The mechanism of activation of IRAK1 and IRAK4 by interleukin-1 and TollLike receptor agonists

We have developed the first assays that measure the protein kinase activities of Interleukin-1 Receptor Associated Kinase 1 (IRAK1) and IRAK4 reliably in human cell extracts, by employing Pellino1 as a substrate in conjunction with specific pharmacological inhibitors of IRAK1 and IRAK4. We exploited these assays to show that IRAK4 was constitutively active and that its intrinsic activity towards Pellino1 was not increased significantly by stimulation with interleukin-1 (IL-1) in IL-1Rexpressing HEK293 cells, Pam3Csk4-stimulated human THP1 monocytes or primary human macrophages. Our results, in conjunction with those of other investigators, suggest that the IL-1-stimulated trans-autophosphorylation of IRAK4 is initiated by the MyD88-induced dimerisation of IRAK4 and is not caused by an increase in the intrinsic catalytic activity of IRAK4. In contrast to IRAK4, we found that IRAK1 was inactive in unstimulated cells and converted to an active protein kinase in response to IL-1 or Pam3Csk4 in human cells. Surprisingly, the IL-1-stimulated activation of IRAK1 was not affected by pharmacological inhibition of IRAK4 and not reversed by dephosphorylation and/or deubiquitylation, suggesting that IRAK1 catalytic activity is not triggered by a covalent modification but by an allosteric mechanism induced by its interaction with IRAK4

Analysis of Expressed Sequence Tags Mapping to the Critical Region of the 5q- Syndrome

The 5q- syndrome is a myelodysplastic syndrome characterised by a macrocytic anaemia, hypolobulated megakaryocytes, a low risk of transformation to AML, and a 5q- chromosome as the sole karyotypic abnormality. The approximate 5Mb critical region of gene loss of the 5q- syndrome has been defined in two patients with the 5q- syndrome at 5q31-q33, flanked by the genes for FGF1 and IL12ß. The frequent loss of genetic material from the long arm of chromosome 5 in association with a malignancy has led to the hypothesis that, by analogy with other malignancies characterised by genetic loss, the 5q- syndrome is caused by loss of function of a gene with tumour suppressor activity. A transcript map of the 5q- syndrome critical region was generated with the aim of identifying the putative tumour suppressor gene associated with this disease. The expressed sequence tag (EST) database, db(EST) was used to isolate novel coding sequences mapping to the critical region of gene loss. Ten novel coding sequences (C5orf4, AF010242, AF156165, Cdy-17a06, Bda-87b11 195312, 4885953 / 143772,120101,195971, and 199067) were localised to the YAC contig spanning the critical region at 5g31-q33. The ten cDNA clones were sequenced, and overlapping clones were identified and sequenced in order to generate complete or partial coding sequences. This included the cloning of novel gene, C5orf4, and the identification of the human synaptopodin and dynactin p62 genes. In addition, the human homologues of the Drosophila melanogaster RMSA-1 and Saccharomyces cerevisiae CDC60 genes, and two known human genes (PP2A and HAH1) were localised to the critical region. Expression in human peripheral blood leukocytes and CD34+ progenitor cells was investigated for each known and novel gene. Genomic localisation, expression patterns and predicted function would suggest these known and novel genes represent putative tumour suppressor genes. Mutation studies were carried out on six known, and two novel candidate genes mapping to the narrowed 1.5Mb critical region of gene loss at 5g31.3-q32. No mutations were found in the coding regions/exons of these genes, suggesting they are not involved in the pathogenesis of the 5q- syndrome

Ab initio calculation of the shock Hugoniot of bulk silicon

We describe how ab initio molecular dynamics can be used to determine the Hugoniot locus (states accessible by a shock wave) for materials with a number of stable phases, and with an approximate treatment of plasticity and yield, without having to simulate these phenomena directly. We consider the case of bulk silicon, with forces from density-functional theory, up to 70 GPa. The fact that shock waves can split into multiple waves due to phase transitions or yielding is taken into account here by specifying the strength of any preceding waves explicitly based on their yield strain. Points corresponding to uniaxial elastic compression along three crystal axes and a number of postshock phases are given, including a plastically yielded state, approximated by an isotropic stress configuration following an elastic wave of predetermined strength. The results compare well to existing experimental data for shocked silicon.We thank Alan Minchinton, Richard Needs, Nikos Nikiforakis, Stephen Walley and David Williamson for useful input and discussions.This research was supported with funding from Orica Ltd. and the following grants: MINECO-Spain’s Plan Nacional Grant No. FIS2012-37549-C05-01, Basque Government Grant No. PI2014-105 CIC07 2014-2016, and EU Grant “ElectronStopping” in the Marie Curie CIG Program. Part of this work was performed using the Darwin Supercomputer of the University of Cambridge High Performance Computing Service [41], provided by Dell Inc. using Strategic Research Infrastructure Funding from the Higher Education Funding Council for England and funding from the Science and Technology Facilities Council

Roles of the TRAF6 and Pellino E3 ligases in MyD88 and RANKL signaling

It is widely accepted that the essential role of TRAF6 in vivo is to generate the Lys63-linked ubiquitin (K63-Ub) chains needed to activate the "master" protein kinase TAK1. Here, we report that TRAF6 E3 ligase activity contributes to but is not essential for the IL-1-dependent formation of K63-Ub chains, TAK1 activation, or IL-8 production in human cells, because Pellino1 and Pellino2 generate the K63-Ub chains required for signaling in cells expressing E3 ligase-inactive TRAF6 mutants. The IL-1-induced formation of K63-Ub chains and ubiquitylation of IRAK1, IRAK4, and MyD88 was abolished in TRAF6/Pellino1/Pellino2 triple-knockout (KO) cells, but not in TRAF6 KO or Pellino1/2 double-KO cells. The reexpression of E3 ligase-inactive TRAF6 mutants partially restored IL-1 signaling in TRAF6 KO cells, but not in TRAF6/Pellino1/Pellino2 triple-KO cells. Pellino1-generated K63-Ub chains activated the TAK1 complex in vitro with similar efficiently to TRAF6-generated K63-Ub chains. The early phase of TLR signaling and the TLR-dependent secretion of IL-10 (controlled by IRAKs 1 and 2) was only reduced modestly in primary macrophages from knockin mice expressing the E3 ligase-inactive TRAF6[L74H] mutant, but the late-phase production of IL-6, IL-12, and TNFα (controlled only by the pseudokinase IRAK2) was abolished. RANKL-induced signaling in macrophages and the differentiation of bone marrow to osteoclasts was similar in TRAF6[L74H] and wild-type cells, explaining why the bone structure and teeth of the TRAF6[L74H] mice was normal, unlike TRAF6 KO mice. We identify two essential roles of TRAF6 that are independent of its E3 ligase activity

Salt-inducible kinases (SIKs) regulate TGFβ-mediated transcriptional and apoptotic responses

The signalling pathways initiated by members of the transforming growth factor-β (TGFβ) family of cytokines control many metazoan cellular processes, including proliferation and differentiation, epithelial-mesenchymal transition (EMT) and apoptosis. TGFβ signalling is therefore strictly regulated to ensure appropriate context-dependent physiological responses. In an attempt to identify novel regulatory components of the TGFβ signalling pathway, we performed a pharmacological screen by using a cell line engineered to report the endogenous transcription of the TGFβ-responsive target gene PAI-1. The screen revealed that small molecule inhibitors of salt-inducible kinases (SIKs) attenuate TGFβ-mediated transcription of PAI-1 without affecting receptor-mediated SMAD phosphorylation, SMAD complex formation or nuclear translocation. We provide evidence that genetic inactivation of SIK isoforms also attenuates TGFβ-dependent transcriptional responses. Pharmacological inhibition of SIKs by using multiple small-molecule inhibitors potentiated apoptotic cell death induced by TGFβ stimulation. Our data therefore provide evidence for a novel function of SIKs in modulating TGFβ-mediated transcriptional and cellular responses.</p

Screening of DUB activity and specificity by MALDI-TOF mass spectrometry

Deubiquitylases (DUBs) are key regulators of the ubiquitin system which cleave ubiquitin moieties from proteins and polyubiquitin chains. Several DUBs have been implicated in various diseases and are attractive drug targets. We have developed a sensitive and fast assay to quantify in vitro DUB enzyme activity using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Unlike other current assays, this method uses unmodified substrates, such as diubiquitin topoisomers. By analyzing 42 human DUBs against all diubiquitin topoisomers we provide an extensive characterization of DUB activity and specificity. Our results confirm the high specificity of many members of the OTU and JAMM DUB families and highlight that all USPs tested display low linkage selectivity. We also demonstrate that this assay can be deployed to assess the potency and specificity of DUB inhibitors by profiling 11 compounds against a panel of 32 DUBs

Activation of the canonical IKK complex by K63/M1-linked hybrid ubiquitin chains

Polyubiquitin (pUb) chains formed between the C terminus of ubiquitin and lysine 63 (K63) or methionine 1 (M1) of another ubiquitin have been implicated in the activation of the canonical IκB kinase (IKK) complex. Here, we demonstrate that nearly all of the M1-pUb chains formed in response to interleukin-1, or the Toll-Like Receptors 1/2 agonist Pam(3)CSK(4), are covalently attached to K63-pUb chains either directly as K63-pUb/M1-pUb hybrids or indirectly by attachment to the same protein. Interleukin-1 receptor (IL-1R)-associated kinase (IRAK) 1 is modified first by K63-pUb chains to which M1-pUb linkages are added subsequently, and myeloid differentiation primary response gene 88 (MyD88) and IRAK4 are also modified by both K63-pUb and M1-pUb chains. We show that the heme-oxidized IRP2 ubiquitin ligase 1 interacting protein (HOIP) component of the linear ubiquitin assembly complex catalyzes the formation of M1-pUb chains in response to interleukin-1, that the formation of K63-pUb chains is a prerequisite for the formation of M1-pUb chains, and that HOIP interacts with K63-pUb but not M1-pUb linkages. These findings identify K63-Ub oligomers as a major substrate of HOIP in cells where the MyD88-dependent signaling network is activated. The TGF-beta–activated kinase 1 (TAK1)-binding protein (TAB) 2 and TAB3 components of the TAK1 complex and the NFκB Essential Modifier (NEMO) component of the canonical IKK complex bind to K63-pUb chains and M1-pUb chains, respectively. The formation of K63/M1-pUb hybrids may therefore provide an elegant mechanism for colocalizing both complexes to the same pUb chain, facilitating the TAK1-catalyzed activation of IKKα and IKKβ. Our study may help to resolve the debate about the relative importance of K63-pUb and M1-pUb chains in activating the canonical IKK complex

Numerical Constitutive Modelling for Continuum Mechanics Simulation

Three investigations are described in this dissertation, on the common theme of obtaining constitutive laws describing bulk properties of crystalline materials. We use ‘first-principles’ techniques where possible, an approach offering results which are predictive, with applicability to a wide class of materials, and with a systematic way to apply the techniques to any particular material of interest. We apply these techniques to silicon. The first investigation aims at developing an equation of state model for temperature dependent, anisotropic non-linear hyperelasticity. A method is presented for finding deformed states of a material on the same isentrope as a given starting configuration. The energies and stresses of a number of elastic deformations are sampled from dft molecular dynamics using this method, over a given range of the seven-dimensional space of deformation and potential temperature. The complete energy surface within this range can then be reliably reconstructed using the technique of Gaussian process regression. This is a machine learning technique that has particular merit here due to its ability to reconstruct a smooth surface without over-fitting. An equation of state model is then constructed for dft silicon, and demonstrated within a finite-volume continuum elasticity simulation for several problems of interest involving shock waves. The second investigation is concerned with the computation of properties of shock waves. We describe a simple annealing procedure to obtain the Hugoniot locus (states accessible by a shock wave) for a given material in a computationally efficient manner, particularly suited to first-principles calculations. We apply this method to determine the Hugoniot locus in bulk silicon from ab initio molecular dynamics with forces from density-functional theory, up to 70 GPa. In addition, we perform direct non-equilibrium molecular dynamics simulations of shock waves using empirical interatomic potentials and compare with our indirect method. We also present a direct ab initio molecular dynamics simulation of an elastic shock-wave in silicon, the first performed, to our knowledge. The third and final investigation is into the computation of thermal conductivity from atomistic simulations. We produce a number of model interatomic potentials for silicon, using the non-parametric, Bayesian approach of Gaussian Approximation Potentials, which are improved systematically through a database of training configurations. We compute the thermal conductivity from these at the level of the phonon-Boltzmann transport equation. The best of these potentials reproduces the dft value of phonon-Boltzmann conductivity to within a few percent, which is itself in good agreement with experiment. We consider several issues relating to computing thermal conductivity from molecular dynamics simulations.Orica Lt