The covalent post-translational modification of selected substrates with the ubiquitin protein has emerged as a central regulatory mechanism, governing protein stability, activity and localisation, and accordingly an array of cellular processes. Ubiquitin signalling versatility arises owing to the diverse nature of (poly)ubiquitin modification, with distinct modifications subsequently transduced in a specific manner by ubiquitin-binding domains found in ubiquitin-binding proteins. In recent years the notion that ubiquitin exerts influence solely via the covalent modification of substrates has been challenged, with unanchored, or substrate-free polyubiquitin chains emerging as key regulators of cellular physiology. The investigations described in this thesis seek to exploit the inherent selectivity of natural and engineered ubiquitin-binding proteins, to afford the purification of endogenous unanchored polyubiquitin, probing the molecular composition and interactions of this biologically significant ubiquitin pool. In chapter 3 by utilising the deubiquitinating enzyme USP5, which contains multiple ubiquitin-binding domains, and is normally responsible for the selective disassembly of unanchored ubiquitin, we purify unanchored polyubiquitin from mammalian cell extracts. Subsequently, we apply both ubiquitin-selective antibodies and mass spectrometry-based analyses to examine the polyubiquitin profile of the mammalian unanchored ubiquitin pool. In chapter 4 we then assess the mechanisms of ubiquitin recognition by USP5, presenting a structural mass spectrometry-based framework to probe and quantify ubiquitin: ubiquitin-binding domain interactions. Finally in chapter 5, based on the conclusions we draw from USP5-ubiquitin recognition in chapter 4, that multiple domains in suitable arrangement yield specificity for polyubiquitin chains, we design and synthesize a synthetic protein to favour the capture of unanchored polyubiquitin chains of defined topology, namely Lys-48 linked diubiquitin (and longer polyubiquitin chains), from mammalian cell extracts. We conclude that strategies for the rational design and engineering of polyubiquitin chain-selective binding in non-biological polymers are possible, paving the way for the generation of reagents to probe the unanchored polyubiquitin chains of defined topology, and more widely the ‘ubiquitome’.

Gastrointestinal (GI) helminths pose a significant threat to the livestock industry and are a recognized cause of global morbidity in humans. Control relies principally on chemotherapy but in the case of nematodes is rapidly losing efficacy through widespread development and spread of resistance to conventional anthelmintics and hence the urgent need for novel classes of anthelmintics. Cysteine proteinases (CPs) from papaya latex have been shown to be effective against three murine nematodes Heligmosomoides bakeri, Protospirura muricola and Trichuris muris in vitro and in vivo and against the economically important nematode parasite of sheep Haemonchus contortus. Preliminary evidence suggests an even broader spectrum of activity with efficacy against the canine hookworm Ancylostoma ceylanicum, juvenile stages of parasitic plant nematodes of the genera Meloidogyne and Globodera and a murine cestode Hymenolepis microstoma in vitro. This project focused on tapeworms. Using 2 different rodent cestodes Hymenolepis diminuta and Hymenolepis microstoma and 1 equine cestode Anoplocephala perfoliata I have been able to show that CPs do indeed affect cestodes whether young newly hatched scoleces in vitro (by causing a significant reduction in motility leading to death of the worms) or mature adult worms in vitro (by causing a significant reduction in motility leading to death of the worms) and in vivo (resulting in a significant, but relatively small, reduction in worm burden and biomass), despite no effects on worm fecundity. Although only minimally efficacious against Hymenolepis microstoma and moderately efficacious against Hymenolepis diminuta in vivo, efficacy was enhanced by the synergistic effects of the immune system demonstrated against Hymenolepis diminuta in the non-permissive host. The results offer the possibility that with further refinement, CPs may be developed into broad spectrum anthelmintics that in addition to their marked effects on nematodes also remove any concurrently residing tapeworms.

Reactive oxygen species (ROS) are linked to a range of degenerative conditions in humans, and may cause damage to an array of cellular components. However, it is unclear which cellular target(s) of ROS may primarily account for toxicity during oxidative stress. The sensitivity of iron-sulphur (Fe-S) clusters to ROS makes these candidate determinants of ROS mediated cell killing. Ribonuclease L inhibitor (Rli1p) is a highly conserved protein that is essential in all tested eukaryotes and archaea, but requires Fe-S clusters for its crucial functions in protein synthesis. Herein, the novel hypothesis that ROS toxicity is caused by loss of Rli1p function was tested. Rli1p activity (in nuclear export of ribosomal subunits) was impaired during mild oxidative stress in yeast. In addition, resistance to pro-oxidants was decreased by RLI1 repression and increased by RLI1 overexpression. This Rli1p-dependency was abolished during anaerobicity and accentuated in cells expressing the Fe-S cluster defective Rli1p construct, rli1C58A. The effects appeared specific to Rli1p as overexpression of other essential Fe-S proteins did not increase stress resistance. Methionine sulphoxide reductases (MSRs) and the Mn-superoxide dismutase (Sod2p) are known to help preserve the integrity of Fe-S clusters in cells. Here, these proteins’ antioxidant actions were shown to be at least partly mediated through Rli1p. Resistance to both chronic and acute oxidative stress was Rli1p-dependent. Further experiments indicated that Rli1p-dependent protein synthesis could be a critical target of ROS and, specifically, that Rli1p function may help to protect against ROS-induced mRNA mistranslation. The study indicated that Rli1p function is a primary biological target of ROS action, owing to its essential nature but dependency on ROS-labile Fe-S clusters. Such insights could offer new approaches for combating oxidative stress-related disease.

The P2Y14 receptor is the most recently identified member of the P2Y family of receptors for adenine and uridine nucleotides and nucleotide sugars. It is activated by UDP, UDP-glucose and its analogues, as well as the synthetic analogue MRS2690, which exhibits greater potency and selectivity at the P2Y14 receptor. The principle aim of this study was to investigate the functional expression of the P2Y14 receptor in porcine pancreatic arteries, and the signalling pathways underlying the vasoconstriction evoked by P2Y14 receptor agonists, together with an examination of the effects of UDP-glucose and MRS2690 on insulin secretion from the rat INS-1 823/13 β-cell line. Segments of porcine pancreatic arteries were prepared for isometric tension recordings in warmed oxygenated Krebs’-Henseleit buffer. Agonists were applied after preconstriction with U46619, a thromboxane A2 mimetic. ATP induced vasoconstriction followed by a vasorelaxation in pancreatic arteries; the contraction was blocked by NF449 (a P2X1 receptor selective antagonist), while the relaxation to ATP was blocked by an adenosine receptor antagonist. Neither the contraction, nor the relaxation to ATP were affected by removal of the endothelium. ADP evoked vasorelaxation, which was inhibited in the presence of SCH58261 (a selective adenosine A2A receptor antagonist). UTP-induced vasoconstriction was attenuated significantly in endothelium-denuded arteries. UDP, UDP-glucose and MRS2690 induced concentration-dependent contractions in porcine pancreatic arteries with a rank order of potency of MRS2690 (10-fold) > UDP-glucose = UDP. The contractions evoked by UDP-glucose and MRS2690 were significantly attenuated in the presence of PPTN (a selective P2Y14 receptor antagonist), indicating actions at P2Y14 receptors. The expression of P2Y14-like receptor was shown by immunohistochemical and contractile studies to be on the endothelium of the pancreatic arteries. UDP-glucose and MRS2690 inhibited forskolin-stimulated cAMP production. UDP-glucose and MRS2690 increased the level of MLC2 phosphorylation; this effect was blocked by PPTN, indicating the involvement of P2Y14 receptors. UDP-glucose increased the level of ERK1/2 phosphorylation. UDP-glucose and MRS2690 inhibited glucose-induced insulin release from the rat INS-1 823/13 β-cell line; this effect was blocked by PPTN, indicating actions through P2Y14 receptors. PPTN itself was able to elevate significantly basal insulin secretion from INS-1 823/13 β-cells, which may suggest a constitutive release of UDP-glucose from these cells. These results suggest that, in porcine pancreatic arteries, ATP induces a vasoconstriction mediated by P2X1 receptors followed by a vasorelaxation evoked by adenosine receptors present on the vascular smooth muscle. ADP induced a relaxation mediated by adenosine A2A receptor. Moreover, my data indicate for the first time, an endothelium-dependent contraction evoked by UTP. A novel vasocontractile role of P2Y14 receptors in porcine pancreatic arteries was also documented. The contractile response was mediated largely by the endothelium. P2Y14-mediated contraction involves a cAMP-dependent mechanism, which is consistent with P2Y14 receptor coupling to Gi protein, and an elevation in phosphorylated MLC2 and ERK1/2. Activation of the P2Y14 receptor evoked a decrease in the level of insulin secreted from the rat pancreas. The current data have identified novel roles of the P2Y14 receptor as a mediator of pancreatic artery contractility and in regulation of insulin secretion. If its role within the vasculature is shown to be more widespread, the P2Y14 receptor may be a novel target for the treatment of cardiovascular disease.

Enteropathogenic Escherichia coli (EPEC) is a diarrheagenic pathogen belonging to the Enterobacteriaceae and considered to be a leading cause of acute infantile diarrhoea in developing nations. During onset of infection, EPEC inject a variety of effector proteins into the host epithelial cells using the type III secretion system (T3SS). These effectors are encoded within the pathogenicity island (PAI) called the locus of enterocyte effacement (LEE). EPEC also secretes the non-LEE encoded serine protease EspC (EPEC secreted protein C) into the extracellular milieu. This autotransporter (AT) exits EPEC through a type V secretion system (T5SS) and is subsequently delivered into host cells by the T3SS. The precise steps by which EspC is secreted are still unknown. By using both traditional and advanced optical microscopes, it is shown here that EspC tagged with two different fluorescent labels locates to a structure that resembles the spiral bacterial cytoskeleton when it has its β-barrel domain attached. It was discounted that the spiral formation was an artefact of aggregation. Moreover, production of another AT (AaaA, derived from Pseudomonas aeruginosa) also generated spiral structures that resemble the ones observed with EspC. Perturbing the structure of the bacterial cytoskeleton actin homologue MreB with the S-(3,4-dichlorobenzyl) isothiourea compound (A22) disrupted the localization of EspC-mCherry. Furthermore, producing EspC-mCherry in an E. coli SecA mutant generated altered localization patterns. Collectively, these results indicate that the spiral localization of EspC is dependent upon its C-terminal β-barrel domain, the Sec translocon and the actin homologue MreB and this spiral secretion pathway seems to be conserved among ATs. Further analysis is required to reveal the molecular mechanism underlying spiral formation to unravel the mystery of AT secretion, thereby enabling the development of new therapeutic targets and treatments.