School of Molecular & Cell Biology (ETDs)
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Item Biophysical evaluation of the kinetics, thermodynamics, and structure-stability relationship of Wuchereria bancrofti glutathione transferase in comparison with human µ and π glutathione transferases(University of the Witwatersrand, Johannesburg, 2024-06) Oyiogu, Blessing Oluebube; Achilonu, Ikechukwu AnthonyLymphatic filariasis is an endemic disease caused mainly by the Wuchereria bancrofti parasite and has been classified as a major neglected tropical disease. The emergence of drug-resistant strains of W. bancrofti and the limited efficacy of the available drugs on adult worms threatens the eradication of the disease. W. bancrofti glutathione S-transferase (WbGST) is a homodimeric enzyme central to detoxifying electrophilic compounds in the parasite due to its lack of cytochrome P-450. Therefore, WbGST is a potential therapeutic target for lymphatic filariasis. Bromosulphophthalein (BSP) and epigallocatechin gallate (EGCG) were previously shown to inhibit glutathione S-transferase activity. In this study, the interaction of WbGST with BSP and EGCG in comparison with human glutathione S-transferase P1-1 (hGSTP1-1) and human glutathione S-transferase M1-1 (hGSTM1-1) isoforms was investigated. Soluble WbGST, hGSTP1-1 and hGSTM1-1 were recombinantly produced and purified successfully to homogeneity. Glutathione and 1-chloro-2,4-dinitrobenzene conjugation assay was employed to analyse the enzyme activity, kinetics and inhibitory potency of the compounds. Spectroscopic studies were employed to investigate the functional and structural impact of ligand binding to the enzymes. Both thermal and chemical stability studies were performed, and binding energetics were analysed using isothermal titration calorimetry. The activity of WbGST was predominantly inhibited, with IC50 values of 5 μM for BSP and 12 μM for EGCG. The EGCG displayed uncompetitive and mixed modes of inhibition towards WbGST with respect to glutathione and hydrophobic binding sites, respectively. Whereas BSP showed a mixed type of inhibition for both active sites of WbGST. Ligands reduced the turnover rates (kcat) and the catalytic efficiencies (kcat/KM) of the enzymes. Upon ligand binding, 8-anilino-1-napthalene sulphonate was displaced from WbGST and hGSTM1-1 by 67%(BSP), 24%(EGCG) and 72%(BSP), 5%(EGCG), respectively; suggesting that the ligands bind to the 8-anilino-1-napthalene sulphonate binding site. Stability studies indicate that WbGST is the least stable of the three enzymes and that glutathione increases its stability. Isothermal titration calorimetry showed that BSP binds to multiple sites in WbGST with binding at site-1 (S1) and site-2 (S2), which are entropically and enthalpically driven, respectively. S1 showed a higher affinity for BSP than S2. EGCG binding to WbGST was entropically driven. BSP had a higher affinity for the enzymes than EGCG. All the results indicated that the ligands significantly impact WbGST more than the human GSTs. Further investigations, such as crystallography and molecular dynamics simulations, will shed more light on the ligan-protein interactions on a molecular level. Overall, this study suggests that BSP and EGCG are efficient inhibitors of WbGST that probably bind to both L and H-sites of WbGST, altering catalytic activity of the enzyme. The unique properties of the L-site are particularly suitable for rational drug design. Therefore, both ligands can be repurposed as new-generation therapeutics against filariasis.Item Identifying Markers of Differentiation in Monocyte-Derived-Macrophages(University of the Witwatersrand, Johannesburg, 2024-08) Gibson, Matthew Leo; Cronjé, Marianne; Gentle, NikkiThe importance of monocytes and monocyte-derived macrophages (MDMs) in both adaptive and innate immunity makes their study a topic of interest. Monocytes differentiate into macrophages through transcriptomic alterations, resulting in extensive changes in gene expression. Macrophage colony stimulating factor (M-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) are the two primary cytokines that stimulate this differentiation, and are known to cause partial polarisation towards the M2 and M1 macrophage subtypes, respectively. However, the degree to which this polarisation takes place is not well-characterised. Therefore, this study aimed to use a computational approach to identify the differences and similarities in gene expression changes in macrophages induced with M-CSF and GM-CSF. RNA sequencing data for three human donors was obtained through EBI and used to quantify gene expression changes associated with M-CSF or GM-CSF treatment. Differential gene expression analysis was performed to identify the genes that were differentially expressed as a result of either treatment relative to the untreated monocytes. Over-representation analysis was used to determine the biological processes in which the differentially expressed genes (DEGs) were involved. Finally, transcription factors were identified within the lists of DEGs, as well as the genes encoding their known protein-protein interacting partners. Treatment with M-CSF and GM-CSF induced 4 072 and 4 399 DEGs, respectively, 2 734 of which were common. An examination of these DEGs revealed that the resultant macrophages lacked changes in expression of genes commonly associated with the M1 and M2 polarisation states. An investigation of the DEGs involved in myeloid cell differentiation and the regulation of inflammatory response revealed CCR2, IGF1 and INHBA to be inversely regulated by the two treatments. Furthermore, nine uniquely differentially expressed transcription factors involved in these biological processes were identified, each of which may be contributing to the lack of complete polarisation following differentiation. These results revealed that M-CSF and GM-CSF-induced macrophages, in the absence of activation, experience highly similar gene expression changes and lack changes in the expression of key polarisation marker genes.Item Exploring temporal changes in the malting barley seed microbiome with meta-omics to understand nitrogen content effects(University of the Witwatersrand, Johannesburg, 2024-10) Tshisekedi, Kalonji Abondance; De Maayer, Pieter; Botes, AngelaBarley (Hordeum vulgare L.) is a critical cereal crop, particularly in beer production, where it plays a significant role in the economy, especially in South Africa. Despite its importance, the barley seed microbiome, which affects seed storage and quality, is not well understood. This research addresses two key questions: (1) how microbial composition and function evolve during storage and (2) how the inherent nitrogen content of the grain affects these dynamics. Using metagenomic and metaproteomic approaches, eight barley samples from the Kadie cultivar, stored for various durations (harvest, three, six, and nine months) with high and low nitrogen content, were analysed. Metagenomic sequencing revealed a predominance of bacterial sequences and minimal fungal presence, with storage time having a greater impact on microbial diversity than nitrogen content. However, specific bacterial genera such as Erwinia, Pantoea, Pseudomonas, and Stenotrophomonas showed nitrogen-dependent prevalence. Metagenome-assembled genomes (MAGs) were reconstructed, representing 26 bacterial genera, with minimal shared orthologues, highlighting taxonomic diversity. Functional analysis identified key metabolic pathways and carbohydrate-active enzymes (CAZymes) essential for microbial adaptation during storage. Metaproteomic analysis further showed the active expression of proteins related to nutrient transport and stress response, indicating functional changes over storage time. Overall, this research enhances the understanding of the barley seed microbiome, providing valuable insights into storage practices that could improve brewing quality and agricultural sustainability.Item Unveiling the biochemical pathway between Type 2 Diabetes Mellitus and early Alzheimer’s disease(University of the Witwatersrand, Johannesburg, 2024-08) Tooray, Shweta; van der Merwe, EloiseResearch related to Alzheimer's Disease (AD) remains a focal point in neurodegeneration studies. This is due to the severity of AD and the clear necessity for non-palliative treatment approaches, as underscored by the high prevalence of the disease. The combined formation of extracellular senile plaques and neurofibrillary tangles (NFTs) plays a crucial role in the development of the cognitive and behavioural symptoms observed in individuals with AD. Despite extensive research efforts, discovering a definitive cure for the disease remains a challenge. Therefore, it is imperative to explore new perspectives and identify the upstream molecular mechanisms that contribute to the onset of the disease. Metabolic disorders are widely recognized as a significant risk factor for AD. Specifically, the metabolic syndrome, Type 2 Diabetes Mellitus (T2DM), is connected to neurodegeneration by promoting the accumulation of neurotoxins, inducing neuronal stress, affecting synaptic communication, and leading to brain atrophy. Individuals with T2DM have an increased risk of developing dementia, with hyperglycaemia exacerbating the impact of AD by causing mitochondrial dysfunction and oxidative stress through reactive oxygen species (ROS) formation, which are also present in AD. Additionally, patients with T2DM exhibit shorter telomeres linked to cell death, which is an associated risk factor for developing AD. These key pathways involved in connecting T2DM and AD were explored in the current study to enhance the understanding of the early events that precede AD. Glucose uptake was measured and observed to decrease over time as a potentially protective response of the cell. Subsequently, mitochondrial activity, assessed using the Alamar blue assay, was found to be heightened as an initial protective mechanism of Aβ42. This was later overwhelmed by the elevated ROS detected through a Total ROS assay kit, induced by the hyperglycaemic state of T2DM. In turn causing the amount of Aβ42 to become toxic and leading to a decline in mitochondrial DNA (mtDNA) over time as measured through qPCR. Additionally, the increases in ROS induced by hyperglycaemia resulted in oxidative damage to telomeres. Simultaneously, Aβ42 physically hinders telomere-telomerase binding, leading to reduced telomerase activity and consequently, shorter telomeres. Furthermore, this study reveals, for the first time, that the novel glucose-lowering drug (GLD) caused an increase in Aβ42 production in the T2DM cell model, whilst effectively decreasing ROS production over a 24-hour period compared to the untreated cell model. The rise in Aβ42 levels caused by GLD could potentially be working to prevent the increase in hyperglycaemia-induced ROS through its metal chelating antioxidant properties by scavenging ROS, in the presence of oxidative stress associated with T2DM. These findings are indicative of an appealing function of GLD by reducing ROS and thereby impeding the progression towards AD. Hence making GLD an attractive therapeutic option for the treatment and/or prevention of AD.Item Immunomodulation of the innate immune system: The role of vitamin D in the context of monocytes and macrophages(University of the Witwatersrand, Johannesburg, 2024-07) Mol, Bronwyn Ashleigh; Gentle, Nikki; Meyer, VanessaMacrophages are widely distributed cells of the innate immune system with essential roles in homeostasis and disease. Despite concerted efforts, several aspects of macrophage origin, biology, and functionality remain poorly understood. To gain a deeper understanding of these cells, a physiologically relevant, but practical model is required. In vitro, macrophages are principally generated from primary monocytes and monocyte-like cell lines through a natural process referred to as monocyte-to-macrophage differentiation. Monocyte-like cell lines have several practical advantages over the use of primary monocytes with the most commonly employed monocyte-like cell lines being THP-1 and U937 cells. Despite their frequent use, no standardised protocol is employed in the differentiation of monocyte-like cell lines to macrophages. Naturally, this results in large discrepancies and a lack of comparability between studies. Furthermore, many of these protocols are not physiologically relevant and produce macrophages that are not responsive to downstream stimuli. 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the biologically active form of vitamin D3, is a recognised immunomodulator that shows pronounced genomic and non-genomic effects in immune cells. It is also reported as an inducer of monocyte-to-macrophage differentiation, though heavily debated, and a potential macrophage polarisation agent. Despite this, there is relatively little information concerning the role of 1,25(OH)2D3 in monocyte-to-macrophage differentiation and macrophage biology. This study aimed to develop a more physiologically relevant differentiation protocol for the monocyte-like THP-1 and U937 cell lines. This model was then used to investigate the role of 1,25(OH)2D3 in monocyte-to-macrophage differentiation and macrophage biology. Assessment of morphological features and the macrophage markers, CD11b and CD14, indicated that in both THP-1 and U937 cells, differentiation induced using a combination of 5 nM of phorbol 12-myristate 13-acetate (PMA) and 10 nM 1,25(OH)2D3 over 96 hours produced the most mature macrophages. It was observed that 1,25(OH)2D3 alone was not capable of inducing differentiation, yet when combined with PMA, greatly enhanced macrophage features. THP-1 cells are the most widely employed monocyte-like cell line, and are proposed to be the most reflective of primary monocytes. In this study these cells were shown to be more responsive to the effects of 1,25(OH)2D3 than their U937 counterparts. As such, RNA-sequencing was used to explore the efficacy of the proposed differentiation protocols and the influence of 1,25(OH)2D3 on macrophage biology in THP-1 cells. Differential gene expression analysis confirmed that the most effective differentiation protocol was the combination of 5 nM PMA with 10 nM 1,25(OH)2D3 when considering macrophage associated features including transcription factor usage, adhesion, phagocytosis, and cytokine and cytokine receptor expression. This protocol also produced THP-1-derived macrophages that showed increased expression of genes considered to be primary macrophage markers. These results also suggested that THP-1 cells differentiated with neither PMA nor PMA with 1,25(OH)2D3 were likely to represent fully polarised macrophages. 1,25(OH)2D3 treatment of THP-1 monocytes and THP-1-derived macrophages produced distinct gene expression profiles with considerably less overlap than expected. Though 1,25(OH)2D3 treatment often affected similar biological processes in both cell types, the genes within these processes found to be differentially expressed in each cell line were often distinct. For example, in THP-1- derived macrophages, but not THP-1 monocytes, 1,25(OH)2D3 treatment resulted in the increased expression of genes encoding numerous antibacterial peptides, several small GTPases and their regulators. Additionally, several type I interferon response related proteins showed decreased expression, while expression of cytokines and cytokine receptors was variable. This, taken together with the morphological work, indicates two potential roles for 1,25(OH)2D3 in macrophages. Firstly, a protective role as it suggests the potential to prime an antibacterial response, while still balancing inflammatory responses and protecting against autoinflammation induced by aberrant type I interferon response. Secondly, a potential role in determining the morphological features, clearly demonstrated through microscopy, and further suggested by the differential expression of a variety of small GTPases and their regulators.Item Knockdown of long non-coding RNA PANDA improves the cytotoxic effects of cisplatin in oesophageal squamous cell carcinoma cell lines(University of the Witwatersrand, Johannesburg, 2024-11) Moonsamy, Sasha Sarasvathee Keshnee; Mavri-Damelin, Demetra; Jivan, RupalOesophageal cancer is one of the leading causes of cancer death worldwide, of which oesophageal squamous cell carcinoma (OSCC) is the major subtype in southern and eastern Africa. Cisplatin is a well-established drug used to treat multiple cancers, including OSCC. Drug resistance is a major impediment to continued cisplatin therapy in numerous cancers. LncRNA P21-associated non-coding RNA DNA damaged activated RNA (PANDA) is known to function in cell cycle regulation in response to DNA damage and is upregulated in OSCC. We aim to determine lncRNA PANDA expression in South African-derived OSCC cells and establish whether down-regulation of this lncRNA can be used to supplement cisplatin therapy. In this study, MTT assays were performed to determine the EC50 concentrations of cisplatin in OSCC (WHCO1, WHCO5, and SNO) cells and HEK293 cells as a non-cancer control. The cytotoxic effects of cisplatin were exerted in all cell lines, with WHCO5 and SNO appearing more responsive to cisplatin than WHCO1 and HEK293. RT-PCR was used to detect if lncRNA PANDA is expressed in untreated and cisplatin-treated cells and was detected in all cell lines. Knockdown of lncRNA PANDA by siRNA was assessed with RT-PCR. Phase contrast microscopy was used to assess whether siRNA reagents altered cell morphology at 5, 24, and 48 hours post treatment. No significant alterations in cell morphology were observed in WHCO1, WHCO5, SNO, and HEK293 cells. MTT assay evaluation after 48 hours of cisplatin exposure, with or without siRNA for lncRNA PANDA, showed a significant reduction in EC50 concentrations in WHCO5, SNO, and HEK293 cell lines, suggesting that knockdown of lncRNA PANDA may improve cisplatin cytotoxicity in some cell lines. However, the EC50 values were higher with lncRNA PANDA knockdown in the WHCO1 cell line, suggesting that not all OSCC cell types may be responsive to this approach. In conclusion, lncRNA PANDA is expressed in response to cisplatin-induced DNA damage, and the down regulation of lncRNA PANDA improves the cytotoxic effects of cisplatin; however, further investigations are warranted in OSCC.Item Elucidating the Structure-Function Relationships of Enterococcus faecium Nicotinate-Nucleotide Adenylyltransferase through X-Ray Crystallography, Computational Modelling and Binding Studies(University of the Witwatersrand, Johannesburg, 2024) Jeje, Olamide Adetomi; Pandian, Ramesh; Achilonu, Ikechukwu A.Nicotinate nucleotide adenylyltransferase (NNAT) is a vital enzyme at the heart of NAD biosynthesis, catalysing a crucial reaction that leads to the formation of pyridine dinucleotides. NAD+ is an essential coenzyme in numerous metabolic processes, DNA repair, and cellular signalling. Given its pivotal role, NNAT has emerged as a compelling drug target, particularly for its potential to disrupt the survival mechanisms of bacterial pathogens. By inhibiting NNAT, it is possible to undermine the metabolic integrity of these pathogens, making NNAT a promising focal point in the fight against bacterial infections and antibiotic resistance. However, understanding the structure-function relationship of Enterococcus faecium NNAT (EfNNAT) has remained elusive. Hence, this study aimed to address this gap bycharacterising EfNNAT and validating its potential as a druggable target. EfNNAT was overexpressed and purified using the Escherichia coli system and IMAC purification technique. Subsequently, biophysical characterisation was performed, followed by the determination of the three-dimensional structure in both apo and liganded forms using X-ray crystallography. High-throughput virtual screening, along with SP and XP docking, was conducted using a library of synthesizable flavonoids. Molecular dynamic simulation and fluorescence studies were employed to establish and validate the binding of identified inhibitors to EfNNAT. Successful expression and purification of EfNNAT yielded approximately 101 mg per 7.8 g of wet E. coli cells, with a purity exceeding 98%. High-resolution crystal structures of EfNNAT in native, adenine-bound, and NMN-bound forms were determined at 1.90 Å, 1.82 Å, and 1.84 Å, respectively. These structures provided insights into EfNNAT's substrate preference and revealed a potential allosteric site at the dimer interface of the NMN-bound structure. Virtual screening identified quercetin 3-O-beta-D-glucose- 7-O-beta-D-gentiobioside as the only potential inhibitor from the flavonoid library used. A 500 ns atomistic molecular dynamics simulation showed the compound interacted through hydrogen bonding and water bridges, albeit unstable within the receptor. ANS and mant-ATP fluorescence spectroscopy confirmed quercetin binding, while thermal shift assay revealed minimal impact of the inhibitor on the protein stability and structure. This study establishes a pipeline from expression and purification to structure solution and potential inhibitor identification for EfNNAT, validating its druggability. The mechanistic insights offer a foundation for advancing drug discovery efforts targeting EfNNAT and other bacterial NNAT enzymes.Item Using ChIP-seq and Gene Expression Microarray data to explore transcriptional dysregulation of PXDN and PXDNL in cardiovascular diseases(University of the Witwatersrand, Johannesburg, 2024) Naidoo, Shiven; Gentle, Nikki; Mavri-Damelin, DemetraBackground: Cardiovascular diseases (CVDs) remain one of the leading causes of death globally. The genes PXDN and PXDNL are both expressed in the cardiovascular system, and their dysregulation has been linked to various disorders, including CVDs, but little is known of their transcriptional regulation in the cardiovascular system or their roles in CVD pathogenesis. Methods: This study developed two custom bioinformatics pipelines in R to mine and analyse ChIP-seq data from ChIP-Atlas and gene expression microarray data from the Gene Expression Omnibus (GEO). The first pipeline used ChIPseeker to identify regulatory transcription factors (TFs) of PXDN and PXDNL in cardiovascular cells and tissues. ChIP-seq data from 400 experiments across 63 TFs was filtered to isolate TFs with high confidence binding peaks in the promoter and first intron of PXDN and PXDNL. The second pipeline used R Bioconductor packages to explore the expression profiles of PXDN, PXDNL, and their TFs in seven microarray datasets across three CVD-related contexts: cardiomyopathies, heart failure and TNF-α stimulation. Results and discussion: This study identified 27 TFs binding to PXDN and 18 TFs binding to PXDNL in cardiovascular cells. Sixteen of these TFs were shared by both PXDN and PXDNL, suggesting potential coregulatory mechanisms in cardiovascular cells where they are both expressed. Unique TFs were also identified for PXDN (11) and PXDNL (2). Differential gene expression analysis revealed no significant change in expression (log2FC > 0.5; p.adj < 0.05) for PXDN, PXDNL and many of their identified TFs in the CVD-related conditions investigated, suggesting that changes at the transcript level may not contribute to the progression of these conditions. Conclusions: This study advances our understanding of the transcriptional regulation of PXDN and PXDNL in healthy cardiovascular cells as well as their expression levels in the investigated CVD-related contexts. This study also contributes a bioinformatics pipeline which can be further developed and applied to analysing data from ChIP-Atlas and GEO. Future research can elucidate the roles of each TF in regulating PXDN and PXDNL in healthy and diseased cell lines