Regulation of tail-anchored ubiquitin conjugating enzymes that are localised to the endoplasmic reticulum

The vast majority of secreted and membrane proteins are translated and folded at the endoplasmic reticulum (ER), where a sophisticated quality control mechanism ensures that only correctly folded proteins exit the ER and traffic to their final destinations. On the other hand, proteins that persistently misfold are eliminated through a process known as ER associated degradation (ERAD). This involves retrotranslocation of the misfolded protein through the ER membrane, and ubiquitination in advance of degradation by cytosolic proteasomes. The process of ERAD is best described in yeast where ubiquitin conjugating enzymes Ubc6p and Ubc7p function with a limited number of E3 ubiquitin ligases to ubiquitinate misfolded proteins. Interestingly, although the mechanistic principles of ERAD have been conserved through evolution, there is increasing evidence that homologues of the yeast enzymes have gained divergent roles and novel regulatory functions in higher eukaryotes, meaning that the process in humans is more complex and involves a larger repertoire of participating proteins. Two homologues of Ubc6p have been described in humans, and have been named as Ubc6 (UBE2J2) and Ubc6e (UBE2J1). However, little work has been done on these enzymes and thus our main objective of this study was to progress the functional characterisation of these ERAD E2 conjugating enzymes. Our studies included a detailed analysis of conditions whereby these proteins are stabilised and degraded. We’ve also explored the different molecular signalling pathways that induced changes on their steady state protein levels. Furthermore, Ubc6e has a phosphorylatable serine residue at position 184. Thus, our studies also involved delineating the signalling kinases that phosphorylate Ubc6e and examining its function in ERAD. Our studies confirm that the E2 Ubc enzymes are regulated posttranslationally and may have important implications in the regulation of ERAD.

Investigation of lantibiotic regulation, immunity and synergy

Due to the increasing incidence of antibiotic resistant strains, the use of novel antimicrobials, such as bacteriocins, has become an ever more likely prospect. Lacticin 3147 (of which there are two components, Ltnα and Ltnβ) and nisin belong to the subgroup of bacteriocins called the lantibiotics, which has attracted much attention in recent years. The lantibiotics are antimicrobial peptides that contain unusual amino acids resulting from a series of enzyme-mediated post translational modifications. Given that there have been relatively few examples of lantibiotic-specific resistance; these antimicrobials appear to represent valid alternatives to classical antibiotics. However, the fact that lantibiotics are naturally only produced in small amounts often hinders their commercialisation. In order to overcome this bottleneck, several approaches can be employed. For example, we can create a situation that reduces the quantity of a lantibiotic required to inhibit a target by combining it with other antimicrobials. Here, following an initial screen involving lacticin 3147 and several classical antibiotics, it was observed between lacticin 3147 and the commercial antibiotics polymyxin B/E function synergistically. This reduced the amounts of the individual antimicrobials required for kill and broadened the spectrum of inhibition of both agents. Upon combination with polymyxins, lacticin 3147, which has been associated with Gram positive targets only, actively targeted Gram negative species such as Escherichia coli and Cronobacter sp. An alternative means of addressing problems associated with lantibiotic yield is to better understand how production is regulated, and ultimately use this information to enhance peptide levels. With this in mind the regulation of lacticin 3147 production from the promoter Pbac was investigated using a green fluorescent protein (GFP) expression reporter system. This revealed that elements within both of the divergent operons of the lacticin 3147 gene cluster are involved in Pbac regulation. That is, LtnR, already established as a negative regulator of itself and the lacticin 3147 associated immunity genes, also acts as an activator of Pbac transcription. In contrast, an enhanced level of expression is observed in the absence of the lacticin 3147 structural genes, ltnA1 and ltnA2, indicating that these genes/gene products are involved in Pbac repression. In fact, through complementation of the ltnA2 gene, it was revealed that this regulation is more likely to be dependent on the presence of the gene transcript rather that the corresponding prepropeptide or modified Ltnβ. It may be that if lacticin 3147 production is successfully enhanced, the ability of the producing cell to protect itself may become an issue. To prepare for such a possibility a bioengineered derivative of the lacticin 3147 immunity protein LtnI (LtnI I81V) which provides enhanced protection was discovered through an in depth investigation involving the site and saturation mutagenesis of this protein. In addition, the creation of truncated forms of LtnI allowed the identification of important and essential regions of this immunity protein. Finally, as mentioned, self-immunity is essential to prevent self-killing. However the discovery of nisin U immunity and regulatory gene homologues (spiFEGRR’K) within the pathogenic strain S. infantarius subsp. infantarius is a cause for concern as it represents an example of immune mimicry, a form of lantibiotic-specific resistance. The ability of spiFEG to confer protection was apparent when they successfully provided protection to nisin A, F, Z, Q and U when expressed heterologously in the nisin sensitive L. lactis HP host. As a consequence of the studies presented in this thesis, it is likely that strategies will emerge that will facilitate the production of greater levels of lacticin 3147 production and lead to enhanced immunity in lactococcal backgrounds. Alternatively the need for enhanced production could be avoided through the use of antimicrobial combinations. In addition, providing awareness of the threats of the emergence of resistance through immune mimicry can allow researchers to develop strategies to prevent this phenomenon from leading to the dissemination of lantibiotic resistance.