Bio21 - Research Publications
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ItemUnravelling the mechanism of neurotensin recognition by neurotensin receptor 1(NATURE PORTFOLIO, 2023-12-09)The conformational ensembles of G protein-coupled receptors (GPCRs) include inactive and active states. Spectroscopy techniques, including NMR, show that agonists, antagonists and other ligands shift the ensemble toward specific states depending on the pharmacological efficacy of the ligand. How receptors recognize ligands and the kinetic mechanism underlying this population shift is poorly understood. Here, we investigate the kinetic mechanism of neurotensin recognition by neurotensin receptor 1 (NTS1) using 19F-NMR, hydrogen-deuterium exchange mass spectrometry and stopped-flow fluorescence spectroscopy. Our results indicate slow-exchanging conformational heterogeneity on the extracellular surface of ligand-bound NTS1. Numerical analysis of the kinetic data of neurotensin binding to NTS1 shows that ligand recognition follows an induced-fit mechanism, in which conformational changes occur after neurotensin binding. This approach is applicable to other GPCRs to provide insight into the kinetic regulation of ligand recognition by GPCRs.
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ItemNo Preview AvailableStructure-activity relationship and target investigation of 2-aryl quinolines with nematocidal activity(ELSEVIER SCI LTD, 2024-04)Within the context of our anthelmintic discovery program, we recently identified and evaluated a quinoline derivative, called ABX464 or obefazimod, as a nematocidal candidate; synthesised a series of analogues which were assessed for activity against the free-living nematode Caenorhabditis elegans; and predicted compound-target relationships by thermal proteome profiling (TPP) and in silico docking. Here, we logically extended this work and critically evaluated the anthelmintic activity of ABX464 analogues on Haemonchus contortus (barber's pole worm) - a highly pathogenic nematode of ruminant livestock. First, we tested a series of 44 analogues on H. contortus (larvae and adults) to investigate the nematocidal pharmacophore of ABX464, and identified one compound with greater potency than the parent compound and showed moderate activity against a select number of other parasitic nematodes (including Ancylostoma, Heligmosomoides and Strongyloides species). Using TPP and in silico modelling studies, we predicted protein HCON_00074590 (a predicted aldo-keto reductase) as a target candidate for ABX464 in H. contortus. Future work aims to optimise this compound as a nematocidal candidate and investigate its pharmacokinetic properties. Overall, this study presents a first step toward the development of a new nematocide.
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ItemNo Preview AvailableFree drug percentage of moxidectin declines with increasing concentrations in the serum of marsupials(ELSEVIER, 2024-04)Moxidectin (MOX) is a macrocyclic lactone used to eliminate endo and ectoparasites in many mammalian species. It is notably the active ingredient of the anti-parasitic drug Cydectin®, manufactured by Virbac, and is frequently used to treat sarcoptic mange in Australian wildlife. Protein binding plays a significant role in the efficacy of a drug, as the unbound/free drug in plasma ultimately reflects the pharmacologically relevant concentration. This study aimed to investigate the free drug percentage of Moxidectin after in vitro spiking into the sera of four sarcoptic mange-susceptible Australian wildlife species; the koala (Phascolarctos cinereus), the bare-nosed wombat (Vombatus ursinus), the eastern grey kangaroo (Macropus giganteus), and the mountain brushtail possum (Trichosurus cunninghami). Three concentration points of MOX were tested for each individual: 20 pg/μL, 100 pg/μL and 500 pg/μL. Serum from five individuals of each species underwent an equilibrium dialysis followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). The results showed an atypical concentration dependent binding across all species, where free drug percentage decreased as MOX concentration increased. In addition, wombats showed significantly lower free drug levels. These findings call for further research into the mechanisms of moxidectin protein binding to help understand MOX pharmacokinetics in marsupials.
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ItemNo Preview AvailableStructure activity relationship and target prediction for ABX464 analogues in Caenorhabditis elegans(PERGAMON-ELSEVIER SCIENCE LTD, 2024-01-15)Global challenges with treatment failures and/or widespread resistance in parasitic worms against commercially available anthelmintics lend impetus to the development of new anthelmintics with novel mechanism(s) of action. The free-living nematode Caenorhabditis elegans is an important model organism used for drug discovery, including the screening and structure-activity investigation of new compounds, and target deconvolution. Previously, we conducted a whole-organism phenotypic screen of the 'Pandemic Response Box' (from Medicines for Malaria Venture, MMV) and identified a hit compound, called ABX464, with activity against C. elegans and a related, parasitic nematode, Haemonchus contortus. Here, we tested a series of 44 synthesized analogues to explore the pharmacophore of activity on C. elegans and revealed five compounds whose potency was similar or greater than that of ABX464, but which were not toxic to human hepatoma (HepG2) cells. Subsequently, we employed thermal proteome profiling (TPP), protein structure prediction and an in silico-docking algorithm to predict ABX464-target candidates. Taken together, the findings from this study contribute significantly to the early-stage drug discovery of a new nematocide based on ABX464. Future work is aimed at validating the ABX464-protein interactions identified here, and at assessing ABX464 and associated analogues against a panel of parasitic nematodes, towards developing a new anthelmintic with a mechanism of action that is distinct from any of the compounds currently-available commercially.
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ItemNo Preview AvailableThe Proteome and Lipidome of Extracellular Vesicles from Haemonchus contortus to Underpin Explorations of Host-Parasite Cross-Talk(MDPI, 2023-07)Many parasitic worms have a major adverse impact on human and animal populations worldwide due to the chronicity of their infections. There is a growing body of evidence indicating that extracellular vesicles (EVs) are intimately involved in modulating (suppressing) inflammatory/immune host responses and parasitism. As one of the most pathogenic nematodes of livestock animals, Haemonchus contortus is an ideal model system for EV exploration. Here, employing a multi-step enrichment process (in vitro culture, followed by ultracentrifugation, size exclusion and filtration), we enriched EVs from H. contortus and undertook the first comprehensive (qualitative and quantitative) multi-omic investigation of EV proteins and lipids using advanced liquid chromatography-mass spectrometry and informatics methods. We identified and quantified 561 proteins and 446 lipids in EVs and compared these molecules with those of adult worms. We identified unique molecules in EVs, such as proteins linked to lipid transportation and lipid species (i.e., sphingolipids) associated with signalling, indicating the involvement of these molecules in parasite-host cross-talk. This work provides a solid starting point to explore the functional roles of EV-specific proteins and lipids in modulating parasite-host cross-talk, and the prospect of finding ways of disrupting or interrupting this relationship to suppress or eliminate parasite infection.
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ItemPhosphoproteomic dysregulation in Huntington's disease mice is rescued by environmental enrichment(OXFORD UNIV PRESS, 2022-11-02)Huntington's disease is a fatal autosomal-dominant neurodegenerative disorder, characterized by neuronal cell dysfunction and loss, primarily in the striatum, cortex and hippocampus, causing motor, cognitive and psychiatric impairments. Unfortunately, no treatments are yet available to modify the progression of the disease. Recent evidence from Huntington's disease mouse models suggests that protein phosphorylation (catalysed by kinases and hydrolysed by phosphatases) might be dysregulated, making this major post-translational modification a potential area of interest to find novel therapeutic targets. Furthermore, environmental enrichment, used to model an active lifestyle in preclinical models, has been shown to alleviate Huntington's disease-related motor and cognitive symptoms. However, the molecular mechanisms leading to these therapeutic effects are still largely unknown. In this study, we applied a phosphoproteomics approach combined with proteomic analyses on brain samples from pre-motor symptomatic R6/1 Huntington's disease male mice and their wild-type littermates, after being housed either in environmental enrichment conditions, or in standard housing conditions from 4 to 8 weeks of age (n = 6 per group). We hypothesized that protein phosphorylation dysregulations occur prior to motor onset in this mouse model, in two highly affected brain regions, the striatum and hippocampus. Furthermore, we hypothesized that these phosphoproteome alterations are rescued by environmental enrichment. When comparing 8-week-old Huntington's disease mice and wild-type mice in standard housing conditions, our analysis revealed 229 differentially phosphorylated peptides in the striatum, compared with only 15 differentially phosphorylated peptides in the hippocampus (statistical thresholds fold discovery rate 0.05, fold change 1.5). At the same disease stage, minor differences were found in protein levels, with 24 and 22 proteins dysregulated in the striatum and hippocampus, respectively. Notably, we found no differences in striatal protein phosphorylation and protein expression when comparing Huntington's disease mice and their wild-type littermates in environmentally enriched conditions. In the hippocampus, only four peptides were differentially phosphorylated between the two genotypes under environmentally enriched conditions, and 22 proteins were differentially expressed. Together, our data indicates that protein phosphorylation dysregulations occur in the striatum of Huntington's disease mice, prior to motor symptoms, and that the kinases and phosphatases leading to these changes in protein phosphorylation might be viable drug targets to consider for this disorder. Furthermore, we show that an early environmental intervention was able to rescue the changes observed in protein expression and phosphorylation in the striatum of Huntington's disease mice and might underlie the beneficial effects of environmental enrichment, thus identifying novel therapeutic targets.
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ItemProteomic analysis of Sarcoptes scabiei reveals that proteins differentially expressed between eggs and female adult stages are involved predominantly in genetic information processing, metabolism and/or host-parasite interactions(PUBLIC LIBRARY SCIENCE, 2022-12)Presently, there is a dearth of proteomic data for parasitic mites and their relationship with the host animals. Here, using a high throughput LC-MS/MS-based approach, we undertook the first comprehensive, large-scale proteomic investigation of egg and adult female stages of the scabies mite, Sarcoptes scabiei-one of the most important parasitic mites of humans and other animals worldwide. In total, 1,761 S. scabiei proteins were identified and quantified with high confidence. Bioinformatic analyses revealed differentially expressed proteins to be involved predominantly in biological pathways or processes including genetic information processing, energy (oxidative phosphorylation), nucleotide, amino acid, carbohydrate and/or lipid metabolism, and some adaptive processes. Selected, constitutively and highly expressed proteins, such as peptidases, scabies mite inactivated protease paralogues (SMIPPs) and muscle proteins (myosin and troponin), are proposed to be involved in key biological processes within S. scabiei, host-parasite interactions and/or the pathogenesis of scabies. These proteomic data will enable future molecular, biochemical and physiological investigations of early developmental stages of S. scabiei and the discovery of novel interventions, targeting the egg stage, given its non-susceptibility to acaricides currently approved for the treatment of scabies in humans.
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ItemRespiratory strategy at birth initiates distinct lung injury phenotypes in the preterm lamb lung(BMC, 2022-12-14)BACKGROUND: A lack of clear trial evidence often hampers clinical decision-making during support of the preterm lung at birth. Protein biomarkers have been used to define acute lung injury phenotypes and improve patient selection for specific interventions in adult respiratory distress syndrome. The objective of the study was to use proteomics to provide a deeper biological understanding of acute lung injury phenotypes resulting from different aeration strategies at birth in the preterm lung. METHODS: Changes in protein abundance against an unventilated group (n = 7) were identified via mass spectrometry in a biobank of gravity dependent and non-dependent lung tissue from preterm lambs managed with either a Sustained Inflation (SI, n = 20), Dynamic PEEP (DynPEEP, n = 19) or static PEEP (StatPEEP, n = 11). Ventilation strategy-specific pathways and functions were identified (PANTHER and WebGestalt Tool) and phenotypes defined using integrated analysis of proteome, physiological and clinical datasets (MixOmics package). RESULTS: 2372 proteins were identified. More altered proteins were identified in the non-dependent lung, and in SI group than StatPEEP and DynPEEP. Different inflammation, immune system, apoptosis and cytokine pathway enrichment were identified for each strategy and lung region. Specific integration maps of clinical and physiological outcomes to specific proteins could be generated for each strategy. CONCLUSIONS: Proteomics mapped the molecular events initiating acute lung injury and identified detailed strategy-specific phenotypes. This study demonstrates the potential to characterise preterm lung injury by the direct aetiology and response to lung injury; the first step towards true precision medicine in neonatology.
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ItemNo Preview AvailableLiver-Secreted Hexosaminidase A Regulates Insulin-Like Growth Factor Signaling and Glucose Transport in Skeletal Muscle(AMER DIABETES ASSOC, 2023-06)Nonalcoholic fatty liver disease (NAFLD) and impaired glycemic control are closely linked; however, the pathophysiological mechanisms underpinning this bidirectional relationship remain unresolved. The high secretory capacity of the liver and impairments in protein secretion in NAFLD suggest that endocrine changes in the liver are likely to contribute to glycemic defects. We identify hexosaminidase A (HEXA) as an NAFLD-induced hepatokine in both mice and humans. HEXA regulates sphingolipid metabolism, converting GM2 to GM3 gangliosides-sphingolipids that are primarily localized to cell-surface lipid rafts. Using recombinant murine HEXA protein, an enzymatically inactive HEXA(R178H) mutant, or adeno-associated virus vectors to induce hepatocyte-specific overexpression of HEXA, we show that HEXA improves blood glucose control by increasing skeletal muscle glucose uptake in mouse models of insulin resistance and type 2 diabetes, with these effects being dependent on HEXA's enzymatic action. Mechanistically, HEXA remodels muscle lipid raft ganglioside composition, thereby increasing IGF-1 signaling and GLUT4 localization to the cell surface. Disrupting lipid rafts reverses these HEXA-mediated effects. In this study, we identify a pathway for intertissue communication between liver and skeletal muscle in the regulation of systemic glycemic control.
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ItemNo Preview AvailableStructural insights into the multifunctionality of rabies virus P3 protein(NATL ACAD SCIENCES, 2023-04-04)Viruses form extensive interfaces with host proteins to modulate the biology of the infected cell, frequently via multifunctional viral proteins. These proteins are conventionally considered as assemblies of independent functional modules, where the presence or absence of modules determines the overall composite phenotype. However, this model cannot account for functions observed in specific viral proteins. For example, rabies virus (RABV) P3 protein is a truncated form of the pathogenicity factor P protein, but displays a unique phenotype with functions not seen in longer isoforms, indicating that changes beyond the simple complement of functional modules define the functions of P3. Here, we report structural and cellular analyses of P3 derived from the pathogenic RABV strain Nishigahara (Nish) and an attenuated derivative strain (Ni-CE). We identify a network of intraprotomer interactions involving the globular C-terminal domain and intrinsically disordered regions (IDRs) of the N-terminal region that characterize the fully functional Nish P3 to fluctuate between open and closed states, whereas the defective Ni-CE P3 is predominantly open. This conformational difference appears to be due to the single mutation N226H in Ni-CE P3. We find that Nish P3, but not Ni-CE or N226H P3, undergoes liquid-liquid phase separation and this property correlates with the capacity of P3 to interact with different cellular membrane-less organelles, including those associated with immune evasion and pathogenesis. Our analyses propose that discrete functions of a critical multifunctional viral protein depend on the conformational arrangements of distant individual domains and IDRs, in addition to their independent functions.
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