Biochemistry and Pharmacology - Research Publications

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    Structural biology of cell surface receptors implicated in Alzheimer's disease
    Hermans, SJ ; Nero, TL ; Morton, CJ ; Gooi, JH ; Crespi, GAN ; Hancock, NC ; Gao, C ; Ishii, K ; Markulic, J ; Parker, MW (SPRINGERNATURE, 2022-02)
    Alzheimer's disease is a common and devastating age-related disease with no effective disease-modifying treatments. Human genetics has implicated a wide range of cell surface receptors as playing a role in the disease, many of which are involved in the production or clearance of neurotoxins in the brain. Amyloid precursor protein, a membrane-bound signaling molecule, is at the very heart of the disease: hereditary mutations in its gene are associated with a greatly increased risk of getting the disease. A proteolytic breakdown product of amyloid precursor protein, the neurotoxic Aβ peptide, has been the target for many drug discovery efforts. Antibodies have been designed to target Aβ production with some success, although they have not proved efficacious in clinical trials with regards to cognitive benefits to date. Many of the recently identified genes associated with late-onset Alzheimer's disease risk are integral to the innate immune system. Some of these genes code for microglial proteins, such as the strongest genetic risk factor for the disease, namely APOE, and the cell surface receptors CD33 and TREM2 which are involved in clearance of the Aβ peptide from the brain. In this review, we show how structural biology has provided key insights into the normal functioning of these cell surface receptors and provided a framework for developing novel treatments to combat Alzheimer's disease.
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    Cholesterol-dependent cytolysins: The outstanding questions
    Johnstone, BA ; Joseph, R ; Christie, MP ; Morton, CJ ; McGuiness, C ; Walsh, JC ; Bocking, T ; Tweten, RK ; Parker, MW (WILEY, 2022-12)
    The cholesterol-dependent cytolysins (CDCs) are a major family of bacterial pore-forming proteins secreted as virulence factors by Gram-positive bacterial species. CDCs are produced as soluble, monomeric proteins that bind specifically to cholesterol-rich membranes, where they oligomerize into ring-shaped pores of more than 30 monomers. Understanding the details of the steps the toxin undergoes in converting from monomer to a membrane-spanning pore is a continuing challenge. In this review we summarize what we know about CDCs and highlight the remaining outstanding questions that require answers to obtain a complete picture of how these toxins kill cells.
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    Heparin Inhibits SARS-CoV-2 Replication in Human Nasal Epithelial Cells
    Lee, LYY ; Suryadinata, R ; McCafferty, C ; Ignjatovic, V ; Purcell, DFJ ; Robinson, P ; Morton, CJ ; Parker, MW ; Anderson, GP ; Monagle, P ; Subbarao, K ; Neil, JA (MDPI, 2022-12)
    SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Vaccination, supported by social and public health measures, has proven efficacious for reducing disease severity and virus spread. However, the emergence of highly transmissible viral variants that escape prior immunity highlights the need for additional mitigation approaches. Heparin binds the SARS-CoV-2 spike protein and can inhibit virus entry and replication in susceptible human cell lines and bronchial epithelial cells. Primary infection predominantly occurs via the nasal epithelium, but the nasal cell biology of SARS-CoV-2 is not well studied. We hypothesized that prophylactic intranasal administration of heparin may provide strain-agnostic protection for household contacts or those in high-risk settings against SARS-CoV-2 infection. Therefore, we investigated the ability of heparin to inhibit SARS-CoV-2 infection and replication in differentiated human nasal epithelial cells and showed that prolonged exposure to heparin inhibits virus infection. Furthermore, we establish a method for PCR detection of SARS-CoV-2 viral genomes in heparin-treated samples that can be adapted for the detection of viruses in clinical studies.
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    Single-molecule analysis of the entire perfringolysin O pore formation pathway
    Mc Guinness, C ; Walsh, JC ; Bayly-Jones, C ; Dunstone, MA ; Christie, MP ; Morton, CJ ; Parker, MW ; Bocking, T (eLIFE SCIENCES PUBL LTD, 2022-08-24)
    The cholesterol-dependent cytolysin perfringolysin O (PFO) is secreted by Clostridium perfringens as a bacterial virulence factor able to form giant ring-shaped pores that perforate and ultimately lyse mammalian cell membranes. To resolve the kinetics of all steps in the assembly pathway, we have used single-molecule fluorescence imaging to follow the dynamics of PFO on dye-loaded liposomes that lead to opening of a pore and release of the encapsulated dye. Formation of a long-lived membrane-bound PFO dimer nucleates the growth of an irreversible oligomer. The growing oligomer can insert into the membrane and open a pore at stoichiometries ranging from tetramers to full rings (~35 mers), whereby the rate of insertion increases linearly with the number of subunits. Oligomers that insert before the ring is complete continue to grow by monomer addition post insertion. Overall, our observations suggest that PFO membrane insertion is kinetically controlled.
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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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    Reaction hijacking of tyrosine tRNA synthetase as a new whole-of-life-cycle antimalarial strategy
    Xie, SC ; Metcalfe, RD ; Dunn, E ; Morton, CJ ; Huang, S-C ; Puhalovich, T ; Du, Y ; Wittlin, S ; Nie, S ; Luth, MR ; Ma, L ; Kim, M-S ; Pasaje, CFA ; Kumpornsin, K ; Giannangelo, C ; Houghton, FJ ; Churchyard, A ; Famodimu, MT ; Barry, DC ; Gillett, DL ; Dey, S ; Kosasih, CC ; Newman, W ; Niles, JC ; Lee, MCS ; Baum, J ; Ottilie, S ; Winzeler, EA ; Creek, DJ ; Williamson, N ; Parker, MW ; Brand, S ; Langston, SP ; Dick, LR ; Griffin, MDW ; Gould, AE ; Tilley, L (AMER ASSOC ADVANCEMENT SCIENCE, 2022-06-03)
    Aminoacyl transfer RNA (tRNA) synthetases (aaRSs) are attractive drug targets, and we present class I and II aaRSs as previously unrecognized targets for adenosine 5'-monophosphate-mimicking nucleoside sulfamates. The target enzyme catalyzes the formation of an inhibitory amino acid-sulfamate conjugate through a reaction-hijacking mechanism. We identified adenosine 5'-sulfamate as a broad-specificity compound that hijacks a range of aaRSs and ML901 as a specific reagent a specific reagent that hijacks a single aaRS in the malaria parasite Plasmodium falciparum, namely tyrosine RS (PfYRS). ML901 exerts whole-life-cycle-killing activity with low nanomolar potency and single-dose efficacy in a mouse model of malaria. X-ray crystallographic studies of plasmodium and human YRSs reveal differential flexibility of a loop over the catalytic site that underpins differential susceptibility to reaction hijacking by ML901.
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    The structure of the extracellular domains of human interleukin 11? receptor reveals mechanisms of cytokine engagement
    Metcalfe, RD ; Aizel, K ; Zlatic, CO ; Nguyen, PM ; Morton, CJ ; Lio, DS-S ; Cheng, H-C ; Dobson, RCJ ; Parker, MW ; Gooley, PR ; Putoczki, TL ; Griffin, MDW (AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2020-06-12)
    Interleukin (IL) 11 activates multiple intracellular signaling pathways by forming a complex with its cell surface α-receptor, IL-11Rα, and the β-subunit receptor, gp130. Dysregulated IL-11 signaling has been implicated in several diseases, including some cancers and fibrosis. Mutations in IL-11Rα that reduce signaling are also associated with hereditary cranial malformations. Here we present the first crystal structure of the extracellular domains of human IL-11Rα and a structure of human IL-11 that reveals previously unresolved detail. Disease-associated mutations in IL-11Rα are generally distal to putative ligand-binding sites. Molecular dynamics simulations showed that specific mutations destabilize IL-11Rα and may have indirect effects on the cytokine-binding region. We show that IL-11 and IL-11Rα form a 1:1 complex with nanomolar affinity and present a model of the complex. Our results suggest that the thermodynamic and structural mechanisms of complex formation between IL-11 and IL-11Rα differ substantially from those previously reported for similar cytokines. This work reveals key determinants of the engagement of IL-11 by IL-11Rα that may be exploited in the development of strategies to modulate formation of the IL-11-IL-11Rα complex.
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    Design of proteasome inhibitors with oral efficacy in vivo against Plasmodium falciparum and selectivity over the human proteasome
    Xie, SC ; Metcalfe, RD ; Mizutani, H ; Puhalovich, T ; Hanssen, E ; Morton, CJ ; Du, Y ; Dogovski, C ; Huang, S-C ; Ciavarri, J ; Hales, P ; Griffin, RJ ; Cohen, LH ; Chuang, B-C ; Wittlin, S ; Deni, I ; Yeo, T ; Ward, KE ; Barry, DC ; Liu, B ; Gillett, DL ; Crespo-Fernandez, BF ; Ottilie, S ; Mittal, N ; Churchyard, A ; Ferguson, D ; Aguiar, ACC ; Guido, RVC ; Baum, J ; Hanson, KK ; Winzeler, EA ; Gamo, F-J ; Fidock, DA ; Baud, D ; Parker, MW ; Brand, S ; Dick, LR ; Griffin, MDW ; Gould, AE ; Tilley, L (NATL ACAD SCIENCES, 2021-09-28)
    The Plasmodium falciparum proteasome is a potential antimalarial drug target. We have identified a series of amino-amide boronates that are potent and specific inhibitors of the P. falciparum 20S proteasome (Pf20S) β5 active site and that exhibit fast-acting antimalarial activity. They selectively inhibit the growth of P. falciparum compared with a human cell line and exhibit high potency against field isolates of P. falciparum and Plasmodium vivax They have a low propensity for development of resistance and possess liver stage and transmission-blocking activity. Exemplar compounds, MPI-5 and MPI-13, show potent activity against P. falciparum infections in a SCID mouse model with an oral dosing regimen that is well tolerated. We show that MPI-5 binds more strongly to Pf20S than to human constitutive 20S (Hs20Sc). Comparison of the cryo-electron microscopy (EM) structures of Pf20S and Hs20Sc in complex with MPI-5 and Pf20S in complex with the clinically used anti-cancer agent, bortezomib, reveal differences in binding modes that help to explain the selectivity. Together, this work provides insights into the 20S proteasome in P. falciparum, underpinning the design of potent and selective antimalarial proteasome inhibitors.
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    A Key Motif in the Cholesterol-Dependent Cytolysins Reveals a Large Family of Related Proteins
    Evans, JC ; Johnstone, BA ; Lawrence, SL ; Morton, CJ ; Christie, MP ; Parker, MW ; Tweten, RK ; McClane, BA (AMER SOC MICROBIOLOGY, 2020-09-29)
    The cholesterol-dependent cytolysins (CDCs) are bacterial, β-barrel, pore-forming toxins. A central enigma of the pore-forming mechanism is how completion of the prepore is sensed to initiate its conversion to the pore. We identified a motif that is conserved between the CDCs and a diverse family of nearly 300 uncharacterized proteins present in over 220 species that span at least 10 bacterial and 2 eukaryotic phyla. Except for this motif, these proteins exhibit little similarity to the CDCs at the primary structure level. Studies herein show this motif is a critical component of the sensor that initiates the prepore-to-pore transition in the CDCs. We further show by crystallography, single particle analysis, and biochemical studies of one of these CDC-like (CDCL) proteins from Elizabethkingia anophelis, a commensal of the malarial mosquito midgut, that a high degree of structural similarity exists between the CDC and CDCL monomer structures and both form large oligomeric pore complexes. Furthermore, the conserved motif in the E. anophelis CDCL crystal structure occupies a nearly identical position and makes similar contacts to those observed in the structure of the archetype CDC, perfringolysin O (PFO). This suggests a common function in the CDCs and CDCLs and may explain why only this motif is conserved in the CDCLs. Hence, these studies identify a critical component of the sensor involved in initiating the prepore-to-pore transition in the CDCs, which is conserved in a large and diverse group of distant relatives of the CDCs.IMPORTANCE The cholesterol-dependent cytolysins' pore-forming mechanism relies on the ability to sense the completion of the oligomeric prepore structure and initiate the insertion of the β-barrel pore from the assembled prepore structure. These studies show that a conserved motif is an important component of the sensor that triggers the prepore-to-pore transition and that it is conserved in a large family of previously unidentified CDC-like proteins, the genes for which are present in a vast array of microbial species that span most terrestrial environments, as well as most animal and human microbiomes. These studies establish the foundation for future investigations that will probe the contribution of this large family of CDC-like proteins to microbial survival and human disease.