ORCID Profile
0000-0003-0195-3949
Current Organisations
International Institute for Population Sciences
,
University of Melbourne
,
Walter and Eliza Hall Institute of Medical Research
,
John Curtin School of Medical Research
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Publisher: Public Library of Science (PLoS)
Date: 26-08-2013
Publisher: Springer Science and Business Media LLC
Date: 20-10-2021
DOI: 10.1038/S41467-021-26394-6
Abstract: The SH2B family of adaptor proteins, SH2-B, APS, and LNK are key modulators of cellular signalling pathways. Whilst SH2-B and APS have been partially structurally and biochemically characterised, to date there has been no such characterisation of LNK. Here we present two crystal structures of the LNK substrate recognition domain, the SH2 domain, bound to phosphorylated motifs from JAK2 and EPOR, and biochemically define the basis for target recognition. The LNK SH2 domain adopts a canonical SH2 domain fold with an additional N-terminal helix. Targeted analysis of binding to phosphosites in signalling pathways indicated that specificity is conferred by amino acids one- and three-residues downstream of the phosphotyrosine. Several mutations in LNK showed impaired target binding in vitro and a reduced ability to inhibit signalling, allowing an understanding of the molecular basis of LNK dysfunction in variants identified in patients with myeloproliferative disease.
Publisher: Elsevier BV
Date: 03-2003
Publisher: Springer Science and Business Media LLC
Date: 18-01-2021
Publisher: Oxford University Press (OUP)
Date: 17-02-2022
DOI: 10.1093/NTR/NTAC049
Abstract: It remains unclear whether electronic cigarette (e-cigarette) use promotes persistent combustible tobacco use or smoking discontinuation over time. Alcohol use is associated with a greater risk of adverse health effects of tobacco, and higher likelihood of e-cigarette use, making drinkers a high-priority subpopulation. This study examined longitudinal patterns of combustible tobacco and e-cigarette use over 24 months in young adult binge drinkers. A pooled dataset of 1002 (58.5% female M age = 22.14) binge drinkers from the United States (60%) and Canada (40%) was used. The primary outcomes were past month combustible tobacco and e-cigarette use. Nicotine dependence was measured using the Fagerström Test of Cigarette Dependence. Alcohol severity was measured using the Young Adult Alcohol Consequences Questionnaire. Latent transition analysis (LTA) was used to identify patterns of cigarette smoking and e-cigarette use over 24 months. The LTA yielded a four-class solution: (1) e-cigarettes-only users (prevalence over time: 7.75%–10.10%), (2) dual-product users (2.61%–9.89%), (3) combustible-only smokers (8.12%–20.70%), and (4) nonusers (61.66%–80.06%). Dual-product users predominantly transitioned to complete abstinence or exclusively e-cigarette use. In combustible-only smokers, the most common transition was to abstinence, followed by persistence of combustible-only status. At 24 months, 63% of e-cigarettes-only users transitioned to abstinence, with 37% continuing e-cigarettes-only use and 0% transitioning to dual or combustible cigarette use. Dual-product use in young adult binge drinkers was associated with discontinuation of combustible tobacco over time, and e-cigarette-only use was not associated with subsequent combustible tobacco use. These findings suggest that concurrent or exclusive e-cigarette use is not a risk factor for the persistence or development of combustible tobacco use in this subpopulation, with dual-product use reflecting a transitional pattern away from combustible use, toward discontinuation.
Publisher: Portland Press Ltd.
Date: 14-09-2021
DOI: 10.1042/BCJ20210572
Abstract: EphB6 and EphA10 are two poorly characterised pseudokinase members of the Eph receptor family, which collectively serves as mediators of contact-dependent cell–cell communication to transmit extracellular cues into intracellular signals. As per their active counterparts, EphB6 and EphA10 deregulation is strongly linked to proliferative diseases. However, unlike active Eph receptors, whose catalytic activities are thought to initiate an intracellular signalling cascade, EphB6 and EphA10 are classified as catalytically dead, raising the question of how non-catalytic functions contribute to Eph receptor signalling homeostasis. In this study, we have characterised the biochemical properties and topology of the EphB6 and EphA10 intracellular regions comprising the juxtamembrane (JM) region, pseudokinase and SAM domains. Using small-angle X-ray scattering and cross-linking-mass spectrometry, we observed high flexibility within their intracellular regions in solution and a propensity for interaction between the component domains. We identified tyrosine residues in the JM region of EphB6 as EphB4 substrates, which can bind the SH2 domains of signalling effectors, including Abl, Src and Vav3, consistent with cellular roles in recruiting these proteins for downstream signalling. Furthermore, our finding that EphB6 and EphA10 can bind ATP and ATP-competitive small molecules raises the prospect that these pseudokinase domains could be pharmacologically targeted to counter oncogenic signalling.
Publisher: American Association for Cancer Research (AACR)
Date: 03-2012
DOI: 10.1158/0008-5472.CAN-12-3019
Abstract: SMCHD1 is an epigenetic modifier of gene expression that is critical to maintain X chromosome inactivation. Here, we show in mouse that genetic inactivation of Smchd1 accelerates tumorigenesis in male mice. Loss of Smchd1 in transformed mouse embryonic fibroblasts increased tumor growth upon transplantation into immunodeficient nude mice. In addition, loss of Smchd1 in Eμ-Myc transgenic mice that undergo lymphomagenesis reduced disease latency by 50% relative to control animals. In premalignant Eμ-Myc transgenic mice deficient in Smchd1, there was an increase in the number of pre-B cells in the periphery, likely accounting for the accelerated disease in these animals. Global gene expression profiling suggested that Smchd1 normally represses genes activated by MLL chimeric fusion proteins in leukemia, implying that Smchd1 loss may work through the same pathways as overexpressed MLL fusion proteins do in leukemia and lymphoma. Notably, we found that SMCHD1 is underexpressed in many types of human hematopoietic malignancy. Together, our observations collectively highlight a hitherto uncharacterized role for SMCHD1 as a candidate tumor suppressor gene in hematopoietic cancers. Cancer Res 73(5) 1591–9. ©2012 AACR.
Publisher: American Society of Hematology
Date: 15-10-2008
DOI: 10.1182/BLOOD-2007-10-119677
Abstract: In an N-nitroso-N-ethylurea (ENU) mutagenesis screen using Mpl−/− mice, we isolated a semidominant suppressor of thrombocytopenia, termed Plt6. The gene mutated in Plt6 mice encodes the transcriptional coregulator p300, and the mutation, a tyrosine to asparagine substitution at amino acid 630 (Y630N), disrupts the interaction between p300 and c-Myb. Mpl−/−p300Plt6/+ mice displayed elevated platelet counts relative to Mpl−/−p300+/+ controls, whereas mice homozygous for the Plt6 mutation produced supraphysiological levels of circulating platelets. On a wild-type genetic background, mice homozygous for the p300Plt6 mutation, or recipients of Mpl+/+p300Plt6/Plt6 bone marrow, also exhibited thrombocytosis as well as deficiencies in B-lymphoid cells. Increased platelet numbers in Plt6 mutant mice were accompanied by significant increases in megakaryocyte progenitor cells within the bone marrow and spleen with concomitantly elevated numbers of megakaryocytes. The expansion of megakaryocytopoiesis and suppression of Mpl−/− thrombocytopenia in Plt6 mutants is highly reminiscent of that observed in mice with mutations affecting the p300 partner protein c-Myb, suggesting an indispensable repressive role for the c-Myb 300 transcriptional regulatory complex in megakaryocyte develop-ment, the inhibition of which allows substantial thrombopoietin (TPO)–independent platelet production.
Publisher: American Chemical Society (ACS)
Date: 09-09-2020
Publisher: American Society of Hematology
Date: 08-04-2010
DOI: 10.1182/BLOOD-2009-08-239210
Abstract: c-Myb is a transcription factor with functions in many hematopoietic lineages. c-Myb–deficient mice display reduced numbers of B cells however, it is unknown what role c-Myb plays in B lymphopoiesis because no critical target genes have been identified in the B-cell lineage. We demonstrate that conditional deletion of c-Myb in B-cell progenitors completely abolishes B-cell development. c-Myb is required for lymphoid progenitors to respond to the cytokines interleukin-7 and thymic stromal lymphopoietin in the absence of sufficient c-Myb activity, mice display a B lymphopenia that closely resembles that observed in interleukin-7 receptor α–deficient animals. Analysis of the multipotent progenitor compartment indicates that c-Myb is also required for up-regulation of multiple lymphoid-associated genes, including Il7r, and for the subsequent development of the common lymphoid progenitor population. These data show that c-Myb plays a critical role in the regulatory pathways governing lymphoid specification and early B-cell differentiation.
Publisher: Humana Press
Date: 04-11-2012
DOI: 10.1007/978-1-62703-242-1_3
Abstract: The discovery that a range of myeloproliferative diseases and leukemias are associated with Janus Kinase (JAK) mutations has highlighted the importance of JAK/STAT signalling in disease and sparked a renewed interest in developing JAK inhibitors. In vitro kinase assays are the most direct and quantitative method to assess mutant forms of JAK for altered enzymatic properties as well as verifying and quantifying the affinity and efficacy of potential inhibitors. Here, we describe protocols for heterologous expression and purification of JAK kinases from insect cells, assays to determine the activity of these purified kinases, and finally inhibition assays to determine the effectiveness of potential inhibitors.
Publisher: Springer Science and Business Media LLC
Date: 23-07-2022
DOI: 10.1038/S41418-022-01042-8
Abstract: Caspase-8 transduces signals from death receptor ligands, such as tumor necrosis factor, to drive potent responses including inflammation, cell proliferation or cell death. This is a developmentally essential function because in utero deletion of endothelial Caspase-8 causes systemic circulatory collapse during embryogenesis. Whether endothelial Caspase-8 is also required for cardiovascular patency during adulthood was unknown. To address this question, we used an inducible Cre recombinase system to delete endothelial Casp8 in 6-week-old conditionally gene-targeted mice. Extensive whole body vascular gene targeting was confirmed, yet the dominant phenotype was fatal hemorrhagic lesions exclusively within the small intestine. The emergence of these intestinal lesions was not a maladaptive immune response to endothelial Caspase-8-deficiency, but instead relied upon aberrant Toll-like receptor sensing of microbial commensals and tumor necrosis factor receptor signaling. This lethal phenotype was prevented in compound mutant mice that lacked the necroptotic cell death effector, MLKL. Thus, distinct from its systemic role during embryogenesis, our data show that dysregulated microbial- and death receptor-signaling uniquely culminate in the adult mouse small intestine to unleash MLKL-dependent necroptotic hemorrhage after loss of endothelial Caspase-8. These data support a critical role for Caspase-8 in preserving gut vascular integrity in the face of microbial commensals.
Publisher: Springer Science and Business Media LLC
Date: 19-06-2020
DOI: 10.1038/S41467-020-16887-1
Abstract: Mixed lineage kinase domain-like (MLKL) is the terminal protein in the pro-inflammatory necroptotic cell death program. RIPK3-mediated phosphorylation is thought to initiate MLKL oligomerization, membrane translocation and membrane disruption, although the precise choreography of events is incompletely understood. Here, we use single-cell imaging approaches to map the chronology of endogenous human MLKL activation during necroptosis. During the effector phase of necroptosis, we observe that phosphorylated MLKL assembles into higher order species on presumed cytoplasmic necrosomes. Subsequently, MLKL co-traffics with tight junction proteins to the cell periphery via Golgi-microtubule-actin-dependent mechanisms. MLKL and tight junction proteins then steadily co-accumulate at the plasma membrane as heterogeneous micron-sized hotspots. Our studies identify MLKL trafficking and plasma membrane accumulation as crucial necroptosis checkpoints. Furthermore, the accumulation of phosphorylated MLKL at intercellular junctions accelerates necroptosis between neighbouring cells, which may be relevant to inflammatory bowel disease and other necroptosis-mediated enteropathies.
Publisher: Portland Press Ltd.
Date: 18-07-2013
DOI: 10.1042/BST20130077
Abstract: JAKs (Janus kinases) are essential mediators of almost all biological signalling events initiated by haemopoietic and immune cytokines. However, aberrant and/or prolonged JAK-induced signalling is detrimental and can give rise to a number of inflammatory and proliferative pathologies. For this reason, the tyrosine kinase activity of the JAKs is carefully regulated at a number of different levels. Primarily, this is achieved by: (i) ensuring that the catalytic domain is ‘switched off’ under basal conditions, and (ii) inhibiting the activity of JAK after it has been switched on. Whereas the first mode of inhibition is mediated by JAK's own pseudokinase domain as well as the action of phosphatases, the second is achieved by the action of the SOCS (suppressor of cytokine signalling) proteins, negative-feedback inhibitors of JAK-mediated signalling. The present review focuses on the mode of action of SOCS1 and SOCS3, the two most potent JAK inhibitors.
Publisher: Elsevier BV
Date: 05-2014
Publisher: Wiley
Date: 17-01-2017
DOI: 10.1038/ICB.2016.125
Abstract: Necroptosis (or 'programmed necrosis') is a caspase-independent cell death pathway that operates downstream of death receptors, including Tumour Necrosis Factor Receptor-1 (TNFR1), and the Toll-like receptors, TLR3 and TLR4. Owing to its immunogenicity, necroptosis has been attributed roles in the pathogenesis of several diseases, including inflammatory bowel disease and the tissue damage arising from ischaemic-reperfusion injuries. Only over the past 7 years has the core machinery of this pathway, the receptor-interacting protein kinase-3 (RIPK3) and the pseudokinase, Mixed Lineage Kinase domain-Like (MLKL), been defined. Our current understanding of the pathway is that RIPK3-mediated phosphorylation activates cytoplasmic MLKL, which is the most terminal known effector in the pathway, leading to MLKL's oligomerisation, translocation to, and permeabilisation of, the plasma membrane. Here, we discuss the insights gleaned from structural and biophysical studies of MLKL and highlight the known unknowns surrounding MLKL's mechanism of action and activation.
Publisher: Portland Press Ltd.
Date: 18-07-2013
DOI: 10.1042/BST20130115
Abstract: Recent studies of proteins containing kinase-like domains that lack catalytic residue(s) classically required for phosphotransfer, termed pseudokinases, have uncovered important roles in cell signalling across the kingdoms of life. Additionally, mutations within pseudokinase domains are known to underlie human diseases, suggesting that these proteins may represent new and unexplored therapeutic targets. To date, few pseudokinases have been studied in intricate detail, but as described in the present article and in the subsequent papers in this issue of Biochemical Society Transactions, several new studies have provided an advanced template and an improved framework for interrogating the roles of pseudokinases in signal transduction. In the present article, we review landmarks in the establishment of this field of study, highlight some experimental challenges and propose a simple scheme for definition of these domains based on their primary sequences, rather than experimentally defined nucleotide-binding or catalytic activities.
Publisher: Springer Science and Business Media LLC
Date: 07-06-2021
DOI: 10.1038/S41467-021-23474-5
Abstract: Necroptosis is a lytic, inflammatory form of cell death that not only contributes to pathogen clearance but can also lead to disease pathogenesis. Necroptosis is triggered by RIPK3-mediated phosphorylation of MLKL, which is thought to initiate MLKL oligomerisation, membrane translocation and membrane rupture, although the precise mechanism is incompletely understood. Here, we show that K63-linked ubiquitin chains are attached to MLKL during necroptosis and that ubiquitylation of MLKL at K219 significantly contributes to the cytotoxic potential of phosphorylated MLKL. The K219R MLKL mutation protects animals from necroptosis-induced skin damage and renders cells resistant to pathogen-induced necroptosis. Mechanistically, we show that ubiquitylation of MLKL at K219 is required for higher-order assembly of MLKL at membranes, facilitating its rupture and necroptosis. We demonstrate that K219 ubiquitylation licenses MLKL activity to induce lytic cell death, suggesting that necroptotic clearance of pathogens as well as MLKL-dependent pathologies are influenced by the ubiquitin-signalling system.
Publisher: Springer Science and Business Media LLC
Date: 09-03-2022
Publisher: Cold Spring Harbor Laboratory
Date: 24-04-2020
DOI: 10.1101/2020.04.22.056440
Abstract: 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. Truncation of an N-terminal extension abolishes cooperative binding. The effector, N -acetylneuraminate, binds NanR and attenuates DNA binding. Crystal structure data show that N -acetylneuraminate binding to NanR causes a domain rearrangement that locks the protein in a conformation that prevents DNA binding. Single-particle cryo-electron microscopy structures of NanR bound to DNA reveal the DNA binding domain is reorganized to engage DNA, while the three dimers assemble in close proximity across the (GGTATA) 3 -repeat operator allowing protein-protein interactions to form via the N-terminal extensions. Our data provides a molecular basis for the regulation of bacterial sialic acid metabolism.
Publisher: Springer Science and Business Media LLC
Date: 22-11-2021
DOI: 10.1038/S41467-021-27032-X
Abstract: The ancestral origins of the lytic cell death mode, necroptosis, lie in host defense. However, the dysregulation of necroptosis in inflammatory diseases has led to widespread interest in targeting the pathway therapeutically. This mode of cell death is executed by the terminal effector, the MLKL pseudokinase, which is licensed to kill following phosphorylation by its upstream regulator, RIPK3 kinase. The precise molecular details underlying MLKL activation are still emerging and, intriguingly, appear to mechanistically- erge between species. Here, we report the structure of the human RIPK3 kinase domain alone and in complex with the MLKL pseudokinase. These structures reveal how human RIPK3 structurally differs from its mouse counterpart, and how human RIPK3 maintains MLKL in an inactive conformation prior to induction of necroptosis. Residues within the RIPK3:MLKL C-lobe interface are crucial to complex assembly and necroptotic signaling in human cells, thereby rationalizing the strict species specificity governing RIPK3 activation of MLKL.
Publisher: Springer Science and Business Media LLC
Date: 11-05-2021
DOI: 10.1038/S41467-021-22979-3
Abstract: Interleukin-1β (IL-1β) is activated by inflammasome-associated caspase-1 in rare autoinflammatory conditions and in a variety of other inflammatory diseases. Therefore, IL-1β activity must be fine-tuned to enable anti-microbial responses whilst limiting collateral damage. Here, we show that precursor IL-1β is rapidly turned over by the proteasome and this correlates with its decoration by K11-linked, K63-linked and K48-linked ubiquitin chains. The ubiquitylation of IL-1β is not just a degradation signal triggered by inflammasome priming and activating stimuli, but also limits IL-1β cleavage by caspase-1. IL-1β K133 is modified by ubiquitin and forms a salt bridge with IL-1β D129. Loss of IL-1β K133 ubiquitylation, or disruption of the K133:D129 electrostatic interaction, stabilizes IL-1β. Accordingly, Il1b K133R/K133R mice have increased levels of precursor IL-1β upon inflammasome priming and increased production of bioactive IL-1β, both in vitro and in response to LPS injection. These findings identify mechanisms that can limit IL-1β activity and safeguard against damaging inflammation.
Publisher: Springer Science and Business Media LLC
Date: 02-12-2021
Publisher: Springer Science and Business Media LLC
Date: 19-06-2020
DOI: 10.1038/S41467-020-16819-Z
Abstract: MLKL is the essential effector of necroptosis, a form of programmed lytic cell death. We have isolated a mouse strain with a single missense mutation, Mlkl D139V , that alters the two-helix ‘brace’ that connects the killer four-helix bundle and regulatory pseudokinase domains. This confers constitutive, RIPK3 independent killing activity to MLKL. Homozygous mutant mice develop lethal postnatal inflammation of the salivary glands and mediastinum. The normal embryonic development of Mlkl D139V homozygotes until birth, and the absence of any overt phenotype in heterozygotes provides important in vivo precedent for the capacity of cells to clear activated MLKL. These observations offer an important insight into the potential disease-modulating roles of three common human MLKL polymorphisms that encode amino acid substitutions within or adjacent to the brace region. Compound heterozygosity of these variants is found at up to 12-fold the expected frequency in patients that suffer from a pediatric autoinflammatory disease, chronic recurrent multifocal osteomyelitis (CRMO).
Publisher: Frontiers Media SA
Date: 09-12-2016
Publisher: Springer Science and Business Media LLC
Date: 15-11-2018
Publisher: The American Association of Immunologists
Date: 08-2019
Abstract: The pyroptotic cell death effector gasdermin D (GSDMD) is required for murine models of hereditary inflammasome-driven, IL-1β–dependent, autoinflammatory disease, making it an attractive therapeutic target. However, the importance of GSDMD for more common conditions mediated by pathological IL-1β activation, such as gout, remain unclear. In this study, we address whether GSDMD and the recently described GSDMD inhibitor necrosulfonamide (NSA) contribute to monosodium urate (MSU) crystal–induced cell death, IL-1β release, and autoinflammation. We demonstrate that MSU crystals, the etiological agent of gout, rapidly activate GSDMD in murine macrophages. Despite this, the genetic deletion of GSDMD or the other lytic effector implicated in MSU crystal killing, mixed lineage kinase domain-like (MLKL), did not prevent MSU crystal–induced cell death. Consequently, GSDMD or MLKL loss did not hinder MSU crystal–mediated release of bioactive IL-1β. Consistent with in vitro findings, IL-1β induction and autoinflammation in MSU crystal–induced peritonitis was not reduced in GSDMD-deficient mice. Moreover, we show that the reported GSDMD inhibitor, NSA, blocks inflammasome priming and caspase-1 activation, thereby preventing pyroptosis independent of GSDMD targeting. The inhibition of cathepsins, widely implicated in particle-induced macrophage killing, also failed to prevent MSU crystal–mediated cell death. These findings 1) demonstrate that not all IL-1β–driven autoinflammatory conditions will benefit from the therapeutic targeting of GSDMD, 2) document a unique mechanism of MSU crystal–induced macrophage cell death not rescued by pan-cathepsin inhibition, and 3) show that NSA inhibits inflammasomes upstream of GSDMD to prevent pyroptotic cell death and IL-1β release.
Publisher: Springer Science and Business Media LLC
Date: 25-09-2023
Publisher: Springer Science and Business Media LLC
Date: 10-2021
DOI: 10.1038/S41591-021-01498-0
Abstract: Anemia is a globally widespread condition in women and is associated with reduced economic productivity and increased mortality worldwide. Here we map annual 2000–2018 geospatial estimates of anemia prevalence in women of reproductive age (15–49 years) across 82 low- and middle-income countries (LMICs), stratify anemia by severity and aggregate results to policy-relevant administrative and national levels. Additionally, we provide subnational disparity analyses to provide a comprehensive overview of anemia prevalence inequalities within these countries and predict progress toward the World Health Organization’s Global Nutrition Target (WHO GNT) to reduce anemia by half by 2030. Our results demonstrate widespread moderate improvements in overall anemia prevalence but identify only three LMICs with a high probability of achieving the WHO GNT by 2030 at a national scale, and no LMIC is expected to achieve the target in all their subnational administrative units. Our maps show where large within-country disparities occur, as well as areas likely to fall short of the WHO GNT, offering precision public health tools so that adequate resource allocation and subsequent interventions can be targeted to the most vulnerable populations.
Publisher: Springer Science and Business Media LLC
Date: 10-03-2017
DOI: 10.1038/NCOMMS14729
Abstract: Remarkably little is known about how intracellular pathogens exit the host cell in order to infect new hosts. Pathogenic chlamydiae egress by first rupturing their replicative niche (the inclusion) before rapidly lysing the host cell. Here we apply a laser ablation strategy to specifically disrupt the chlamydial inclusion, thereby uncoupling inclusion rupture from the subsequent cell lysis and allowing us to dissect the molecular events involved in each step. Pharmacological inhibition of host cell calpains inhibits inclusion rupture, but not subsequent cell lysis. Further, we demonstrate that inclusion rupture triggers a rapid necrotic cell death pathway independent of BAK, BAX, RIP1 and caspases. Both processes work sequentially to efficiently liberate the pathogen from the host cytoplasm, promoting secondary infection. These results reconcile the pathogen's known capacity to promote host cell survival and induce cell death.
Publisher: Portland Press Ltd.
Date: 14-02-2014
DOI: 10.1042/BJ20131516
Abstract: JAK2 (Janus kinase 2) initiates the intracellular signalling cascade downstream of cell surface receptor activation by cognate haemopoietic cytokines, including erythropoietin and thrombopoietin. The pseudokinase domain (JH2) of JAK2 negatively regulates the catalytic activity of the adjacent tyrosine kinase domain (JH1) and mutations within the pseudokinase domain underlie human myeloproliferative neoplasms, including polycythaemia vera and essential thrombocytosis. To date, the mechanism of JH2-mediated inhibition of JH1 kinase activation as well as the susceptibility of pathological mutant JAK2 to inhibition by the physiological negative regulator SOCS3 (suppressor of cytokine signalling 3) have remained unclear. In the present study, using recombinant purified JAK2JH1-JH2 proteins, we demonstrate that, when activated, wild-type and myeloproliferative neoplasm-associated mutants of JAK2 exhibit comparable enzymatic activity and inhibition by SOCS3 in in vitro kinase assays. SAXS (small-angle X-ray scattering) showed that JAK2JH1-JH2 exists in an elongated configuration in solution with no evidence for interaction between JH1 and JH2 domains in cis. Collectively, these data are consistent with a model in which JAK2's pseudokinase domain does not influence the activity of JAK2 once it has been activated. Our data indicate that, in the absence of the N-terminal FERM domain and thus cytokine receptor association, the wild-type and pathological mutants of JAK2 are enzymatically equivalent and equally susceptible to inhibition by SOCS3.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 02-02-2021
DOI: 10.1126/SCISIGNAL.ABC6178
Abstract: Signaling events in distinct subcellular locations determine whether MLKL mediates necroptosis.
Publisher: Elsevier BV
Date: 06-2017
Publisher: Springer Science and Business Media LLC
Date: 27-10-2017
DOI: 10.1038/S41467-017-01279-9
Abstract: The mammalian pseudokinase SgK223, and its structurally related homologue SgK269, are oncogenic scaffolds that nucleate the assembly of specific signalling complexes and regulate tyrosine kinase signalling. Both SgK223 and SgK269 form homo- and hetero-oligomers, a mechanism that underpins a ersity of signalling outputs. However, mechanistic insights into SgK223 and SgK269 homo- and heterotypic association are lacking. Here we present the crystal structure of SgK223 pseudokinase domain and its adjacent N- and C-terminal helices. The structure reveals how the N- and C-regulatory helices engage in a novel fold to mediate the assembly of a high-affinity dimer. In addition, we identified regulatory interfaces on the pseudokinase domain required for the self-assembly of large open-ended oligomers. This study highlights the ersity in how the kinase fold mediates non-catalytic functions and provides mechanistic insights into how the assembly of these two oncogenic scaffolds is achieved in order to regulate signalling output.
Publisher: Proceedings of the National Academy of Sciences
Date: 04-09-2007
Abstract: The Par-1/MARK protein kinases play a pivotal role in establishing cellular polarity. This family of kinases contains a unique domain architecture, in which a ubiquitin-associated (UBA) domain is located C-terminal to the kinase domain. We have used a combination of x-ray crystallography and NMR dynamics experiments to understand the interaction of the human (h) MARK3 UBA domain with the adjacent kinase domain as compared with ubiquitin. The x-ray crystal structure of the linked hMARK3 kinase and UBA domains establishes that the UBA domain forms a stable intramolecular interaction with the N-terminal lobe of the kinase domain. However, solution-state NMR studies of the isolated UBA domain indicate that it is highly dynamic, undergoing conformational transitions that can be explained by a folding–unfolding equilibrium. NMR titration experiments indicated that the hMARK3 UBA domain has a detectable but extremely weak affinity for mono ubiquitin, which suggests that conformational instability of the isolated hMARK3 UBA domain attenuates binding to ubiquitin despite the presence of residues typically involved in ubiquitin recognition. Our data identify a molecular mechanism through which the hMARK3 UBA domain has evolved to bind the kinase domain, in a fashion that stabilizes an open conformation of the N- and C-terminal lobes, at the expense of its capacity to engage ubiquitin. These results may be relevant more generally to the 30% of UBA domains that lack significant ubiquitin-binding activity, and they suggest a unique mechanism by which interaction domains may evolve new binding properties.
Publisher: Portland Press Ltd.
Date: 18-07-2013
DOI: 10.1042/BST20130075
Abstract: Approximately 10% of the human kinome has been classified as pseudokinases due to the absence of one or more of three motifs known to play key roles in the catalytic activities of protein kinases. Structural and functional studies are now emerging, reclassifying this ‘dead’ kinase family as essential signalling molecules that act as crucial modulators of signal transduction. This raises the prospect that pseudokinases may well represent an as-yet-unexplored class of drug targets. However, the extent to which nucleotide binding and catalytic activity contribute to the biological functions of pseudokinases remains an area of great controversy. In the present review, we discuss the advantages and disadvantages of the different methods employed to characterize the nucleotide-binding properties and activity of pseudokinases.
Publisher: Elsevier
Date: 2022
DOI: 10.1016/BS.MIE.2022.03.029
Abstract: Pseudokinase domains are found throughout the kingdoms of life and serve myriad roles in cell signaling. These domains, which resemble protein kinases but are catalytically-deficient, have been described principally as protein interaction domains. Broadly, pseudokinases have been reported to function as: allosteric regulators of conventional enzymes scaffolds to nucleate assembly and/or localization of signaling complexes molecular switches or competitors of signaling complex assembly. From detailed structural and biochemical studies of in idual pseudokinases, a picture of how they mediate protein interactions is beginning to emerge. Many such studies have relied on recombinant protein production in insect cells, where endogenous chaperones and modifying enzymes favor bona fide folding of pseudokinases. Here, we describe methods for co-expression of pseudokinases and their interactors in insect cells, as exemplified by the MLKL pseudokinase, which is the terminal effector in the necroptosis cell death pathway, and its upstream regulator kinase RIPK3. These methods are broadly applicable to co-expression of other pseudokinases with their interaction partners from bacmids using the baculovirus-insect cell expression system.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 05-01-2018
Abstract: Human malaria is caused by half a dozen species of Plasmodium protozoan parasites, each with distinctive biology. P. vivax , which causes relapsing malaria, specifically parasitizes immature red blood cells called reticulocytes. Gruszczyk et al. identified TfR1 (host transferrin receptor 1) as an alternative receptor for P. vivax . TfR1 binds to a specific P. vivax surface protein. However, the parasite that causes cerebral malaria, P. falciparum , does not share TfR1 as a receptor: P. falciparum could still infect cells in which TfR1 expression was knocked down, but P. vivax could not. Monoclonal antibodies to the P. vivax protein successfully hindered P. vivax infection of red blood cells. Science , this issue p. 48
Publisher: Springer Science and Business Media LLC
Date: 19-04-2022
DOI: 10.1038/S41467-022-29670-1
Abstract: Modulation of protein abundance using t ag- T argeted P rotein D egrader (tTPD) systems targeting FKBP12 F36V (dTAGs) or HaloTag7 (HaloPROTACs) are powerful approaches for preclinical target validation. Interchanging tags and tag-targeting degraders is important to achieve efficient substrate degradation, yet limited degrader/tag pairs are available and side-by-side comparisons have not been performed. To expand the tTPD repertoire we developed catalytic Nano Luc-targeting PRO TACs (NanoTACs) to hijack the CRL4 CRBN complex and degrade NanoLuc tagged substrates, enabling rapid luminescence-based degradation screening. To benchmark NanoTACs against existing tTPD systems we use an interchangeable reporter system to comparatively test optimal degrader/tag pairs. Overall, we find the dTAG system exhibits superior degradation. To align tag-induced degradation with physiology we demonstrate that NanoTACs limit MLKL-driven necroptosis. In this work we extend the tTPD platform to include NanoTACs adding flexibility to tTPD studies, and benchmark each tTPD system to highlight the importance of comparing each system against each substrate.
Publisher: American Society of Hematology
Date: 17-04-2014
DOI: 10.1182/BLOOD-2013-06-510743
Abstract: cIAPs and XIAP negatively regulate cytokine production, including TNF to disrupt myeloid lineage differentiation. IAPs prevent RIPK1 and RIPK3 activity to limit cytokine production prior to cell death.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 16-06-2020
DOI: 10.1126/SCISIGNAL.AAZ5599
Abstract: Solving the structure of the hinge domain of an epigenetic regulator highlights residues involved in dimerization and function.
Publisher: Springer Science and Business Media LLC
Date: 27-06-2018
DOI: 10.1038/S41586-018-0249-1
Abstract: Plasmodium vivax is the most widely distributed malaria parasite that infects humans
Publisher: Springer Science and Business Media LLC
Date: 26-09-2018
DOI: 10.1038/S41467-018-06186-1
Abstract: Attachment of ubiquitin to lysine 119 of Histone 2A (H2AK119Ub) is an epigenetic mark characteristic of repressed developmental genes, which is removed by the Polycomb Repressive-Deubiquitinase (PR-DUB) complex. Here we report the crystal structure of the Drosophila PR-DUB, revealing that the deubiquitinase Calypso and its activating partner ASX form a 2:2 complex. The bidentate Calypso–ASX complex is generated by dimerisation of two activated Calypso proteins through their coiled-coil regions. Disrupting the Calypso dimer interface does not affect inherent catalytic activity, but inhibits removal of H2AK119Ub as a consequence of impaired recruitment to nucleosomes. Mutating the equivalent surface on the human counterpart, BAP1, also compromises activity on nucleosomes. Together, this suggests that high local concentrations drive assembly of bidentate PR-DUB complexes on chromatin—providing a mechanistic basis for enhanced PR-DUB activity at specific genomic foci, and the impact of distinct classes of PR-DUB mutations in tumorigenesis.
Publisher: Elsevier BV
Date: 07-2010
Publisher: Portland Press Ltd.
Date: 15-06-2017
DOI: 10.1042/BST20160331
Abstract: Over the past decade, our understanding of the mechanisms by which pseudokinases, which comprise ∼10% of the human and mouse kinomes, mediate signal transduction has advanced rapidly with increasing structural, biochemical, cellular and genetic studies. Pseudokinases are the catalytically defective counterparts of conventional, active protein kinases and have been attributed functions as protein interaction domains acting variously as allosteric modulators of conventional protein kinases and other enzymes, as regulators of protein trafficking or localisation, as hubs to nucleate assembly of signalling complexes, and as transmembrane effectors of such functions. Here, by categorising mammalian pseudokinases based on their known functions, we illustrate the mechanistic ersity among these proteins, which can be viewed as a window into understanding the non-catalytic functions that can be exerted by conventional protein kinases.
Publisher: Elsevier BV
Date: 02-2012
Publisher: eLife Sciences Publications, Ltd
Date: 02-12-2014
DOI: 10.7554/ELIFE.03464
Abstract: SHARPIN regulates immune signaling and contributes to full transcriptional activity and prevention of cell death in response to TNF in vitro. The inactivating mouse Sharpin cpdm mutation causes TNF-dependent multi-organ inflammation, characterized by dermatitis, liver inflammation, splenomegaly, and loss of Peyer's patches. TNF-dependent cell death has been proposed to cause the inflammatory phenotype and consistent with this we show Tnfr1, but not Tnfr2, deficiency suppresses the phenotype (and it does so more efficiently than Il1r1 loss). TNFR1-induced apoptosis can proceed through caspase-8 and BID, but reduction in or loss of these players generally did not suppress inflammation, although Casp8 heterozygosity significantly delayed dermatitis. Ripk3 or Mlkl deficiency partially ameliorated the multi-organ phenotype, and combined Ripk3 deletion and Casp8 heterozygosity almost completely suppressed it, even restoring Peyer's patches. Unexpectedly, Sharpin, Ripk3 and Casp8 triple deficiency caused perinatal lethality. These results provide unexpected insights into the developmental importance of SHARPIN.
Publisher: Springer Science and Business Media LLC
Date: 23-11-2021
Publisher: American Chemical Society (ACS)
Date: 06-11-2018
DOI: 10.1021/ACS.JNATPROD.8B00494
Abstract: Four new β-triketone monoterpene hybrids, intermediones A-D (1-4), have been identified from the flowers of the Australian eucalypt tree Corymbia intermedia. Intermediones A-D are β-triketones that incorporate a pinene moiety attached via a benzyl group to a syncarpic acid. The structures of 1-4, including relative configurations, were elucidated from the analysis of 1D/2D NMR and MS data. The absolute configurations of intermediones A and B were determined by comparison of experimental and predicted ECD spectra. Intermedione D possesses a tetracyclic ring system that is related to that found in the meroterpenes, guadials B and C. Low to moderate antiplasmodial activity toward the chloroquine-sensitive (3D7) strain of Plasmodium falciparum, with IC
Publisher: EMBO
Date: 27-01-2014
Publisher: Elsevier BV
Date: 05-2008
DOI: 10.1016/J.CYTO.2008.02.010
Abstract: Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3 and IL-5 are related cytokines that play key roles in regulating the differentiation, proliferation, survival and activation of myeloid blood cells. The cell surface receptors for these cytokines are composed of cytokine-specific alpha-subunits and a common beta-receptor (betac), a shared subunit that is essential for receptor signaling in response to GM-CSF, IL-3 and IL-5. Previous studies have reached conflicting conclusions as to whether N-glycosylation of the betac-subunit is necessary for functional GM-CSF, IL-3 and IL-5 receptors. We sought to clarify whether betac N-glycosylation plays a role in receptor function, since all structural studies of human betac to date have utilized recombinant protein lacking N-glycosylation at Asn(328). Here, by eliminating in idual N-glycans in human betac and the related murine homolog, beta(IL-3), we demonstrate unequivocally that ligand-binding and receptor activation are not critically dependent on in idual N-glycosylation sites within the beta-subunit although the data do not preclude the possibility that N-glycans may exert some sort of fine control. These studies support the biological relevance of the X-ray crystal structures of the human betac domain 4 and the complete ectodomain, both of which lack N-glycosylation at Asn(328).
Publisher: Cold Spring Harbor Laboratory
Date: 05-03-2020
DOI: 10.1101/2020.03.05.978056
Abstract: Mitotic catastrophe (MC) is an important oncosuppressive mechanism that serves to eliminate cells that become polyploid or aneuploidy due to aberrant mitosis. Previous studies have demonstrated that the activation and catalytic function of caspase-2 are key steps in MC to trigger apoptosis and/or cell cycle arrest of mitotically defective cells. However, the molecular mechanisms that regulate caspase-2 activation and its function are unclear. Here we identify six new phosphorylation sites in caspase-2 and show that a key mitotic kinase, Aurora B kinase (AURKB), phosphorylates caspase-2 at the highly conserved residue S384. We demonstrate that phosphorylation at S384 blocks caspase-2 catalytic activity and apoptosis function in response to mitotic insults, without affecting caspase-2 dimerisation. Moreover, molecular modelling suggests that phosphorylation at S384 may affect substrate binding by caspase-2. We propose that caspase-2 S384 phosphorylation by AURKB is a key mechanism that controls caspase-2 activation during mitosis.
Publisher: Cold Spring Harbor Laboratory
Date: 08-06-2018
DOI: 10.1101/342212
Abstract: The regulation of higher order chromatin structure is complex and dynamic however we do not yet understand the full suite of mechanisms governing architecture. Here we reveal the non-canonical SMC protein Smchd1 as a novel regulator of long-range chromatin interactions, and add it to the canon of epigenetic proteins required for Hox gene regulation. The effect of losing Smchd1-dependent chromatin interactions has varying outcomes dependent on chromatin context. At autosomal targets transcriptionally sensitive to Smchd1 deletion, we find increased short-range interactions and ectopic enhancer activation. By contrast, the inactive X chromosome is transcriptionally refractive to Smchd1 ablation, despite chromosome-wide increases in short-range interactions. There we observe spreading of H3K27me3 domains into regions not normally decorated by this mark. Together these data suggest Smchd1 has the capacity to insulate the chromatin, thereby limiting access to other chromatin modifying proteins.
Publisher: Elsevier BV
Date: 12-2022
DOI: 10.1053/J.GASTRO.2022.08.040
Abstract: Necroptosis is a highly inflammatory mode of cell death that has been implicated in causing hepatic injury including steatohepatitis/ nonalcoholic steatohepatitis (NASH) however, the evidence supporting these claims has been controversial. A comprehensive, fundamental understanding of cell death pathways involved in liver disease critically underpins rational strategies for therapeutic intervention. We sought to define the role and relevance of necroptosis in liver pathology. Several animal models of human liver pathology, including diet-induced steatohepatitis in male mice and erse infections in both male and female mice, were used to dissect the relevance of necroptosis in liver pathobiology. We applied necroptotic stimuli to primary mouse and human hepatocytes to measure their susceptibility to necroptosis. Paired liver biospecimens from patients with NASH, before and after intervention, were analyzed. DNA methylation sequencing was also performed to investigate the epigenetic regulation of RIPK3 expression in primary human and mouse hepatocytes. Identical infection kinetics and pathologic outcomes were observed in mice deficient in an essential necroptotic effector protein, MLKL, compared with control animals. Mice lacking MLKL were indistinguishable from wild-type mice when fed a high-fat diet to induce NASH. Under all conditions tested, we were unable to induce necroptosis in hepatocytes. We confirmed that a critical activator of necroptosis, RIPK3, was epigenetically silenced in mouse and human primary hepatocytes and rendered them unable to undergo necroptosis. We have provided compelling evidence that necroptosis is disabled in hepatocytes during homeostasis and in the pathologic conditions tested in this study.
Publisher: Springer Science and Business Media LLC
Date: 14-01-2016
Abstract: Necroptosis is a caspase-independent form of regulated cell death that has been implicated in the development of a range of inflammatory, autoimmune and neurodegenerative diseases. The pseudokinase, Mixed Lineage Kinase Domain-Like (MLKL), is the most terminal known obligatory effector in the necroptosis pathway, and is activated following phosphorylation by Receptor Interacting Protein Kinase-3 (RIPK3). Activated MLKL translocates to membranes, leading to membrane destabilisation and subsequent cell death. However, the molecular interactions governing the processes downstream of RIPK3 activation remain poorly defined. Using a phenotypic screen, we identified seven heat-shock protein 90 (HSP90) inhibitors that inhibited necroptosis in both wild-type fibroblasts and fibroblasts expressing an activated mutant of MLKL. We observed a modest reduction in MLKL protein levels in human and murine cells following HSP90 inhibition, which was only apparent after 15 h of treatment. The delayed reduction in MLKL protein abundance was unlikely to completely account for defective necroptosis, and, consistent with this, we also found inhibition of HSP90 blocked membrane translocation of activated MLKL. Together, these findings implicate HSP90 as a modulator of necroptosis at the level of MLKL, a function that complements HSP90’s previously demonstrated modulation of the upstream necroptosis effector kinases, RIPK1 and RIPK3.
Publisher: Portland Press Ltd.
Date: 10-10-2014
DOI: 10.1042/BJ20140863
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 haemopoietic 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 granulocyte/macrophage colony-stimulating factor (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. In the present study, we have solved the crystal structure of the βIL-3 ectodomain at 3.45 Å (1 Å=0.1 nm) 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 with 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 ergent evolution of both βIL-3 and IL-3 underlies their unique capacity for direct interaction and specificity.
Publisher: Proceedings of the National Academy of Sciences
Date: 19-06-2015
Abstract: Structural maintenance of chromosomes flexible hinge domain containing 1 (Smchd1) is a protein that plays an important role in maintaining gene silencing in many biological circumstances, including facioscapulohumeral muscular dystrophy however, how it brings about gene silencing is unknown. Understanding the molecular mechanism by which Smchd1 contributes to stable transcriptional silencing is critical to appreciate how it functions in normal biology and when it is mutated in facioscapulohumeral muscular dystrophy. This study reveals, for the first time to our knowledge, where Smchd1 binds genome-wide, its hitherto unappreciated functional interaction with chromatin organizer CCCTC-binding factor in gene regulation, and which part of the protein is required for chromatin binding. These data lead to a new model of Smchd1 function, where it directly binds DNA to mediate 3D chromatin architecture.
Publisher: American Chemical Society (ACS)
Date: 13-02-2023
Publisher: Informa UK Limited
Date: 07-12-2009
DOI: 10.3109/08977190903443048
Abstract: Mouse interleukin-3 (mIL-3) is a critical cytokine regulator of myeloid cell differentiation, survival and activation, and consequently this cytokine has become a key reagent for hematological studies in the laboratory. Although bacterial expression has been used for the preparation of recombinant mIL-3 for more than 20 years, the resultant cytokine is known to exhibit poor solubility, be prone to aggregation, and may contain mispaired disulfide bonds. As a result, little structural characterization of mIL-3 has been possible to date. In the present work, we describe a convenient, inexpensive, and scalable protocol for preparing an mIL-3 analog with wild-type bioactivity from Escherichia coli via a simple purification scheme. This analog is typically expressed at >1 mg/l of shaking Super broth culture and, owing to solubility >5 mg/ml, structural studies in solution by nuclear magnetic resonance spectroscopy are feasible for mIL-3 for the first time.
Publisher: Elsevier BV
Date: 08-2010
Publisher: EMBO
Date: 17-01-2023
Abstract: Genetic lesions in X‐linked inhibitor of apoptosis (XIAP) pre‐dispose humans to cell death–associated inflammatory diseases, although the underlying mechanisms remain unclear. Here, we report that two patients with XIAP deficiency–associated inflammatory bowel disease display increased inflammatory IL‐1β maturation as well as cell death–associated caspase‐8 and Gasdermin D (GSDMD) processing in diseased tissue, which is reduced upon patient treatment. Loss of XIAP leads to caspase‐8‐driven cell death and bioactive IL‐1β release that is only abrogated by combined deletion of the apoptotic and pyroptotic cell death machinery. Namely, extrinsic apoptotic caspase‐8 promotes pyroptotic GSDMD processing that kills macrophages lacking both inflammasome and apoptosis signalling components (caspase‐1, ‐3, ‐7, ‐11 and BID), while caspase‐8 can still cause cell death in the absence of both GSDMD and GSDME when caspase‐3 and caspase‐7 are present. Neither caspase‐3 and caspase‐7‐mediated activation of the pannexin‐1 channel, or GSDMD loss, prevented NLRP3 inflammasome assembly and consequent caspase‐1 and IL‐1β maturation downstream of XIAP inhibition and caspase‐8 activation, even though the pannexin‐1 channel was required for NLRP3 triggering upon mitochondrial apoptosis. These findings uncouple the mechanisms of cell death and NLRP3 activation resulting from extrinsic and intrinsic apoptosis signalling, reveal how XIAP loss can co‐opt dual cell death programs, and uncover strategies for targeting the cell death and inflammatory pathways that result from XIAP deficiency.
Publisher: Cold Spring Harbor Laboratory
Date: 15-03-2023
DOI: 10.1101/2023.03.14.532682
Abstract: Cell death frequently occurs in the pathogenesis of obesity and non-alcoholic fatty liver disease (NAFLD). However, the exact contribution of core cell death machinery to disease manifestations remains ill-defined. Here, we show via the direct comparison of mice genetically deficient in apoptotic caspase-8 in myeloid cells, or the essential necroptotic regulators, Receptor-interacting protein kinase-3 (RIPK3) and Mixed lineage kinase domain-like (MLKL), that RIPK3-caspase-8 signaling regulates macrophage inflammatory responses and drives adipose tissue inflammation and NAFLD upon high-fat diet feeding. In contrast, MLKL, ergent to RIPK3, contributes to both obesity and NAFLD in a manner largely independent of inflammation. We also uncover that MLKL regulates the expression of molecules involved in lipid uptake, transport and metabolism and, congruent with this, we discover a shift in the hepatic lipidome upon MLKL deletion. Collectively, these findings highlight MLKL as an attractive therapeutic target to combat the growing obesity pandemic and metabolic disease.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 10-03-2020
DOI: 10.1126/SCISIGNAL.AAY6318
Abstract: SAM domains mediate heterotypic and homotypic interactions between ASK family kinases.
Publisher: Elsevier BV
Date: 10-2009
DOI: 10.1016/J.JMB.2009.08.049
Abstract: FF domains are poorly understood protein interaction modules that are present within eukaryotic transcription factors, such as CA150 (TCERG-1). The CA150 FF domains have been shown to mediate interactions with the phosphorylated C-terminal domain of RNA polymerase II (phosphoCTD) and a multitude of transcription factors and RNA processing proteins, and may therefore have a central role in organizing transcription. FF domains occur in tandem arrays of up to six domains, although it is not known whether they adopt higher-order structures. We have used the CA150 FF1+FF2 domains as a model system to examine whether tandem FF domains form higher-order structures in solution using NMR spectroscopy. In the solution structure of FF1 fused to the linker that joins FF1 to FF2, we observed that the highly conserved linker peptide is ordered and forms a helical extension of helix alpha3, suggesting that the interdomain linker might have a role in orientating FF1 relative to FF2. However, examination of the FF1+FF2 domains using relaxation NMR experiments revealed that although these domains are not rigidly orientated relative to one another, they do not tumble independently. Thus, the FF1+FF2 structure conforms to a dumbbell-shape in solution, where the helical interdomain linker maintains distance between the two dynamic FF domains without cementing their relative orientations. This model for FF domain organization within tandem arrays suggests a general mechanism by which in idual FF domains can manoeuvre to achieve optimal recognition of flexible binding partners, such as the intrinsically-disordered phosphoCTD.
Publisher: Proceedings of the National Academy of Sciences
Date: 31-03-2020
Abstract: The necroptosis cell death pathway has been implicated in host defense and in the pathology of inflammatory diseases. While phosphorylation of the necroptotic effector pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) by the upstream protein kinase RIPK3 is a hallmark of pathway activation, the precise checkpoints in necroptosis signaling are still unclear. Here we have developed monobodies, synthetic binding proteins, that bind the N-terminal four-helix bundle (4HB) “killer” domain and neighboring first brace helix of human MLKL with nanomolar affinity. When expressed as genetically encoded reagents in cells, these monobodies potently block necroptotic cell death. However, they did not prevent MLKL recruitment to the “necrosome” and phosphorylation by RIPK3, nor the assembly of MLKL into oligomers, but did block MLKL translocation to membranes where activated MLKL normally disrupts membranes to kill cells. An X-ray crystal structure revealed a monobody-binding site centered on the α4 helix of the MLKL 4HB domain, which mutational analyses showed was crucial for reconstitution of necroptosis signaling. These data implicate the α4 helix of its 4HB domain as a crucial site for recruitment of adaptor proteins that mediate membrane translocation, distinct from known phospholipid binding sites.
Publisher: Portland Press Ltd.
Date: 02-10-2015
DOI: 10.1042/BJ20150678
Abstract: The pseudokinase MLKL (mixed lineage kinase domain-like), has recently emerged as a critical component of the necroptosis cell death pathway. Although 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 MS, Ser158, Ser228 and Ser248. Expression of a phosphomimetic 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.
Publisher: Springer International Publishing
Date: 2014
Publisher: Elsevier BV
Date: 07-2016
DOI: 10.1016/J.IMMUNI.2016.06.029
Abstract: RIPK1 and RIPK3 are well-known signaling traffic cops in innate immunity. In this issue of Immunity, Degterev and colleagues show that when they blow the whistle on bacterial infection, they quickly point a white-gloved hand down the express route to inflammatory cytokine production.
Publisher: Informa UK Limited
Date: 20-01-2014
DOI: 10.3109/08977194.2013.874347
Abstract: Activation of the cell surface receptor, c-Mpl, by the cytokine, thrombopoietin (TPO), underpins megakaryocyte and platelet production in mammals. In humans, mutations in c-Mpl have been identified as the molecular basis of Congenital Amegakaryocytic Thrombocytopenia (CAMT). Here, we show that CAMT-associated mutations in c-Mpl principally lead to defective receptor presentation on the cell surface. In contrast, one CAMT mutant c-Mpl, F104S, was expressed on the cell surface, but showed defective TPO binding and receptor activation. Using mutational analyses, we examined which residues adjacent to F104 within the membrane-distal cytokine receptor homology module (CRM) of c-Mpl comprise the TPO-binding epitope, revealing residues within the predicted Domain 1 E-F and A-B loops and Domain 2 F'-G' loop as key TPO-binding determinants. These studies underscore the importance of the c-Mpl membrane-distal CRM to TPO-binding and suggest that mutations within this CRM that perturb TPO binding could give rise to CAMT.
Publisher: Public Library of Science (PLoS)
Date: 21-08-2013
Publisher: Proceedings of the National Academy of Sciences
Date: 29-12-2016
Abstract: Plasmodium vivax is responsible for the most widely distributed recurring human malaria infections whereas Plasmodium falciparum inflicts the most mortality and morbidity in human populations. Malaria parasites enter our blood cells by making proteins that recognize and bind to their cognate receptors on the red blood cell surface. Our research describes, to our knowledge, the first crystal structure of PvRBP2a, an erythrocyte-binding protein from P. vivax, which revealed a structural scaffold similar to that of PfRh5, the essential erythrocyte-binding protein in P. falciparum . Structural comparisons between PvRBP2a and PfRh5 provide an important foundation toward understanding how P. vivax and P. falciparum parasites use a homologous erythrocyte-binding protein family to engage alternate erythrocyte receptors and ultimately govern host cell specificity.
Publisher: Elsevier BV
Date: 12-2011
Abstract: Epigenetic aberrations are increasingly regarded as key factors in cancer progression. Recently, deregulation of histone acetyltransferases (HATs) has been linked to several types of cancer. Monocytic leukemia zinc finger protein (MOZ) is a member of the MYST family of HATs, which regulate gene expression in cell proliferation and differentiation. Deregulation of these processes through constitutively active MOZ fusion proteins gives rise to the formation of leukemic stem cells, rendering MOZ an excellent target for treating myeloid leukemia. The authors implemented a hit discovery c aign to identify small-molecule inhibitors of MOZ-HAT activity. They developed a robust, homogeneous assay measuring the acetylation of synthetic histone peptides. In a primary screening c aign testing 243 000 lead-like compounds, they identified inhibitors from several chemical classes. Secondary assays were used to eliminate assay-interfering compounds and prioritize confirmed hits. This study establishes a new high-throughput assay for HAT activity and could provide the foundation for the development of a new class of drugs for the treatment of leukemias.
Publisher: Springer Science and Business Media LLC
Date: 02-07-2020
DOI: 10.1038/S41591-020-0972-7
Abstract: An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Publisher: Portland Press Ltd.
Date: 22-01-2016
DOI: 10.1042/BSR20150246
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 (aa) of human MLKL is sufficient to cause cell death, whereas expression of the first 154 aa is not. Given that aa 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.
Publisher: Springer Science and Business Media LLC
Date: 03-01-2023
Publisher: Informa UK Limited
Date: 23-01-2015
Publisher: Portland Press Ltd.
Date: 24-02-2010
DOI: 10.1042/BJ20091745
Abstract: GM-CSF (granulocyte/macrophage colony-stimulating factor) is an important mediator of inducible haemopoiesis and inflammation, and has a critical role in the function of alveolar macrophages. Its clinical applications include the mobilization of haemopoietic progenitors, and a role as an immune stimulant and vaccine adjuvant in cancer patients. GM-CSF signals via a specific α receptor (GM-CSFRα) and the shared hβc (human common β-subunit). The present study has investigated the role of the Ig-like domain of GM-CSFRα in GM-CSF binding and signalling. Deletion of the Ig-like domain abolished direct GM-CSF binding and decreased growth signalling in the presence of hβc. To locate the specific residues in the Ig-like domain of GM-CSFRα involved in GM-CSF binding, a structural alignment was made with a related receptor, IL-13Rα1 (interleukin-13 receptor α1), whose structure and mode of interaction with its ligand has recently been elucidated. Mutagenesis of candidate residues in the predicted region of interaction identified Val51 and Cys60 as having critical roles in binding to the α receptor, with Arg54 and Leu55 also being important. High-affinity binding in the presence of hβc was strongly affected by mutation of Cys60 and was also reduced by mutation of Val51, Arg54 and Leu55. Of the four key residues, growth signalling was most severely affected by mutation of Cys60. The results indicate a previously unrecognized role for the Ig-like domain, and in particular Cys60, of GM-CSFRα in the binding of GM-CSF and subsequent activation of cellular signalling.
Publisher: Springer Science and Business Media LLC
Date: 13-01-2017
Publisher: Springer Science and Business Media LLC
Date: 04-2022
DOI: 10.1038/S41419-022-04740-W
Abstract: Necroptosis is a form of caspase-independent programmed cell death that arises from disruption of cell membranes by the mixed lineage kinase domain-like (MLKL) pseudokinase after its activation by the upstream kinases, receptor interacting protein kinase (RIPK)-1 and RIPK3, within a complex known as the necrosome. Dysregulated necroptosis has been implicated in numerous inflammatory pathologies. As such, new small molecule necroptosis inhibitors are of great interest, particularly ones that operate downstream of MLKL activation, where the pathway is less well defined. To better understand the mechanisms involved in necroptosis downstream of MLKL activation, and potentially uncover new targets for inhibition, we screened known kinase inhibitors against an activated mouse MLKL mutant, leading us to identify the lymphocyte-specific protein tyrosine kinase (Lck) inhibitor AMG-47a as an inhibitor of necroptosis. We show that AMG-47a interacts with both RIPK1 and RIPK3, that its ability to protect from cell death is dependent on the strength of the necroptotic stimulus, and that it blocks necroptosis most effectively in human cells. Moreover, in human cell lines, we demonstrate that AMG-47a can protect against cell death caused by forced dimerisation of MLKL truncation mutants in the absence of any upstream signalling, validating that it targets a process downstream of MLKL activation. Surprisingly, however, we also found that the cell death driven by activated MLKL in this model was completely dependent on the presence of RIPK1, and to a lesser extent RIPK3, although it was not affected by known inhibitors of these kinases. Together, these results suggest an additional role for RIPK1, or the necrosome, in mediating human necroptosis after MLKL is phosphorylated by RIPK3 and provide further insight into reported differences in the progression of necroptosis between mouse and human cells.
Publisher: Elsevier
Date: 2022
DOI: 10.1016/BS.MIE.2022.03.054
Abstract: The non-catalytic cousins of protein kinases, the pseudokinases, have grown to prominence as indispensable signaling entities over the past decade, despite their lack of catalytic activity. Because their importance has only been fully embraced recently, many of the 10% of the human kinome categorized as pseudokinases are yet to be attributed biological functions. The advent of CRISPR-Cas9 editing to genetically delete pseudokinases in a cell line of interest has proven invaluable to dissecting many functions and remains the method of choice for gene knockout. Here, using the terminal effector pseudokinase in the necroptosis cell death pathway, MLKL, as an exemplar, we describe a method for genetic knockout of pseudokinases in cultured cells. This method does not retain the CRISPR guide sequence in the edited cells, which eliminates possible interference in subsequent reconstitution studies where mutant forms of the pseudokinase can be reintroduced into cells exogenously for detailed mechanistic characterization.
Publisher: Springer Science and Business Media LLC
Date: 19-11-2019
Publisher: Springer Science and Business Media LLC
Date: 26-10-2023
Publisher: Springer Science and Business Media LLC
Date: 18-08-2020
DOI: 10.1038/S41418-020-00604-Y
Abstract: Mitotic catastrophe (MC) is an important oncosuppressive mechanism that serves to eliminate cells that become polyploid or aneuploid due to aberrant mitosis. Previous studies have demonstrated that the activation and catalytic function of caspase-2 are key steps in MC to trigger apoptosis and/or cell cycle arrest of mitotically defective cells. However, the molecular mechanisms that regulate caspase-2 activation and its function are unclear. Here, we identify six new phosphorylation sites in caspase-2 and show that a key mitotic kinase, Aurora B kinase (AURKB), phosphorylates caspase-2 at the highly conserved residue S384. We demonstrate that phosphorylation at S384 blocks caspase-2 catalytic activity and apoptosis function in response to mitotic insults, without affecting caspase-2 dimerisation. Moreover, molecular modelling suggests that phosphorylation at S384 may affect substrate binding by caspase-2. We propose that caspase-2 S384 phosphorylation by AURKB is a key mechanism that controls caspase-2 activation during mitosis.
Publisher: Springer Science and Business Media LLC
Date: 23-06-2022
DOI: 10.1038/S41419-022-05009-Y
Abstract: Necroptosis is a caspase-independent, pro-inflammatory mode of programmed cell death which relies on the activation of the terminal effector, MLKL, by the upstream protein kinase RIPK3. To mediate necroptosis, RIPK3 must stably interact with, and phosphorylate the pseudokinase domain of MLKL, although the precise molecular cues that provoke RIPK3 necroptotic signaling are incompletely understood. The recent finding that RIPK3 S227 phosphorylation and the occurrence of a stable RIPK3:MLKL complex in human cells prior to exposure to a necroptosis stimulus raises the possibility that additional, as-yet-unidentified phosphorylation events activate RIPK3 upon initiation of necroptosis signaling. Here, we sought to identify phosphorylation sites of RIPK3 and dissect their regulatory functions. Phosphoproteomics identified 21 phosphorylation sites in HT29 cells overexpressing human RIPK3. By comparing cells expressing wild-type and kinase-inactive D142N RIPK3, autophosphorylation sites and substrates of other cellular kinases were distinguished. Of these 21 phosphosites, mutational analyses identified only pT224 and pS227 as crucial, synergistic sites for stable interaction with MLKL to promote necroptosis, while the recently reported activation loop phosphorylation at S164/T165 negatively regulate the kinase activity of RIPK3. Despite being able to phosphorylate MLKL to a similar or higher extent than wild-type RIPK3, mutation of T224, S227, or the RHIM in RIPK3 attenuated necroptosis. This finding highlights the stable recruitment of human MLKL by RIPK3 to the necrosome as an essential checkpoint in necroptosis signaling, which is independent from and precedes the phosphorylation of MLKL.
Publisher: Springer Science and Business Media LLC
Date: 06-06-2014
DOI: 10.1038/CDD.2014.70
Publisher: EMBO
Date: 26-10-2021
Publisher: Springer Science and Business Media LLC
Date: 14-02-2018
Publisher: Springer Science and Business Media LLC
Date: 12-02-2016
DOI: 10.1038/CDD.2015.169
Publisher: Elsevier BV
Date: 08-2011
DOI: 10.1016/J.JMR.2011.05.013
Abstract: Conformational or chemical exchange can cause significant sensitivity loss in NMR spectroscopy through resonance broadening for nuclear spins involved in these processes. While this effect may sometimes be alleviated by manipulating experimental conditions such as temperature, pH, and buffers, conditions optimal for all resonances are not always achievable. As a consequence, any means of recovering or minimizing this exchange-induced sensitivity loss is potentially of significant value in regaining information otherwise lost. We report the experimental observation of significant sensitivity gain for nuclear spins undergoing chemical exchange with solvent (water) at exchange rates ca 1-10 s(-1) in (1)H-(15)N correlation spectra of proteins acquired with band-selective pulses (the SOFAST-HMQC sequence).
Publisher: Springer Science and Business Media LLC
Date: 13-04-2021
DOI: 10.1038/S41467-021-22400-Z
Abstract: Phosphorylation of the MLKL pseudokinase by the RIPK3 kinase leads to MLKL oligomerization, translocation to, and permeabilization of, the plasma membrane to induce necroptotic cell death. The precise choreography of MLKL activation remains incompletely understood. Here, we report Monobodies, synthetic binding proteins, that bind the pseudokinase domain of MLKL within human cells and their crystal structures in complex with the human MLKL pseudokinase domain. While Monobody-32 constitutively binds the MLKL hinge region, Monobody-27 binds MLKL via an epitope that overlaps the RIPK3 binding site and is only exposed after phosphorylated MLKL disengages from RIPK3 following necroptotic stimulation. The crystal structures identified two distinct conformations of the MLKL pseudokinase domain, supporting the idea that a conformational transition accompanies MLKL disengagement from RIPK3. These studies provide further evidence that MLKL undergoes a large conformational change upon activation, and identify MLKL disengagement from RIPK3 as a key regulatory step in the necroptosis pathway.
Publisher: Springer Science and Business Media LLC
Date: 28-01-2021
DOI: 10.1038/S41419-021-03418-Z
Abstract: Necroptosis is a pro-inflammatory cell death program executed by the terminal effector, mixed lineage kinase domain-like (MLKL). Previous studies suggested a role for the necroptotic machinery in platelets, where loss of MLKL or its upstream regulator, RIPK3 kinase, impacted thrombosis and haemostasis. However, it remains unknown whether necroptosis operates within megakaryocytes, the progenitors of platelets, and whether necroptotic cell death might contribute to or diminish platelet production. Here, we demonstrate that megakaryocytes possess a functional necroptosis signalling cascade. Necroptosis activation leads to phosphorylation of MLKL, loss of viability and cell swelling. Analyses at steady state and post antibody-mediated thrombocytopenia revealed that platelet production was normal in the absence of MLKL, however, platelet activation and haemostasis were impaired with prolonged tail re-bleeding times. We conclude that MLKL plays a role in regulating platelet function and haemostasis and that necroptosis signalling in megakaryocytes is dispensable for platelet production.
Publisher: Springer Science and Business Media LLC
Date: 15-02-2013
Publisher: Springer Science and Business Media LLC
Date: 14-09-2020
Publisher: Portland Press Ltd.
Date: 13-04-2017
DOI: 10.1042/BST20160400
Abstract: Pseudoenzymes are catalytically dead counterparts of enzymes. Despite their first description some 50 years ago, the importance and functional ersity of these ‘fit-for-purpose’ polypeptides is only now being appreciated. Pseudoenzymes have been identified throughout all the kingdoms of life and, owing to predicted deficits in enzyme activity due to the absence of catalytic residues, have been variously referred to as pseudoenzymes, non-enzymes, dead enzymes, prozymes or ‘zombie’ proteins. An important goal of the recent Biochemical Society Pseudoenzymes-focused meeting was to explore the functional and evolutionary ersity of pseudoenzymes and to begin to evaluate their functions in biology, including cell signalling and metabolism. Here, we summarise the impressive breadth of enzyme classes that are known to have pseudoenzyme counterparts and present ex les of known cellular functions. We predict that the next decades will represent golden years for the analysis of pseudoenzymes.
Publisher: Portland Press Ltd.
Date: 10-06-2016
DOI: 10.1042/BCJ20160189
Abstract: Structural maintenance of chromosomes flexible hinge domain containing 1 (Smchd1) is an epigenetic regulator that plays critical roles in gene regulation during development. Mutations in SMCHD1 were recently implicated in the pathogenesis of facioscapulohumeral muscular dystrophy (FSHD), although the mechanistic basis remains of outstanding interest. We have previously shown that Smchd1 associates with chromatin via its homodimeric C-terminal hinge domain, yet little is known about the function of the putative GHKL (gyrase, Hsp90, histidine kinase, MutL)-type ATPase domain at its N-terminus. To formally assess the structure and function of Smchd1’s ATPase domain, we have generated recombinant proteins encompassing the predicted ATPase domain and the adjacent region. Here, we show that the Smchd1 N-terminal region exists as a monomer and adopts a conformation resembling that of monomeric full-length heat shock protein 90 (Hsp90) protein in solution, even though the two proteins share only ∼8% overall sequence identity. Despite being monomeric, the N-terminal region of Smchd1 exhibits ATPase activity, which can be antagonized by the reaction product, ADP, or the Hsp90 inhibitor, radicicol, at a nanomolar concentration. Interestingly, introduction of an analogous mutation to that identified in SMCHD1 of an FSHD patient compromised protein stability, suggesting a possible molecular basis for loss of protein function and pathogenesis. Together, these results reveal important structure–function characteristics of Smchd1 that may underpin its mechanistic action at the chromatin level.
Publisher: Elsevier BV
Date: 06-2004
Publisher: Springer Science and Business Media LLC
Date: 27-05-2017
Publisher: American Association for the Advancement of Science (AAAS)
Date: 13-08-2019
DOI: 10.1126/SCISIGNAL.AAT9797
Abstract: A roadmap for identifying and analyzing pseudoenzymes is proposed for this rapidly expanding field.
Publisher: Springer Science and Business Media LLC
Date: 21-06-2018
DOI: 10.1038/S41467-018-04714-7
Abstract: Necroptotic cell death is mediated by the most terminal known effector of the pathway, MLKL. Precisely how phosphorylation of the MLKL pseudokinase domain activation loop by the upstream kinase, RIPK3, induces unmasking of the N-terminal executioner four-helix bundle (4HB) domain of MLKL, higher-order assemblies, and permeabilization of plasma membranes remains poorly understood. Here, we reveal the existence of a basal monomeric MLKL conformer present in human cells prior to exposure to a necroptotic stimulus. Following activation, toggling within the MLKL pseudokinase domain promotes 4HB domain disengagement from the pseudokinase domain αC helix and pseudocatalytic loop, to enable formation of a necroptosis-inducing tetramer. In contrast to mouse MLKL, substitution of RIPK3 substrate sites in the human MLKL pseudokinase domain completely abrogated necroptotic signaling. Therefore, while the pseudokinase domains of mouse and human MLKL function as molecular switches to control MLKL activation, the underlying mechanism differs between species.
Publisher: Oxford University Press (OUP)
Date: 30-01-2019
DOI: 10.1093/NAR/GKZ005
Publisher: Springer Science and Business Media LLC
Date: 13-02-2017
Publisher: American Chemical Society (ACS)
Date: 20-02-2003
DOI: 10.1021/JO0267091
Abstract: A versatile, stereoselective synthesis of 5-hydroxypiperidinones with substituents at N1, C3, and C6 has been developed. The sequence involves ring-closing metathesis of a diene amide and epoxidation of the resulting alkene, followed by base-mediated elimination, and finally hydrogenation.
Publisher: Elsevier BV
Date: 07-2022
Publisher: Springer Science and Business Media LLC
Date: 20-02-2010
DOI: 10.1007/S12104-010-9213-1
Abstract: Interleukin-3 (IL-3) is a cytokine that acts as a critical mediator of inflammation and immune responses to infections. IL-3, like interleukin-5 (IL-5) and granulocyte-macrophage colony stimulating factor (GM-CSF), exerts its effects on target cells via receptors composed of cytokine-specific alpha-subunits and a common beta-subunit (betac-subunit, shared with IL-5 and GM-CSF). In contrast to humans, mice also possess an additional beta-receptor, beta(IL-3), that can specifically bind IL-3. Except for a study carried out on an analogue of human IL-3 that contains 14 mutations, structure-related studies of IL-3 have been very limited, largely because of its poor solution behaviour. Here we report (1)H, (13)C, and (15)N chemical shift assignments of murine IL-3 comprising residues 33-156 (SWISS-PROT accession number: P01586), in which the only mutation is an alanine substitution of Cys105. The mIL-3 construct used in the present study was engineered by eliminating residues 27-32 of the N-terminus (the first 26 residues of the primary sequence of mIL-3 are cleaved in vivo during secretion), the C-terminal 10 residues (157-166), and a disulfide bond between Cys105 and Cys166 that is poorly conserved in orthologue sequences. The new construct vastly improves the solubility of murine IL-3 while maintaining its wild-type biological activity.
Publisher: Elsevier BV
Date: 06-2018
Publisher: Springer Science and Business Media LLC
Date: 17-03-2015
DOI: 10.1038/NCOMMS7442
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.
Publisher: Springer Science and Business Media LLC
Date: 21-02-2023
Publisher: Elsevier
Date: 2006
Publisher: Springer Science and Business Media LLC
Date: 04-2021
DOI: 10.1038/S41419-021-03636-5
Abstract: Maturity-onset diabetes of the young, MODY, is an autosomal dominant disease with incomplete penetrance. In a family with multiple generations of diabetes and several early onset diabetic siblings, we found the previously reported P33T PDX1 damaging mutation. Interestingly, this substitution was also present in a healthy sibling. In contrast, a second very rare heterozygous damaging mutation in the necroptosis terminal effector, MLKL, was found exclusively in the diabetic family members. Aberrant cell death by necroptosis is a cause of inflammatory diseases and has been widely implicated in human pathologies, but has not yet been attributed functions in diabetes. Here, we report that the MLKL substitution observed in diabetic patients, G316D, results in diminished phosphorylation by its upstream activator, the RIPK3 kinase, and no capacity to reconstitute necroptosis in two distinct MLKL −/− human cell lines. This MLKL mutation may act as a modifier to the P33T PDX1 mutation, and points to a potential role of impairment of necroptosis in diabetes. Our findings highlight the importance of family studies in unraveling MODY’s incomplete penetrance, and provide further support for the involvement of dysregulated necroptosis in human disease.
Publisher: F1000 Research Ltd
Date: 19-11-2015
DOI: 10.12688/F1000RESEARCH.7046.1
Abstract: Several programmed lytic and necrotic-like cell death mechanisms have now been uncovered, including the recently described receptor interacting protein kinase-3 (RIPK3)-mixed lineage kinase domain-like (MLKL)-dependent necroptosis pathway. Genetic experiments have shown that programmed necrosis, including necroptosis, can play a pivotal role in regulating host-resistance against microbial infections. Alternatively, excess or unwarranted necroptosis may be pathological in autoimmune and autoinflammatory diseases. This review highlights the recent advances in our understanding of the post-translational control of RIPK3-MLKL necroptotic signaling. We discuss the critical function of phosphorylation in the execution of necroptosis, and highlight the emerging regulatory roles for several ubiquitin ligases and deubiquitinating enzymes. Finally, based on current evidence, we discuss the potential mechanisms by which the essential, and possibly terminal, necroptotic effector, MLKL, triggers the disruption of cellular membranes to cause cell lysis.
Publisher: Springer Science and Business Media LLC
Date: 17-01-2013
Publisher: Springer Science and Business Media LLC
Date: 27-02-2014
Publisher: Springer Science and Business Media LLC
Date: 03-03-2013
DOI: 10.1038/NSMB.2519
Publisher: Springer Science and Business Media LLC
Date: 09-03-2017
DOI: 10.1038/SREP43827
Abstract: Devil facial tumour disease (DFTD) is a transmissible cancer devastating the Tasmanian devil ( Sarcophilus harrisii ) population. The cancer cell is the ‘infectious’ agent transmitted as an allograft by biting. Animals usually die within a few months with no evidence of antibody or immune cell responses against the DFTD allograft. This lack of anti-tumour immunity is attributed to an absence of cell surface major histocompatibility complex (MHC)-I molecule expression. While the endangerment of the devil population precludes experimentation on large experimental groups, those examined in our study indicated that immunisation and immunotherapy with DFTD cells expressing surface MHC-I corresponded with effective anti-tumour responses. Tumour engraftment did not occur in one of the five immunised Tasmanian devils, and regression followed therapy of experimentally induced DFTD tumours in three Tasmanian devils. Regression correlated with immune cell infiltration and antibody responses against DFTD cells. These data support the concept that immunisation of devils with DFTD cancer cells can successfully induce humoral responses against DFTD and trigger immune-mediated regression of established tumours. Our findings support the feasibility of a protective DFTD vaccine and ultimately the preservation of the species.
Publisher: Portland Press Ltd.
Date: 23-01-2018
DOI: 10.1042/BCJ20170427
Abstract: The retinoic acid-inducible gene-I (RIG-I) receptor recognizes short 5′-di- and triphosphate base-paired viral RNA and is a critical mediator of the innate immune response against viruses such as influenza A, Ebola, HIV and hepatitis C. This response is reported to require an orchestrated interaction with the tripartite motif 25 (TRIM25) B30.2 protein-interaction domain. Here, we present a novel second RIG-I-binding interface on the TRIM25 B30.2 domain that interacts with CARD1 and CARD2 (caspase activation and recruitment domains) of RIG-I and is revealed by the removal of an N-terminal α-helix that mimics dimerization of the full-length protein. Further characterization of the TRIM25 coiled-coil and B30.2 regions indicated that the B30.2 domains move freely on a flexible tether, facilitating RIG-I CARD recruitment. The identification of a dual binding mode for the TRIM25 B30.2 domain is a first for the SPRY/B30.2 domain family and may be a feature of other SPRY/B30.2 family members.
Publisher: The Company of Biologists
Date: 2018
DOI: 10.1242/JEB.192245
Abstract: Bacterial and viral pathogens can weaken epithelial barriers by targeting and disrupting tight junction (TJ) proteins. Comparatively, however, little is known about the direct effects of fungal pathogens on TJ proteins and their expression. The disease, chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), is threatening hibian populations worldwide. Bd is known to infect hibian skin and disrupt cutaneous osmoregulation. However, exactly how this occurs is poorly understood. This study considered the impact of Bd infection on the barrier properties of the Australian green tree frog (Litoria caerulea) epidermis by examining how inoculation of animals with Bd influenced the paracellular movement of FITC-dextran (4 kDa, FD-4) across the skin in association with alterations in the mRNA and protein abundance of select TJ proteins of the epidermal TJ complex. It was observed that Bd infection increased paracellular movement of FD-4 across the skin linearly with fungal infection load. In addition, Bd infection increased transcript abundance of the tricellular TJ (tTJ) protein tricellulin (tric) as well as the bicellular TJ (bTJ) proteins occludin (ocln), claudin (cldn) -1, -4 and the scaffolding TJ protein zonula occludens-1 (zo-1). However, while Tric protein abundance increased in accord with changes in transcript abundance, protein abundance of Cldn-1 was significantly reduced and Ocln protein abundance was unchanged. Data indicate that disruption of cutaneous osmoregulation in L. caerulea following Bd infection occurs, at least in part, by an increase in epidermal paracellular permeability in association with compromised integrity of the epidermal TJ complex.
Publisher: Proceedings of the National Academy of Sciences
Date: 17-01-2017
Abstract: Necroptotic cell death is mediated by activation of the mixed-lineage kinase domain-like protein (MLKL). The inflammation associated with this form of cell death is thought to be due to the release of proinflammatory cellular contents after plasma membrane rupture. In contrast to this prevailing view, we show that MLKL activates the innate immune receptor nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) in a cell-intrinsic manner. Importantly, we show that MLKL-mediated NLRP3 and caspase-1 activation and the secretion of the proinflammatory cytokine IL-1β is a major determinant of necroptotic-derived inflammatory signals. These findings suggest that NLRP3 and IL-1β may be relevant therapeutic targets in MLKL-driven diseases.
Publisher: Portland Press Ltd.
Date: 10-01-2014
DOI: 10.1042/BJ20131270
Abstract: The pseudokinase MLKL (mixed lineage kinase domain-like) was identified recently as an essential checkpoint in the programmed necrosis or ‘necroptosis’ cell death pathway. In the present study, we report the crystal structure of the human MLKL pseudokinase domain at 1.7 Å (1 Å=0.1 nm) resolution and probe its nucleotide-binding mechanism by performing structure-based mutagenesis. By comparing the structures and nucleotide-binding determinants of human and mouse MLKL orthologues, the present study provides insights into the evolution of nucleotide-binding mechanisms among pseudokinases and their mechanistic ergence from conventional catalytically active protein kinases.
Publisher: Elsevier BV
Date: 10-2020
Publisher: Cold Spring Harbor Laboratory
Date: 03-05-2021
DOI: 10.1101/2021.05.03.442385
Abstract: Necroptosis is a lytic programmed cell death pathway with origins in innate immunity that is frequently dysregulated in inflammatory diseases. The terminal effector of the pathway, MLKL, is licensed to kill following phosphorylation of its pseudokinase domain by the upstream regulator, RIPK3 kinase. Phosphorylation provokes the unleashing of MLKL’s N-terminal four-helix bundle (4HB or HeLo) domain, which binds and permeabilizes the plasma membrane to cause cell death. The precise mechanism by which the 4HB domain permeabilizes membranes, and how the mechanism differs between species, remains unclear. Here, we identify the membrane binding epitope of mouse MLKL using NMR spectroscopy. Using liposome permeabilization and cell death assays, we validate K69 in the α3 helix, W108 in the α4 helix, and R137/Q138 in the first brace helix as crucial residues for necroptotic signaling. This epitope differs from the phospholipid binding site reported for human MLKL, which comprises basic residues primarily located in the α1 and α2 helices. In further contrast to human and plant MLKL orthologs, in which the α3-α4 loop forms a helix, this loop is unstructured in mouse MLKL in solution. Together, these findings illustrate the versatility of the 4HB domain fold, whose lytic function can be mediated by distinct epitopes in different orthologs.
Publisher: Springer Science and Business Media LLC
Date: 08-02-2023
DOI: 10.1038/S41418-023-01121-4
Abstract: MLKL and RIPK3 are the core signaling proteins of the inflammatory cell death pathway, necroptosis, which is a known mediator and modifier of human disease. Necroptosis has been implicated in the progression of disease in almost every physiological system and recent reports suggest a role for necroptosis in aging. Here, we present the first comprehensive analysis of age-related histopathological and immunological phenotypes in a cohort of Mlkl –/– and Ripk3 –/– mice on a congenic C57BL/6 J genetic background. We show that genetic deletion of Mlkl in female mice interrupts immune system aging, specifically delaying the age-related reduction of circulating lymphocytes. -Seventeen-month-old Mlkl –/– female mice were also protected against age-related chronic sterile inflammation in connective tissue and skeletal muscle relative to wild-type littermate controls, exhibiting a reduced number of immune cell infiltrates in these sites and fewer regenerating myocytes. These observations implicate MLKL in age-related sterile inflammation, suggesting a possible application for long-term anti-necroptotic therapy in humans.
Publisher: Springer Science and Business Media LLC
Date: 09-01-2017
DOI: 10.1038/NG.3765
Abstract: Bosma arhinia microphthalmia syndrome (BAMS) is an extremely rare and striking condition characterized by complete absence of the nose with or without ocular defects. We report here that missense mutations in the epigenetic regulator SMCHD1 mapping to the extended ATPase domain of the encoded protein cause BAMS in all 14 cases studied. All mutations were de novo where parental DNA was available. Biochemical tests and in vivo assays in Xenopus laevis embryos suggest that these mutations may behave as gain-of-function alleles. This finding is in contrast to the loss-of-function mutations in SMCHD1 that have been associated with facioscapulohumeral muscular dystrophy (FSHD) type 2. Our results establish SMCHD1 as a key player in nasal development and provide biochemical insight into its enzymatic function that may be exploited for development of therapeutics for FSHD.
Publisher: Portland Press Ltd.
Date: 09-07-2021
DOI: 10.1042/BCJ20210278
Abstract: Structural maintenance of chromosomes flexible hinge domain-containing 1 (SMCHD1) is an epigenetic regulator that mediates gene expression silencing at targeted sites across the genome. Our current understanding of SMCHD1's molecular mechanism, and how substitutions within SMCHD1 lead to the diseases, facioscapulohumeral muscular dystrophy (FSHD) and Bosma arhinia microphthalmia syndrome (BAMS), are only emerging. Recent structural studies of its two component domains — the N-terminal ATPase and C-terminal SMC hinge — suggest that dimerization of each domain plays a central role in SMCHD1 function. Here, using biophysical techniques, we demonstrate that the SMCHD1 ATPase undergoes dimerization in a process that is dependent on both the N-terminal UBL (Ubiquitin-like) domain and ATP binding. We show that neither the dimerization event, nor the presence of a C-terminal extension past the transducer domain, affect SMCHD1's in vitro catalytic activity as the rate of ATP turnover remains comparable to the monomeric protein. We further examined the functional importance of the N-terminal UBL domain in cells, revealing that its targeted deletion disrupts the localization of full-length SMCHD1 to chromatin. These findings implicate UBL-mediated SMCHD1 dimerization as a crucial step for chromatin interaction, and thereby for promoting SMCHD1-mediated gene silencing.
Publisher: Elsevier BV
Date: 09-2019
Publisher: Springer Science and Business Media LLC
Date: 20-04-2020
DOI: 10.1038/S41591-020-0807-6
Abstract: A double burden of malnutrition occurs when in iduals, household members or communities experience both undernutrition and overweight. Here, we show geospatial estimates of overweight and wasting prevalence among children under 5 years of age in 105 low- and middle-income countries (LMICs) from 2000 to 2017 and aggregate these to policy-relevant administrative units. Wasting decreased overall across LMICs between 2000 and 2017, from 8.4% (62.3 (55.1–70.8) million) to 6.4% (58.3 (47.6–70.7) million), but is predicted to remain above the World Health Organization’s Global Nutrition Target of % in over half of LMICs by 2025. Prevalence of overweight increased from 5.2% (30 (22.8–38.5) million) in 2000 to 6.0% (55.5 (44.8–67.9) million) children aged under 5 years in 2017. Areas most affected by double burden of malnutrition were located in Indonesia, Thailand, southeastern China, Botswana, Cameroon and central Nigeria. Our estimates provide a new perspective to researchers, policy makers and public health agencies in their efforts to address this global childhood syndemic.
Publisher: Portland Press Ltd.
Date: 23-08-2023
DOI: 10.1042/BST20230828
Abstract: Biochemical Society Transactions is the reviews journal of the Biochemical Society. Publishing concise reviews written by experts in the field it provides a timely snapshot of the latest developments across all areas of the molecular and cellular biosciences. Elevating authors’ ideas and expertise, each review includes a perspectives section where authors offer comment on the latest advances, a glimpse of future challenges and highlighting the importance of associated research areas in far broader contexts. As Biochemical Society Transactions celebrates its 50th birthday, the current Editor-in-Chief looks back on the journal's history, and looks forward to the next 50 years.
Publisher: Elsevier BV
Date: 02-2009
Publisher: Cold Spring Harbor Laboratory
Date: 26-11-2999
DOI: 10.1101/2020.10.26.356063
Abstract: Necroptosis is a lytic, inflammatory cell death pathway that is dysregulated in many human pathologies. The pathway is executed by a core machinery comprising the RIPK1 and RIPK3 kinases, which assemble into necrosomes in the cytoplasm, and the terminal effector pseudokinase, MLKL. RIPK3-mediated phosphorylation of MLKL induces oligomerization and translocation to the plasma membrane where MLKL accumulates as hotspots and perturbs the lipid bilayer to cause death. The precise choreography of events in the pathway, where they occur within cells, and pathway differences between species, are of immense interest. However, they have been poorly characterized due to a dearth of validated antibodies for microscopy studies. Here, we describe a toolbox of antibodies for immunofluorescent detection of the core necroptosis effectors, RIPK1, RIPK3 and MLKL, and their phosphorylated forms, in human and mouse cells. By comparing reactivity with endogenous proteins in wild-type cells and knockout controls in basal and necroptosis-inducing conditions, we characterise the specificity of frequently-used commercial and recently-developed antibodies for detection of necroptosis signaling events. Importantly, our findings demonstrate that not all frequently-used antibodies are suitable for monitoring necroptosis by immunofluorescence microscopy, and methanol-is preferable to paraformaldehyde-fixation for robust detection of specific RIPK1, RIPK3 and MLKL signals.
Publisher: Hindawi Limited
Date: 13-06-2017
DOI: 10.1111/CMI.12750
Abstract: Pathogenic microbes have evolved countless sophisticated mechanisms to subvert host immune responses and cause disease. Understanding evasion strategies employed by pathogens has led to numerous discoveries on specific host cell processes that are critical for controlling infection. Programmed cell death (PCD) is a key host defence to microbial infection, as well as being critical for organ development and cellular homeostasis in multicellular organisms. Much of our current understanding of PCD as a host response to infection has stemmed from the discovery and study of viral inhibitors of apoptosis, and more recently viral inhibition of the newly characterised from of PCD termed necroptosis, the mechanisms of which are still under intense investigation. Many bacterial pathogens also encode inhibitors of PCD, yet these discoveries are relatively more recent and thus the biological significance of such mechanisms is still under debate. In this viewpoint article, we will argue the concept that necroptosis is merely a "back-up" mechanism in the event that apoptosis is inhibited, or whether it is a true host innate response to infection that has evolved in response to a growing arsenal of microbial evasion strategies.
Publisher: Springer Science and Business Media LLC
Date: 19-04-2018
DOI: 10.1038/S41467-018-04013-1
Abstract: The SOCS family of proteins are negative-feedback inhibitors of signalling induced by cytokines that act via the JAK/STAT pathway. SOCS proteins can act as ubiquitin ligases by recruiting Cullin5 to ubiquitinate signalling components however, SOCS1, the most potent member of the family, can also inhibit JAK directly. Here we determine the structural basis of both these modes of inhibition. Due to alterations within the SOCS box domain, SOCS1 has a compromised ability to recruit Cullin5 however, it is a direct, potent and selective inhibitor of JAK catalytic activity. The kinase inhibitory region of SOCS1 targets the substrate binding groove of JAK with high specificity and thereby blocks any subsequent phosphorylation. SOCS1 is a potent inhibitor of the interferon gamma (IFNγ) pathway, however, it does not bind the IFNγ receptor, making its mode-of-action distinct from SOCS3. These findings reveal the mechanism used by SOCS1 to inhibit signalling by inflammatory cytokines.
Publisher: Elsevier BV
Date: 10-2020
Publisher: Research Square Platform LLC
Date: 26-09-2022
DOI: 10.21203/RS.3.RS-2064774/V1
Abstract: EphB6 is an understudied ephrin receptor tyrosine pseudokinase, the expression of which is downregulated in multiple types of metastatic cancers. Compared to its kinase-active counterparts, how EphB6 regulates signal transduction in the absence of intrinsic kinase activity remains unknown. Here, we unveil the molecular details of key signal transduction mechanisms driven by EphB6. We identify ephrinB1 as a cognate ligand of EphB6 and demonstrate EphB6’s ability to form extensive co-clusters at the plasma membrane upon binding ephrinB1 in trans across cell-cell contacts, which are resistant to endocytosis. Using super resolution microscopy, correlative light and electron microscopy and cryo-electron tomography, we reveal that co clustering of EphB6 and ephrinB1 promotes the formation of unprecedented double membrane tubular structure interconnecting cells. Importantly, phenotypically, co-clustering of EphB6 and ephrinB1 and the resulting tubular structures suppress cancer cell invasion by prolonging cell-cell contacts, rationalising a role for EphB6 pseudokinase as a tumor suppressor when interacting with its ligands in trans .
Publisher: Springer Science and Business Media LLC
Date: 19-06-2020
DOI: 10.1038/S41467-020-16823-3
Abstract: The MLKL pseudokinase is the terminal effector in the necroptosis cell death pathway. Phosphorylation by its upstream regulator, RIPK3, triggers MLKL’s conversion from a dormant cytoplasmic protein into oligomers that translocate to, and permeabilize, the plasma membrane to kill cells. The precise mechanisms underlying these processes are incompletely understood, and were proposed to differ between mouse and human cells. Here, we examine the ergence of activation mechanisms among nine vertebrate MLKL orthologues, revealing remarkable specificity of mouse and human RIPK3 for MLKL orthologues. Pig MLKL can restore necroptotic signaling in human cells while horse and pig, but not rat, MLKL can reconstitute the mouse pathway. This selectivity can be rationalized from the distinct conformations observed in the crystal structures of horse and rat MLKL pseudokinase domains. These studies identify important differences in necroptotic signaling between species, and suggest that, more broadly, ergent regulatory mechanisms may exist among orthologous pseudoenzymes.
Publisher: American Society for Clinical Investigation
Date: 17-10-2016
DOI: 10.1172/JCI87545
Publisher: Elsevier BV
Date: 02-2023
DOI: 10.1016/J.TCB.2022.05.008
Abstract: The past decade has seen the emergence of the necroptosis programmed cell death pathway as an important contributor to the pathophysiology of myriad diseases. The receptor interacting protein kinase (RIPK)1 and RIPK3, and the pseudokinase executioner protein, mixed lineage kinase domain-like (MLKL), have grown to prominence as the core pathway components. Depending on cellular context, these proteins also serve as integrators of signals, such as post-translational modifications and protein or metabolite interactions, adding layers of complexity to pathway regulation. Here, we describe the emerging picture of the web of proteins that tune necroptotic signal transduction and how these events have erged across species, presumably owing to selective pressures of pathogens upon the RIPK3-MLKL protein pair.
Publisher: Springer Science and Business Media LLC
Date: 12-02-2021
DOI: 10.1038/S41467-021-21191-7
Abstract: The life cycle of Baculoviridae family insect viruses depends on the viral protein kinase, PK-1, to phosphorylate the regulatory protein, p6.9, to induce baculoviral genome release. Here, we report the crystal structure of Cydia pomenella granulovirus PK-1, which, owing to its likely ancestral origin among host cell AGC kinases, exhibits a eukaryotic protein kinase fold. PK-1 occurs as a rigid dimer, where an antiparallel arrangement of the αC helices at the dimer core stabilizes PK-1 in a closed, active conformation. Dimerization is facilitated by C-lobe:C-lobe and N-lobe:N-lobe interactions between protomers, including the domain-swapping of an N-terminal helix that crowns a contiguous β-sheet formed by the two N-lobes. PK-1 retains a dimeric conformation in solution, which is crucial for catalytic activity. Our studies raise the prospect that parallel, side-to-side dimeric arrangements that lock kinase domains in a catalytically-active conformation could function more broadly as a regulatory mechanism among eukaryotic protein kinases.
Publisher: Springer Science and Business Media LLC
Date: 12-08-2019
DOI: 10.1038/S41388-019-0931-2
Abstract: Eph receptors, the largest subfamily of receptor tyrosine kinases, are linked with proliferative disease, such as cancer, as a result of their deregulated expression or mutation. Unlike other tyrosine kinases that have been clinically targeted, the development of therapeutics against Eph receptors remains at a relatively early stage. The major reason is the limited understanding on the Eph receptor regulatory mechanisms at a molecular level. The complexity in understanding Eph signalling in cells arises due to following reasons: (1) Eph receptors comprise 14 members, two of which are pseudokinases, EphA10 and EphB6, with relatively uncharacterised function (2) activation of Eph receptors results in dimerisation, oligomerisation and formation of clustered signalling centres at the plasma membrane, which can comprise different combinations of Eph receptors, leading to erse downstream signalling outputs (3) the non-catalytic functions of Eph receptors have been overlooked. This review provides a structural perspective of the intricate molecular mechanisms that drive Eph receptor signalling, and investigates the contribution of intra- and inter-molecular interactions between Eph receptors intracellular domains and their major binding partners. We focus on the non-catalytic functions of Eph receptors with relevance to cancer, which are further substantiated by exploring the role of the two pseudokinase Eph receptors, EphA10 and EphB6. Throughout this review, we carefully analyse and reconcile the existing/conflicting data in the field, to allow researchers to further the current understanding of Eph receptor signalling.
Publisher: Springer Science and Business Media LLC
Date: 28-09-2023
Publisher: American Chemical Society (ACS)
Date: 27-07-2015
DOI: 10.1021/ACS.BIOCHEM.5B00619
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 preformed 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.
Publisher: Proceedings of the National Academy of Sciences
Date: 06-10-2014
Abstract: The four-helix bundle (4HB) domain of Mixed Lineage Kinase Domain-Like (MLKL) bears two clusters of residues that are required for cell death by necroptosis. Mutations within a cluster centered on the α4 helix of the 4HB domain of MLKL prevented its membrane translocation, oligomerization, and ability to induce necroptosis. This cluster is composed principally of acidic residues and therefore challenges the idea that the 4HB domain engages negatively charged phospholipid membranes via a conventional positively charged interaction surface. The importance of membrane translocation to MLKL-mediated death is supported by our identification of a small molecule that binds the MLKL pseudokinase domain and retards membrane translocation to inhibit necroptotic signaling.
Publisher: Springer Science and Business Media LLC
Date: 20-08-2018
DOI: 10.1038/S41594-018-0111-Z
Abstract: The regulation of higher-order chromatin structure is complex and dynamic, and a full understanding of the suite of mechanisms governing this architecture is lacking. Here, we reveal the noncanonical SMC protein Smchd1 to be a novel regulator of long-range chromatin interactions in mice, and we add Smchd1 to the canon of epigenetic proteins required for Hox-gene regulation. The effect of losing Smchd1-dependent chromatin interactions has varying outcomes that depend on chromatin context. At autosomal targets transcriptionally sensitive to Smchd1 deletion, we found increased short-range interactions and ectopic enhancer activation. In contrast, the inactive X chromosome was transcriptionally refractive to Smchd1 ablation, despite chromosome-wide increases in short-range interactions. In the inactive X, we observed spreading of trimethylated histone H3 K27 (H3K27me3) domains into regions not normally decorated by this mark. Together, these data suggest that Smchd1 is able to insulate chromatin, thereby limiting access to other chromatin-modifying proteins.
Publisher: Cold Spring Harbor Laboratory
Date: 12-09-2022
DOI: 10.1101/2022.09.08.507056
Abstract: Across the globe, 2-3% of humans carry the p.Ser132Pro single nucleotide polymorphism in MLKL , the terminal effector protein of the inflammatory form of programmed cell death, necroptosis. We show that this substitution confers a gain in necroptotic function in human cells, with more rapid accumulation of activated MLKL S132P in biological membranes and MLKL S132P overriding pharmacological and endogenous inhibition of MLKL. In mouse cells, the equivalent Mlkl S131P mutation confers a gene dosage dependent reduction in sensitivity to TNF-induced necroptosis in both hematopoietic and non-hematopoietic cells, but enhanced sensitivity to IFN-β induced death in non-hematopoietic cells. In vivo , Mlkl S131P homozygosity reduces the capacity to clear Salmonella from major organs and retards recovery of hematopoietic stem cells. Thus, by dysregulating necroptosis, the S131P substitution impairs the return to homeostasis after systemic challenge. Present day carriers of the MLKL S132P polymorphism may be the key to understanding how MLKL and necroptosis modulate the progression of complex polygenic human disease.
Publisher: Elsevier BV
Date: 09-2020
Publisher: Wiley
Date: 25-09-2015
DOI: 10.1111/FEBS.13504
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 ischaemia 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. We review the current understanding and gaps in knowledge relating to how MLKL can be activated by receptor interacting protein kinase (RIPK)3 downstream of tumour necrosis factor receptor 1:RIPK1, Toll like receptor-3:TRIF and viral DNA: DAI (DNA-dependent activator of interferon regulatory factors)/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.
Publisher: Springer New York
Date: 2017
DOI: 10.1007/978-1-4939-7154-1_7
Abstract: The protocol herein describes a robust and proven method for the measurement of pseudokinase-ligand interaction using a fluorescence-based thermal shift assay (TSA). Pseudokinases are kinase-like proteins that have recently emerged as crucial regulatory modules of signal transduction pathways and may well represent a novel class of drug targets. However, unlike kinases, the regulatory activity of pseudokinases is mainly conferred through protein-protein interactions. Understanding the mechanisms that underlie pseudokinase conformational changes through ligand binding and how such conformational changes can tune signaling pathways is a necessary step to unravel their biological functions.Thermal denaturation-based methods have proven to be a powerful method for determining the capacity of purified recombinant pseudokinases to bind ligands and can simultaneously inform on the potential druggability of the nucleotide-binding site. This assay takes advantage of a change in fluorescence arising when the dye, SYPRO Orange, binds to hydrophobic patches that become exposed when a protein undergoes thermal unfolding. Ligand binding to a protein is known to increase its thermal stability, which is reflected by a shift between the thermal denaturation curves of the unliganded protein and the liganded protein. Here, we illustrate the utility of the method with the pseudokinases, ErbB3/HER3, ILK, ROP5Bi, JAK1, JAK2, TYK2, MLKL, STRAD, TRIB1, VRK3, and ROR1. This method can also be used to determine optimal buffer conditions that may increase protein stability and can be tailored to other protein families.
Publisher: Wiley
Date: 02-08-2013
DOI: 10.1111/FEBS.12419
Abstract: Both of the TNF superfamily ligands, TNF and LTα, can bind and signal through TNFR1 and TNFR2, yet mice mutant for each have different phenotypes. Part of this difference is because LTα but not TNF can activate Herpes Virus Entry Mediator and also heterotrimerise with LTβ to activate LTβR, which is consistent with the similar phenotypes of the LTα and LTβR deficient mice. However, it has also been reported that the LTα3 homotrimer signals differently than TNF through TNFR1, and has unique roles in initiation and exacerbation of some inflammatory diseases. Our modeling of the TNF/TNFR1 interface compared to the LTα3/TNFR1 structure revealed some differences that could affect signalling by the two ligands. To determine whether there were any functional differences in the ability of TNF and LTα3 to induce TNFR1-dependent apoptosis or necroptosis, and if there were different requirements for cIAPs and Sharpin to transmit the TNFR1 signal, we compared the ability of cells to respond to TNF and LTα3. Contrary to our hypothesis, we were unable to discover differences in signalling by TNFR1 in response to TNF and LTα3. Our results imply that the reasons for the conservation of LTα are most likely due either to differential regulation, the ability to signal through Herpes Virus Entry Mediator or the ability of LTα to form heterotrimers with LTβ.
Publisher: Humana Press
Date: 2009
DOI: 10.1007/978-1-60327-394-7_6
Abstract: Understanding protein-protein interactions is a key step in unravelling the roles proteins play in cellular function. The ability to analyse protein-protein interactions rapidly and economically is a powerful research tool. Using peptide SPOT arrays, peptides of known sequence can be synthesized directly in discrete spots on a cellulose membrane and assayed for an interaction with a protein of interest. Several hundred peptides can be synthesized on each cellulose membrane therefore, this method is amenable to designing high-throughput peptide binding studies. SPOT arrays are particularly well suited for deducing peptidic binding motifs within proteins that are difficult to purify in sufficient quantities for traditional biochemical analyses, as well as for determining binding specificities and targets for proteins of undefined function. Peptide SPOT arrays have been used extensively to define protein-protein interaction surfaces. In this chapter, we will outline the steps involved in designing and probing a peptide SPOT array to identify peptide binding motifs for a protein of interest.
Publisher: The American Association of Immunologists
Date: 03-2018
Abstract: Neutrophil extracellular traps (NETs) and the cell death associated with it (NETosis) have been implicated in numerous diseases. Mechanistic studies of NETosis have typically relied on nonphysiological stimuli, such as PMA. The human disease of gout is caused by monosodium urate (MSU) crystals. We observed that DNA consistent with NETs is present in fluid from acutely inflamed joints of gout patients. NETs also coat the crystals found in uninflamed tophi of chronic gout patients. We developed a quantitative, live cell imaging assay, which measures the key features of NETosis, namely, cell death and chromatin decondensation. We show that MSU and other physiologically relevant crystals induce NETosis through a molecular pathway that is distinct from PMA and Candida hyphae. Crystals interact with lysosomes to induce NADPH oxidase-independent cell death, with postmortem chromatin decondensation mediated by neutrophil elastase. The resulting MSU-induced NETs are enriched for actin and are resistant to serum and DNase degradation. These findings demonstrate a distinct physiological NETosis pathway in response to MSU crystals, which coats MSU crystals in DNA that persists in tissues as gouty tophi.
Publisher: Portland Press Ltd.
Date: 12-05-2023
DOI: 10.1042/BCJ20230035
Abstract: Necroptosis is a mode of programmed, lytic cell death that is executed by the mixed lineage kinase domain-like (MLKL) pseudokinase following activation by the upstream kinases, receptor-interacting serine/threonine protein kinase (RIPK)-1 and RIPK3. Dysregulated necroptosis has been implicated in the pathophysiology of many human diseases, including inflammatory and degenerative conditions, infectious diseases and cancers, provoking interest in pharmacological targeting of the pathway. To identify small molecules impacting on the necroptotic machinery, we performed a phenotypic screen using a mouse cell line expressing an MLKL mutant that kills cells in the absence of upstream death or pathogen detector receptor activation. This screen identified the vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) tyrosine kinase inhibitor, ABT-869 (Linifanib), as a small molecule inhibitor of necroptosis. We applied a suite of cellular, biochemical and biophysical analyses to pinpoint the apical necroptotic kinase, RIPK1, as the target of ABT-869 inhibition. Our study adds to the repertoire of established protein kinase inhibitors that additionally target RIPK1 and raises the prospect that serendipitous targeting of necroptosis signalling may contribute to their clinical efficacy in some settings.
Publisher: Elsevier BV
Date: 11-2022
DOI: 10.1016/J.STR.2022.08.006
Abstract: Tribbles proteins (TRIB1-3) are pseudokinases that recruit substrates to the COP1 ubiquitin ligase. TRIB2 was the first Tribbles ortholog to be implicated as a myeloid leukemia oncogene, because it recruits the C/EBPα transcription factor for ubiquitination by COP1. Here we report identification of nanobodies that bind the TRIB2 pseudokinase domain with low nanomolar affinity. A crystal structure of the TRIB2-Nb4.103 complex identified the nanobody to bind the N-terminal lobe of TRIB2, enabling specific recognition of TRIB2 in an activated conformation that is similar to the C/EBPα-bound state of TRIB1. Characterization in solution revealed that Nb4.103 can stabilize a TRIB2 pseudokinase domain dimer in a face-to-face manner. Conversely, a distinct nanobody (Nb4.101) binds through a similar epitope but does not readily promote dimerization. In combination, this study identifies features of TRIB2 that could be exploited for the development of inhibitors and nanobody tools for future investigation of TRIB2 function.
Publisher: Elsevier BV
Date: 12-2017
DOI: 10.1016/J.SBI.2017.07.004
Abstract: Pseudoenzymes were first described more than 50 years ago, when it was recognised that a subset of proteins that are structurally homologous to active enzymes lack amino acids necessary for catalytic activity. Recently, interest in pseudoenzymes has surged as it has become apparent that they constitute ∼10% of proteomes and perform essential metabolic and signalling functions that can be experimentally distinguished from catalytic outputs of enzymes. Here, we highlight recent structural studies of pseudoenzymes, which have revealed the molecular basis for roles as allosteric regulators of conventional enzymes, as molecular switches and integrators, as hubs for assembling protein complexes, and as competitors of substrate availability and holoenzyme assembly. As structural studies continue to illuminate pseudoenzyme molecular mechanisms, we anticipate that our knowledge of the breadth of their biological functions will expand in parallel.
Publisher: Portland Press Ltd.
Date: 10-03-2016
DOI: 10.1042/BJ20151049
Abstract: The structural maintenance of chromosomes (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 non-canonical member of the SMC family. Although 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 the 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 organized with respect to the hinge region. In the present study, by employing SAXS we demonstrate that the hinge domain of Smchd1 probably adopts an unconventional homodimeric arrangement augmented by an intermolecular 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.
Publisher: Elsevier BV
Date: 2001
DOI: 10.1016/S0092-8674(01)00213-6
Abstract: The receptor systems for the hemopoietic cytokines GM-CSF, IL-3, and IL-5 consist of ligand-specific alpha receptor subunits that play an essential role in the activation of the shared betac subunit, the major signaling entity. Here, we report the structure of the complete betac extracellular domain. It has a structure unlike any class I cytokine receptor described thus far, forming a stable interlocking dimer in the absence of ligand in which the G strand of domain 1 hydrogen bonds into the corresponding beta sheet of domain 3 of the dimer-related molecule. The G strand of domain 3 similarly partners with the dimer-related domain 1. The structure provides new insights into receptor activation by the respective alpha receptor:ligand complexes.
Publisher: Springer Science and Business Media LLC
Date: 31-03-2021
DOI: 10.1038/S41467-021-22253-6
Abstract: 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.
Publisher: Elsevier BV
Date: 09-2013
DOI: 10.1016/J.IMMUNI.2013.06.018
Abstract: Mixed lineage kinase domain-like (MLKL) is a component of the "necrosome," the multiprotein complex that triggers tumor necrosis factor (TNF)-induced cell death by necroptosis. To define the specific role and molecular mechanism of MLKL action, we generated MLKL-deficient mice and solved the crystal structure of MLKL. Although MLKL-deficient mice were viable and displayed no hematopoietic anomalies or other obvious pathology, cells derived from these animals were resistant to TNF-induced necroptosis unless MLKL expression was restored. Structurally, MLKL comprises a four-helical bundle tethered to the pseudokinase domain, which contains an unusual pseudoactive site. Although the pseudokinase domain binds ATP, it is catalytically inactive and its essential nonenzymatic role in necroptotic signaling is induced by receptor-interacting serine-threonine kinase 3 (RIPK3)-mediated phosphorylation. Structure-guided mutation of the MLKL pseudoactive site resulted in constitutive, RIPK3-independent necroptosis, demonstrating that modification of MLKL is essential for propagation of the necroptosis pathway downstream of RIPK3.
Publisher: Wiley
Date: 11-12-2006
DOI: 10.1016/J.FEBSLET.2006.11.080
Abstract: The family of polo like kinases (Plks) regulate cell cycle progression through key functional roles in mitosis. While the four mammalian family members, Plk1-4, share overlapping functions, each member possesses unique functions that may be dictated in part by their ability to phosphorylate different substrates. Numerous cellular substrates for Plk1, 2, and 3 have been characterized, but the protein targets for Plk4/Sak remain unknown. We have purified the kinase domain of Sak and demonstrated that it has robust kinase activity in vitro. Using in vitro kinase assays on peptide spots arrays, we determined the consensus phosphorylation motif for Sak to be yen-[Ile/Leu/Val]-Ser/Thr-phi-phi-X- yen/Pro (where phi denotes a large hydrophobic residue, yen is a charged residue dependent on the context of the surrounding sequence, and residues in brackets are unfavoured). This consensus phosphorylation motif differs from that of Plk1, and provides a basis for future studies to identify in vivo substrates of Sak.
Publisher: Elsevier BV
Date: 04-2017
DOI: 10.1016/J.TIG.2017.01.007
Abstract: It has very recently become clear that the epigenetic modifier SMCHD1 has a role in two distinct disorders: facioscapulohumoral muscular dystrophy (FSHD) and Bosma arhinia and micropthalmia (BAMS). In the former there are heterozygous loss-of-function mutations, while both gain- and loss-of-function mutations have been proposed to underlie the latter. These findings have led to much interest in SMCHD1 and how it works at the molecular level. We summarise here current understanding of the mechanism of action of SMCHD1, its role in these diseases, and what has been learnt from study of mouse models null for Smchd1 in the decade since the discovery of SMCHD1.
Publisher: Humana Press
Date: 2009
DOI: 10.1007/978-1-60327-394-7_7
Abstract: The human proteome is known to contain >500 protein kinases, which regulate almost all facets of cellular biology by the post-translational attachment of a phosphate moiety to serine, threonine, or tyrosine residues within a substrate protein. Most protein kinases remain poorly characterized and, as a result, current studies are directed toward defining their target substrates experimentally to gain a comprehensive view of the signaling proteins and pathways modulated by these kinases. Herein, we describe a rapid and convenient method for elucidating the consensus substrate motif for phosphorylation by a protein kinase using peptide SPOT arrays that are custom-synthesized on a cellulose membrane support. The definition of the target consensus motif provides an important starting point for the identification of physiologically relevant kinase substrates.
Publisher: Springer Science and Business Media LLC
Date: 20-09-2023
Publisher: Portland Press Ltd.
Date: 20-12-2013
DOI: 10.1042/BJ20131174
Abstract: Protein kinase-like domains that lack conserved residues known to catalyse phosphoryl transfer, termed pseudokinases, have emerged as important signalling domains across all kingdoms of life. Although predicted to function principally as catalysis-independent protein-interaction modules, several pseudokinase domains have been attributed unexpected catalytic functions, often amid controversy. We established a thermal-shift assay as a benchmark technique to define the nucleotide-binding properties of kinase-like domains. Unlike in vitro kinase assays, this assay is insensitive to the presence of minor quantities of contaminating kinases that may otherwise lead to incorrect attribution of catalytic functions to pseudokinases. We demonstrated the utility of this method by classifying 31 erse pseudokinase domains into four groups: devoid of detectable nucleotide or cation binding cation-independent nucleotide binding cation binding and nucleotide binding enhanced by cations. Whereas nine pseudokinases bound ATP in a alent cation-dependent manner, over half of those examined did not detectably bind nucleotides, illustrating that pseudokinase domains predominantly function as non-catalytic protein-interaction modules within signalling networks and that only a small subset is potentially catalytically active. We propose that henceforth the thermal-shift assay be adopted as the standard technique for establishing the nucleotide-binding and catalytic potential of kinase-like domains.
Publisher: Elsevier BV
Date: 2021
Publisher: Elsevier BV
Date: 02-2014
DOI: 10.1016/J.COI.2013.10.017
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.
Publisher: Elsevier BV
Date: 11-2015
DOI: 10.1016/J.STR.2015.08.017
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.
Publisher: American Chemical Society (ACS)
Date: 02-03-2011
DOI: 10.1021/BI101810F
Abstract: Interleukin-3 (IL-3), a cytokine produced primarily by activated T-cells during immune responses, is a crucial regulator of allergic inflammation. The three-dimensional structure of murine IL-3 (mIL-3) has remained elusive owing to its poor solubility and strong tendency toward aggregation under solution conditions typically used for structural studies. Here we describe the solution properties and structure of mIL-3 determined by NMR using an engineered construct of mIL-3 (mIL-3(33-156)). mIL-3 adopts a four-helical bundle fold, typical of proteins belonging to the short-chain cytokine family, and features a core of highly conserved hydrophobic residues. While significant line broadening and peak disappearance were observed in NMR spectra at higher temperatures, there was no evidence for temperature-dependent changes of the oligomeric state of mIL-3(33-156). Further analysis of the temperature dependence of amide (1)H chemical shifts and backbone (15)N relaxation parameters, including (15)N relaxation dispersion, revealed the presence of significant conformational exchange and local conformational heterogeneity. Residues recently shown by mutagenesis to play key roles in β(IL-3) receptor recognition and activation, which are located within the α(A) and α(C) helices and aligned on one face of the mIL-3(33-156) structure, are relatively rigid. In contrast, pronounced conformational heterogeneity was observed for a cluster of residues located on the opposite side of mIL-3, which corresponds spatially to sites in the related cytokines human IL-3, IL-5, and GM-CSF that are known to mediate interactions with their respective α-receptor subunits. Such conformational heterogeneity may facilitate the interaction of mIL-3 with each of two naturally occurring mIL-3Rα isoforms, leading to structurally distinct high-affinity complexes.
Publisher: Proceedings of the National Academy of Sciences
Date: 27-02-2017
Abstract: Phosphorylation catalyzed by protein kinases governs many aspects of cellular behavior. Apoptosis signal-regulating kinases (ASK1–3) trigger responses to stress, but the structural basis of their regulation remains unclear. Here, we show that a domain directly adjacent to the ASK1 kinase domain promotes activity of ASK1 on a key substrate and also orients an additional ASK1 domain nearby to suppress kinase activity. The structure of this regulatory domain appears to be shared by all ASK kinases and provides a versatile mechanism to control ASK activity in response to various stress stimuli.
Publisher: Portland Press Ltd.
Date: 24-07-2014
DOI: 10.1042/BJ20140712
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 ersity of regulatory mechanisms utilized by the JAK family to maintain signalling fidelity, and suggest alternative therapeutic strategies to complement existing ATP-competitive kinase inhibitors.
Location: Australia
Start Date: 2019
End Date: 2022
Funder: Marsden Fund
View Funded ActivityStart Date: 2018
End Date: 2020
Funder: Australian Research Council
View Funded ActivityStart Date: 2015
End Date: 2018
Funder: Marsden Fund
View Funded ActivityStart Date: 2017
End Date: 2019
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2018
End Date: 2020
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2017
End Date: 2019
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2008
End Date: 2010
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2014
End Date: 2017
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2018
End Date: 2020
Funder: National Health and Medical Research Council
View Funded Activity