1,431 research outputs found

    Identifying alterations in adipose tissue-derived islet GPCR peptide ligand mRNAs in obesity: implications for islet function

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    In addition to acting as an energy reservoir, white adipose tissue is a vital endocrine organ involved in the modulation of cellular function and the maintenance of metabolic homeostasis through the synthesis and secretion of peptides, known as adipokines. It is known that some of these secretory peptides play important regulatory roles in glycaemic control by acting directly on islet β-cells or on insulin-sensitive tissues. Excess adiposity causes alterations in the circulating levels of some adipokines which, depending on their mode of action, can have pro-inflammatory, pro-diabetic or anti-inflammatory, anti-diabetic properties. Some adipokines that are known to act at β-cells have actions that are transduced by binding to G protein- coupled receptors (GPCRs). This large family of receptors represents ~35% of all current drug targets for the treatment of a wide range of diseases, including type 2 diabetes (T2D). Islets express ~300 GPCRs, yet only one islet GPCR is currently directly targeted for T2D treatment. This deficit represents a therapeutic gap that could be filled by the identification of adipose tissue-derived islet GPCR peptide ligands that increase insulin secretion and overall β-cell function. Thus, by defining their mechanisms of action, there is potential for the development of new pharmacotherapies for T2D. Therefore, this thesis describes experiments which aimed to compare the expression profiles of adipose tissue-derived islet GPCR peptide ligand mRNAs under lean and obese conditions, and to characterise the functional effects of a selected candidate of interest on islet cells. Visceral fat depots were retrieved from high-fat diet-induced and genetically obese mouse models, and from human participants. Fat pads were either processed as whole tissue, or mature adipocyte cells were separated from the stromal vascular fraction (SVF) which contains several other cell populations, including preadipocytes and macrophages. The expression levels of 155 islet GPCR peptide ligand mRNAs in whole adipose tissue or in isolated mature adipocytes were quantified using optimised RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) protocols. Comparisons between lean and obese states in mice models and humans revealed significant modifications in the expression levels of several adipokine mRNAs. As expected, mRNAs encoding the positive control genes, Lep and AdipoQ were quantifiable, with the expression of Lep mRNA increasing and that of AdipoQ mRNA decreasing in obesity. Expression of Ccl4 mRNA, encoding chemokine (C-C motif) ligand 4, was significantly upregulated in whole adipose tissue across all models of obesity compared to their lean counterparts. This coincided with elevated circulating Ccl4 peptide levels. This increase was not replicated in isolated mature adipocytes, indicating that the source of upregulated Ccl4 expression in obesity was the SVF of adipose tissue. Based on this significant increase in Ccl4 mRNA expression within visceral fat and its undetermined effects on β-cell function, Ccl4 was selected for further investigation in MIN6 β-cells and mouse islets. PRESTO-Tango β-arrestin reporter assays were performed to determine which GPCRs were activated by exogenous Ccl4. Experiments using HTLA cells expressing a protease-tagged β- arrestin and transfected with GPCR plasmids of interest indicated that 100ng/mL Ccl4 significantly activated Cxcr1 and Cxcr5, but it was not an agonist at the previously identified Ccl4-target GPCRs Ccr1, Ccr2, Ccr5, Ccr9 and Ackr2. RNA extraction and RT-qPCR experiments using MIN6 β-cells and primary islets from lean mice revealed the expression of Cxcr5 mRNA in mouse islets, but it was absent in MIN6 β-cells. The remaining putative Ccl4 receptors (Ccr1, Ccr2, Ccr5, Ccr9, Cxcr1 and Ackr2) were either absent or present at trace levels in mouse islets and MIN6 β-cells. Recombinant mouse Ccl4 protein was used for functional experiments at concentrations of 5, 10, 50 and 100ng/mL, based on previous reports of biological activities at these concentrations. Trypan blue exclusion testing was initially performed to assess the effect of exogenous Ccl4 on MIN6 β-cell viability and these experiments indicated that all concentrations (5-100ng/mL) were well-tolerated. Since β-cells have a low basal rate of apoptosis, cell death was induced by exposure to the saturated free fatty acid, palmitate, or by a cocktail of pro-inflammatory cytokines (interleukin-1β, tumour necrosis factor-α and interferon-γ). In MIN6 β-cells, Ccl4 demonstrated concentration-dependent protective effects against palmitate-induced and cytokine-induced apoptosis. Conversely, while palmitate and cytokines also increased apoptosis of mouse islets, Ccl4 did not protect islets from either inducer. Quantification of bromodeoxyuridine (BrdU) incorporation into β-cell DNA indicated that Ccl4 caused a concentration-dependent reduction in proliferation of MIN6 β-cells in response to 10% fetal bovine serum (FBS). In contrast, immunohistochemical quantification of Ki67-positive mouse islet β-cells showed no differences in β-cell proliferation between control- and Ccl4-treated islets. Whilst the number of β-cells and δ-cells were unaffected, α- cells were significantly depleted by Ccl4 treatment. Exogenous Ccl4 had no effect on nutrient- stimulated insulin secretion from both MIN6 β-cells and primary mouse islets. The 3T3-L1 preadipocyte cell line was used to assess potential Ccl4-mediated paracrine and/or autocrine signalling within adipose tissue. Ccl4 did not alter the mRNA expression of Pparγ, a master regulator of adipocyte differentiation, but did significantly downregulate the mRNA expression of the crucial adipogenic gene, adiponectin. Oil Red O staining and Western blotting were performed to assess lipid accumulation, and insulin and lipolytic signalling, respectively, and these experiments indicated that the observed Ccl4-induced decrease in adiponectin expression failed to correlate with any changes in adipocyte function. In summary, these data demonstrated anti-apoptotic and anti-proliferative actions of the adipokine, Ccl4, on MIN6 β-cells that were not replicated in mouse islets. The absence of any anti-apoptotic, insulin secretory and/or pro-proliferative effects of Ccl4 in islet β-cells suggests that it is unlikely to play a role in regulating β-cell function via crosstalk between adipose tissue and islets. The divergent functional effects highlight that whilst MIN6 cells are a useful primary β-cell surrogate for some studies, primary islets should always be used to confirm physiological relevance. On the other hand, significant α-cell depletion following Ccl4 treatment suggests a cell-specific function within the islets. Furthermore, Ccl4 impaired adiponectin mRNA expression in adipocytes, although, how adipocyte function is affected as a result requires further investigation. Collectively, these data have contributed increased understanding of the role of obesity in modifying the expression of adipose tissue-derived islet GPCR peptide ligands

    Functional characterization of the Ustilago maydis effector genes UMAG_11060 and UMAG_05306

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    Ustilago maydis causes corn smut and triggers tumor formation in all aerial parts of maize. To adapt to the host plant and promote disease progression, U. maydis uses effector proteins that exhibit organ-specific expression and adaptation during infection. This study focuses on two of these effectors, UMAG_11060 and UMAG_05306. This study characterizes UMAG_11060 (Chapter 2), which encodes the effector protein TOPLESS (TPL) interacting protein 6 (Tip6). The study shows that Tip6 interacts with the N-terminal region of ZmTPL2 through its two EAR (ethylene-responsive element binding factor-associated amphiphilic repression) motifs. These motifs are crucial for virulence function and alter the nuclear distribution pattern of ZmTPL2, disrupting host transcriptional regulation. This disruption leads to the down-regulation of 13 transcription factors in the AP2/ERF B1 subfamily. This study proposes a regulatory mechanism in which Tip6 uses repressive domains to recruit the corepressor ZmTPL2, thereby disrupting the transcriptional networks of the host plant. The second part of the thesis focuses on the characterization of UMAG_05306 (Chapter 3), which exhibits highly specific subcellular localization and appears as thick and twisted filament-like structures. The study shows that UMAG_05306 interacts with four maize dynamin related proteins (DRPs) and is able to interact with both the N- terminal and C-terminal of ZmDRP5. Three DRPs are found to interact with maize tubulin. Furthermore, UMAG_05306 directly interacts with tubulin. These findings shed light on their potential roles in U. maydis infection. In conclusion, this study provides insight into the molecular mechanisms underlying U. maydis infection and reveals the importance of UMAG_11060 and UMAG_05306 effectors for virulence and tumor formation

    Computational Approaches to Drug Profiling and Drug-Protein Interactions

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    Despite substantial increases in R&D spending within the pharmaceutical industry, denovo drug design has become a time-consuming endeavour. High attrition rates led to a long period of stagnation in drug approvals. Due to the extreme costs associated with introducing a drug to the market, locating and understanding the reasons for clinical failure is key to future productivity. As part of this PhD, three main contributions were made in this respect. First, the web platform, LigNFam enables users to interactively explore similarity relationships between ‘drug like’ molecules and the proteins they bind. Secondly, two deep-learning-based binding site comparison tools were developed, competing with the state-of-the-art over benchmark datasets. The models have the ability to predict offtarget interactions and potential candidates for target-based drug repurposing. Finally, the open-source ScaffoldGraph software was presented for the analysis of hierarchical scaffold relationships and has already been used in multiple projects, including integration into a virtual screening pipeline to increase the tractability of ultra-large screening experiments. Together, and with existing tools, the contributions made will aid in the understanding of drug-protein relationships, particularly in the fields of off-target prediction and drug repurposing, helping to design better drugs faster

    A mechanistic approach to understand the role of p90 ribosomal S6 kinases in Prostate Cancer

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    The RSKs are known to be involved in many cancers, including Prostate Cancer (PCa), however the representation of the Ribosomal S6 Kinase (RSK) isoforms is skewed and there is little investigation into the RSK regulation of the Androgen Receptor (AR). Here we show in model African Green Monkey Kidney Fibroblast (COS-1 cells) that all RSKs regulate the AR transcriptional activity and interact with the AR in proximity. Analysis of PCa cell lines highlighted that all RSKs exhibited higher expression than the control but most significantly RSK2. We then transitioned to lymph node carcinoma of the prostate (LNCaP) cells as they are more biologically relevant, siRNA knockdown coupled with qPCR showed that key AR target genes was differentially regulated by the RSKs; RSK1 and RSK4 knockdown caused increased expression of NDRG1; RSK1 and RSK2 knockdown induced a decrease in TMPRSS2; RSK2 knockdown produced a significant decrease in PSA, respectively. To understand if these effects would translate to a cellular change we investigated proliferation, siRNA knockdown of RSK2 and RSK4 caused a significant decrease in LNCaP cells. Interestingly, investigation of RSK4 is also underrepresented in structural biology despite having a different mechanism from other isoforms. Here, we attempted to express, purify, and crystalize different domains of RSK4 to understand its mechanism of activation. Crystallization attempts were unsuccessful, potentially due proteolysis and chaperone contaminations. Kinetic assays showed that phospho-mimetic mutations were successful in creating a constitutively active mutant construct. Pull down assays experimentally showed that the AGC region of RSK4 is essential for the PDK1 independent activation. The multiple techniques used in this study strongly suggest that RSK are in fact involved in PCa signalling and warrant further investigation and highlight the nonredundant functions of the RSK isoforms and a novel relationship between AR and RSK4. Additionally, the pull-down assay highlighted that the AGC region of RSK facilitates interaction between the N- And C-terminal domains which has not been previously experimentally demonstrated thus supporting previous speculation around its activation mechanism

    Machine Learning Small Molecule Properties in Drug Discovery

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    Machine learning (ML) is a promising approach for predicting small molecule properties in drug discovery. Here, we provide a comprehensive overview of various ML methods introduced for this purpose in recent years. We review a wide range of properties, including binding affinities, solubility, and ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity). We discuss existing popular datasets and molecular descriptors and embeddings, such as chemical fingerprints and graph-based neural networks. We highlight also challenges of predicting and optimizing multiple properties during hit-to-lead and lead optimization stages of drug discovery and explore briefly possible multi-objective optimization techniques that can be used to balance diverse properties while optimizing lead candidates. Finally, techniques to provide an understanding of model predictions, especially for critical decision-making in drug discovery are assessed. Overall, this review provides insights into the landscape of ML models for small molecule property predictions in drug discovery. So far, there are multiple diverse approaches, but their performances are often comparable. Neural networks, while more flexible, do not always outperform simpler models. This shows that the availability of high-quality training data remains crucial for training accurate models and there is a need for standardized benchmarks, additional performance metrics, and best practices to enable richer comparisons between the different techniques and models that can shed a better light on the differences between the many techniques.Comment: 46 pages, 1 figur

    PROTEOMIC APPROACHES TO IDENTIFY UNIQUE AND SHARED SUBSTRATES AMONG KINASE FAMILY MEMBERS

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    Protein phosphorylation is a reversible post-translational modification that is a critical component of almost all signaling pathways. Kinases regulate substrate proteins through phosphorylation, and nearly all proteins are phosphorylated to some extent. Crucially, breakdown in phosphorylation signaling is an underlying factor in many diseases, including cancer. Understanding how phosphorylation signaling mediates cellular pathways is crucial for understanding cell biology and human disease. Targeted protein degradation (TPD) is a strategy to rapidly deplete a protein of interest (POI) and is applicable to any gene that is amenable to CRISPR-Cas9 editing. One TPD approach is the auxin-inducible degron (AID) system, which relies on the expression of an AID fusion protein and the F-box protein Tir1. Addition of auxin drives binding of the AID-POI and Tir1, resulting in rapid ubiquitination and degradation. Recently, we demonstrated that this approach can be used to study kinase-substrate relationships in a manner analogous to small-molecule inhibition using the kinase Plk1 as a proof-of-concept. Based on the results of this study, we applied AID-Tir1 protein degradation to interrogate kinase-substrate relationships for the Polo-like kinase (Plk), p21-activated kinase (PAK), and Aurora kinase families. Additionally, we made significant improvements to the CRISPR-Cas9 workflow and improved efficiency of AID-Tir1 cell line generation for kinases of interest. Targeted degradation of PAK1 revealed low PAK1 activity in HEK293 cells. Follow-up experiments showed that, while many phosphorylation sites are regulated by the group 1 PAKs, PAK1 does not regulate these pathways alone and likely has overlapping functions with the closely related kinase, PAK2. We applied AID-Tir1 to Aurora B and observed high correlation between Aurora B degradation and inhibition by the Aurora B inhibitor AZD1152, demonstrating that protein degradation is a selective approach to identify direct Aurora B substrates. We identified an uncharacterized truncated Aurora B isoform that is sufficient for Aurora B signaling in the absence of full-length Aurora B. Finally, we used fluorescent reporter proteins and Fluorescence Activated Cell Sorting (FACS) to greatly improve the efficiency of AID-Tir1 cell line generation for kinases of interest. These improvements make strides towards widespread implementation of targeted degradation as a tool to study kinase-substrate relationships

    The genetic interactions of PKHD1 and ATMIN in autosomal recessive polycystic kidney disease (ARPKD)

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    A thesis submitted in partial fulfilment of the requirements of the University of Wolverhampton for the degree of Doctor of Philosophy.The main gene associated with Autosomal Recessive Polycystic Kidney Disease (ARPKD) is PKHD1 which encodes a ciliary protein associated with planar cell polarity. In mice, mutations in the transcription factor Atmin can present with an ARPKD-like phenotype with kidney disease similar to an early manifestation of ARPKD. Like the mouse gene Pkhd1, mutations in Atmin are associated with altered WNT/PCP expression. Previous work has suggested that Atmin and Pkhd1 do not physically interact, but Atmin may modulate Pkhd1 expression. However, the mechanisms governing this relationship are unknown. ARPKD is a rare disorder typically associated with severe kidney and liver disease in children. The disease has considerable clinical and familial variability, but little is known regarding genotype-phenotype relationships. It has been proposed that genetic modifiers may influence disease severity. Next-generation sequencing (NGS) using ChIP-Seq and RNA-Seq techniques in mouse kidneys and intermedullary collecting duct (mIMCD3) cells identified new transcriptional targets of Atmin, which did not include Pkhd1 but included genes associated with cystic kidneys in animal models (Camk2g and G6pc). NGS in Atmin and Pkhd1 KDs identified a common transcriptional network between the two genes. Gene enrichment analysis suggests this common network is associated with immune system processes. Dysregulated genes associated with double KDs showed greater enrichment of processes associated with the actin cytoskeleton, cell cycle and energy metabolism. Loss of Atmin expression negatively impacts the ciliary localisation of Fibrocystin, suggesting that Atmin may be needed for the proper localisation of Fibrocystin to the cilium. NGS in ARPKD kidneys highlights mutations in ATMIN as a potential regulator of disease severity, associated with reduced ARPKD severity. Expression differences in WNT genes may be present between severe and moderate ARPKD and transcriptomic profiling identified candidate diagnostic markers in ARPKD which included MSC, FGA, WNT4, WNT9B and KIF26B. This work indicates that Atmin and Pkhd1 interact in a similar transcriptional network in mice. Atmin is not a transcription factor of Pkhd1 but may modulate its function by governing its ciliary localisation by a yet unknown mechanism. Additionally, ATMIN mutations may modulate ARPKD disease severity, and the amount of differential expression in WNT/PCP genes may be a marker of disease severity.PKD Charity, Arran Brown Rainbow Foundation, and the University of Wolverhampton
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