the ligands in an enantiopure form for catalyst preparation. Chapter Three describes the synthesis of both racemic mandelamide and enantiopure (S)-mandelamide. In addition, a pair of diastereomerically pure mandelamides with α-methylbenzylamine were synthesised and characterised. The mandelamides were prepared to enable exploration of their crystal landscape in collaboration with another research group in UCC. Chapter Four contains the full experimental details and spectroscopic and analytical characterisation of all compounds synthesised in this project, while details of chiral stationary phase HPLC analysis are included in Appendix 1.Item Interrogating regulated intramembrane proteolysis of IGF-1R, and its role in cell migration(University College Cork, 2023) O'Donoghue, Jordan Christopher; McCarthy, Justin V.; O'Connor, Rosemary; University College CorkThe IGF-1R has long been implicated in numerous malignant characteristics of cancers including enhanced proliferation, insensitivity to apoptosis, chemoresistance, increased migratory and invasive capacity, epithelial-to-mesenchymal transition, and metabolic augmentation. IGF-1R, much like other receptor tyrosine kinases, has been characterised as a substrate for gamma-secretase-mediated regulated intramembrane proteolysis (RIP). This proteolytic pathway begins with ectodomain shedding, mediated by sheddases, which produces a soluble fragment in the extracellular space. Following ectodomain shedding, a membrane anchored C-terminal fragment (CTF) is produced which is then cleaved by gamma-secretase at the internal juxtamembrane region to produce a soluble intracellular domain (ICD). Classically, RIP was thought to be a means by which cells would terminate receptor signalling, however contemporary research indicates a more nuanced role. It is now understood that the fragments generated through RIP can retain signalling of the full-form receptors and, in some instances, may acquire novel functionality. Although the RIP of IGF-1R has been outlined, the underlying dynamics of this pathway and the consequences of such remain to be elucidated. With this in mind, we endeavoured to identify the regulatory mechanisms underlying IGF-1R RIP and subsequently aimed to identify potential functions of the fragments produced. Firstly, we generated a GFP-tagged K1003 IGF-1R point mutant, termed a kinase dead mutant, which lacks the ability to catalyse trans-autophosphorylation of the IGF-1R kinase domains thus preventing downstream signal transduction. Trans-autophosphorylation is a critical step in the activation of receptor tyrosine kinases during which the kinase domain of one intracellular domain catalyses the phosphorylation of tyrosine residues within the other intracellular domain. With this tool, we validated the cleavage of IGF-1R and the production of its CTF via transient transfection of HEK293T cells with plasmids expressing either wild-type or K1003R kinase dead mutant forms of IGF-1R C-terminally tagged with GFP. We subsequently identified receptor kinase activation as a potential driver of IGF-1R RIP and determined that ligand stimulation significantly catalyses the accumulation of IGF-1R CTF. This was accomplished via transient transfection of HEK293T cells with plasmids expressing the aforementioned IGF-1R forms. Following this, we elucidated clathrin-mediated endocytosis as a probable regulatory pre-requisite step for IGF-1R RIP by gamma-secretase. Furthermore, our data indicates that ectodomain shedding of IGF-1R is likely catalysed in a metalloprotease dependent fashion, consistent with other receptor tyrosine kinases. We also confirmed that the CTF can undergo nuclear translocation in a clathrin-mediated endocytosis dependent manner, consistent with the translocation of the holoreceptor. Utilising wound healing assays, conducted in Hela cells, our data indicates that gamma-secretase inhibition by DAPT (a small molecule gamma-secretase inhibitor) alone does not antagonise IGF-1-induced cell migration. Similarly, the administration of Pitstop alone does not impinge upon IGF-1 induced cell migration. Interestingly, the administration of batimistat (a broad spectrum metalloprotease inhibitor) significantly suppresses cell migration in the presence of IGF-1. Collectively, our data provides a framework for the construction of the IGF-1R RIP cascade. Following ligand-binding it appears that IGF-1R undergoes metalloprotease-dependent ectodomain shedding leading to loss of the extracellular alpha-chains. The CTF, which remains in the plasma membrane, then undergoes clathrin-mediated endocytosis and packaging into early endosomes. Following internalisation, the CTF is cleaved by gamma-secretase to generate a soluble ICD. Our data also demonstrates that IGF-1R CTF undergoes nuclear translocation in a clathrin-mediated endocytosis dependent fashion. Lastly, data gathered from our wound healing assays indicates that IGF-1R ectodomain shedding is likely a required event for the efficient IGF-1-induced migration of cells, possibly through the proteolytic augmentation of focal adhesion complexes containing IGF-1R.