Biochemistry and Molecular Biology - Theses
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A structural understanding of interleukin- 11 signalling
Interleukin (IL)-11 is an IL-6 family cytokine with several described roles in normal human physiology, haematopoiesis and disease. IL-11 signals by forming a hexameric signalling complex with its two receptors, the IL-11 specific receptor IL- 11R alpha, and the receptor glycoprotein (gp) 130, which is also utilised by approximately 15 other cytokines. IL-11 signalling has been shown to have roles in several diseases, including gastrointestinal cancer and cardiovascular fibrosis. Despite the growing importance that has been assigned to IL-11 signalling in disease, little is known about the structure of IL-11, the structural mechanisms of IL-11 signalling, or the structural basis of existing IL-11 signalling inhibitors. Overall, this thesis aims to develop a structural and mechanistic understanding of the formation of the IL-11 signalling complex, and the mechanism of action of an existing IL-11 signalling inhibitor. Here, we present the structures of IL-11 and IL-11R alpha. The new high-resolution structure of IL-11 uncovers details not resolved in previous structures, including in a loop which has previously been directly implicated in binding to IL-11R alpha. The structure of IL-11R alpha shows structural differences to related cytokine receptors. Combined with biophysical analysis of the interaction between IL-11 and IL-11R alpha, our structures allow us to propose a model for the recruitment of IL-11 by IL-11R alpha. We also used our structure to investigate the mechanism of action of known disease mutations in IL-11R alpha. These mutations were distal to cytokine-binding regions in IL-11R alpha and appear to act through destabilisation of IL-11R alpha. We also solved the structure of the IL-11 signalling complex using cryo-electron microscopy and X-ray crystallography. The complex is a hexameric complex consisting of two copies each of IL-11, IL-11R alpha and gp130. The resolution of the map is sufficiently high to define in detail of the binding sites on IL-11 and the receptors and uncovers mechanistic differences in complex formation by IL-11 and the related cytokines IL-6 and viral IL-6. Using solution methods, we also showed that the IL-11 signalling complex is hexameric in solution, and forms in three high- affinity steps, combined with our knowledge of the IL-11/IL-11R alpha interaction, this allows us a detailed, mechanistic understanding of the formation of the IL-11 signalling complex. We also studied the lead IL-11 signalling inhibitor, IL-11 Mutein. IL-11 Mutein was developed from an existing antagonistic mutant of IL-11, IL-11 W147A, which removed a key residue required for hexameric complex formation. IL-11 Mutein was developed from IL-11 W147A using phage display, to identify novel mutations which increased the potency of IL-11 W147A. We solved structures of IL-11 Mutein and the IL-11 W147A mutant, showing that the mutations in IL-11 Mutein disrupt a loop in proximity to the IL-11R alpha binding site. We showed that IL-11 Mutein is an effective IL-11 antagonist and prevents the assembly of the IL-11 signalling complex. Supported with extensive biochemical and biophysical analysis, we propose a model for the antagonistic action of IL-11 Mutein, which will guide the development of novel cytokine-like inhibitors. Overall, the work presented here allows a detailed understanding of the structural mechanisms underpinning IL-11 signalling. The results will allow the development of novel antagonists targeting the IL-11 signalling complex, which we hope will be of therapeutic benefit in cancers and other diseases.
Investigating the Epitranscriptome of Plasmodium falciparum
Recent years have seen an increasing identification and awareness of post-transcriptional modifications of RNA. Nucleotide modifications have long been known to be important in diverse non-coding RNAs, but post-transcriptional modifications of mRNA are now also recognised as playing an important role in regulation of gene expression. One of the most abundant mRNA modifications is N6-methyladenosine (m6 A), the presence of which guides mRNA metabolism including maturation, nuclear export, translation and decay. In model eukaryotes, m6A is produced by the action of METTL-family methyltransferases and recognised by a family of YTH proteins. These enzymes play a key role in cellular differentiation and development. I have identified several putative METTL methyltransferases and several YTH reader proteins in the Plasmodium falciparum genome, and have investigated the m6A modification in the transcriptome of P. falciparum. I have characterised two methyltransferase proteins, Mettl3 and Mettl14-like proteins and two reader YTHDC1-like proteins (PfYTHDC1-a and PfYTHDC1-b)in P. falciparum through localisation, overexpression experiments and knock-sideways studies. Initial studies using epitope tagging and GFP fusion proteins revealed a predominantly nuclear localisation for both the methyltransferase and reader proteins, with some signal in the cytoplasm. Knock-sideways studies revealed complete inducible mis-localisation by 24 hours for PfMETTL3 and within 7 hours for PfYTHDC1-a. Additionally, under induced mis-localisation, both transfectant lines underwent morphological changes as evident by microscopy, with clear spots within the digestive vacuole, changes not seen in the 3D7 parental line. A pilot RNA-seq study was conducted, wherein differential transcript expression was analysed as a means to characterise the impact of perturbing m6A modifications by overexpressing and disrupting METTL methyltransferases. Future experiments should analyse the distribution of m6A in mRNA by nanopore direct-RNA sequencing. Additional investigations are also warranted on the influence of the METTL and YTHDC1-like proteins on splicing patterns in the parasite as a means to test the role of these modifications on proliferation and differentiation.
Neuroinflammation, microglia and the cell biology of Alzheimer's Disease
The pathology of Alzheimer’s disease (AD) is characterised by progressive accumulation of misfolded proteins, which form senile plaques and neurofibrillary tangles, and chronic inflammation in the brain associated with inflammatory mediators by the activation of innate immune responses. There has been considerable interest in the role of neuroinflammation in directly contributing to the progression of AD. Studies in mice and humans have identified a role for microglial cells, the resident innate immune cells of the CNS, in AD. Activated microglia are a key hallmark of the disease and the secretion of pro-inflammatory cytokines by microglia may result in a positive feedback loop between neurons and microglia, resulting in ongoing low-grade inflammation and associated neurotoxicity. The underlying mechanisms however are poorly understood. Here the role of microglia was investigated, especially their link to ApoE – the strongest risk factor for late onset Alzheimer’s disease – and the relationship between microglia, neurons and neuroinflammation. Target replacement mice were used, where the human ApoE2, ApoE3 or ApoE4 allele replaces the mouse ApoE allele. Microglia were activated in a two- step setup. Initially cells were primed with LPS, followed by a secondary stimulus, such as ATP or Ab. The system was used to characterise the cytokines secreted by activated microglia and to assess the impact of conditioned medium from stimulated and Ab treated microglia on neuronal morphology. The first results Chapter (Chapter 3) established the system – mouse microglia were isolated from brains of neonatal mice and characterised by CD11b staining. Microglia from all three ApoE genotypes were directly compared and the data from ELISA and mass spectrometry revealed an enhanced pro- inflammatory response by ApoE4 microglia and the least efficient at internalizing amyloid b. Chapter 4 analysed the impact of conditional medium from the microglia ApoE variants on neurons and the results showed an ApoE-dependent effect on dendrite morphology. Conditioned media from immunostimulated and Ab microglia were incubated with cortical neurons from wt animals. Both the dendrite length and number of dendrites were significantly reduced in neurons treated with conditioned medium from ApoE4 microglia. TNFa was identified as a major cytokine and was responsible for modifying neuron morphology in cell assays. Neutralising the cytokine, with an anti-TNFa antibody abrogated the majority of morphological changes induced by the conditioned media from activated microglia. Hence the data suggests that TNFa may have a major role in mediating neuroinflammation. The third results Chapter (Chapter 5) compared aspects of macrophage function with microglia. Here it was shown, that microglia do not require SNX5 for macropinocytosis, and most likely utilise peripheral mediated macropinocytosis as the main form of macropinocytic internalisation. A major finding was the ability of sodium chloride to augment a pro-inflammatory response not only by immunostimulated macrophages by also microglia. Inhibition of the p38 MAPK signalling pathway partially ameliorated the NaCl- induced inflammatory responses in both macrophages and microglia, together with high levels of secreted IL-1b, indicating activation of the NLRP3 inflammasome. Overall the studies highlight a role for APoE4 allele to promote an enhanced inflammatory response by microglia cells.
Delayed death by plastid inhibition in Plasmodium falciparum
Apicomplexan parasites possess a plastid organelle called the apicoplast. Inhibitors that selectively target apicoplast housekeeping functions, including DNA replication and protein translation, are lethal for the parasite, and several (doxycycline, clindamycin, and azithromycin) are in clinical use as antimalarials. A major limitation of such drugs is that treated parasites only arrest one intraerythrocytic development cycle (approximately 48 hours) after treatment commences, a phenotype known as the ‘delayed death’ effect. The molecular basis of delayed death is a long-standing mystery in parasitology and establishing the mechanism would aid rational clinical implementation of apicoplast-targeted drugs. Parasites undergoing delayed death transmit defective apicoplasts to their daughter cells and cannot produce the sole, blood-stage essential metabolic product of the apicoplast: the isoprenoid precursor isopentenyl pyrophosphate. How the isoprenoid precursor depletion kills the parasite remains unknown. We investigated the requirements for the range of isoprenoids in the human malaria parasite Plasmodium falciparum and characterised the molecular and morphological phenotype of parasites experiencing delayed death. Metabolomic profiling reveals disruption of digestive vacuole function in the absence of apicoplast derived isoprenoids. Three-dimensional electron microscopy reveals digestive vacuole fragmentation and the accumulation of cytostomal invaginations, characteristics common in digestive vacuole disruption. We show that DV disruption results from a defect in the trafficking of vesicles to the digestive vacuole. The loss of prenylation of vesicular trafficking proteins abrogates their membrane attachment and function and prevents the parasite from feeding. Our data show that the proximate cause of delayed death is an interruption of protein prenylation and consequent cellular trafficking defects.
Teaching STEM for conceptual change: Lessons from large classes in biomedical science
Despite the best efforts of academics, some students leave the undergraduate science courses with uncorrected misconceptions. There are several possible explanations for the persistence of misconceptions through and beyond undergraduate education, one of which is due to large class sizes. It seems, however, that large class teaching will be a continued response to the expansion of higher education. Therefore, it is imperative for academics to introduce teaching methods that are suited to large class settings and that will arrest problems that lead to uncorrected misconceptions. Conceptual change theory is one of the prevailing theoretical approaches to transforming misconceptions into conventional scientific understanding by reorganising or modifying the ideas underlying the misconception. One of the best approaches academics can take to fostering conceptual change is to infuse active learning into their teaching practice. In this thesis, I propose an active learning intervention that potentially fosters conceptual change in a large class setting, as well as presenting the theoretical underpinnings of the development of this intervention. I started by enquiring into the current pedagogical practices of Australian biomedical science academics in order to understand how they cope with the mass education context, and whether there are specific obstacles to the introduction of active learning into their classrooms. Findings indicate that, despite identifying themselves as traditional teachers, the current pedagogical practices of Australian biomedical science academics exhibit potential in ‘teaching for conceptual change’ by being reflective teachers, by shifting to non-traditional teaching, and by collaborating with their colleagues to design the curriculum. Next, I probed the precursors for conceptual change learning experienced by interviewing students. By capitalising on the cognitive disequilibrium generated by misconceptions, I was able to further inform the development of the intervention. A contradictory understanding of current knowledge triggers cognitive disequilibrium because students are unable to bridge the gap between their prior knowledge and an incoming information. Findings show that the amount of prior knowledge, regardless of the quality, appeared to predict students’ degree of confidence in it. In turn, the degree of confidence in prior knowledge appeared to predict a student’s decision to change their mind in a collaborative setting; and the degree of confidence in prior knowledge also appeared to predict learning and retention of concepts. Finally, drawing from the notion that cognitive disequilibrium, if properly induced, can lead to conceptual change, I propose a misconception-driven intervention that potentially induces cognitive disequilibrium by using intentionally flawed scientific manuscripts. This active learning intervention showed comparable improvements with a didactic approach in terms of students’ conceptual understanding and student self-reported engagement, however, independent classroom observers observed that this intervention improved student engagement. A review of the extant literature on student engagement suggests that engagement is predominantly linked to improved learning outcomes. I would argue further that for the learning process to ultimately lead to conceptual change, the responsibility does not only depend on the improved pedagogical practices of academics, but also more importantly, on the students themselves. Our role as academics is to integrate active learning methods that promote self-regulation, which will enable students to reflect on their own understanding and become more metaconceptually aware for them to recognise a potential state of cognitive disequilibrium. To be able to do this, academics should attend education conferences or fora and professional development opportunities to help them improve their pedagogical practice.
Immune defense mechanisms against Legionella longbeachae
The pulmonary epithelial barrier is the first line of defense against pathogens invading the lungs. If those are able to overcome this first barrier, myeloid cells of the innate immune system are instrumental for the antimicrobial defense and can directly eliminate invading microorganisms. This work aimed to identify novel mechanisms by which pulmonary epithelial cells and myeloid cells eliminate invading bacteria from the lungs. For this, infections with Legionella longbeachae were used to investigate a severe and often fatal form of pneumonia in humans known as Legionnaires’ disease in a mouse model. Following infection, infiltration of immune cells was dominated by neutrophils and, to a lesser extent, by monocytes. In addition to this, a significantly higher fraction of neutrophils contained L. longbeachae bacteria compared with other myeloid immune cells. Within host cells, bacteria translocated effector proteins mostly into neutrophils, and were residing in a vacuole resembling the Legionella-containing vacuole, as known from infections with L. pneumophila. However, neutrophils played an important role in the in vivo clearance of L. longbeachae, as mice depleted of this cell type exhibited significantly higher bacterial burden in the lungs. Besides neutrophils, monocytes also contributed to the control of pulmonary L. longbeachae infections, while lymphoid immune cells had no effect on the clearance of the bacteria. Molecularly, it is well known that IL18 is important in anti-bacterial defense by inducing lymphocytes to release IFN gamma. However, IL18 receptor (IL18R) expression on lymphoid cells did apparently not promote L. longbeachae clearance. Instead, expression by pulmonary stromal cells was required and sufficient for elimination of the bacteria. Stromal expression of the IL18 receptor was almost confined to the ciliated epithelial cell compartment in the bronchioles. IL18R signaling in those cells did not promote mucus production but it rather enhanced the anti-bactericidal activity of neutrophils. Therefore, these results indicate a non-canonical role of IL18 in the defense against pulmonary L. longbeachae infection, linking non-immune pulmonary epithelial cells with inflammatory neutrophils.
Structure and conformational dynamics studies of α1A-adrenoceptor
G-Protein Coupled Receptors (GPCRs) are integral membrane proteins representing one of the most important class of drug targets. alpha1-adrenoceptors (alpha1-ARs) comprise three GPCR subtypes that stimulate smooth muscle contraction in response to binding adrenaline and noradrenaline. alpha1A-AR and alpha1B-AR are clinically targeted for treating hypertension and benign prostatic hyperplasia but are putative drug targets for neurodegenerative diseases. The subtype-selective antibodies and tool compounds are required to probe the role of these receptors in the brain and to validate them as drug targets for neurodegenerative diseases, where the structure studies of alpha1-ARs would assist. So far, no structures of alpha1-ARs have been published which may due to their low stabilities when purified. Thus, in this study, we selected an ultra-stable alpha1A-AR through directed evolution to pave way for the structure determination of alpha1A-AR. GPCRs typically bind to an extracellular ligand and transmit signals across the cell membrane via an allosteric network from the ligand-binding site to the G-protein binding site via a series of conserved microswitches. Crystal structures of GPCRs provide snapshots of inactive and active states, but poorly describe the conformational dynamics of the allosteric network that underlies GPCR activation. We analyse the structural correlation between ligand binding and the allosteric network of the alpha1A-AR. NMR of 13CH3-methionine labelled alpha1A-AR mutants, each exhibiting differing signalling capacities, revealed how different ligands modulate receptor conformational equilibria. 13CH3-methionine residues near the microswitches revealed distinct states that correlated with ligand efficacies, supporting a conformational selection mechanism. Restoration of functional microswitches revealed that while the conformational states of individual microswitches are loosely independent, for alpha1A-AR activation, complete flexibility of all microswitches is necessary for full receptor function. Our work deepens the understanding of the activation mechanism of alpha1A-AR and how it works in human body. This is important for the development of novel drugs to target alpha1A-AR and to understand its physiological functions.
Mechanism of activation of the relaxin family peptide receptors RXFP1 and RXFP2
The relaxin family peptide receptors RXFP1 and RXFP2 are the cognate receptors for the peptide hormones relaxin and INSL3 respectively, best known for their roles in reproductive biology. Being GPCRs, these receptors are members of the largest family of membrane-bound receptors known in the human genome, but they are unique within this family due to the existence of a single low-density lipoprotein type A (LDLa) module on the N-terminus of their large ectodomain. The LDLa module is imperative to normal receptor signalling and is hypothesised to be a tethered ligand, interacting with the receptor transmembrane domain (TMD) to bring about an active conformation. This module is connected to the leucine-rich repeats that make up the remainder of their extracellular domain by a stretch of amino acids 32 long in RXFP1 and 25 in RXFP2. These linking residues have been termed the linker, and a series of accidental and intentional discoveries led to the notion that the linker plays an important role within the activation mechanism of both RXFP1 and RXFP2 in response to their peptide ligands. The work presented within this thesis delves into the details of this activity, exploring the various regions of the linker, as well as the LDLa modules, with the use of mutagenesis and the construction of chimeras of the two receptors. The mutants and chimeras were systematically tested in stably or transiently transfected HEK293T cells in a series of ligand binding, activity (focussing on their ability to prompt an accumulation of intracellular cAMP) and cell surface/total expression assays. By comparing the behaviour of the mutant and chimeric receptors to that of their wild-type counterparts we have been able to paint a detailed picture of the binding and activation mechanisms of RXFP1 and RXFP2 in response to either active peptide ligand, adding our findings to a growing understanding of their activity quickly emerging from the lab and the field at large. Complementing the data generated from cell-based assays are nuclear magnetic resonance (NMR) studies performed concurrently using recombinantly expressed and purified RXFP1 and RXFP2 LDLa-linker proteins in titrations with relaxin as well as the receptor extracellular loops, as expressed on a soluble scaffold based on thermostabilized protein GB1. While the NMR experiments were carried out by a collaborating student, the RXFP2 LDLa-linker and soluble scaffold proteins were designed and characterized for use in NMR during this PhD project, and the details are outlined herein. In Chapter 2 the LDLa modules of the two receptors were swapped, such that RXFP1 contained the LDLa module from RXFP2 and vice versa. We found that while ligand-induced activity was weakened in the chimeric receptors, they were able to produce a robust signal, and for RXFP2 (but not RXFP1) the signal was slightly closer to wild-type levels upon subsequent swapping of the TMDs, such that they would match the non-native LDLa module . The result contradicted previous findings in which RXFP1 with the LDLa module from RXFP2 was shown to be incapable of signalling upon relaxin stimulation. We rationalized this discrepancy as being due to the differing design in the cloning of the chimeric constructs. While our versions swapped only the LDLa modules themselves, the previous versions had also swapped a large portion of the neighbouring linker residues. This alerted us to a possible function being carried out by the linker and guided our future work. We proceeded to mutate residues of the linker to alanine, carrying out an extensive scan of the RXFP1 linker that is presented in Chapter 3. We discovered that the initial residues formed something of a motif with the sequence GDNNGW, and when mutated – the residues Asp2, Gly5 and Trp6 in particular – the receptors lost their ability to stimulate cAMP production and bind relaxin effectively. This information along with NMR data led to the conclusion that this motif made up an activation region that was involved in the tethered ligand activation mechanism of RXFP1. Of note, we observed that the fold-difference in affinity for relaxin exhibited by the mutants of the activation domain was not commensurate with the enormous weakening in potency in relaxin signalling assays shown by the same mutants. This contrasted with mutants of the central part of the linker, in particular Phe54 and Tyr58, which when mutated individually or together displayed similar fold-differences from wild-type in relaxin potency and affinity. Coupled with compelling NMR data we concluded from this evidence that the central portion of the linker constituted a relaxin binding site that had hitherto never been described . We pursued a similar investigation focussing on the linker of RXFP2 in Chapter 4. The supposed activation region is largely conserved in the similar receptor, with the initial sequence of the linker GDTSGW. Indeed, mutation to alanine of these residues and the Phe found three residues along showed that both relaxin and INSL3 activation was dependent on equivalent residues to those identified in the RXFP1 mutagenesis campaign. Similarly, relaxin binding was severely compromised in the mutant receptors, while INSL3 binding was not. This result mirrored prior knowledge coming from mutagenesis and A-chain truncations of the relaxin and INSL3 peptides and highlighted a differing mode of action on RXFP2 in response to the two similar agonists. We postulated that the activation region of RXFP2 consisted of the linker residues from the GDxxGW motif found in both receptors, but they were assisted or accompanied by the actions of the N-terminal residues of the INSL3 A-chain . We further investigated the relaxin binding site from the RXFP1 linker by creating another two chimeras, in which the implicated residues were inserted into an equivalent position in the RXFP2 linker. The linker of RXFP2 is shorter than that of RXFP1 and hence a relaxin binding site had not been identified at a similar position, but by including the residues we supposed to be contributing to this binding site we were able to increase the potency and affinity for relaxin of the chimera to more closely resemble the behaviour of RXFP1 with its native ligand. In addition, the dissociation kinetics of the interaction, measured using a NanoBRET assay, resembled more closely the case of RXFP1 than RXFP2. This information helped us to confirm the existence of the relaxin binding site and highlight major differences between the two receptors with a focus on the linker . The work presented in this thesis gives a deep and detailed look at an integral part of the binding and activation mechanisms of RXFP1 and RXFP2 in response to the peptide hormones relaxin and INSL3, paving the way for their use in a therapeutic setting. The resolution of the role that the LDLa module plays has altered the prevailing view about receptor activation in a number of aspects. Firstly, the complex binding mode of both relaxin and INSL3 has been defined more thoroughly, and secondly, we now know to focus on the linker when defining TM interactions. It therefore highlights the utility of using a combination of approaches – cell-based assays partnered with mutagenesis and chimeras alongside protein expression and NMR – to reach valid conclusions about molecular systems.
Determination of ligand binding conformations at α1-adrenergic receptor subtypes based on NMR and MD studies
Adrenergic receptor (AR) subtypes (α1A, α1B, α1D, α2A, α2B, α2C, β1, β2, β3) are G-protein coupled receptors (GPCRs) activated by the same endogenous catecholamines, adrenaline and nor-adrenaline. The two subtypes α1A- and α1B-AR maintain a complex balance in modulating the functions of the sympathetic and central nervous systems, whereby chronic activity can be either detrimental or protective for both heart and brain function. Regulation is believed to be mediated through the distinct activation of individual α1-AR subtypes and thus, subtype selective activation or blocking may have major clinical implications. Despite having tremendous clinical importance, there are no approved α1A- or α1B-AR selective marketed drugs. Firstly, the conservation of the orthosteric binding site within a GPCR family makes it challenging to achieve receptor subtype selectivity for competitive compounds. In such a case, allosteric modulators, which interact with binding sites that are topographically distinct from the binding site of the endogenous ligand, offer an alternative. Secondly, lack of structural information of a majority of GPCR members makes it difficult to predict ligand binding. Intrinsic instability of these receptors make crystallisation challenging and as yet, no crystal structure has been reported for α1-AR subtypes while crystallising weakly binding ligands to GPCRs is difficult. The broad aim of the thesis was to gain molecular insight into the structures of weakly binding GPCR ligands by applying atomic resolution NMR methods. In this study, we have used variants of α1A- and α1B-AR which were thermostabilised using the directed evolution method, Cellular high throughput encapsulation, solubilisation and screening (CHESS). The long-term stability of these receptors in detergents has enabled us to investigate binding of a variety of ligands including native agonists, adrenaline and noradrenaline, an α1A-AR selective agonist A-61603, as well as allosteric modulators, benzodiazepines, by employing solution-based ligand-observed Nuclear Magnetic Resonance methods including STD-NMR (saturation transfer difference NMR), Water-LOGSY (water-ligand observed via gradient spectroscopy), Tr-NOESY (transferred nuclear overhauser effect spectroscopy) and INPHARMA (interligand noes for pharmacophore mapping) experiments. These are nuclear overhauser effect based methods, which rely on the transfer of magnetisation from the target protein or other molecules (such as bulk water and ligand) to ligands through dipole–dipole interactions. STD-NMR and Water-LOGSY experiments are most commonly used to detect weak ligand binding and in fragment screening projects, while Tr-NOESY and INPHARMA are used to detect and map ligand binding conformations. The INPHARMA experiment takes advantage of known or expected orientations of a bound ligand, for example adrenaline bound to its GPCRs, to determine the respective orientation of novel ligands for which there are no known structures. We obtained experimental NMR data for the ligand A-61603 (α1A-AR selective agonist) with respect to adrenaline for both α1A- and α1B-AR. These data were compared to the back-calculated spectra obtained from molecular dynamics simulations. The results helped mechanistically explain the selectivity of A-61603 towards α1A-AR. Overall, we have shown that this solution-based methodology provides valuable information on ligand-binding poses inside the highly conserved orthosteric binding site of ARs, dissecting out subtle structural variations across the subtypes and thereby may aid in future subtype selective drug development.
Determining the role of doublecortin X (DCX) in cytoskeleton organisation
X-linked Doublecortin (DCX) is a 40 kDa neuron-specific microtubule (MT)-associated protein. The initial identification of inheritable dcx gene mutations and their disruption of cortical layering and brain development in lissencephaly and subcortical band heterotopia have prompted subsequent evaluation of the functions of the DCX protein. Previous biochemical studies have revealed that DCX binds between four tubulin monomers within the polymerised MT lattice to stabilise the lateral and longitudinal contacts along the MT protofilaments. Although DCX has been proposed to regulate MT-related cellular events, many aspects of its mechanism of actions and its regulation of functions are yet to be determined. The DCX protein consists of two MT-interacting doublecortin (DC) domains, DC1 (the N-terminal DC domain) and DC2 (the C-terminal DC domain), linked in tandem via a flexible unstructured region (linker) and additionally flanked by a likely unstructured N-terminal and S/P-rich C-terminal sequences. Since many DCX pathogenic mutations have been predominantly mapped to within the DC domains to thus disrupt the interaction of DCX with MTs, studies of the regulation of the DCX-MT interaction have been undertaken to improve understanding of the functions of DCX. In addition, the DCX termini that flank the DC domains may act as regulators of DCX function, but whereas the S/P-rich DCX C-terminus is known for its phosphoregulatory role in DCX’s functions, a regulatory role of the DCX N-terminus region is largely unknown. The studies presented in this thesis address the critical roles of the DCX N- and C-termini in DCX function. Immunoprecipitation and live-imaging approaches using monkey fibroblastoma COS-1 and human neuroblastoma SH-SY5Y cells have been employed to identify the effects of the DCX N- and C-termini on the association of DCX with the cytoskeleton. The results were combined with live-imaging fluorescence recovery after photobleaching (FRAP) protocol findings to define the regulatory impact of DCX termini on dynamics of DCX in association with the cytoskeletal components, MTs and F-ACT. These studies have been the first to show the dynamic association of DCX with MTs in living cells. The role of the DCX C-terminus has been evaluated by examining the features of DCX lacking its C-terminus. Whereas full length, wildtype DCX shows rapid and complete exchange within the MT network, the exchange of the truncated DCX protein is slowed significantly. Moreover, dynamics of exchange of the C-terminal truncated DCX was unaltered in the presence of a MT-stabilising agent, taxol, or a hyperosmotic stress stimulus, sorbitol, both of which were shown to slow wildtype DCX exchanges rate within the MT network. Thus, DCX dynamically associates with MTs in living cells and its C-terminal region plays important roles in the association of DCX with MTs. To explore the regulatory role of DCX N-terminus, the influence of the only identified phosphorylation site within the DCX N-terminus, DCX S28, on association of DCX with MTs and F-ACT was assessed. Thus, both DCX S28A (phospho-resistant) and DCX S28E (phospho-mimetic) mutants were examined alongside wildtype DCX. For DCX S28E, decreased interaction with MTs but a shift to favour association with both F-ACT and the ACT-binding protein, spinophilin (Spn) was observed. FRAP studies showed that DCX S28E increased the dynamics of association with MTs. Compared to DCX S28E, the associations with MTs and F-ACT were reversed in the presence of DCX S28A. Therefore, these results highlight a new role for DCX S28 as a regulatory switch for cytoskeletal organisation and thus highlight a contribution by DCX-N in the phosphoregulation of DCX function. The impact of a pathogenic mutant within the DCX N-terminus, DCX E2K, on the cytoskeletal association was also studied. Unlike most DC domain pathogenic mutants of DCX, the DCX E2K mutant protein retained its ability to interact with MTs. However, MTs in association with DCX E2K showed a reduced sensitivity to nocodazole-induced depolymerisation as well as slower α-tubulin exchanges rate. Furthermore, the DCX E2K mutant showed increased association with the F-ACT. These results highlight the importance of the N-terminus of DCX in regulating the association with, and the coordination of, the MT and F-ACT networks. Taken together, the studies presented in this thesis have revealed several new features of the regulatory roles of the DCX termini in the association of the DCX protein with MTs and F-ACT. The findings should aid a better understanding of the DCX function in MTs and F-ACT organisation during MT-related cellular events including neuronal cell migration.
Detecting horizontal co-transfer of antimicrobial resistance genes in bacteria: a network approach
Antimicrobials have been widely using as a major resource to treat bacterial infections for almost a century. However, it is not unusual to see antimicrobial resistance emerges in a bacterial species due to natural selection under the usage of antimicrobials. Moreover, numerous studies show that bacteria can accumulate genes encoding resistance to different classes of antimicrobials and share them with other bacteria regardless of ancestry via a biological process called horizontal gene transfer, causing emergence and fast transmission of multidrug resistance. As such, antimicrobial resistance becomes an urgent and global threat to public health, pushing us backwards to the pre-antimicrobial era. In this thesis, I focus on horizontal co-transfer of resistance genes between bacteria of the same species, which is usually caused by co-localisation of resistance genes in mobile genetic elements, also known as physical linkage between these genes. This kind of linkage plays a pivotal role in the evolution of multidrug resistance, because the mobile elements can translocate, recombine and aggregate, rapidly rendering their host bacteria resistant to a wide spectrum of antimicrobials. By far there is nonetheless not an approach identifying horizontally co-transferred genes in a single bacterial species. Yet most authors of literature reported a few co-mobilised resistance genes each time following biological experiments, and some researchers only applied simple association analysis to representative bacterial isolates of distinct species so as to minimise the possibility that a specific combination of genes is inherited from their most-recent common ancestor. In contrast, intra-species association analysis is severely confounded by strong sample relatedness because of bacterial clonal reproduction. This obstacle leaves a gap between the known high frequency of intra-species horizontal gene transfer and our understandings of this process. This thesis presents a scalable computational approach that uses whole-genome sequencing data to identify co-transferred antimicrobial resistance genes in bacteria collected in a few decades from the same species. Moreover, it demonstrates applications of the approach to three clinically important pathogens and reports key players, patterns and dynamics underlying the horizontal co-transfer of resistance genes within each species. In the first chapter, I provide a background to antimicrobial resistance, horizontal gene transfer, whole-genome sequencing and contemporary bioinformatic techniques. I also summarise outcomes of horizontal gene co-transfer for characteristics that we can utilise for inference of physical linkage. Finally, I compare several statistical approaches determining pairwise association between presence-absence status of genes or alleles in bacteria to justify the necessity of controlling for sample relatedness in association analysis. For the second chapter, I derived a methodology inferring co-transferred genes by integrating gene detection, de novo genome assembly, core-genome and phylogenetic analysis, linear mixed models, hypothesis tests for effects of sample relatedness and evaluation of consistency in pairwise physical distances between resistance alleles in bacterial genomes. This methodology is designed to overcome limitations of existing methods summarised in the first chapter. Moreover, I show interpretations of expected outcomes and discuss constraints of this approach. The next three chapters present an implementation of my methodology and its applications on antimicrobial resistance genes in three clinically important species of Enterobacteriaceae. First, I conducted an empirical study following a simulation-and-validation strategy on finished-grade full genomes of six strains of Klebsiella pneumoniae to find out an optimal method that measures the pairwise physical distances between alleles in de novo genome assemblies. I found that for each assembly graph, the most accurate measurements are obtained via setting up constraints for both the number of nodes in the graph and the maximum of distance measurements. Second, I developed GeneMates, a computational and integrative software package that implements my methods proposed in the second chapter for the identification of physically linked resistance alleles or for analysing associations between a large number of resistance alleles when controlling for individual relatedness. In particular, GeneMates leverages network topology to identify potential physical linkage between the alleles. For validation, I applied this tool to whole-genome sequencing data of Escherichia coli and Salmonella Typhimurium, whose acquired resistance genes and relevant mobile genetic elements have been well characterised in publications. In result sections, I illustrate clusters of physically linked resistance alleles and discoveries of their vectors. For the last result chapter, I applied GeneMates to genomes of a large global collection of K.pneumoniae strains, which are adept to uptake DNA from various environments. Furthermore, I compared structure and contents of co-localised allele clusters across time and geography and discovered patterns underlying the evolution of multidrug resistance in this species. To conclude, I have developed and implemented a network approach that performs association tests on presence-absence of resistance alleles in a large collection of bacterial isolates of the same species and infers potential horizontally co-transferred alleles. I have validated this approach using known co-mobilisable resistance genes and the approach showed higher statistical power than existing methods. The GeneMates package will become a powerful tool contributing to routine surveillance of antimicrobial resistance and identifications of known and novel mobile genetic elements. In addition, applications of this package to other kinds of bacterial genes is also feasible and convenient.
Assembly of the Plasmodium falciparum virulence complex
After invading a human red blood cell (RBC), Plasmodium falciparum modifies the host RBC surface by exporting proteins that traffic to and interact with the RBC membrane skeleton. The physical properties of the RBC are altered and parasite derived structures called knobs arrive at the cell periphery. These knobs are comprised primarily of the knob-associated histidine rich protein (KAHRP), which acts as a scaffold for the presentation of the major virulence protein, P. falciparum erythrocyte membrane protein 1 (PfEMP1), at the membrane. Knobs are of particular interest as PfEMP1-mediated cytoadhesion is a key contributor to parasite virulence and to the complications that arise in cases of severe malaria. In this study I used a method for exposing the inner surface of the infected RBC membrane and visualised the physical organisation of the membrane proteins by scanning electron microscopy (SEM). The locations of particular proteins were also mapped using direct stochastic optical reconstruction microscopy (dSTORM). In a major advance, I combined these two imaging modalities in a correlative light and electron microscopy based approach (STORM-SEM) to investigate membrane remodelling and virulence complex assembly. I investigated how RBC membrane skeleton remodelling facilitates knob assembly upon the arrival of KAHRP at the RBC periphery and how PfEMP1 arrives at the membrane and assembles into knobs. I showed that KAHRP is delivered to the RBC membrane as modular units. As the asexual parasite matures, the KAHRP modules re-arranged into five-membered ring-shaped oligomers. Correlative imaging revealed that the KAHRP modules are located at the perimeter of the physical knob. Parasites expressing C-terminally truncated KAHRP proteins were generated to analyse the regions of KAHRP required for oligomeric ring formation and membrane skeleton binding. I showed that the KAHRP spectrin-binding domain is needed for knob assembly and that the KAHRP 3’ repeat region is needed for formation of the canonical knob structure. Electron tomography of the mutant parasites revealed that correct formation of the ring-shaped KAHRP oligomer is required to maintain the correct arrangement of a spiral structure found at knob complexes. Arrival of knobs at the RBC membrane has been linked to decreased RBC deformability. Interruption of remodelling of actin from the membrane skeleton using the mycotoxin cytochalasin D resulted in increased infected RBC deformability and a reduction in the number of knobs at the membrane. This indicates that remodelling of the host actin is required for the assembly of knobs. Recovery of cellular rigidity after removal of cytochalasin D highlighted the importance of membrane skeleton remodelling in the physical RBC changes that occur during infection. The mechanism by which the major virulence protein, PfEMP1, is delivered to the virulence complex is not well understood. Using STORM-SEM, I provided evidence that PfEMP1 is delivered to the RBC membrane at regions away from the knobs. Temporal analysis suggests that PfEMP1 associates with the periphery of the physical knob structure before lateral insertion into the knob complex. By combining STORM-SEM with cellular biology and biophysical measurements, this work has revealed new details of the remodelling events that underpin parasite virulence. Based on the data presented in this study I propose a new model for virulence complex assembly. In this model KAHRP is delivered to the RBC membrane skeleton as modular units. Actin mining and subsequent rearrangement of the spectrin meshwork facilitates self-association of KAHRP into a five-segment oligomer, enabling the assembly of an underlying spiral structure. Separately, PfEMP1 is inserted into the membrane, before lateral insertion into the virulence complex. It is hoped that the findings in this study will point to novel strategies to target parasite virulence machinery and to fight malaria infection.