James M Murphy

University of Melbourne, Melbourne, Victoria, Australia

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Publications (60)371.92 Total impact

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    Kelan Chen · Peter E Czabotar · Marnie E Blewitt · James M Murphy
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    ABSTRACT: The structural maintenance of chromosome (SMC) proteins are fundamental to chromosome organization. They share a characteristic domain structure, featuring a central SMC hinge domain that is critical for forming SMC dimers and interacting with nucleic acids. The structural maintenance of chromosomes flexible hinge domain containing 1 (Smchd1) is a noncanonical member of the SMC family. While it has been well established that Smchd1 serves crucial roles in epigenetic silencing events implicated in development and disease, much less is known about the structure and function of Smchd1 protein. Recently, we demonstrated that the C-terminal hinge domain of Smchd1 forms a nucleic acid-binding homodimer, however, it is unclear how the protomers are assembled within the hinge homodimer and how the full-length Smchd1 protein is organised with respect to the hinge region. Here, by employing small-angle X-ray scattering (SAXS) we demonstrate that the hinge domain of Smchd1 likely adopts an unconventional homodimeric arrangement augmented by a coiled-coil formed between the two monomers. Such a dimeric structure differs markedly from that of archetypical SMC proteins, raising the possibility that Smchd1 binds chromatin in an unconventional manner.
    Preview · Article · Jan 2016 · Biochemical Journal
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    ABSTRACT: The pseudokinase mixed lineage kinase domain-like (MLKL) is an essential effector of necroptotic cell death. Two distinct human MLKL isoforms have previously been reported, but their capacities to trigger cell death have not been compared directly. Herein, we examine these two MLKL isoforms, and further probe the features of the human MLKL N-terminal domain that are required for cell death. Expression in HEK293T cells of the N-terminal 201 amino acids of human MLKL is sufficient to cause cell death, whereas expression of the first 154 amino acids is not. Given that amino acids 1-125 are able to initiate necroptosis, our findings indicate that the helix that follows this region restrains necroptotic activity, which is again restored in longer constructs. Furthermore, MLKL isoform 2 (MLKL2), which lacks much of the regulatory pseudokinase domain, is a much more potent inducer of cell death than MLKL isoform 1 (MLKL1) in ectopic expression studies in HEK293T cells. Modelling predicts that a C-terminal helix constrains the activity of MLKL1, but not MLKL2. Although both isoforms are expressed by human-monocyte derived macrophages at the mRNA level, MLKL2 is expressed at much lower levels. We propose that it may have a regulatory role in controlling macrophage survival, either in the steady state or in response to specific stimuli.
    Full-text · Article · Dec 2015 · Bioscience Reports
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    James M. Murphy · James E. Vince

    Preview · Article · Nov 2015 · F1000 Research
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    ABSTRACT: CCAAT-enhancer binding proteins (C/EBPs) are transcription factors that play a central role in the differentiation of myeloid cells and adipocytes. Tribbles pseudokinases govern levels of C/EBPs by recruiting them to the COP1 ubiquitin ligase for ubiquitination. Here, we present the first crystal structure of a Tribbles protein, which reveals a catalytically inactive TRIB1 pseudokinase domain with a unique adaptation in the αC helix. A second crystal structure and biophysical studies of TRIB1 with its C-terminal extension, which includes the COP1-binding motif, show that the C-terminal extension is sequestered at a site formed by the modified TRIB1 αC helix. In addition, we have identified and characterized the TRIB1 substrate-recognition sequence within C/EBPα, which is evolutionarily conserved in C/EBP transcription factors. Binding studies indicate that C/EBPα recruitment is weaker in the presence of the C-terminal COP1-binding motif, but the magnitude of this effect suggests that the two bind distinct rather directly overlapping binding sites.
    No preview · Article · Oct 2015 · Structure
  • Peter E Czabotar · James M Murphy
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    ABSTRACT: Recently, the programmed necrosis or "necroptosis" cell death pathway has attracted much interest because of its implication in multiple pathologies, including inflammatory diseases and the cell death arising from ischemia reperfusion injuries. Pharmacologically, necroptosis is an attractive target, because, unlike the counterpart pathway, apoptosis, it is dispensable for mammalian development. In particular, the most terminal-known obligate effector in the necroptosis pathway, the pseudokinase MLKL (mixed lineage kinase domain-like), holds particular appeal because, thus far, its only known function is as a mediator of necroptotic cell death. Here, we review the current understanding and gaps in knowledge relating to how MLKL can be activated by RIPK3 downstream of TNF receptor 1:RIPK1, Toll like receptor-3:TRIF and viral DNA:DAI/ZBF1. We also discuss the potential mechanism(s) by which MLKL induces necroptotic cell death, with particular emphasis on insights arising from structural studies of mouse and human MLKL. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    No preview · Article · Sep 2015 · FEBS Journal
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    ABSTRACT: The pseudokinase, MLKL (mixed lineage kinase domain-like), has recently emerged as a critical component of the necroptosis cell death pathway. While it is clear that phosphorylation of the activation loop in the MLKL pseudokinase domain by the upstream protein kinase, RIPK3 (receptor interacting protein kinase-3), is crucial to trigger MLKL activation, it has remained unclear whether other phosphorylation events modulate MLKL function. By reconstituting Mlkl(-/-) , Ripk3(-/-) and Mlkl(-/-)Ripk3(-/-) cells with MLKL phospho-site mutants, we compared the function of known MLKL phosphorylation sites in regulating necroptosis with three phospho-sites that we identified by mass spectrometry, S158, S228 and S248. Expression of a phospho-mimetic S345D MLKL activation loop mutant induced stimulus-independent cell death in all knockout cells, demonstrating that RIPK3 phosphorylation of the activation loop of MLKL is sufficient to induce cell death. Cell death was also induced by S228A, S228E and S158A MLKL mutants in the absence of death stimuli, but was most profound in Mlkl (-/-) Ripk3(-/-) double knockout fibroblasts . These data reveal a potential role for RIPK3 as a suppressor of MLKL activation and indicate that phosphorylation can fine-tune the ability of MLKL to induce necroptosis. Copyright 2015 The Author(s).
    No preview · Article · Aug 2015 · Biochemical Journal

  • No preview · Conference Paper · Aug 2015
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    ABSTRACT: SOCS5 can negatively regulate both JAK/STAT and EGF-receptor pathways and has therefore been implicated in regulating both the immune response and tumorigenesis. Understanding the molecular basis for SOCS5 activity may reveal novel ways to target key components of these signaling pathways. The N-terminal region of SOCS5 coordinates critical protein interactions involved in inhibition of JAK/STAT signaling, and a conserved region within the N-terminus of SOCS5 mediates direct binding to the JAK kinase domain. Here we have characterized the solution conformation of this conserved JAK interaction region (JIR) within the largely disordered N-terminus of SOCS5. Using nuclear magnetic resonance (NMR) chemical shift analysis, relaxation measurements and NOE analysis, we demonstrate the presence of pre-formed structural elements in the JIR of mouse SOCS5 (mSOCS5175-244), consisting of an α-helix encompassing residues 224-233, preceded by a turn and an extended structure. We have identified a phosphorylation site (Ser211) within the JIR of mSOCS5 and have investigated the role of phosphorylation in modulating JAK binding using site-directed mutagenesis.
    Full-text · Article · Jul 2015 · Biochemistry
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    ABSTRACT: Structural maintenance of chromosomes flexible hinge domain containing 1 (Smchd1) is an epigenetic repressor with described roles in X inactivation and genomic imprinting, but Smchd1 is also critically involved in the pathogenesis of facioscapulohumeral dystrophy. The underlying molecular mechanism by which Smchd1 functions in these instances remains unknown. Our genome-wide transcriptional and epigenetic analyses show that Smchd1 binds cis-regulatory elements, many of which coincide with CCCTC-binding factor (Ctcf) binding sites, for example, the clustered protocadherin (Pcdh) genes, where we show Smchd1 and Ctcf act in opposing ways. We provide biochemical and biophysical evidence that Smchd1-chromatin interactions are established through the homodimeric hinge domain of Smchd1 and, intriguingly, that the hinge domain also has the capacity to bind DNA and RNA. Our results suggest Smchd1 imparts epigenetic regulation via physical association with chromatin, which may antagonize Ctcf-facilitated chromatin interactions, resulting in coordinated transcriptional control.
    Full-text · Article · Jun 2015 · Proceedings of the National Academy of Sciences
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    ABSTRACT: Intracellular nucleotide binding and oligomerization domain (NOD) receptors recognize antigens including bacterial peptidoglycans and initiate immune responses by triggering the production of pro-inflammatory cytokines through activating NF-κB and MAP kinases. Receptor interacting protein kinase 2 (RIPK2) is critical for NOD-mediated NF-κB activation and cytokine production. Here we develop and characterize a selective RIPK2 kinase inhibitor, WEHI-345, which delays RIPK2 ubiquitylation and NF-κB activation downstream of NOD engagement. Despite only delaying NF-κB activation on NOD stimulation, WEHI-345 prevents cytokine production in vitro and in vivo and ameliorates experimental autoimmune encephalomyelitis in mice. Our study highlights the importance of the kinase activity of RIPK2 for proper immune responses and demonstrates the therapeutic potential of inhibiting RIPK2 in NOD-driven inflammatory diseases.
    No preview · Article · Mar 2015 · Nature Communications
  • Joanne M Hildebrand · Isabelle S Lucet · James M Murphy
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    ABSTRACT: The pseudokinase domain of the necroptosis effector mixed lineage kinase domain-like (MLKL) functions as a latch to restrain the unleashing of its N-terminal 4-helix bundle (4HB) domain. Cell death mediated by the 4HB domain relies on membrane association and oligomerization, which can be inhibited by an ATP-mimetic small molecule that binds the pseudokinase domain of MLKL.
    No preview · Article · Jan 2015
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    ABSTRACT: eLife digest In response to an injury or infection, areas of the body can become inflamed as the immune system attempts to repair the damage and/or destroy any microbes or toxins that have entered the body. At the level of individual cells inflammation can involve cells being programmed to die in one of two ways: apoptosis and necroptosis. Apoptosis is a highly controlled process during which the contents of the cell are safely destroyed in order to prevent damage to surrounding cells. Necroptosis, on the other hand, is not controlled: the cell bursts and releases its contents into the surroundings. Inflammation is activated by a protein called TNFR1, which is controlled by a complex that includes a protein called SHARPIN. Mice that lack the SHARPIN protein develop inflammation on the skin and internal organs, even in the absence of injury or infection. However, it is not clear how SHARPIN controls TNFR1 to prevent inflammation. Rickard et al. and, independently Kumari et al. have now studied this process in detail. Rickard et al. cross bred mice that lack SHARPIN with mice lacking other proteins involved in inflammation and cell death. The experiments show that apoptosis is the main form of cell death in skin inflammation, but necroptosis has a bigger role in the inflammation of internal organs. Mice that lack both the apoptotic and necroptotic cell-death pathways can develop relatively normally, but they die shortly after birth if they also lack SHARPIN. Experiments on these mice could help us to understand how SHARPIN works. DOI: http://dx.doi.org/10.7554/eLife.03464.002
    Full-text · Article · Dec 2014 · eLife Sciences

  • No preview · Conference Paper · Nov 2014
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    ABSTRACT: Necroptosis is considered to be complementary to the classical caspase-dependent programmed cell death pathway, apoptosis. The pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) is an essential effector protein in the necroptotic cell death pathway downstream of the protein kinase Receptor Interacting Protein Kinase-3 (RIPK3). How MLKL causes cell death is unclear, however RIPK3-mediated phosphorylation of the activation loop in MLKL trips a molecular switch to induce necroptotic cell death. Here, we show that the MLKL pseudokinase domain acts as a latch to restrain the N-terminal four-helix bundle (4HB) domain and that unleashing this domain results in formation of a high-molecular-weight, membrane-localized complex and cell death. Using alanine-scanning mutagenesis, we identified two clusters of residues on opposing faces of the 4HB domain that were required for the 4HB domain to kill cells. The integrity of one cluster was essential for membrane localization, whereas MLKL mutations in the other cluster did not prevent membrane translocation but prevented killing; this demonstrates that membrane localization is necessary, but insufficient, to induce cell death. Finally, we identified a small molecule that binds the nucleotide binding site within the MLKL pseudokinase domain and retards MLKL translocation to membranes, thereby preventing necroptosis. This inhibitor provides a novel tool to investigate necroptosis and demonstrates the feasibility of using small molecules to target the nucleotide binding site of pseudokinases to modulate signal transduction.
    Preview · Article · Oct 2014 · Proceedings of the National Academy of Sciences
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    ABSTRACT: Interleukin-3 (IL-3) is a cytokine secreted by mast cells and activated T-cells known to be an important regulator of differentiation, survival, proliferation and activation of a range of hematopoietic lineages. The effects of IL-3 on target cells are mediated by a transmembrane receptor system composed of a cytokine-specific α-subunit and a β-subunit, the principal signalling entity. In the mouse, two β-subunits have co-evolved: a common β-subunit (βc) shared between IL-3 and the related cytokines, IL-5 and GM-CSF; and an IL-3-specific β-subunit (βIL-3). βIL‑3 differs from βc in its specificity for IL-3 and its capacity to bind IL-3 directly in the absence of an α-subunit and, in the absence of structural information, the basis for these properties has remained enigmatic. Here, we present the crystal structure of the βIL-3 ectodomain at 3.45 Å resolution. This structure provides the first evidence that βIL-3 adopts an arch-shaped, intertwined homodimer with similar topology to the paralogous βc structure. In contrast to apo-βc, however, the ligand-binding interface of βIL‑3 appears to pre-exist in a conformation receptive to IL-3 engagement. Molecular modelling of the IL-3:βIL‑3 interface, in conjunction with previous mutational studies, suggests that divergent evolution of both βIL‑3 and IL-3 underlies their unique capacity for direct interaction and specificity.
    No preview · Article · Aug 2014 · Biochemical Journal
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    ABSTRACT: The JAK (Janus kinase) family members serve essential roles as the intracellular signalling effectors of cytokine receptors. This family, comprising JAK1, JAK2, JAK3 and TYK2 (tyrosine kinase 2), was first described more than 20 years ago, but the complexities underlying their activation, regulation and pleiotropic signalling functions are still being explored. Here, we review the current knowledge of their physiological functions and the causative role of activating and inactivating JAK mutations in human diseases, including haemopoietic malignancies, immunodeficiency and inflammatory diseases. At the molecular level, recent studies have greatly advanced our knowledge of the structures and organization of the component FERM (4.1/ezrin/radixin/moesin)-SH2 (Src homology 2), pseudokinase and kinase domains within the JAKs, the mechanism of JAK activation and, in particular, the role of the pseudokinase domain as a suppressor of the adjacent tyrosine kinase domain's catalytic activity. We also review recent advances in our understanding of the mechanisms of negative regulation exerted by the SH2 domain-containing proteins, SOCS (suppressors of cytokine signalling) proteins and LNK. These recent studies highlight the diversity of regulatory mechanisms utilized by the JAK family to maintain signalling fidelity, and suggest alternative therapeutic strategies to complement existing ATP-competitive kinase inhibitors.
    No preview · Article · Aug 2014 · Biochemical Journal
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    ABSTRACT: Upon ligand binding, RIPK1 is recruited to tumor necrosis factor receptor superfamily (TNFRSF) and Toll-like receptor (TLR) complexes promoting prosurvival and inflammatory signaling. RIPK1 also directly regulates caspase-8-mediated apoptosis or, if caspase-8 activity is blocked, RIPK3-MLKL-dependent necroptosis. We show that C57BL/6 Ripk1(-/-) mice die at birth of systemic inflammation that was not transferable by the hematopoietic compartment. However, Ripk1(-/-) progenitors failed to engraft lethally irradiated hosts properly. Blocking TNF reversed this defect in emergency hematopoiesis but, surprisingly, Tnfr1 deficiency did not prevent inflammation in Ripk1(-/-) neonates. Deletion of Ripk3 or Mlkl, but not Casp8, prevented extracellular release of the necroptotic DAMP, IL-33, and reduced Myd88-dependent inflammation. Reduced inflammation in the Ripk1(-/-)Ripk3(-/-), Ripk1(-/-)Mlkl(-/-), and Ripk1(-/-)Myd88(-/-) mice prevented neonatal lethality, but only Ripk1(-/-)Ripk3(-/-)Casp8(-/-) mice survived past weaning. These results reveal a key function for RIPK1 in inhibiting necroptosis and, thereby, a role in limiting, not only promoting, inflammation.
    Full-text · Article · May 2014 · Cell
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    James M Murphy · John Silke
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    ABSTRACT: When our time comes to die most people would probably opt for a quick, peaceful and painless exit. But the manner and timing are rarely under our direct control. Hence the Ars moriendi, literally, "The Art of Dying", two texts written in Latin around the 15th century that offered advice on how to die well according to the Christian ideals of the time. In contrast, for individual cells, the death process is frequently under their control and several signaling pathways that cause cell death, including apoptosis, pyroptosis and necroptosis, have been described. Furthermore the manner in which cells die can have good or bad consequences for the organism. In this review we will discuss how cells die via the necroptotic signaling pathway, with emphasis on recent structural work and place this work in a biological context by discussing relevant studies with knock-out animals.
    Preview · Article · Feb 2014 · EMBO Reports
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    ABSTRACT: Loss of Inhibitor of Apoptosis (IAP) proteins, particularly cIAP1, can promote production of tumor necrosis factor (TNF) and sensitize cancer cell lines to TNF induced necroptosis by promoting formation of a death inducing signaling complex containing receptor-interacting serine/threonine-protein kinase 1 and 3 (RIPK1, RIPK3). To define the role of IAPs in myelopoiesis, we generated a mouse with cIAP1, cIAP2 and XIAP deleted in the myeloid lineage. Loss of cIAPs and XIAP in the myeloid lineage caused over-production of many pro-inflammatory cytokines, resulting in granulocytosis and severe sterile inflammation. In vitro differentiation of macrophages from bone marrow in the absence of cIAPs and XIAP led to detectable levels of TNF and resulted in reduced numbers of mature macrophages. The cytokine production and consequent cell death caused by IAP depletion was attenuated by loss or inhibition of TNF or TNFR1. The loss of RIPK1 or RIPK3, but not the RIPK3 substrate MLKL, attenuated TNF secretion and thereby prevented apoptotic cell death and not necrosis. Our results demonstrate that cIAPs and XIAP together restrain RIPK1 and RIPK3 dependent cytokine production in myeloid cells to critically regulate myeloid homeostasis.
    Full-text · Article · Feb 2014 · Blood
  • Nufail Khan · Kate E Lawlor · James M Murphy · James E Vince
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    ABSTRACT: Necroptosis describes a pro-inflammatory form of cell death governed by the kinases RIP1 and RIP3. Necroptosis can occur following stimulation of the DNA receptor, DAI, or activation of death receptor, Toll-like receptor, T-cell antigen receptor, or interferon receptor signaling. Analysis of RIP3 deficient mice has implicated necroptosis in several inflammatory-driven diseases, including atherosclerosis, alcoholic liver disease and retinal degeneration. Although studies have demonstrated that mixed lineage kinase domain-like (MLKL) is the only substrate of RIP3 kinase that is essential for necroptotic death, the molecular determinants acting downstream of MLKL remain ambiguous. In addition, RIP3 can signal necroptosis independent of RIP1, may induce apoptosis, and can directly promote pro-inflammatory cytokine production. Therefore it will be important to determine if non-necroptotic RIP3 signaling influences RIP3 dependent pathologies.
    No preview · Article · Feb 2014 · Current opinion in immunology

Publication Stats

1k Citations
371.92 Total Impact Points

Institutions

  • 2011-2015
    • University of Melbourne
      Melbourne, Victoria, Australia
  • 2009-2014
    • The Walter and Eliza Hall Institute of Medical Research
      • Division of Cancer and Haematology
      Melbourne, Victoria, Australia
  • 2008-2014
    • Royal Melbourne Hospital
      Melbourne, Victoria, Australia
  • 2013
    • University of Liverpool
      Liverpool, England, United Kingdom
  • 2001-2010
    • Australian National University
      • Molecular Bioscience Department
      Canberra, Australian Capital Territory, Australia
  • 2007-2009
    • Samuel Lunenfeld Research Institute
      Toronto, Ontario, Canada
  • 2006
    • University of Toronto
      • Department of Molecular Genetics
      Toronto, Ontario, Canada