Biochemistry and Pharmacology - Theses
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ItemProteomic analysis of mHttex1 expression in Huntington’s disease( 2021)Huntington’s disease (HD) is a fatal neurodegenerative disorder caused by CAG trinucleotide repeat expansion in exon 1 of the Huntingtin (Htt) gene. This sequence encodes an abnormally elongated polyglutamine (polyQ) tract within the Huntingtin (Htt) protein that is directly involved in aggregation and Htt-mediated cytotoxicity. The key pathological signature of HD is the aggregation of mutant Htt protein into punctate aggregates. However, the mechanism by which polyQ-expanded mutant Httex1 (mHttex1) causes toxicity remains elusive. Previous research has indicated that mHttex1 can exert toxicity to cell models through two distinct phases. The first is when the protein is soluble and the second is when it is aggregated into inclusion bodies, which are the major pathological signature of HD brain. I hypothesized that apoptosis is caused by an unresolved quality control mechanism that oversees mHttex1 at synthesis. The goal of this project was to develop and implement a novel proteomics strategy to specifically detect the proteins that engage with mutant Htt during protein synthesis. I compared pathogenic huntingtin (Q97) and non-pathogenic huntingtin (Q25) using a proteomics-based approach. Firstly, a self-cleaving NS3 viral protease system called TimeSTAMP was employed, which can efficiently cleave epitopes from newly synthesized proteins and be potently inhibited using a viral protease inhibitor. The goal was to inhibit the cleavage across different-time windows to “pulse” label newly synthesized Htt and at the end of the pulse steps, proteins were crossed-linked with disuccinimidyl sulfoxide (DSSO) to preserve transient interactions. We also wanted to examine the changes in the global proteome and phosphoproteome across these mutant form and wild-type counterpart. After transfection of Neuro2a cells with TimeSTAMP-Httex1 constructs of differing poly-Q length, cells were lysed using RIPA lysis buffer. Proteins were then treated with a label-free relative quantitative phosphoproteomics workflow: i.e., samples were denatured (e.g., in 8 M urea), reduced and alkylated, then subjected to tryptic digestion. Next, a phosphopeptide enrichment step was performed before samples analyzed using LC-MS/MS. However, there was no significant difference observed between pathogenic and non-pathogenic Huntingtin, indicating a lack of polyQ-length dependence. To further probe the toxicity of the pathogenic huntingtin, I investigated its protein-protein interactions using a different proteomics-based approach. After transfection of Neuro2a cells with the Q25-GFPEm or Q97-GFPEm constructs, proteins were cross-linked with disuccinimidyl sulfoxide (DSSO) and then protein interactors were pulled down using anti-GFP VHH coupled magnetic agarose beads. We also examined the changes in the global proteome and phosphoproteome across the mutant form and wild-type counterpart. I did not find any significant difference between pathogenic and non-pathogenic Huntingtin which further indicates that the result was not polyQ dependent. Determination of these mechanisms are anticipated to be important for the design of new therapeutic strategies that mitigate toxicity of soluble mHttex1.
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ItemDoxycycline has a dual mode of action against malaria parasites( 2021)Traditionally used as a broad-spectrum antibiotic, doxycycline is frequently used for malaria prophylaxis and treatment - the latter in combination with artemisinin derivatives. Its mechanism of action in Plasmodium spp. has not yet been fully elucidated, though there is substantial evidence that ribosomes in the apicoplast - a relict plastid - are the primary target, with doxycycline causing delayed death (a phenotype associated with inhibitors of apicoplast housekeeping). Inhibition of the apicoplast depletes isoprenoids, synthesised via a pathway housed in the organelle, perturbing the prenyl-dependent trafficking mechanism for haemoglobin uptake and trafficking. This same uptake of haemoglobin is required for activation of artemisinin derivatives. Here, we show that apicoplast-targeting antibiotics, such as doxycycline, reduce the abundance of the catalyst of artemisinin activation (free haem) in P. falciparum, likely through diminished haemoglobin digestion. We demonstrate antagonism between dihydroartemisinin and these antibiotics, likely because apicoplast inhibitors reduce artemisinin activation. Separately, we identify a secondary, more immediate target of doxycycline that exists at clinically relevant concentrations. We show that supplementation with the apicoplast-derived isoprenoid precursor, isopentenyl pyrophosphate, only rescues parasites from delayed death, demonstrating independence of the first cycle target from the relict plastid. Instead, we show that doxycycline depletes mitochondrial electron transport and selectively reduces the abundance of proteins encoded by the mitochondrion in the related apicomplexan parasite, Toxoplasma gondii, suggesting that inhibition of mitochondrial protein synthesis could underpin the immediate death phenotype caused by doxycycline. These data have potential clinical significance when considering the reliance on - and widespread use of - doxycycline and other apicoplast-targeting antibiotics in malaria endemic regions. They reinforce the strategic importance of rational choice of antimalarial combinations; and lay the groundwork for further exploration of the underlying mechanisms of drug resistance in Plasmodium parasites.
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ItemThe cell biology of the FcRn-albumin recycling system( 2021)Human serum albumin (HSA) is the most abundant protein in plasma and has an exceptionally long circulatory half-life of around three weeks in humans. The enhanced half-life properties of HSA result from the selective interaction with the neonatal Fc receptor (FcRn) in acidic endosomes, which protects endocytosed albumin from lysosomal degradation and mediates recycling back to the plasma membrane. Endothelial and innate immune cells are considered the most relevant cells for FcRn-mediated albumin homeostasis in vivo. However, little is known about FcRn-albumin cell biology in physiologically relevant primary cells and the spatiotemporal aspects of the FcRn-albumin interaction within intracellular endosomes. My studies have used cell biological and biophysical approaches to examine FcRn-albumin interactions and trafficking in primary macrophages and endothelial cells. Here, I used two independent biophysical approaches to visualise the intracellular receptor-ligand interactions within globular endosomes and tubular transport carriers of primary macrophages. Firstly, fluorescence lifetime imaging microscopy (FLIM) of Foerster resonance energy transfer (FRET) and secondly, raster image correlation spectroscopy (RICS) to monitor the diffusion kinetics of single fluorescent-labelled HSA molecules. Based on these analyses, I identified an interaction between FcRn and albumin within intracellular endosomes, and emerging tubules, in human FcRn-expressing macrophages. Furthermore, I detected a higher population of immobile, FcRn-bound wildtype HSA molecules within the lumen of endosomal structures compared to the non-FcRn binding rHSAH464Q mutant. My findings revealed the kinetics of FcRn-albumin binding within endosomal structures for recruitment into transport carriers for recycling. To investigate FcRn-albumin cell biology in physiologically relevant primary endothelial cells, I established cell lines of primary human vascular endothelial cells from the outgrowth in culture of blood endothelial precursors known as blood outgrowth endothelial cells (BOECs). My observations show that these endothelial cell lines internalised fluorescent-labelled HSA efficiently via fluid phase macropinocytosis. Intracellular HSA molecules co-localised with FcRn in endosomal structures potentially allowing the interaction of the receptor with its ligand. Wildtype HSA, but not the non-FcRn binding rHSAH464Q mutant, was sorted into FcRn-positive tubular transport carriers, that are likely to mediate recycling of endocytosed HSA back to the plasma membrane. These findings support the proposed contribution of vascular endothelial cells to albumin homeostasis in vivo. Understanding the underlying mechanisms of FcRn-albumin cell biology and the contribution of different cell types to albumin homeostasis is important for the design and generation of half-life extended albumin fusion proteins for the treatment of serum protein-related diseases such as hemophilia A (HemA). Despite exhibiting enhanced pharmacological properties, to date, very few albumin fusion protein therapeutics have been approved for the treatment of human patients. In particular for HemA, the treatment using recombinant coagulation factor VIII (FVIII) products is aggravated by the frequent development of inhibitory antibodies against FVIII in HemA patients which subsequently have to undergo highly expensive and burdensome immune tolerance induction protocols. In this study, I have established an imaging flow cytometry-based antigen uptake assay to investigate the internalisation of FVIII-Albumin fusion proteins by FVIII-specific B cells expanding the knowledge about how albumin fusion proteins might contribute to immune tolerance induction towards FVIII in vivo. Additionally, I established two in vitro protocols which, in combination, allow the generation of high numbers of FVIII-specific regulatory T cells. These antigen-specific Tregs have the potential to suppress immune responses against recombinant FVIII in vivo and represent an alternative approach to facilitate immune tolerance towards FVIII in HemA patients. In summary, this thesis has revealed the fundamental aspects of FcRn-albumin cell biology and trafficking in primary macrophages and endothelial cells, and potential strategies for immune tolerance induction using FVIII-Albumin fusion proteins in the context of HemA.
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ItemThe human TIM22 complex: functions, substrates, and connections to disease( 2021)Mitochondria are hubs of metabolic activity in cells. In addition to supporting ATP production through nutrient catabolism, mitochondria also house many crucial biosynthetic pathways, including iron-sulphur cluster synthesis and heme synthesis. The crucial role of mitochondria in cellular function means that their dysfunction is associated with a variety of human pathologies, including cancer, neurodegenerative disease, and mitochondrial disease. The diversity of mitochondrial functions necessitates a large and versatile proteome, which in human cells contains over 1000 proteins. Targeting and import of mitochondrial proteins to the correct mitochondrial subcompartment is mediated by multi-subunit complexes called translocases. Insertion of proteins with multiple transmembrane domains into the inner mitochondrial membrane is mediated by a translocase called the Translocase of the Inner Membrane 22 (TIM22) complex. The human TIM22 complex contains two subunits, AGK and Tim29, which are not present in the more extensively characterised Saccharomyces cerevisiae TIM22 complex. AGK had been previously described as a lipid kinase, although its kinase activity is dispensable for its function at the TIM22 complex. Mutations in the AGK gene cause a mitochondrial disease called Sengers syndrome, characterised by congenital cataracts, hypertrophic cardiomyopathy, lactic acidosis, and exercise intolerance. Despite recent advances in understanding of AGK function, the extent to which the functions of AGK in lipid and protein biogenesis contribute to the pathogenesis of Sengers syndrome were unclear, and the impact of AGK mutations on protein function had not been assessed. We set out to define the impact of AGK and TIM22 complex dysfunction on mitochondrial biology to provide much needed information on how perturbations at the TIM22 complex cause mitochondrial disease. Using unbiased proteomics approaches we characterised multiple Sengers syndrome patient fibroblast and disease-model cell lines, identifying mitochondrial one-carbon metabolism as a dysregulated pathway and potential therapeutic target in Sengers syndrome. One-carbon metabolism supports redox homeostasis and translation within the mitochondria, and its dysregulation might contribute to the pathology of Sengers syndrome. This analysis also identified Sideroflexins as novel substrates of the TIM22 complex. Sideroflexins are serine transporters required for one-carbon metabolism, providing a direct link between AGK/TIM22 complex dysfunction and the observed one-carbon metabolism defect. We also used mitochondrial proteomics and biochemistry to analyse several AGK mutations, revealing that AGK mutations vary in their functionality, and that partial functionality of certain mutations might underpin phenotypic variability observed for Sengers syndrome. Our focus on sideroflexins as TIM22 complex substrates drew our attention to SFXN4, a poorly characterised and divergent sideroflexin that cannot transport serine and causes a mitochondrial disease when mutated. We demonstrated that deletion of SFXN4 results in a severe isolated complex I defect, and that SFXN4 interacts with extensively with complex I assembly factors, establishing it as a bona-fide complex I assembly factor. Overall, this project demonstrates the power of unbiased approaches for defining protein function and identifying targetable pathways in mitochondrial disease. This study highlights the importance of fundamental biology for generating foundational knowledge that forms the basis of our understanding of human disease.
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ItemRegulation of dendritic cell function and immunity via O-GlcNAc glycosylation( 2021)Dendritic cells (DCs) have become steadily recognized as attractive drug targets due to their unique position in the immune system as initiators of primary immune responses. Their function and immunological properties are regulated by a myriad of proteins, which themselves are regulated by post-translational modifications. Among these, O-GlcNAcylation of nucleocytosolic proteins through the actions of the O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) has been found to regulate the activity of cells of the adaptive immune system. In T cells O-GlcNAcylation of transcription factors has been shown to be essential for their activation, while a lack of OGT in B cells enhances the apoptosis of mature B cells and causes an impaired B cell activation response as well. Any role of O-GlcNAcylation has not been explored in DCs yet. Although O-GlcNAcylation is highly conserved among all metazoans that have been studied so far, little is known about the cellular and physiological role of this PTM and the regulatory mechanisms behind it are only poorly understood. This project investigates, how the O-GlcNAc cycling enzymes OGT and OGA are regulated and affect the function of dendritic cells. The first aim explores the role of O-GlcNAcylation in immunological activities. I have observed that murine tumour derived DCs (Mutu DCs) deficient in OGT or OGA display an altered immunological phenotype, with enhanced activation as determined by the maturation markers CD40, CD86 and MHC II as well as increased secretion of pro-inflammatory cytokines in a setting mimicking infection. In the second and third aims, I seek to identify mechanisms that underpin the regulation of OGT and OGA activity. We hypothesised that several proteins act as co-factors involved in the regulation of both enzymes. Furthermore, we proposed that various other proteins interact with OGT and OGA according to cellular and environmental cues to potentially direct them to their target proteins. Immunoprecipitation of OGT and OGA and analysis of co-immunoprecipitated proteins by tandem liquid chromatography mass spectrometry led to the identification of the OGT and OGA interactomes in DCs. These included both known and novel interactors. The association of OGT with the potential co-factor Ras-related protein Rab-8A (Rab8a) was validated, and subsequent analysis revealed that Rab8a is a potential regulator of OGT. While Rab8a knockout (KO) Mutu DCs displayed unaltered OGT expression, total O-GlcNAc levels were reduced and a change in the substrate selectivity of OGT was observed. Interaction between OGA and the Cyclase Associated Actin Cytoskeleton Regulatory Protein 1 (Cap1) was also further characterized following co-immunoprecipitation experiments. Although validation of the association with independent approaches could not be confirmed, reduced expression of Cap1 was detected in OGA KO Mutu DCs. Lastly, the crosstalk between phosphorylation and O-GlcNAcylation was studied. Analysing the phosphoproteome in wildtype (WT) and OGA KO Mutu DCs revealed a relative abundance difference of 69 phosphopeptides in OGA KO Mutu DCs in comparison to WT Mutu DCs suggesting that the activity of OGA affects the overall phosphorylation status of the cell hinting towards a regulatory crosstalk between phosphorylation and O-GlcNAcylation. In summary, the presented data suggests that DC need to finely regulate O-GlcNAcylation to fulfil their immunological functions and that the activity of OGT and OGA is regulated through the association with other proteins. These findings will allow the exploration of the role of these interactors to further understand if these regulate mechanisms that can favour either an immunogenic or tolerogenic phenotype of DCs.
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ItemStructure and dynamic studies of AAA+ ATPase p97 molecular machine( 2021)The superfamily of AAA+ ATPases (ATPases Associated with diverse cellular Activities) is a conserved and vital family of proteins in all living organisms. AAA+ ATPases hydrolyse ATP and convert the chemical energy to mechanical force. These mechanochemical enzymes perform a variety of cellular functions. Members of this superfamily most commonly assemble as homohexamers, the functional form of these macromolecules. A prominent member of this family is the protein named p97, or VCP. In all eukaryotic cells, p97 is one of the most abundant cytosolic proteins where it plays a central role in organelle and protein homeostasis in ubiquitin-dependent and independent pathways. It acts as an unfoldase to unfold its substrate. To play a role as a multifunctional protein in the cell, p97 interacts with a vast number of cofactors. The monomer of the homohexameric AAA ATPase p97 contains the cofactor or substrate-binding domain (N) and two ATPase domains (D1 and D2), and a disordered C terminal region. A single amino acid substitution in this protein causes neurodegenerative diseases, and an increased expression level of p97 has been identified in several cancers. It also plays a role in viral infections. Therefore, it is an important therapeutic protein. The structure of p97 has been widely investigated, and the high-resolution X-ray and cryo-EM structural information of full-length p97 became available in recent decades. However, structural knowledge and detailed insight into the disease-associated mutant, and the interaction of p97 with cofactors have limited our understanding of how disease-associated mutation and cofactors regulate or modify the functions of p97. The work presented here attempts to characterise how one of the most clinically relevant mutations of p97 (R155P) and one of the best-characterized cofactors (p47) modify the dynamic properties of p97. My approach has been to combine a series of structural and biochemical techniques such as single-particle cryo-EM, HDX-MS, X-link MS, and 19F NMR. In chapter 2, I describe how I have determined the high-resolution cryo-EM structures of the R155P and wild-type p97 in interaction with ATPgS. In chapter 3, using a combination of the structural and dynamical approaches, I show that the mutant stabilizes the up conformation of the N domain and affects the intra and inter subunit communication of the p97. During these studies, I have captured the wild-type p97 protein in a novel conformation and identified that the conformation of the N domain is not solely determined by the nucleotide occupancy of the D1 and D2 domains. In chapter 4, I present evidence I collected using SEC-MALS, AUC, and SPR that support the fact that the cofactor p47 is a monomer in solution. This analysis addresses controversy in the field. Using HDX-MS and X-link MS, I have shown changes in the conformations and interacting domains of both p97 and p47 upon complex formation with and without adding ATPgS. In chapter 5, I present preliminary studies on how 19F NMR would be an appropriate approach to study the dynamic flexibility of the p97 N domain. Together, these studies provide additional knowledge to the molecular mechanisms that regulate p97 function, our work will lead to progress in using hybrid structural biology techniques to understand the mechanism of macromolecules’ function.
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ItemMeasuring intolerance to missense variation within the human genome and proteome( 2021)This thesis summarises an experimental investigation of the measurement and application of intolerance to missense variation in the human genome, and its use in predicting the functional consequences of variants, as well as its ability to identify novel functionally relevant protein features. Using gnomAD, currently the largest publicly available dataset of sequenced human exomes, as well as UK Biobank and DiscovEHR population variation databases, I have systematically measured the proportion of missense variation across over 18,000 human genes and 80,000 gene transcripts over a sliding window of 31 codons, named the Missense Tolerance Ratio (MTR), and observed that known pathogenic variants in epilepsy patients preferentially exist in regions estimated as intolerant. We further validated the MTR using the set of known pathogenic variants in ClinVar and observed a significant difference in the MTR distribution between these and novel control missense variant datasets. Intolerant regions within a gene’s sequence have also been observed to cluster within the protein tertiary structure. We anticipate that the MTR therefore has extraordinary potential in identifying important functional domains within protein structures. Current methods of estimating the functional importance of regions within structures largely rely on conservation, however this is heavily dependent on the depth and appropriateness of the alignment where functionality is often not fully preserved between species. Missense intolerance within tertiary structures was measured using the MTR3D and shown to provide complementary information to the MTR. By combining the MTR and MTR3D with additional structural properties such as residue depth, we also developed the MTRX, a combined measure of intolerance that incorporates the predictions from the different scores. This was shown to have high predictive power towards known pathogenic variants. To assist in the prediction of variant consequences and inform on research in drug design, protein biochemistry and gene analyses, we are providing these intolerance estimates in their sequence-based and structure-based formulations to be freely available as user-friendly web-servers.
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ItemMicroglia and type-I interferons: emerging actors in Alzheimer’s disease( 2021)Alzheimer’s disease (AD) remains the most common cause of dementia worldwide. Post-mortem brains of AD individuals reveal classical pathological hallmarks of amyloid plaques and hyper-phosphorylated tau throughout the regions of the cortex and hippocampus. However, there have been few successes in therapeutics targeting these pathologies to treat AD, therefore suggesting new approaches are required. Recent evidence suggests that neuroinflammation, once thought inconsequential, contributes to AD progression. AD brains display enhanced gliosis surrounding plaques and elevated levels of pro-inflammatory cytokines. The characterisation of central nervous system (CNS) cell types and specific mediators of this neuroinflammatory response is critical in the identification of much- needed therapeutic targets to limit the damage in the AD brain. Our laboratory has previously identified that the type-I interferons (IFNs) are critical mediators of neuroinflammation contributing to the cognitive decline in a murine AD model. This thesis aimed to further characterise the specific effects of the type-I IFNs on microglia, a resident CNS immune cell. This thesis posits that type-I IFN mediated neuroinflammation modulates microglial phenotype contributing to the progression of AD pathology. This thesis aimed to investigate how type-I IFNs alter microglial phenotype and function in response to AB1-42, the key component of amyloid plaques, using both in vitro and in vivo murine models. From this study it was confirmed that microglia mount both pro-inflammatory and type-I IFN responses to AB1-42, of which both are ablated in Ifnar1-/-microglia, which lack the type-I IFN receptor. This finding was then extended to investigate if this phenotypic change manifested in altered functional changes in the ability of microglia to phagocytose or take up and internalise particles. Both IFNa and IFNb decreased the ability of microglia in vitro to phagocytose both bio-particles and fluorescently tagged AB1-42. This was investigated further in vivo, in mice that had received intra-hippocampal injections of AB1-42. No changes were observed in measures of AB uptake at 1-, 2- or 4-week post injection between wildtype and Ifnar1-/- mice. Mice with reduced type-I IFN signalling did, however, display decreased expression levels of Il6 and an altered astrocytic response within the hippocampus, suggestive of a lessened inflammatory response to AB1-42. Type-I IFNs are known to be elevated in aging, the largest risk factor for AD. This thesis took an in silico approach to compare type-I IFN expression between “normal” ageing and AD to gain a greater understanding of its role in driving the pathological changes in the AD brain. Using publicly available RNASeq datasets, this confirmed significant overlaps between type-I IFN related genes upregulated throughout both ageing and AD. This was most evident when examining a dataset containing purified cell types where there is a distinct lack of differences between controls and AD individuals. One of these genes, ISG20 was validated in a post-mortem human AD cohort and found to be significantly upregulated by QPCR and western blot analyses, supporting future investigations into its role in the disease pathogenesis. This thesis has built on previous findings from both our lab and others on the diverse roles of the type-I IFNs within the CNS and in AD. Specifically, it has identified and characterised a novel role for the type-I IFNs in modulating phagocytosis, a cellular process important in not only AD but several other neurodegenerative disorders. This thesis took a bioinformatic approach to identify a novel gene involved in AD, highlighting the increasing power of these tools and data when combined with traditional wet lab approaches to identify potential new therapeutic targets. Ultimately, this thesis forms a solid foundation to build upon and aid in the development of novel and much needed therapeutic approaches to slow AD progression.
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ItemStructural and functional characterization of TACAN, a novel protein involved in sensing pain( 2021)Ion-transport proteins, including ion channels, transporters, and pumps, play an essential role in many crucial processes including signal transduction and maintaining cell equilibrium in all living organisms. Signal transduction is defined as the process in which chemical or physical signals generate specific series of events in the cell to initiate appropriate responses. Determining the structure of ion-transport proteins and proteins that regulate their function is necessary to understand the underlying mechanism and regulation of ion transportation across membrane bilayers. Due to their immense importance in cell function and their accessibility on the cell surface, ion channels are extremely important drug targets. Moreover, malfunction of ion channels has been linked to a wide range of diseases affecting heart, brain, nervous system. Examples of such diseases are periodic paralysis, mixed arrhythmias associated with dilated cardiomyopathy, peripheral nerve hyperexcitability and chronic pain. The ion channels involved in sensing mechanical pain, however, are poorly characterised. TACAN, also known as TMEM120A, has been proposed to be an ion channel that contributes to pain sensation in nociceptors. TACAN has been shown to be involved in detection of painful mechanical stimuli in vivo and its deletion impairs the detection of painful mechanical stimuli in mice. When TACAN is expressed in cells in vitro, an increase of mechanically evoked currents has been reported. Electrophysiology studies of TACAN in planar lipid bilayer confirmed the pore-forming ability and functionality of TACAN. At the start of this study, there were no structural information of TACAN and the mechanism of function of this ion channel was unknown. Based on secondary structure predictions, TACAN is expected to comprise 5 or 6 transmembrane helices, a 138-residue N-terminal domain and a 14-residue C-terminal domain that includes a cluster of nine basic residues. In this project, TACAN has been purified from bacteria in detergent (DDM) and incorporated in three different membrane mimetic systems including Peptidisc, Saposin lipid nanoparticle (SLnP) and nanodisc. TACAN-SLnP and TACAN-nanodisc were more promising systems for visualising TACAN in cryo grid preparation. A cryo EM dataset has been collected from TACAN purified in SLnP system. Due to the unexpected publication of the structure of TACAN in June 2021, the nanodisc data collection was stopped. We focussed on determining proteins interacting with TACAN in HEK-293S GnTI- cells using pull-down assays and LC-MS/MS. To shed light on the potential importance of lipids for TACAN function, lipids that interact with TACAN were also determined using a lipidomics approach. This study provided foundational insights into the structure and function of TACAN. Our results show that TACAN forms a dimer in membrane bilayer. TACAN specifically interacts with poly-unsaturated glycerophospholipids, such as phosphatidyl inositol (PI), phosphatidyl choline (PC), phosphatidyl ethanolamine (PE) and ether phosphatidyl ethanolamine (Ether PE). Co-IP of TACAN, revealed that TACAN interacts with proteins involved in lipid metabolism, such as Ethanolamine phosphotransferase 1 (EPT1) and Steoryl-CoA Desaturase (SCD). Our results are consistent with a role of TACAN in sensing mechanical pain by regulating the membrane structure and altering the mechanical force afflicted on other mechanosensitive ion channels.
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ItemStructural characterization and mechanism of binding of pregnancy hormone H2 relaxin to the Glucocorticoid Receptor( 2021)H2 relaxin was discovered as a pregnancy hormone in 1926. Over the next decades, subsequent studies aimed at understanding the functional properties of this hormone unravelled a plethora of diverse physiological functions like vasodilatory actions, collagen turnover, anti-inflammatory actions, renal functions amongst others. These physiological actions slowly garnered pharmacological interest and catapulted relaxin as a new-age therapeutic for the treatment of diseases such as scleroderma and acute heart failure. Relaxin, like insulin, is composed of two chains connected by three disulfide bonds. Chemical and recombinant methods of relaxin synthesis have been widely patented and used by research groups to obtain pure relaxin for studies. While chemical synthesis method is not cost effective for large scale production, recombinant production is often limited by poor yield in a regular laboratory setting where expression is carried out in shaker flasks. Further, there is a need to produce relaxin that does not self-associate into dimers as the dimerization interface effectively obstructs the active site for binding. Previous studies have demonstrated that relaxin signals by binding to and activating two receptors belonging to different families: the G-protein coupled receptor (GPCR) RXFP1 and the nuclear receptor cum transcription factor Glucocorticoid receptor (GR). While relaxin binding to RXFP1 has been heavily studied and most characterized, studies focused on relaxin binding to GR has been neglected. It is partly due to the inability to recombinantly express and purify glucocorticoid receptor in an active soluble form stable enough for structural and functional studies. This has limited our understanding of relaxin mediated GR activation pathway despite its crucial role in anti-inflammatory actions, autoregulation of relaxin gene expression and protective actions in hepatic and pancreatic cells from ischemia damage. Since most pharmacological effects of relaxin cannot be explained by RXFP1/relaxin interaction alone, GR/relaxin pathway becomes an extremely important are of study to fully understand the pharmacological effects of relaxin. The work presented in this thesis attempts to resolve the issues discussed above. First, a method was designed, developed and optimized for production of high yields of fully active mature monomeric relaxin in bacterial expression system. The method does not require the use of a fermenter and can be utilized in any laboratory setting. The three different relaxin analogues (relaxin H2A(Q1G), mini relaxin and relaxin K2R), were regularly purified in high yields from E. coli cells. This method has been demonstrated as suitable for production of isotopically labelled proteins for nuclear magnetic resonance (NMR) studies through successful purification of 15N isotope labelled relaxin analogues. In Chapter 3, a simple strategy was developed to recombinantly express and purify the ligand binding domain of human glucocorticoid receptor (GR-LBD) in E. coli expression system to produce high quantity of soluble protein stable for biophysical characterization. The recombinantly produced GR-LBD (called GR-LBDF602S/C638G in this thesis) shows lesser aggregation than previously reported and has been shown to be fully functional. Owing to the successful purification of stable and functional GR-LBD, the next step was to determine the binding mode of relaxin and a possible mechanism of activation of GR by relaxin, described in Chapter 4. A combination of biophysical and structural biology techniques such as microscale thermophoresis (MST), differential scanning fluorimetry (DSF), hydrogen deuterium exchange mass spectrometry (HDX-MS) and NMR were used to identify relaxin binding site of glucocorticoid receptor, which was identified to be the steroid binding pocket of GR-LBD. The effects of relaxin binding on GR-LBD structure were then evaluated to determine if relaxin binding activates the receptor like an agonist or suppresses the transcriptional activity by behaving like an antagonist. GR-LBD has an activation function 2 (AF-2) site which essentially recruits a cofactor on ligand binding, the cofactor (can be coactivator or corepressor) being specific to the type of ligand bound and the cellular environment. Binding of both coactivator and corepressor peptide motifs to GR-LBD/ H2 relaxin complex were tested and it was discovered that relaxin can bind to both cofactors to bring about distinct conformational changes to the receptor. Therefore, it is hypothesized that relaxin behaves like a classical GR agonist but may also possess partial antagonist activity. Studies presented here provide new evidence to confirm that relaxin directly binds to the human glucocorticoid receptor ligand binding domain and provides new insights into the complex activation mechanism of GR by relaxin hormone. This study is crucial for understanding the anti-inflammatory effects of relaxin and can be used to translate relaxin into a novel anti-inflammatory drug in future.