Biochemistry and Pharmacology - Research Publications

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    Distinct Assemblies of Heterodimeric Cytokine Receptors Govern Stemness Programs in Leukemia
    Kan, WL ; Dhagat, U ; Kaufmann, KB ; Hercus, TR ; Nero, TL ; Zeng, AGX ; Toubia, J ; Barry, EF ; Broughton, SE ; Gomez, GA ; Benard, BA ; Dottore, M ; Shing, KS ; Boutzen, H ; Samaraweera, SE ; Simpson, KJ ; Jin, L ; Goodall, GJ ; Begley, CG ; Thomas, D ; Ekert, PG ; Tvorogov, D ; D'Andrea, RJ ; Dick, JE ; Parker, MW ; Lopez, AF (AMER ASSOC CANCER RESEARCH, 2023-08-04)
    UNLABELLED: Leukemia stem cells (LSC) possess distinct self-renewal and arrested differentiation properties that are responsible for disease emergence, therapy failure, and recurrence in acute myeloid leukemia (AML). Despite AML displaying extensive biological and clinical heterogeneity, LSC with high interleukin-3 receptor (IL3R) levels are a constant yet puzzling feature, as this receptor lacks tyrosine kinase activity. Here, we show that the heterodimeric IL3Rα/βc receptor assembles into hexamers and dodecamers through a unique interface in the 3D structure, where high IL3Rα/βc ratios bias hexamer formation. Importantly, receptor stoichiometry is clinically relevant as it varies across the individual cells in the AML hierarchy, in which high IL3Rα/βc ratios in LSCs drive hexamer-mediated stemness programs and poor patient survival, while low ratios mediate differentiation. Our study establishes a new paradigm in which alternative cytokine receptor stoichiometries differentially regulate cell fate, a signaling mechanism that may be generalizable to other transformed cellular hierarchies and of potential therapeutic significance. SIGNIFICANCE: Stemness is a hallmark of many cancers and is largely responsible for disease emergence, progression, and relapse. Our finding that clinically significant stemness programs in AML are directly regulated by different stoichiometries of cytokine receptors represents a hitherto unexplained mechanism underlying cell-fate decisions in cancer stem cell hierarchies. This article is highlighted in the In This Issue feature, p. 1749.
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    A novel phosphocholine-mimetic inhibits a pro-inflammatory conformational change in C-reactive protein
    Zeller, J ; Shing, KSCT ; Nero, TL ; McFadyen, JD ; Krippner, G ; Bogner, B ; Kreuzaler, S ; Kiefer, J ; Horner, VK ; Braig, D ; Danish, H ; Baratchi, S ; Fricke, M ; Wang, X ; Kather, MG ; Kammerer, B ; Woollard, KJ ; Sharma, P ; Morton, CJ ; Pietersz, G ; Parker, MW ; Peter, K ; Eisenhardt, SU (WILEY, 2023-01-11)
    C-reactive protein (CRP) is an early-stage acute phase protein and highly upregulated in response to inflammatory reactions. We recently identified a novel mechanism that leads to a conformational change from the native, functionally relatively inert, pentameric CRP (pCRP) structure to a pentameric CRP intermediate (pCRP*) and ultimately to the monomeric CRP (mCRP) form, both exhibiting highly pro-inflammatory effects. This transition in the inflammatory profile of CRP is mediated by binding of pCRP to activated/damaged cell membranes via exposed phosphocholine lipid head groups. We designed a tool compound as a low molecular weight CRP inhibitor using the structure of phosphocholine as a template. X-ray crystallography revealed specific binding to the phosphocholine binding pockets of pCRP. We provide in vitro and in vivo proof-of-concept data demonstrating that the low molecular weight tool compound inhibits CRP-driven exacerbation of local inflammatory responses, while potentially preserving pathogen-defense functions of CRP. The inhibition of the conformational change generating pro-inflammatory CRP isoforms via phosphocholine-mimicking compounds represents a promising, potentially broadly applicable anti-inflammatory therapy.
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    C-reactive protein, immunothrombosis and venous thromboembolism
    Dix, C ; Zeller, J ; Stevens, H ; Eisenhardt, SU ; Shing, KSCT ; Nero, TL ; Morton, CJ ; Parker, MW ; Peter, K ; McFadyen, JD (FRONTIERS MEDIA SA, 2022-09-13)
    C-reactive protein (CRP) is a member of the highly conserved pentraxin superfamily of proteins and is often used in clinical practice as a marker of infection and inflammation. There is now increasing evidence that CRP is not only a marker of inflammation, but also that destabilized isoforms of CRP possess pro-inflammatory and pro-thrombotic properties. CRP circulates as a functionally inert pentameric form (pCRP), which relaxes its conformation to pCRP* after binding to phosphocholine-enriched membranes and then dissociates to monomeric CRP (mCRP). with the latter two being destabilized isoforms possessing highly pro-inflammatory features. pCRP* and mCRP have significant biological effects in regulating many of the aspects central to pathogenesis of atherothrombosis and venous thromboembolism (VTE), by directly activating platelets and triggering the classical complement pathway. Importantly, it is now well appreciated that VTE is a consequence of thromboinflammation. Accordingly, acute VTE is known to be associated with classical inflammatory responses and elevations of CRP, and indeed VTE risk is elevated in conditions associated with inflammation, such as inflammatory bowel disease, COVID-19 and sepsis. Although the clinical data regarding the utility of CRP as a biomarker in predicting VTE remains modest, and in some cases conflicting, the clinical utility of CRP appears to be improved in subsets of the population such as in predicting VTE recurrence, in cancer-associated thrombosis and in those with COVID-19. Therefore, given the known biological function of CRP in amplifying inflammation and tissue damage, this raises the prospect that CRP may play a role in promoting VTE formation in the context of concurrent inflammation. However, further investigation is required to unravel whether CRP plays a direct role in the pathogenesis of VTE, the utility of which will be in developing novel prophylactic or therapeutic strategies to target thromboinflammation.
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    The mechanism of GM-CSF inhibition by human GM-CSF auto-antibodies suggests novel therapeutic opportunities
    Dhagat, U ; Hercus, TR ; Broughton, SE ; Nero, TL ; Shing, KSCT ; Barry, EF ; Thomson, CA ; Bryson, S ; Pai, EF ; McClure, BJ ; Schrader, JW ; Lopez, AF ; Parker, MW (TAYLOR & FRANCIS INC, 2018)
    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor that can stimulate a variety of cells, but its overexpression leads to excessive production and activation of granulocytes and macrophages with many pathogenic effects. This cytokine is a therapeutic target in inflammatory diseases, and several anti-GM-CSF antibodies have advanced to Phase 2 clinical trials in patients with such diseases, e.g., rheumatoid arthritis. GM-CSF is also an essential factor in preventing pulmonary alveolar proteinosis (PAP), a disease associated with GM-CSF malfunction arising most typically through the presence of GM-CSF neutralizing auto-antibodies. Understanding the mechanism of action for neutralizing antibodies that target GM-CSF is important for improving their specificity and affinity as therapeutics and, conversely, in devising strategies to reduce the effects of GM-CSF auto-antibodies in PAP. We have solved the crystal structures of human GM-CSF bound to antigen-binding fragments of two neutralizing antibodies, the human auto-antibody F1 and the mouse monoclonal antibody 4D4. Coordinates and structure factors of the crystal structures of the GM-CSF:F1 Fab and the GM-CSF:4D4 Fab complexes have been deposited in the RCSB Protein Data Bank under the accession numbers 6BFQ and 6BFS, respectively. The structures show that these antibodies bind to mutually exclusive epitopes on GM-CSF; however, both prevent the cytokine from interacting with its alpha receptor subunit and hence prevent receptor activation. Importantly, identification of the F1 epitope together with functional analyses highlighted modifications to GM-CSF that would abolish auto-antibody recognition whilst retaining GM-CSF function. These results provide a framework for developing novel GM-CSF molecules for PAP treatment and for optimizing current anti-GM-CSF antibodies for use in treating inflammatory disorders.
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    EPO does not promote interaction between the erythropoietin and beta-common receptors (vol 8, 12457, 2018)
    Shing, KSCT ; Broughton, SE ; Nero, TL ; Gillinder, K ; Ilsley, MD ; Ramshaw, H ; Lopez, AF ; Griffin, MDW ; Parker, MW ; Perkins, AC ; Dhagat, U (NATURE PUBLISHING GROUP, 2019-05-21)
    A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
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    EPO does not promote interaction between the erythropoietin and beta-common receptors
    Shing, KSCT ; Broughton, SE ; Nero, TL ; Gillinder, K ; Ilsley, MD ; Ramshaw, H ; Lopez, AF ; Griffin, MDW ; Parker, MW ; Perkins, AC ; Dhagat, U (NATURE PUBLISHING GROUP, 2018-08-20)
    A direct interaction between the erythropoietin (EPOR) and the beta-common (βc) receptors to form an Innate Repair Receptor (IRR) is controversial. On one hand, studies have shown a functional link between EPOR and βc receptor in tissue protection while others have shown no involvement of the βc receptor in tissue repair. To date there is no biophysical evidence to confirm a direct association of the two receptors either in vitro or in vivo. We investigated the existence of an interaction between the extracellular regions of EPOR and the βc receptor in silico and in vitro (either in the presence or absence of EPO or EPO-derived peptide ARA290). Although a possible interaction between EPOR and βc was suggested by our computational and genomic studies, our in vitro biophysical analysis demonstrates that the extracellular regions of the two receptors do not specifically associate. We also explored the involvement of the βc receptor gene (Csf2rb) under anaemic stress conditions and found no requirement for the βc receptor in mice. In light of these studies, we conclude that the extracellular regions of the EPOR and the βc receptor do not directly interact and that the IRR is not involved in anaemic stress.
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    A dual role for the N-terminal domain of the IL-3 receptor in cell signalling
    Broughton, SE ; Hercus, TR ; Nero, TL ; Kan, WL ; Barry, EF ; Dottore, M ; Shing, KSCT ; Morton, CJ ; Dhagat, U ; Hardy, MP ; Wilson, NJ ; Downton, MT ; Schieber, C ; Hughes, TP ; Lopez, AF ; Parker, MW (NATURE PUBLISHING GROUP, 2018-01-26)
    The interleukin-3 (IL-3) receptor is a cell-surface heterodimer that links the haemopoietic, vascular and immune systems and is overexpressed in acute and chronic myeloid leukaemia progenitor cells. It belongs to the type I cytokine receptor family in which the α-subunits consist of two fibronectin III-like domains that bind cytokine, and a third, evolutionarily unrelated and topologically conserved, N-terminal domain (NTD) with unknown function. Here we show by crystallography that, while the NTD of IL3Rα is highly mobile in the presence of IL-3, it becomes surprisingly rigid in the presence of IL-3 K116W. Mutagenesis, biochemical and functional studies show that the NTD of IL3Rα regulates IL-3 binding and signalling and reveal an unexpected role in preventing spontaneous receptor dimerisation. Our work identifies a dual role for the NTD in this cytokine receptor family, protecting against inappropriate signalling and dynamically regulating cytokine receptor binding and function.