Vaccinia virus immunomodulator A46 : a lipid and protein-binding scaffold for sequestering host TIR-domain proteins

TS received Austrian Science Fund (FWF) grants P24038, W1221 and W1258. GAB is a member of Max F. Perutz Laboratories and the Vienna International PostDoctoral Program (VIPS). TKS is a holder of Wellcome Trust grant 097831. IU has Spanish Ministry of Economy and Competitiveness grant BIO2013-49604-EXP.Vaccinia virus interferes with early events of the activation pathway of the transcriptional factor NF-kB by binding to numerous host TIR-domain containing adaptor proteins. We have previously determined the X-ray structure of the A46 C-terminal domain; however, the structure and function of the A46 N-terminal domain and its relationship to the C-terminal domain have remained unclear. Here, we biophysically characterize residues 1-83 of the N-terminal domain of A46 and present the X-ray structure at 1.55 Å. Crystallographic phases were obtained by a recently developed ab initio method entitled ARCIMBOLDO_BORGES that employs tertiary structure libraries extracted from the Protein Data Bank; data analysis revealed an all β-sheet structure. This is the first such structure solved by this method which should be applicable to any protein composed entirely of β-sheets. The A46(1-83) structure itself is a β-sandwich containing a co-purified molecule of myristic acid inside a hydrophobic pocket and represents a previously unknown lipid-binding fold. Mass spectrometry analysis confirmed the presence of long-chain fatty acids in both N-terminal and full-length A46; mutation of the hydrophobic pocket reduced the lipid content. Using a combination of high resolution X-ray structures of the N-and C-terminal domains and SAXS analysis of full-length protein A46(1-240), we present here a structural model of A46 in a tetrameric assembly. Integrating affinity measurements and structural data, we propose how A46 simultaneously interferes with several TIR-domain containing proteins to inhibit NF-κB activation and postulate that A46 employs a bipartite binding arrangement to sequester the host immune adaptors TRAM and MyD88.Publisher PDFPeer reviewe

Thrombin-induced Ca2+ mobilization in vascular smooth muscle utilizes a slowly ribosylating pertussis toxin-sensitive G protein: evidence for the involvement of a G protein in inositol trisphosphate-dependent Ca2+ release

The role of pertussis toxin (PT)-sensitive and -insensitive guanine nucleotide-binding proteins (G proteins) in the stimulation of Ca mobilization by thrombin was investigated in cultured rat aortic smooth muscle cells. Characterization using immunoblotting with specific antisera indicated the presence in isolated membranes of the Gα(i2), Gα(i3), Gα(s), Gβ, and Gβ protein subunits as well as a lower molecular weight species of unknown identity. To assess the importance of G proteins in the coupling of thrombin receptors to Ca mobilization, we investigated the effect of PT on Ca responses using fluorescence spectroscopy and the Ca indicator dye Fura-2. Pretreatment of cells for 2 h with PT (1 μg/ml), which produced 91.3% ADP-ribosylation of PT-sensitive G proteins, did not affect the magnitude of thrombin-induced release of Ca from internal stores, suggesting that the residual 8.7% of PT-sensitive G proteins, or PT-insensitive mechanisms, was responsible for Ca release. However, after an 18-h pretreatment with PT, which produced ADP-ribosylation of the total complement of PT-sensitive G proteins, the thrombin-induced peak Ca response was inhibited by approximately 72%, suggesting that the major fraction of the Ca response was mediated by a slowly ribosylating component. The delayed effect of the toxin was not caused by down-regulation of the β-subunit of G proteins because quantitative immunoblots showed that levels of the β-subunit remained constant throughout the period of PT pretreatment. It was also not caused by a reduction in the size of the thrombin-releasable Ca pool because Ca release induced by agents that release Ca directly from internal stores, 2,5-di-tert-butylhydroquinone or thapsigargin, was not affected. In addition, the delayed effect of PT could not be explained in terms of differences in thrombin-induced [H]inositol trisphosphate (IP) formation because the level of inhibition of IP formation after a 2-h PT treatment was similar to that present after an 18-h pretreatment. The results indicate that a slowly ribosylating PT- sensitive species is the major G protein pathway that couples thrombin- receptor activation to Ca mobilization. This G protein appears to be involved not in the mechanisms that generate IP but rather possibly in coupling at the level of the intracellular Ca store

Hematopoietic Cell–Restricted Deletion of CD36 Reduces High-Fat Diet–Induced Macrophage Infiltration and Improves Insulin Signaling in Adipose Tissue

OBJECTIVE: The fatty acid translocase and scavenger receptor CD36 is important in the recognition and uptake of lipids. Accordingly, we hypothesized that it plays a role in saturated fatty acid-induced macrophage lipid accumulation and proinflammatory activation. RESEARCH DESIGN AND METHODS: In vitro, the effect of CD36 inhibition and deletion in lipid-induced macrophage inflammation was assessed using the putative CD36 inhibitor, sulfosuccinimidyl oleate (SSO), and bone marrow-derived macrophages from mice with (CD36KO) or without (wild-type) global deletion of CD36. To investigate whether deletion of macrophage CD36 would improve insulin sensitivity in vivo, wild-type mice were transplanted with bone marrow from CD36KO or wild-type mice and then fed a standard or high-fat diet (HFD) for 20 weeks. RESULTS: SSO treatment markedly reduced saturated fatty acid-induced lipid accumulation and inflammation in RAW264.7 macrophages. Mice harboring CD36-specific deletion in hematopoietic-derived cells (HSC CD36KO) fed an HFD displayed improved insulin signaling and reduced macrophage infiltration in adipose tissue compared with wild-type mice, but this did not translate into protection against HFD-induced whole-body insulin resistance. Contrary to our hypothesis and our results using SSO in RAW264.7 macrophages, neither saturated fatty acid-induced lipid accumulation nor inflammation was reduced when comparing CD36KO with wild-type bone marrow-derived macrophages. CONCLUSIONS: Although CD36 does not appear important in saturated fatty acid-induced macrophage lipid accumulation, our study uncovers a novel role for CD36 in the migration of proinflammatory phagocytes to adipose tissue in obesity, with a concomitant improvement in insulin action

QM/MM description of newly selected catalytic bioscavengers against organophosphorus compounds revealed reactivation stimulus mediated by histidine residue in the acyl-binding loop

© 2018 Zlobin, Mokrushina, Terekhov, Zalevsky, Bobik, Stepanova, Aliseychik, Kartseva, Panteleev, Golovin, Belogurov, Gabibov and Smirnov. Butyrylcholinesterase (BChE) is considered as an efficient stoichiometric antidote against organophosphorus (OP) poisons. Recently we utilized combination of calculations and ultrahigh-throughput screening (uHTS) to select BChE variants capable of catalytic destruction of OP pesticide paraoxon. The purpose of this study was to elucidate the molecular mechanism underlying enzymatic hydrolysis of paraoxon by BChE variants using hybrid quantum mechanical/molecular mechanical (QM/MM) calculations. Detailed analysis of accomplished QM/MM runs revealed that histidine residues introduced into the acyl-binding loop are always located in close proximity with aspartate residue at position 70. Histidine residue acts as general base thus leading to attacking water molecule activation and subsequent SN2 inline hydrolysis resulting in BChE reactivation. This combination resembles canonical catalytic triad found in active centers of various proteases. Carboxyl group activates histidine residue by altering its pKa, which in turn promotes the activation of water molecule in terms of its nucleophilicity. Observed re-protonation of catalytic serine residue at position 198 from histidine residue at position 438 recovers initial configuration of the enzyme's active center, facilitating next catalytic cycle. We therefore suggest that utilization of uHTS platform in combination with deciphering of molecular mechanisms by QM/MM calculations may significantly improve our knowledge of enzyme function, propose new strategies for enzyme design and open new horizons in generation of catalytic bioscavengers against OP poisons

Characterisation of PduS, the pdu Metabolosome Corrin Reductase, and Evidence of Substructural Organisation within the Bacterial Microcompartment

PduS is a corrin reductase and is required for the reactivation of the cobalamin-dependent diol dehydratase. It is one component encoded within the large propanediol utilisation (pdu) operon, which is responsible for the catabolism of 1,2-propanediol within a self-assembled proteinaceous bacterial microcompartment. The enzyme is responsible for the reactivation of the cobalamin coenzyme required by the diol dehydratase. The gene for the cobalamin reductase from Citrobacter freundii (pduS) has been cloned to allow the protein to be overproduced recombinantly in E. coli with an N-terminal His-tag. Purified recombinant PduS is shown to be a flavoprotein with a non-covalently bound FMN that also contains two coupled [4Fe-4S] centres. It is an NADH-dependent flavin reductase that is able to mediate the one-electron reductions of cob(III)alamin to cob(II)alamin and cob(II)alamin to cob(I)alamin. The [4Fe-4S] centres are labile to oxygen and their presence affects the midpoint redox potential of flavin. Evidence is presented that PduS is able to bind cobalamin, which is inconsistent with the view that PduS is merely a flavin reductase. PduS is also shown to interact with one of the shell proteins of the metabolosome, PduT, which is also thought to contain an [Fe-S] cluster. PduS is shown to act as a corrin reductase and its interaction with a shell protein could allow for electron passage out of the bacterial microcompartment

Analysis of Immunogenicity of Intracellular CTAR Fragments of Epstein—Barr Virus Latent Phase Protein LMP1

© 2017, Springer Science+Business Media, LLC. Intracellular fragments of latent phase protein LMP1 of Epstein—Barr virus, denoted as CTAR1/2/3, can trigger a variety of cell cascades and contribute to the transforming potential of the virus. Generation of recombinant proteins CTAR1/2/3 is expected to yield more ample data on functional and immunogenic characteristics of LMP1. We created genetic constructs for prokaryotic expression of LMP1 CTAR fragments and selected optimal conditions for their production and purification. Using a new library of LMP1 CTAR fragments, we carried out epitope mapping of a diagnostic anti-LMP1 antibody S12. Analysis of polyclonal serum antibodies from mice immunized with full-length LMP1 confirmed immunogenicity of CTAR elements comparable with that of full-length protein

Performances of JEM-EUSO

In this paper we describe the requirements and the expected performances of JEM-EUSO. Designed as the first mission to explore the Ultra High Energy Universe from space, JEM-EUSO will monitor the earth's atmosphere at night to record the UV (300–400 nm) tracks generated by the Extensive Air Showers produced by Ultra High Energy primaries propagating in the atmosphere. After briefing summarizing the main aspects of the JEM-EUSO Instrument and mission baseline, we will present, in details, our studies of the expected trigger rate, the estimated exposure, as well as on the expected angular, energy, and Xmax resolution. Eventually, the obtained results will be discussed in the context of the scientific requirements of the mission

An evaluation of the exposure in nadir observation of the JEM-EUSO mission

We evaluate the exposure during nadir observations with JEM-EUSO, the Extreme Universe Space Observatory,on-board the Japanese Experiment Module of the International Space Station. Designed as a mission to explore the extreme energy Universe from space, JEM-EUSO will monitor the Earth's nighttime atmosphere to record the ultraviolet light from tracks generated by extensive air showers initiated by ultra-high energy cosmic rays. In the present work, we discuss the particularities of space-based observation and we compute the annual exposure in nadir observation. The results are based on studies of the expected trigger aperture and observational duty cycle, as well as, on the investigations of the effects of clouds and different types of background light. We show that the annual exposure is about one order of magnitude higher than those of the presently operating ground-based observatories.Fil: Adams, J. H.. University of Alabama in Huntsville; Estados UnidosFil: Ahmad, S.. Universite Paris Sud; FranciaFil: Albert, J. N..Fil: Allard, D.. Universite Paris Diderot - Paris 7; FranciaFil: Ambrosio, M.. Istituto Nazionale di Fisica Nucleare; ItaliaFil: Anchordoqui, L.. Medical College Of Wisconsin; Estados UnidosFil: Anzalone, A.. INAF; ItaliaFil: Arai, Y.. High Energy Accelerator Research Organization (KEK); JapónFil: Aramo, C..Fil: Asano, K.. Interactive Research Center of Science, Tokyo Institute of Technology; JapónFil: Ave, M.. Universidad de Santiago de Compostela; EspañaFil: Barrillon, P.. Universite de Paris; FranciaFil: Batsch, T.. National Centre for Nuclear Research; PoloniaFil: Bayer, J.. University of Tubingen; AlemaniaFil: Belenguer, T.. j Instituto Nacional de Técnica Aeroespacial (INTA); EspañaFil: Bellotti, R.. Universita’ degli Studi di Bari Aldo Moro and INFN; ItaliaFil: Berlind, A. A.. Vanderbilt University; Estados UnidosFil: Bertaina, M.. Universita di Torino; ItaliaFil: Biermann, P. L.. Karlsruhe Institute of Technology (KIT); AlemaniaFil: Biktemerova,. Joint Institute for Nuclear Research; RusiaFil: Blaksley, C.. Universite de la Sorbona Nouvelle; FranciaFil: Blecki, J.. Space Research Centre of the Polish Academy of Sciences (CBK); PoloniaFil: Blin-Bondil, S.. Universite de Paris; FranciaFil: Blumer, J.. Karlsruhe Institute of Technology (KIT),; AlemaniaFil: Bobik, P.. Institute of Experimental Physics; EslovaquiaFil: Bogomilov, M.. St. Kliment Ohridski University of Sofia; BulgariaFil: Bonamente, M.. University of Alabama in Huntsville; Estados UnidosFil: Briz, S.. Universidad Carlos III de Madrid,; EspañaFil: Supanitsky, Alberto Daniel. Consejo Nacional de Investigaciónes Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Astronomía y Física del Espacio. - Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Astronomía y Física del Espacio; Argentin