Biochemistry and Pharmacology - Research Publications
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ItemNo Preview AvailableMolecular Basis of Functional Effects of Phosphorylation of the C-Terminal Domain of the Rabies Virus P Protein(AMER SOC MICROBIOLOGY, 2022-05-11)The rabies virus (RABV) phosphoprotein (P protein) is expressed as several isoforms, which differ in nucleocytoplasmic localization and microtubule (MT) association, mediated by several sequences, including nuclear localization (NLS) and export (NES) sequences. This appears to underpin a functional diversity enabling multiple functions in viral replication and modulation of host biology. Mechanisms regulating trafficking are poorly defined, but phosphorylation by protein kinase C (PKC) in the P protein C-terminal domain (PCTD) regulates nuclear trafficking, mediated by PCTD-localized NLS/NES sequences, indicating that phosphorylation contributes to functional diversity. The molecular mechanism underlying the effects of PKC, and potential roles in regulating other host-cell interactions are unresolved. Here, we assess effects of phosphorylation on the P3 isoform, which differs from longer isoforms through an ability to localize to the nucleus and associate with MTs, which are associated with antagonism of interferon (IFN) signaling. We find that phosphomimetic mutation of the PKC site S210 inhibits nuclear accumulation and MT association/bundling. Structural analysis indicated that phosphomimetic mutation induces no significant structural change to the NLS/NES but results in the side chain of N226 switching its interactions from E228, within the NES, to E210. Intriguingly, N226 is the sole substituted residue between the PCTD of the pathogenic IFN-resistant RABV strain Nishigahara and a derivative attenuated IFN-sensitive strain Ni-CE, inhibiting P3 nuclear localization and MT association. Thus, S210 phosphorylation appears to impact on N226/E228 to regulate P protein localization, with N226 mutation in Ni-CE mimicking a constitutively phosphorylated state resulting in IFN sensitivity and attenuation. IMPORTANCE Rabies virus P protein is a multifunctional protein with critical roles in replication and manipulation of host-cell processes, including subversion of immunity. This functional diversity involves interactions of several P protein isoforms with the cell nucleus and microtubules. Previous studies showed that phosphorylation of the P protein C-terminal domain (PCTD) at S210, near nuclear trafficking sequences, regulates nucleocytoplasmic localization, indicating key roles in functional diversity. The molecular mechanisms of this regulation have remained unknown. Here, we show that phosphomimetic mutation of S210 regulates nuclear localization and MT association. This regulation does not appear to result from disrupted PCTD structure, but rather from a switch of specific side chain interactions of N226. Intriguingly, N226 was previously implicated in P protein nuclear localization/MT association, immune evasion, and RABV pathogenesis, through undefined mechanisms. Our data indicate that the S210-N226 interface is a key regulator of virus-host interactions, which is significant for pathogenesis.
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ItemThe Monomeric α-Crystallin Domain of the Small Heat-shock Proteins αB-crystallin and Hsp27 Binds Amyloid Fibril Ends(ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD, 2022-08-30)Small heat-shock proteins (sHSPs) are ubiquitously expressed molecular chaperones present in all kingdoms of life that inhibit protein misfolding and aggregation. Despite their importance in proteostasis, the structure-function relationships of sHSPs remain elusive. Human sHSPs are characterised by a central, highly conserved α-crystallin domain (ACD) and variable-length N- and C-terminal regions. The ACD forms antiparallel homodimers via an extended β-strand, creating a shared β-sheet at the dimer interface. The N- and C-terminal regions mediate formation of higher order oligomers that are thought to act as storage forms for chaperone-active dimers. We investigated the interactions of the ACD of two human sHSPs, αB-crystallin (αB-C) and Hsp27, with apolipoprotein C-II amyloid fibrils using analytical ultracentrifugation and nuclear magnetic resonance spectroscopy. The ACD was found to interact transiently with amyloid fibrils to inhibit fibril elongation and naturally occurring fibril end-to-end joining. This interaction was sensitive to the concentration of fibril ends indicating a 'fibril-capping' interaction. Furthermore, resonances arising from the ACD monomer were attenuated to a greater extent than those of the ACD dimer in the presence of fibrils, suggesting that the monomer may bind fibrils. This hypothesis was supported by mutagenesis studies in which disulfide cross-linked ACD dimers formed by both αB-C and Hsp27 were less effective at inhibiting amyloid fibril elongation and fibril end-to-end joining than ACD constructs lacking disulfide cross-linking. Our results indicate that sHSP monomers inhibit amyloid fibril elongation, highlighting the importance of the dynamic oligomeric nature of sHSPs for client binding.
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ItemNo Preview AvailableEmerging roles for IL-11 in inflammatory diseases(ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD, 2022-01)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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ItemReaction hijacking of tyrosine tRNA synthetase as a new whole-of-life-cycle antimalarial strategy(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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ItemThe structure of the extracellular domains of human interleukin 11? receptor reveals mechanisms of cytokine engagement(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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ItemDesign of proteasome inhibitors with oral efficacy in vivo against Plasmodium falciparum and selectivity over the human proteasome(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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ItemMechanism of NanR gene repression and allosteric induction of bacterial sialic acid metabolism(NATURE PORTFOLIO, 2021-03-31)Bacteria respond to environmental changes by inducing transcription of some genes and repressing others. Sialic acids, which coat human cell surfaces, are a nutrient source for pathogenic and commensal bacteria. The Escherichia coli GntR-type transcriptional repressor, NanR, regulates sialic acid metabolism, but the mechanism is unclear. Here, we demonstrate that three NanR dimers bind a (GGTATA)3-repeat operator cooperatively and with high affinity. Single-particle cryo-electron microscopy structures reveal the DNA-binding domain is reorganized to engage DNA, while three dimers assemble in close proximity across the (GGTATA)3-repeat operator. Such an interaction allows cooperative protein-protein interactions between NanR dimers via their N-terminal extensions. The effector, N-acetylneuraminate, binds NanR and attenuates the NanR-DNA interaction. The crystal structure of NanR in complex with N-acetylneuraminate reveals a domain rearrangement upon N-acetylneuraminate binding to lock NanR in a conformation that weakens DNA binding. Our data provide a molecular basis for the regulation of bacterial sialic acid metabolism.
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ItemProbing the correlation between ligand efficacy and conformational diversity at the ?(1A)-adrenoreceptor reveals allosteric coupling of its microswitches(American Society for Biochemistry and Molecular Biology, 2020-05-22)G protein–coupled receptors (GPCRs) use a series of conserved microswitches to transmit signals across the cell membrane via an allosteric network encompassing the ligand-binding site and the G protein-binding site. Crystal structures of GPCRs provide snapshots of their inactive and active states, but poorly describe the conformational dynamics of the allosteric network that underlies GPCR activation. Here, we analyzed the correlation between ligand binding and receptor conformation of the α1A-adrenoreceptor, a GPCR that stimulates smooth muscle contraction in response to binding noradrenaline. NMR of [13CϵH3]methionine-labeled α1A-adrenoreceptor variants, each exhibiting differing signaling capacities, revealed how different classes of ligands modulate the conformational equilibria of this receptor. [13CϵH3]Methionine residues near the microswitches exhibited distinct states that correlated with ligand efficacies, supporting a conformational selection mechanism. We propose that allosteric coupling among the microswitches controls the conformation of the α1A-adrenoreceptor and underlies the mechanism of ligand modulation of GPCR signaling in cells.
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ItemEmerging roles for the IL-6 family of cytokines in pancreatic cancer(PORTLAND PRESS LTD, 2020-08)Pancreatic cancer has one of the poorest prognoses of all malignancies, with little improvement in clinical outcome over the past 40 years. Pancreatic ductal adenocarcinoma is responsible for the vast majority of pancreatic cancer cases, and is characterised by the presence of a dense stroma that impacts therapeutic efficacy and drives pro-tumorigenic programs. More specifically, the inflammatory nature of the tumour microenvironment is thought to underlie the loss of anti-tumour immunity and development of resistance to current treatments. Inflammatory pathways are largely mediated by the expression of, and signalling through, cytokines, chemokines, and other cellular messengers. In recent years, there has been much attention focused on dual targeting of cancer cells and the tumour microenvironment. Here we review our current understanding of the role of IL-6, and the broader IL-6 cytokine family, in pancreatic cancer, including their contribution to pancreatic inflammation and various roles in pancreatic cancer pathogenesis. We also summarise potential opportunities for therapeutic targeting of these pathways as an avenue towards combating poor patient outcomes.
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ItemStructural Understanding of Interleukin 6 Family Cytokine Signaling and Targeted Therapies: Focus on Interleukin 11(FRONTIERS MEDIA SA, 2020-07-16)Cytokines are small signaling proteins that have central roles in inflammation and cell survival. In the half-century since the discovery of the first cytokines, the interferons, over fifty cytokines have been identified. Amongst these is interleukin (IL)-6, the first and prototypical member of the IL-6 family of cytokines, nearly all of which utilize the common signaling receptor, gp130. In the last decade, there have been numerous advances in our understanding of the structural mechanisms of IL-6 family signaling, particularly for IL-6 itself. However, our understanding of the detailed structural mechanisms underlying signaling by most IL-6 family members remains limited. With the emergence of new roles for IL-6 family cytokines in disease and, in particular, roles of IL-11 in cardiovascular disease, lung disease, and cancer, there is an emerging need to develop therapeutics that can progress to clinical use. Here we outline our current knowledge of the structural mechanism of signaling by the IL-6 family of cytokines. We discuss how this knowledge allows us to understand the mechanism of action of currently available inhibitors targeting IL-6 family cytokine signaling, and most importantly how it allows for improved opportunities to pharmacologically disrupt cytokine signaling. We focus specifically on the need to develop and understand inhibitors that disrupt IL-11 signaling.