Characterisation of Lentiviral Vpr Function and Mechanism

Genetic conflict between viruses and their hosts has driven an ‘arms race’, forcing the evolution of both immune defencesdefenses and multiple viral strategies to counteract and evade them. In the case of HIV many of these processes are well characterised – the virus carries with it a set of accessory proteins which target specific host restriction factors. Of these accessory proteins Vpr is the least well understood, with no described role that adequately explains its conservation across all known primate lentiviruses. Unpublished data from our lab indicate that Vpr is able to rescue infection in macrophages from addition of cGAMP, a second messenger protein produced by the cytosolic DNA sensor cGAS which activates antiviral immune signalling pathways. This study first sought to test the hypothesis that Vpr has evolved to counteract cGAS/STING mediated cytosolic DNA sensing using a co-transfection assay to test Vpr proteins from all groups of primate lentiviruses. Initial observations appeared to demonstrate specific degradation of innate immune signalling proteins. It was subsequently shown that HIV-1 M Vpr antagonises expression from all tested co-transfected plasmids. This phenotype was demonstrated to be species specific, and to correlate with both the history of zoonotic transmission and localisation of Vpr to the nuclear rim. Additionally, it was shown that Vpr antagonises NFB signalling activated by TNF, independent of an effect on expression from transfected plasmids, but with the same dependence on nuclear localisation, putatively by the same mechanism. Next, this study characterised an observation that the Vpr from the lentivirus infecting a mona monkey (SIVmon) stimulates NFB signalling. It was hypothesised that the SIVmon Vpr might have molecular binding partners in common with the HIV-1 M Vpr and conditions were optimised for proteomics studies to determine these binding partners. This study provides insights into the role of Vpr in antagonising innate immune sensing. Additionally, overexpression assays have been used widely in the literature describing Vpr. The data presented here indicate that observations using these assays, apparently demonstrating specific degradation of host cellular proteins, should be interpreted cautiously

Regulation of Chromatin Modifier Genes by Microrna Vis-À-Vis Regulation of Microrna by DNA Methylation and Histone Modifications in Human Cancer

This thesis deals with the role of microRNA (miRNA) regulating other epigenetic modifiers like DNA methyltransferase 1 (DNMT1), and histone methyltransferase myeloid/lymphoid or mixed-lineage leukaemia (MLL1) also known as Histone-lysine N-methyltransferase 2A. It also divulges the reason for aberrant expression of miRNAs (miR-152, miR-148a, and miR-193a) in breast and prostate cancer. Silencing of the miR-152 gene due to promoter DNA methylation alter the expression pattern of several other genes. E-cadherin (CDH1) forms the core of adherent junctions between surrounding epithelial cells, link with the actin cytoskeleton and affects cell signalling. CDH1 gene is downregulated by promoter DNA methylation during cancer progression. In this investigation, we attempt to elucidate the correlation of miR-152 and CDH1 function, as it is well known that the loss of CDH1 function is one of the primary reasons for cancer metastasis and aggressiveness of spreading. For the first time here it has been shown that loss of CDH1 expression is directly proportional to the loss of miR-152 function in breast cancer cells. mRNA and protein expression profile of DNMT1 implicate that miR-152 targets DNMT1 mRNA and inhibits its protein expression. Tracing the molecular marks on DNA and histone 3 for understanding the mechanism of gene regulation by ChIP analyses leads to a paradoxical result that shows DNA methylation adjacent to active histone marking (enrichment of H3K4me3) silence miR- 152 gene. This thesis also demonstrated that miR-148a remains downregulated in hormone-refractory prostate cancer compared to other healthy cells and its upregulation induce apoptosis in hormone-refractory and metastatic prostate cancer cells. Here for the first time, it was analyzed the role of miR-148a in the regulation of DNMT1 in prostate cancer cells. The ectopic expression of miR-148a shows a noticeable amount of programmed cell death and repression of cancer cell proliferation. It also revealed the silencing of miR-148a in prostate cancer cells was done by DNMT1. This finding gives a new avenue to targeting prostate cancer cells and proved the role of miR-148a as a therapeutic. Moreover, other experiments also demonstrate the regulation of MLL1 by miR-193a. MiR-193a has been downregulated in prostate cancer by DNA methylation and help in MLL1 overexpression during prostate cancer progression. Most importantly it was found by inhibiting MLL1 it changes the global H3K4 methylation pattern increasing the monomethylation and decreasing trimethylation at H3K4 positions. H3K4 trimethylation is an active gene mark present in various oncogenes during cancer progression. By inhibiting H3K4, tri-methylation cancer progression can be repressed. Ectopic expression of miR- 193a results in cell death, inhibition of cellular migration, and anchorage-independent growth of cancer cells. All together this thesis supports that miR-152, miR-148a, and miR-193a are regulated by DNA methylation, and they affect the expression of the various epigenetic modifiers. Hence these can be targeted for therapeutic intervention for breast and prostate cancer

An investigation into the biosynthesis of proximicins

PhD ThesisThe proximicins are a family of three compounds – A-C – produced by two marine Actinomycete Verrucosispora strains – V. maris AB18-032 and V. sp. str. 37 - and are characterised by the presence of 2,4-disubstituted furan rings. Proximicins demonstrate cell-arresting and antimicrobial ability, making them interesting leads for clinical drug development. Proximicin research has been largely overshadowed by other Verrucosispora strain secondary metabolites (SM), and despite the publication of the V. maris AB18-032 draft, the enzymatic machinery responsible for their production has not been established. It has been noted in related research into a pyrrole-containing homolog – congocidine –due to the structural similarity exhibited, proximicins likely utilise a similar biosynthetic route. The initial aim of this research was to confirm the presumed pathway to proximicin biosynthesis. Following the sequencing, assembly and annotation of the second proximicin producer, Verrucosispora sp. str. MG37, and genome mining of V. maris AB18-032, no common clusters mimicked that of congocidine, casting doubt on the previously assumed analogous biosynthetic routes. A putative proximicin biosynthesis (ppb) cluster was identified, containing non-ribosomal peptide synthetase (NRPS) enzymes, exhibiting some homology with congocidine. NRPSsystems represent a network of interacting proteins, which act as a SM assembly line: crucially, adenylation (A)- domain enzymes act as the ‘gate-keeper’, determining which precursors are included into the elongating peptide. To elucidate the route to proximicins, activity characterisation of the four A-domains present in ppb cluster was attempted. The A-domain Ppb120 was shown to possess novel activity, demonstrating a high promiscuity towards heterocycle containing precursors, in addition to the absence of an apparent essential domain. This discovery refutes previous work outlining the core residues which dictate A-domain activity, while also presenting a facile route to novel heterocycle-containing compounds. Despite extensive work, A-domains ppb195 and ppb210, were ineffectively purified in the active form – informing future work into A-domains activity characterisation. Finally, the ppb220 A-domain which lies at the border of ppb, was inactive suggesting over-estimation of the cluster margins. To confirm ppb220 redundancy and confirm ppb boundaries, CRISPR/Cas gene editing studies were done. The gene responsible for the orange pigment of Verrucosispora strains was initially targeted and successfully deleted, and ppb studies commenced. The research here refutes the previously presumed route to proximicin biosynthesis; the ppb cluster instead comprises enzymes exhibiting unique activity and structure. The findings represent the foundations for allowing exploitation of chemistry exhibited within the proximicin family. The novelty exhibited can be utilised in the search for antimicrobial clinical leads, by allowing the production of compounds containing previously inaccessible heterocycle chemistry

The development of single-molecule approaches to study molecular chaperone function

Protein misfolding and aggregation are associated with the pathogenesis of a wide range of neurodegenerative and systemic diseases, including Alzheimer’s disease, Parkinson’s disease and cataracts. Consequently, cells have evolved a vast network of molecular chaperones, which include the intracellular heat-shock proteins (Hsp), that act to prevent protein misfolding and aggregation as part of protein homeostasis (proteostasis). However, the precise molecular mechanisms by which these chaperones prevent protein aggregation are unclear, mostly due to the significant heterogeneity and transient nature by which they interact with client proteins and co-chaperones. This heterogeneity has made these proteins difficult to study using conventional ensemble-averaging approaches, as rare and transient interactions between individual species cannot be observed. Recently, single-molecule approaches have emerged as a useful tool to study chaperone function since individual protein trajectories can be monitored in real time, enabling the characterization of events typically masked in ensemble measurements. The work described in this thesis aimed to utilize and further develop single-molecule approaches for the study of protein misfolding, aggregation and molecular chaperone function. A critical aspect of chaperone function is how they modify the conformation of their client proteins to promote folding and prevent misfolding. To address this, two well-established client proteins, firefly luciferase and rhodanese, were developed into protein-folding sensors that could be used to monitor chaperone-induced conformational changes in real time using single-molecule fluorescence resonance energy transfer (smFRET). To do so, both luciferase and rhodanese were modified such that they could be specifically immobilized to a functionalized coverslip surface to monitor structural changes in a temporally-resolved manner and over extended periods via total internal reflection fluorescence (TIRF) microscopy. For the first time, the conformation of a client protein as it was being folded by the bacterial or human Hsp70 chaperone machinery (i.e., Hsp40, Hsp70 and a nucleotide-exchange factor) was monitored in real time. From these data, it was demonstrated that the Hsp40 chaperone binds to and conformationally remodels misfolded client proteins for delivery to Hsp70. Following binding by Hsp70, the conformation of the client becomes significantly expanded but remains conformationally dynamic, thereby resolving misfolded states. Release of the client protein by Hsp70 enables an opportunity for spontaneous refolding of the client to the native state or collapse to a misfolded conformer. The latter can undergo additional rounds of chaperone action until the native state is acquired. Thus, the luciferase and rhodanese protein-folding sensors developed in this work were used to elucidate key aspects of chaperone function and represent ideal tools for the continued study of other chaperone systems

Design and engineering genetic tools for Desulfovibrio alaskensis

Microorganisms, such as the anaerobic bacterium Desulfovibrio alaskensis, have evolved various mechanisms to resist high concentrations of toxic heavy metals; one of these mechanisms involves the synthesis of nanoparticles (NPs). It may be possible to utilise this ability to both reclaim heavy metals from contaminated effluents and to convert them into industrially useful NPs. By engineering a genetically modified D. alaskensis, through synthetic biology, cell surface engineering and by designing a modular cloning (MoClo) toolkit, there is a further opportunity to tailor nanoparticle synthesis. DNA assembly techniques have revolutionised biotechnology research and innovation. However, despite many advances in molecular biology, the assembly of DNA parts into new constructs remains cumbersome and unpredictable. The innovation of cloning toolkits and standards such as MoClo have standardised the process of DNA assembly, making it easier, faster, modular and cost-effective. The D. alaskensis MoClo toolkit developed in this work consists of characterised oxygen-independent reporters, synthetic promoters and ribosome binding site (RBS) libraries. The D. alaskensis MoClo toolkit was utilised to assemble a combinatorial library of transcriptional units (TUs) expressing the NiFe hydrogenase small subunit. Platinum NPs were synthesised by the combinatorial library, and examined for their oxidative and reduction catalytic activities were tested. To enhance D. alaskensis resistance to Cu, Pt and Pd, cell surface engineering was used to express synthetic phytochelatin EC20 on the outer membrane. Tests of Escherichia coli expressing EC20/IgA to Cu, Pt and Pd concluded that EC20 confers a higher resistance to all metals

Towards optical and potentiometric measurements of double layer structures and dynamics

My doctoral research pertains with developing accessible and technically simple methods to study the electric double layer. A complete and correct description of double layers formed when wetting charged surfaces with electrolytic solutions is of great practical importance. This phase boundary governs charge transport, energy storage and lubricating properties of interfaces. Currently, all techniques available to probe double layer structures and dynamics are rather technically demanding (e.g. electrochemical impedance spectroscopy, AFM and STM), or carry intrinsic limitations, such as when metal probes are employed, a situation where the probe element inevitably bears its own double layer making hard to extract data on the sample under analysis

Doctor of Philosophy

dissertationIt is well documented that more than 50% of all human cancers have a mutated p53 gene status, rendering it inactive. The resulting tumor-derived p53 variants, similar to wild-type (wt) p53, retain their ability to oligomerize via the tetramerization domain. Upon hetero-oligomerization, mutant p53 enforces a dominant negative effect over active wt-p53 in cancer cells. To overcome this barrier, we have designed a chimeric superactive p53 (p53-CC) with an alternative oligomerization domain (CC) from breakpoint cluster region (Bcr). This approach led to the hypothesis that swapping the oligomerization domain of p53 with an alternative oligomerization domain will prevent hetero-oligomerization and transdominant inhibition by mutant p53 in cancer cells. The tumor suppressor activity of the chimeric p53-CC was evaluated in vitro and found to be similar to that of wt-p53 regardless of cancer type or endogenous p53 status. However, co-immunoprecipitation and viral transduction of p53-CC and wt-p53 into a breast cancer cell line that harbors a tumor derived transdominant mutant p53 validated that p53-CC indeed evades sequestration and consequent transdominant inhibition by endogenous mutant p53. Following proof-of-concept studies, the superior tumor suppressor activity of p53-CC and its ability to cause tumor regression of the MDA-MB-468 aggressive p53-dominant negative breast cancer tumor model was demonstrated in vivo. In addition, the underlying differential mechanisms of activity for p53-CC and wt-p53 delivered using viral-mediated gene therapy approach in the MDA-MB-468 tumor model were investigated. Finally, since domain swapping to create p53-CC could result in p53-CC interacting with endogenous Bcr, which is ubiquitous in cells, modifications on the CC domain were necessary to minimize potential interactions with Bcr. Hence, the possible design of mutations that will improve homo-dimerization of CC mutants and disfavor hetero-oligomerization with wild-type CC (CCwt) were investigated, with the goal of minimizing potential interactions with endogenous Bcr in cells. Indeed, the resulting lead candidate p53-CCmutE34K-R55E avoided binding to endogenous Bcr and retained p53 tumor suppressor activity. Although breast cancer was the main focus of this dissertation, the application of this research extends to many other types of cancer, including the deadliest cancers (pancreatic, lung, and ovarian), which currently lack effective treatments