Investigation of p75 neurotrophin receptor and the role of its post-translational modifications in tumour cell motility and invasiveness

The p75 neurotrophin receptor (p75NTR) is a member of the tumour necrosis factor superfamily, which relies on the recruitment of cytosolic protein partners - including the TNF receptor associated factor 6 (TRAF6) E3 ubiquitin ligase - to produce cellular responses such as apoptosis, survival, and inhibition of neurite outgrowth. Recently,p75NTR was also shown to undergo γ-secretase-mediated regulated intramembrane proteolysis, and the receptor ICD was found to migrate to the nucleus where it regulates gene transcription. Moreover, γ-secretase-mediated proteolysis was shown to be involved in glioblastoma cell migration and invasion. In this study we report that TRAF6-mediated K63-linked polyubiquitination at multiple or alternative lysine residues influences p75NTR-ICD stability in vitro. In addition, we found that TRAF6-mediated ubiquitination of p75NTR is not influenced by inhibition of dynamin. Moreover, we report beta-transducin repeats-containing protein (β-TrCP) as a novel E3- ligase that ubiquitinates p75NTR, which is independent of serine phosphorylation of the p75NTR destruction motif. In contrast to its influence on other substrates, co-expression of β-TrCP did not reduce p75NTR stability. We created U87-MG glioblastoma cell lines stably expressing wild type, γ-secretaseresistant and constitutively cleaved receptor, as well as the ICD-stabilized mutant K301R. Interestingly, only wild-type p75NTR induces increased glioblastoma cell migration, which could be reversed by application of γ-secretase inhibitor. Microarray and qRT-PCR analysis of mRNA transcripts in these cell lines yielded several promising genes that might be involved in glioblastoma cell migration and invasion, such as cadherin 11 and matrix metalloproteinase 12. Analysis of potential transcription factor binding sites revealed that transcription of these genes might be regulated by well known p75NTR signalling cascades such as NF-κB or JNK signalling, which are independent of γ-secretase-mediated cleavage of the receptor. In contrast, while p75NTR overexpression was confirmed in melanoma cell lines and a patient sample of melanoma metastasis to the brain, inhibition of γ-secretase did not influence melanoma cell migration. Collectively, this study provides several avenues to better understand the physiological importance of posttranslational modifications of p75NTR and the significance of the receptor in glioblastoma cell migration and invasion.

FLT3-driven redox signalling in acute myeloid leukaemia

Acute myeloid leukaemia refers to cancer of the blood and bone marrow characterised by the rapid expansion of immature blasts of the myeloid lineage. The aberrant proliferation of these blasts interferes with normal haematopoiesis, resulting in symptoms such as anaemia, poor coagulation and infections. The molecular mechanisms underpinning acute myeloid leukaemia are multi-faceted and complex, with a range of diverse genetic and cytogenetic abnormalities giving rise to the acute myeloid leukaemia phenotype. Amongst the most common causative factors are mutations of the FLT3 gene, which codes for a growth factor receptor tyrosine kinase required by developing haematopoietic cells. Disruptions to this gene can result in constitutively active FLT3, driving the de-regulated proliferation of undifferentiated precursor blasts. FLT3-targeted drugs provide the opportunity to inhibit this oncogenic receptor, but over time can give rise to resistance within the blast population. The identification of targetable components of the FLT3 signalling pathway may allow for combination therapies to be used to impede the emergence of resistance. However, the intracellular signal transduction pathway of FLT3 is relatively obscure. The objective of this study is to further elucidate this pathway, with particular focus on the redox signalling element which is thought to be involved. Signalling via reactive oxygen species is becoming increasingly recognised as a crucial aspect of physiological and pathological processes within the cell. The first part of this study examined the effects of NADPH oxidase-derived reactive oxygen species on the tyrosine phosphorylation levels of acute myeloid leukaemia cell lines. Using two-dimensional phosphotyrosine immunoblotting, a range of proteins were identified as undergoing tyrosine phosphorylation in response to NADPH oxidase activity. Ezrin, a cytoskeletal regulatory protein and substrate of Src kinase, was selected for further study. The next part of this study established that NADPH oxidase is subject to regulation by FLT3. Both wild type and oncogenic FLT3 signalling were shown to affect the expression of a key NADPH oxidase subunit, p22phox, and FLT3 was also demonstrated to drive intracellular reactive oxygen species production. The NADPH oxidase target protein, Ezrin, undergoes phosphorylation on two tyrosine residues downstream of FLT3 signalling, an effect which was shown to be p22phox-dependent and which was attributed to the redox regulation of Src. The cytoskeletal associations of Ezrin and its established role in metastasis prompted the investigation of the effects of FLT3 and NADPH oxidase activity on the migration of acute myeloid leukaemia cell lines. It was found that inhibition of either FLT3 or NADPH oxidase negatively impacted on the motility of acute myeloid leukaemia cells. The final part of this study focused on the relationship between FLT3 signalling and phosphatase activity. It was determined, using phosphatase expression profiling and real-time PCR, that several phosphatases are subject to regulation at the levels of transcription and post-translational modification downstream of oncogenic FLT3 activity. In summary, this study demonstrates that FLT3 signal transduction utilises a NADPH oxidase-dependent redox element, which affects Src kinase, and modulates leukaemic cell migration through Ezrin. Furthermore, the expression and activity of several phosphatases is tightly linked to FLT3 signalling. This work reveals novel components of the FLT3 signalling cascade and indicates a range of potential therapeutic targets.

Characterization of TRAF6 mediated ubiquitination of presenilins and γ-secretase substrates

Post-translational modification of the γ-secretase protease complexes and their substrates has an important role in controlling receptor-initiated signalling events, which are critically important in the pathogenesis of cancer, inflammatory and Alzheimer’s disease. Our lab has previously characterised an interaction between TRAF6 and presenilin-1, which lead to the identification of interleukin-1 (IL-1) receptor type 1 (IL-1R1) and Toll-like receptor-4 (TLR4) as novel γ-secretase substrates. Subsequently our group showed that TRAF6 promoted ubiquitination and γ-secretase cleavage of IL-1R1. The aim of this project is to study the association between TRAF6 and the presenilins, the critical γ-secretase complex components, and to determine the functional importance of TRAF6-mediated ubiquitination of γ-secretase substrates. Firstly, we show that the full-length presenilins are novel substrates of TRAF6-mediated Lysine-63-linked polyubiquitination. Secondly, we show that co-expression of TRAF6 and the presenilins increases the stability and alters the turnover of the presenilins. Thirdly, we reveal that TRAF6-mediated ubiquitination of presenilin does not affect γ-secretase enzyme activity, but may regulate the full-length presenilin functions such as ER Ca2+ signalling. Previously, we have reported IL-1R1 as a novel substrate of TRAF6-mediated ubiquitination. In this study, we identified five lysine residues in the IL-1R1 intracellular domain targeted by TRAF6-mediated polyubiquitination. Furthermore, mutagenesis of these five lysine residues led to decreased IL-1R1 cell surface expression, precluded the ectodomain shedding and attenuated the responsiveness to IL-1β stimulation, demonstrating the critical role of TRAF6 in IL-1R1 trafficking.

The analysis of Bcr-Abl—Nox signalling in chronic myeloid leukaemia

Chronic Myeloid Leukaemia (CML) is a myeloproliferative disorder characterised by increased proliferation of haematopoietic stem cells. CML results following generation of the chimeric protein Bcr-Abl, a constitutively active tyrosine kinase which induces oncogenesis in part by promoting increased cell survival and proliferation. Since the development of Bcr-Abl-specific tyrosine kinase inhibitors (TKIs) there has been a substantial improvement in the clinical treatment of CML. Unfortunately, residual disease and the development of TKI resistance has become an ever growing concern, resulting in the need for a greater understanding of the disease in order to develop new treatment strategies. Interestingly, constitutive expression of the Bcr-Abl in CML is known to produce elevated levels of Reactive Oxygen Species (ROS) which are known to influence a variety of cellular processes. Previous studies have demonstrated that NADPH oxidase (Nox) activity contributes to intracellular-ROS levels in Bcr-Abl-positive cells, enhancing survival signalling. The objective of this study was to elucidate how Nox protein activity was influenced downstream of Bcr-Abl while examining how Nox-derived ROS influenced CML disease phenotype to identify the potential in targeting these proteins to improve CML treatment. These studies demonstrated that inhibition of Bcr-Abl signalling, led to a significant reduction in ROS levels which was concurrent with the GSK-3dependent, post-translational down-regulation of the small membrane-bound protein p22phox, an essential component of the Nox complex. siRNA knockdown of p22phox identified it to have a significant role in cellular proliferation and cell viability, demonstrating the importance of Nox protein activity in CML disease phenotype. Furthermore, removal of p22phox was demonstrated to make cells significantly more susceptible to Bcr-Abl-specific TKI treatment, while pharmacological silencing of Nox activity in combination with TKIs was demonstrated to produce substantial, synergistic increases in cell death through augmentation of apoptosis, demonstrating the therapeutic potential of targeting Nox proteins in combination with Bcr-Abl inhibition.

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.

Bioengineering strategies to improve functional qualities of nisin

The abuse of antibiotics and the emergence of multi-drug resistant bacterial strains have created the need to explore alternative methods of controlling microbial pathogens. The bacteriocin family of antimicrobial peptides has been proposed as one such alternative to classic antibiotics. Nisin A belongs to the subgroup of bacteriocins called the lantibiotics, which contain several unusual amino acids as a consequence of enzyme-mediated post-translational modifications. As nisin is produced by generally regarded as safe (GRAS) microorganisms, it could potentially be applied in a clinical setting. However, as lantibiotics are naturally produced in such small quantities, this can hinder their industrial potential. In order to overcome this, several approaches can be utilised. For example, given the gene encoded nature of lantibiotics, genetic engineering approaches can be implemented in order to yield variants with enhanced properties. Here, the use of mutagenesis-based strategies was employed to obtain a derivative of nisin with enhanced bioactivity in vitro. Investigations with purified peptide highlighted the enhanced specific activity of this variant, nisin M21V, against food-borne Listeria monocytogenes strains. Furthermore, this specific enhanced bioactivity was evident in a mouse model of listeriosis. Reductions in bioluminescence and microbial counts in organs from infected mice were observed following treatment with nisin M21V compared to that of wild-type nisin A. Peptide bioengineering approaches were also implemented to obtain additional novel derivatives of nisin. The generation of “S5X” and “S33X” banks (representing a change of natural serines at positions 5 and 33 to all possible alternative residues) by a combination of site-saturation and site-directed mutagenesis led to the identification of several derivatives exhibiting improved stability. This allowed the rational design of variants with enhanced stability compared to that of wild type nisin. Another means of tackling issues associated with lantibiotic yield is to combine lantibiotics with other antimicrobials. This could circumvent the need for enhanced production while also reducing concentrations of the peptide antimicrobials. We observed that combinations of nisin variants and low levels of plant essential oils (thymol, carvacrol, trans-cinnamaldehyde) significantly controlled Gram negative foodborne pathogens in in vitro assays compared to nisin A-essential oil combinations. This enhanced control was also evident in model food systems. Nisin variants used in conjunction with carvacrol significantly reduced numbers of E. coli O157:H7 in apple juice while a commercial nisin preparation used in combination with citric acid significantly controlled C. sakazakii in infant milk formula. It is noteworthy that while nisin is generally associated with Gram positive targets, upon combination with plant essential oils the spectrum of inhibition was broadened to Gram negative targets.

The synthetic progestin norgestrel modulates Nrf2 signaling and acts as an antioxidant in a model of retinal degeneration

Retinitis pigmentosa (RP) is one of the most common retinal degenerative conditions affecting people worldwide, and is currently incurable. It is characterized by the progressive loss of photoreceptors, in which the death of rod cells leads to the secondary death of cone cells; the cause of eventual blindness. As rod cells die, retinal-oxygen metabolism becomes perturbed, leading to increased levels of reactive oxygen species (ROS) and thus oxidative stress; a key factor in the secondary death of cones. In this study, norgestrel, an FDA-approved synthetic analog of progesterone, was found to be a powerful neuroprotective antioxidant, preventing light-induced ROS in photoreceptor cells, and subsequent cell death. Norgestrel also prevented light-induced photoreceptor morphological changes that were associated with ROS production, and that are characteristic of RP. Further investigation showed that norgestrel acts via post-translational modulation of the major antioxidant transcription factor Nrf2; bringing about its phosphorylation, subsequent nuclear translocation, and increased levels of its effector protein superoxide dismutase 2 (SOD2). In summary, these results demonstrate significant protection of photoreceptor cells from oxidative stress, and underscore the potential of norgestrel as a therapeutic option for RP.

Bifidobacterium longum 1714 as a translational psychobiotic: modulation of stress, electrophysiology and neurocognition in healthy volunteers

The emerging concept of psychobiotics—live microorganisms with a potential mental health benefit—represents a novel approach for the management of stress-related conditions. The majority of studies have focused on animal models. Recent preclinical studies have identified the B. longum 1714 strain as a putative psychobiotic with an impact on stress-related behaviors, physiology and cognitive performance. Whether such preclinical effects could be translated to healthy human volunteers remains unknown. We tested whether psychobiotic consumption could affect the stress response, cognition and brain activity patterns. In a within-participants design, healthy volunteers (N=22) completed cognitive assessments, resting electroencephalography and were exposed to a socially evaluated cold pressor test at baseline, post-placebo and post-psychobiotic. Increases in cortisol output and subjective anxiety in response to the socially evaluated cold pressor test were attenuated. Furthermore, daily reported stress was reduced by psychobiotic consumption. We also observed subtle improvements in hippocampus-dependent visuospatial memory performance, as well as enhanced frontal midline electroencephalographic mobility following psychobiotic consumption. These subtle but clear benefits are in line with the predicted impact from preclinical screening platforms. Our results indicate that consumption of B. longum 1714 is associated with reduced stress and improved memory. Further studies are warranted to evaluate the benefits of this putative psychobiotic in relevant stress-related conditions and to unravel the mechanisms underlying such effects.