Biochemistry and Pharmacology - Research Publications

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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.
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    Transitional changes in the CRP structure lead to the exposure of proinflammatory binding sites
    Braig, D ; Nero, TL ; Koch, H-G ; Kaiser, B ; Wang, X ; Thiele, JR ; Morton, CJ ; Zeller, J ; Kiefer, J ; Potempa, LA ; Mellett, NA ; Miles, LA ; Du, X-J ; Meikle, PJ ; Huber-Lang, M ; Stark, GB ; Parker, MW ; Peter, K ; Eisenhardt, SU (NATURE PUBLISHING GROUP, 2017-01-23)
    C-reactive protein (CRP) concentrations rise in response to tissue injury or infection. Circulating pentameric CRP (pCRP) localizes to damaged tissue where it leads to complement activation and further tissue damage. In-depth knowledge of the pCRP activation mechanism is essential to develop therapeutic strategies to minimize tissue injury. Here we demonstrate that pCRP by binding to cell-derived microvesicles undergoes a structural change without disrupting the pentameric symmetry (pCRP*). pCRP* constitutes the major CRP species in human-inflamed tissue and allows binding of complement factor 1q (C1q) and activation of the classical complement pathway. pCRP*-microvesicle complexes lead to enhanced recruitment of leukocytes to inflamed tissue. A small-molecule inhibitor of pCRP (1,6-bis(phosphocholine)-hexane), which blocks the pCRP-microvesicle interactions, abrogates these proinflammatory effects. Reducing inflammation-mediated tissue injury by therapeutic inhibition might improve the outcome of myocardial infarction, stroke and other inflammatory conditions.
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    Potent hepatitis C inhibitors bind directly to NS5A and reduce its affinity for RNA
    Ascher, DB ; Wielens, J ; Nero, TL ; Doughty, L ; Morton, CJ ; Parker, MW (NATURE PUBLISHING GROUP, 2014-04-23)
    Hepatitis C virus (HCV) infection affects more than 170 million people. The high genetic variability of HCV and the rapid development of drug-resistant strains are driving the urgent search for new direct-acting antiviral agents. A new class of agents has recently been developed that are believed to target the HCV protein NS5A although precisely where they interact and how they affect function is unknown. Here we describe an in vitro assay based on microscale thermophoresis and demonstrate that two clinically relevant inhibitors bind tightly to NS5A domain 1 and inhibit RNA binding. Conversely, RNA binding inhibits compound binding. The compounds bind more weakly to known resistance mutants L31V and Y93H. The compounds do not affect NS5A dimerisation. We propose that current NS5A inhibitors act by favouring a dimeric structure of NS5A that does not bind RNA.