Biochemistry and Pharmacology - Research Publications

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    G-CSFR antagonism reduces mucosal injury and airways fibrosis in a virus-dependent model of severe asthma
    Wang, H ; Aloe, C ; McQualter, J ; Papanicolaou, A ; Vlahos, R ; Wilson, N ; Bozinovski, S (WILEY, 2021-04-01)
    BACKGROUND AND PURPOSE: Asthma is a chronic disease that displays heterogeneous clinical and molecular features. A phenotypic subset of late-onset severe asthmatics has debilitating fixed airflow obstruction, increased neutrophilic inflammation and a history of pneumonia. Influenza A virus (IAV) is an important viral cause of pneumonia and asthmatics are frequently hospitalised during IAV epidemics. This study aims to determine whether antagonising granulocyte colony stimulating factor receptor (G-CSFR) prevents pneumonia-associated severe asthma. EXPERIMENTAL APPROACH: Mice were sensitised to house dust mite (HDM) to establish allergic airway inflammation and subsequently infected with IAV (HKx31/H3N2 subtype). A neutralising monoclonal antibody against G-CSFR was therapeutically administered. KEY RESULTS: In IAV-infected mice with prior HDM sensitisation, a significant increase in airway fibrotic remodelling and airways hyper-reactivity was observed. A mixed granulocytic inflammatory profile consisting of neutrophils, macrophages and eosinophils was prominent and at a molecular level, G-CSF expression was significantly increased in HDMIAV-treated mice. Blockage of G-CSFR reduced neutrophilic inflammation in the bronchoalveolar and lungs by over 80% in HDMIAV-treated mice without altering viral clearance. Markers of NETosis (dsDNA and myeloperoxidase in bronchoalveolar), tissue injury (LDH activity in bronchoalveolar) and oedema (total bronchoalveolar-fluid protein) were also significantly reduced with anti-G-CSFR treatment. In addition, anti-G-CSFR antagonism significantly reduced bronchoalveolar gelatinase activity, active TFGβ lung levels, collagen lung expression, airways fibrosis and airways hyper-reactivity in HDMIAV-treated mice. CONCLUSIONS AND IMPLICATIONS: We have shown that antagonising G-CSFR-dependent neutrophilic inflammation reduced pathological disruption of the mucosal barrier and airways fibrosis in an IAV-induced severe asthma model.
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    Activated microglia drive demyelination via CSF1R signaling
    Marzan, DE ; Brugger-Verdon, V ; West, BL ; Liddelow, S ; Samanta, J ; Salzer, JL (WILEY, 2021-02-23)
    Microgliosis is a prominent pathological feature in many neurological diseases including multiple sclerosis (MS), a progressive auto-immune demyelinating disorder. The precise role of microglia, parenchymal central nervous system (CNS) macrophages, during demyelination, and the relative contributions of peripheral macrophages are incompletely understood. Classical markers used to identify microglia do not reliably discriminate between microglia and peripheral macrophages, confounding analyses. Here, we use a genetic fate mapping strategy to identify microglia as predominant responders and key effectors of demyelination in the cuprizone (CUP) model. Colony-stimulating factor 1 (CSF1), also known as macrophage colony-stimulating factor (M-CSF) - a secreted cytokine that regulates microglia development and survival-is upregulated in demyelinated white matter lesions. Depletion of microglia with the CSF1R inhibitor PLX3397 greatly abrogates the demyelination, loss of oligodendrocytes, and reactive astrocytosis that results from CUP treatment. Electron microscopy (EM) and serial block face imaging show myelin sheaths remain intact in CUP treated mice depleted of microglia. However, these CUP-damaged myelin sheaths are lost and robustly phagocytosed upon-repopulation of microglia. Direct injection of CSF1 into CNS white matter induces focal microgliosis and demyelination indicating active CSF1 signaling can promote demyelination. Finally, mice defective in adopting a toxic astrocyte phenotype that is driven by microglia nevertheless demyelinate normally upon CUP treatment implicating microglia rather than astrocytes as the primary drivers of CUP-mediated demyelination. Together, these studies indicate activated microglia are required for and can drive demyelination directly and implicate CSF1 signaling in these events.
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    3, 2, 1, go! Cryptosporidium counts down to sex.
    Jex, AR ; Tonkin, CJ ; Ralph, SA (Public Library of Science (PLoS), 2022-05)
    Cryptosporidium is a leading cause of death from childhood diarrhea, but its biology is poorly understood. A recent study in PLOS Biology reveals hitherto unknown aspects of the parasite's life cycle that may lead to improvements in ex vivo culture.
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    Linear plasmids in Klebsiella and other Enterobacteriaceae
    Hawkey, J ; Cottingham, H ; Tokolyi, A ; Wick, RR ; Judd, LM ; Cerdeira, L ; Garcia, DDO ; Wyres, KL ; Holt, KE (MICROBIOLOGY SOC, 2022-04-01)
    Linear plasmids are extrachromosomal DNA elements that have been found in a small number of bacterial species. To date, the only linear plasmids described in the family Enterobacteriaceae belong to Salmonella, first found in Salmonella enterica Typhi. Here, we describe a collection of 12 isolates of the Klebsiella pneumoniae species complex in which we identified linear plasmids. Screening of assembly graphs assembled from public read sets identified linear plasmid structures in a further 13 K. pneumoniae species complex genomes. We used these 25 linear plasmid sequences to query all bacterial genome assemblies in the National Center for Biotechnology Information database, and discovered an additional 61 linear plasmid sequences in a variety of Enterobacteriaceae species. Gene content analysis divided these plasmids into five distinct phylogroups, with very few genes shared across more than two phylogroups. The majority of linear plasmid-encoded genes are of unknown function; however, each phylogroup carried its own unique toxin-antitoxin system and genes with homology to those encoding the ParAB plasmid stability system. Passage in vitro of the 12 linear plasmid-carrying Klebsiella isolates in our collection (which include representatives of all five phylogroups) indicated that these linear plasmids can be stably maintained, and our data suggest they can transmit between K. pneumoniae strains (including members of globally disseminated multidrug-resistant clones) and also between diverse Enterobacteriaceae species. The linear plasmid sequences, and representative isolates harbouring them, are made available as a resource to facilitate future studies on the evolution and function of these novel plasmids.
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    Chromosome-level genome of Schistosoma haematobium underpins genome-wide explorations of molecular variation
    Stroehlein, AJ ; Korhonen, PK ; Lee, VV ; Ralph, SA ; Mentink-Kane, M ; You, H ; McManus, DP ; Tchuente, L-AT ; Stothard, JR ; Kaur, P ; Dudchenko, O ; Aiden, EL ; Yang, B ; Yang, H ; Emery, AM ; Webster, BL ; Brindley, PJ ; Rollinson, D ; Chang, BCH ; Gasser, RB ; Young, ND ; Zamanian, M (PUBLIC LIBRARY SCIENCE, 2022-02-01)
    Urogenital schistosomiasis is caused by the blood fluke Schistosoma haematobium and is one of the most neglected tropical diseases worldwide, afflicting > 100 million people. It is characterised by granulomata, fibrosis and calcification in urogenital tissues, and can lead to increased susceptibility to HIV/AIDS and squamous cell carcinoma of the bladder. To complement available treatment programs and break the transmission of disease, sound knowledge and understanding of the biology and ecology of S. haematobium is required. Hybridisation/introgression events and molecular variation among members of the S. haematobium-group might effect important biological and/or disease traits as well as the morbidity of disease and the effectiveness of control programs including mass drug administration. Here we report the first chromosome-contiguous genome for a well-defined laboratory line of this blood fluke. An exploration of this genome using transcriptomic data for all key developmental stages allowed us to refine gene models (including non-coding elements) and annotations, discover 'new' genes and transcription profiles for these stages, likely linked to development and/or pathogenesis. Molecular variation within S. haematobium among some geographical locations in Africa revealed unique genomic 'signatures' that matched species other than S. haematobium, indicating the occurrence of introgression events. The present reference genome (designated Shae.V3) and the findings from this study solidly underpin future functional genomic and molecular investigations of S. haematobium and accelerate systematic, large-scale population genomics investigations, with a focus on improved and sustained control of urogenital schistosomiasis.
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    Known allosteric proteins have central roles in genetic disease
    Abrusan, G ; Ascher, DB ; Inouye, M ; Haliloglu, T (PUBLIC LIBRARY SCIENCE, 2022-02-01)
    Allostery is a form of protein regulation, where ligands that bind sites located apart from the active site can modify the activity of the protein. The molecular mechanisms of allostery have been extensively studied, because allosteric sites are less conserved than active sites, and drugs targeting them are more specific than drugs binding the active sites. Here we quantify the importance of allostery in genetic disease. We show that 1) known allosteric proteins are central in disease networks, contribute to genetic disease and comorbidities much more than non-allosteric proteins, and there is an association between being allosteric and involvement in disease; 2) they are enriched in many major disease types like hematopoietic diseases, cardiovascular diseases, cancers, diabetes, or diseases of the central nervous system; 3) variants from cancer genome-wide association studies are enriched near allosteric proteins, indicating their importance to polygenic traits; and 4) the importance of allosteric proteins in disease is due, at least partly, to their central positions in protein-protein interaction networks, and less due to their dynamical properties.
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    The structure and function of modular Escherichia coli O157:H7 bacteriophage FTBEc1 endolysin, LysT84: defining a new endolysin catalytic subfamily.
    Love, MJ ; Coombes, D ; Ismail, S ; Billington, C ; Dobson, RCJ (Portland Press Ltd., 2022-01-28)
    Bacteriophage endolysins degrade peptidoglycan and have been identified as antibacterial candidates to combat antimicrobial resistance. Considering the catalytic and structural diversity of endolysins, there is a paucity of structural data to inform how these enzymes work at the molecular level - key data that is needed to realize the potential of endolysin-based antibacterial agents. Here, we determine the atomic structure and define the enzymatic function of Escherichia coli O157:H7 phage FTEBc1 endolysin, LysT84. Bioinformatic analysis reveals that LysT84 is a modular endolysin, which is unusual for Gram-negative endolysins, comprising a peptidoglycan binding domain and an enzymatic domain. The crystal structure of LysT84 (2.99 Å) revealed a mostly α-helical protein with two domains connected by a linker region but packed together. LysT84 was determined to be a monomer in solution using analytical ultracentrifugation. Small-angle X-ray scattering data revealed that LysT84 is a flexible protein but does not have the expected bimodal P(r) function of a multidomain protein, suggesting that the domains of LysT84 pack closely creating a globular protein as seen in the crystal structure. Structural analysis reveals two key glutamate residues positioned on either side of the active site cavity; mutagenesis demonstrating these residues are critical for peptidoglycan degradation. Molecular dynamic simulations suggest that the enzymatically active domain is dynamic, allowing the appropriate positioning of these catalytic residues for hydrolysis of the β(1-4) bond. Overall, our study defines the structural basis for peptidoglycan degradation by LysT84 which supports rational engineering of related endolysins into effective antibacterial agents.
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    microRNA-21-mediated SATB1/S100A9/NF-kappa B axis promotes chronic obstructive pulmonary disease pathogenesis
    Kim, RY ; Sunkara, KP ; Bracke, KR ; Jarnicki, AG ; Donovan, C ; Hsu, AC ; Ieni, A ; Beckett, EL ; Galvao, I ; Wijnant, S ; Ricciardolo, FL ; Di Stefano, A ; Haw, TJ ; Liu, G ; Ferguson, AL ; Palendira, U ; Wark, PA ; Conickx, G ; Mestdagh, P ; Brusselle, GG ; Caramori, G ; Foster, PS ; Horvat, JC ; Hansbro, PM (AMER ASSOC ADVANCEMENT SCIENCE, 2021-11-24)
    Chronic obstructive pulmonary disease (COPD) is the third leading cause of morbidity and death worldwide. Inhalation of cigarette smoke (CS) is the major cause in developed countries. Current therapies have limited efficacy in controlling disease or halting its progression. Aberrant expression of microRNAs (miRNAs) is associated with lung disease, including COPD. We performed miRNA microarray analyses of the lungs of mice with CS-induced experimental COPD. miR-21 was the second highest up-regulated miRNA, particularly in airway epithelium and lung macrophages. Its expression in human lung tissue correlated with reduced lung function in COPD. Prophylactic and therapeutic treatment with a specific miR-21 inhibitor (Ant-21) inhibited CS-induced lung miR-21 expression in mice; suppressed airway macrophages, neutrophils, and lymphocytes; and improved lung function, as evidenced by decreased lung hysteresis, transpulmonary resistance, and tissue damping in mouse models of COPD. In silico analyses identified a potential miR-21/special AT-rich sequence–binding protein 1 (SATB1)/S100 calcium binding protein A9 (S100A9)/nuclear factor κB (NF-κB) axis, which was further investigated. CS exposure reduced lung SATB1 in a mouse model of COPD, whereas Ant-21 treatment restored SATB1 and reduced S100A9 expression and NF-κB activity. The beneficial effects of Ant-21 in mice were reversed by treatment with SATB1-targeting small interfering RNA. We have identified a pathogenic role for a miR-21/SATB1/S100A9/NF-κB axis in COPD and defined miR-21 as a therapeutic target for this disease.
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    Emerging roles for IL-11 in inflammatory diseases
    Fung, KY ; Louis, C ; Metcalfe, RD ; Kosasih, CC ; Wicks, IP ; Griffin, MDW ; Putoczki, TL (ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD, 2021-10-22)
    Interleukin-11 (IL-11) is a cytokine that has been strongly implicated in the pathogenesis of fibrotic diseases and solid malignancies. Elevated IL-11 expression is also associated with several non-malignant inflammatory diseases where its function remains less well-characterized. Here, we summarize current literature surrounding the contribution of IL-11 to the pathogenesis of autoimmune inflammatory diseases, including rheumatoid arthritis, multiple sclerosis, diabetes and systemic sclerosis, as well as other chronic inflammatory conditions such as periodontitis, asthma, chronic obstructive pulmonary disease, psoriasis and colitis.
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    Orexin receptors in GtoPdb v.2021.3.
    Coleman, P ; de Lecea, L ; Gotter, A ; Hagan, J ; Hoyer, D ; Kilduff, T ; Kukkonen, JP ; Porter, R ; Renger, J ; Siegel, JM ; Sutcliffe, G ; Upton, N ; Winrow, CJ (Edinburgh University Library, 2021)
    Orexin receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Orexin receptors [42]) are activated by the endogenous polypeptides orexin-A and orexin-B (also known as hypocretin-1 and -2; 33 and 28 aa) derived from a common precursor, preproorexin or orexin precursor, by proteolytic cleavage and some typical peptide modifications [109]. Currently the only orexin receptor ligands in clinical use are suvorexant and lemborexant, which are used as hypnotics. Orexin receptor crystal structures have been solved [134, 133, 54, 117, 46].