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

Resveratrol inhibits benzo[a]pyrene–DNA adduct formation in human bronchial epithelial cells

Resveratrol ( trans-3,4’,5-trihydroxystilbene), a phytoalexin present in various plants and foods, has in several in vitro and in vivo studies demonstrated cancer chemopreventive and chemotherapeutic potential. We investigated the in vitro effect of resveratrol on benzo[ a] pyrene ( B[ a] P)-induced DNA adducts in human bronchial epithelial cells. This was compared to the effect of resveratrol on the expression of the cytochrome P450 (CYP) genes CYP1A1 and CYP1B1 and the formation of B[ a] P metabolites. Exposure of BEAS-2B and BEP2D cells to B[ a] P and increasing concentrations of resveratrol resulted in a dose- and time-dependent inhibition of DNA adduct formation quantified by P-32-postlabelling. Supporting this result, resveratrol was shown to inhibit CYP1A1 and CYP1B1 gene expression, as measured by real-time reverse transcriptase - polymerase chain reaction. Also, a significant correlation was found between the number of DNA adducts and the mRNA levels of these genes. Using HPLC analysis, a concomitant decrease in the formation of B[ a]P-derived metabolic products was detected. In conclusion, these data lend support to a chemopreventive role of resveratrol in polycyclic aromatic hydrocarbon-induced carcinogenesis

Homology modeling and molecular dynamics simulations of MUC1-9/H-2Kb complex suggest novel binding interactions

International audienceHuman MUC1 is over-expressed in human adenocarcinomas and has been used as a target for immunotherapy studies. The 9-mer MUC1-9 peptide has been identified as one of the peptides which binds to murine MHC class I H-2K. The structure of MUC1-9 in complex with H-2K has been modeled and simulated with classical molecular dynamics, based on the x-ray structure of the SEV9 peptide/H-2K complex. Two independent trajectories with the solvated complex (10 ns in length) were produced. Approximately 12 hydrogen bonds were identified during both trajectories to contribute to peptide/MHC complex, as well as 1-2 water mediated hydrogen bonds. Stability of the complex was also confirmed by buried surface area analysis, although the corresponding values were about 20% lower than those of the original x-ray structure. Interestingly, a bulged conformation of the peptide's central region, partially characterized as a -turn, was found exposed form the binding groove. In addition, P1 and P9 residues remained bound in the A and F binding pockets, even though there was a suggestion that P9 was more flexible. The complex lacked numerous water mediated hydrogen bonds that were present in the reference peptide x-ray structure. Moreover, local displacements of residues Asp4, Thr5 and Pro9 resulted in loss of some key interactions with the MHC molecule. This might explain the reduced affinity of the MUC1-9 peptide, relatively to SEV9, for the MHC class I H-2K

Identification of HLA-DRPheβ47 as the susceptibility marker of hypersensitivity to beryllium in individuals lacking the berylliosis-associated supratypic marker HLA-DPGluβ69

BACKGROUND: Susceptibility to beryllium (Be)-hypersensitivity (BH) has been associated with HLA-DP alleles carrying a glutamate at position 69 of the HLA-DP β-chain (HLA-DPGlu69) and with several HLA-DP, -DQ and -DR alleles and polymorphisms. However, no genetic associations have been found between BH affected subjects not carrying the HLA-DPGlu69 susceptibility marker. METHODS: In this report, we re-evaluated an already described patient populations after 7 years of follow-up including new 29 identified BH subjects. An overall population 36 berylliosis patients and 38 Be-sensitization without lung granulomas and 86 Be-exposed controls was analysed to assess the role of the individual HLA-class II polymorphisms associated with BH-susceptibility in HLA-DPGlu69 negative subjects by univariate and multivariate analysis. RESULTS: As previously observed in this population the HLA-DPGlu69 markers was present in higher frequency in berylliosis patients (31 out of 36, 86%) than in Be-sensitized (21 out of 38, 55%, p = 0.008 vs berylliosis) and 41 out of 86 (48%, p < 0.0001 vs berylliosis, p = 0.55 vs Be-sensitized) Be-exposed controls. However, 22 subjects presenting BH did not carry the HLA-DPGlu69 marker. We thus evaluated the contribution of all the HLA-DR, -DP and -DQ polymorphisms in determining BH susceptibility in this subgroup of HLA-Glu69 subjects. In HLA-DPGlu69-negatives a significant association with BH was found for the HLA-DQLeu26, for the HLA-DRB1 locus residues Ser13, Tyr26, His32, Asn37, Phe47 and Arg74 and for the HLA-DRB3 locus clusterized residues Arg11, Tyr26, Asp28, Leu38, Ser60 and Arg74. HLA-DRPhe47 (OR 2.956, p < 0.05) resulting independently associated with BH. Further, Be-stimulated T-cell proliferation in the HLA-DPGlu69-negative subjects (all carrying HLA-DRPhe47) was inhibited by the anti-HLA-DR antibody (range 70–92% inhibition) significantly more than by the anti-HLA-DP antibody (range: 6–29%; p < 0.02 compared to anti-HLA-DR) while it was not affected by the anti-HLA-DQ antibody. CONCLUSION: We conclude that HLA-DPGlu69 is the primary marker of Be-hypersensitivity and HLA-DRPhe47 is associated with BH in Glu69-negative subjects, likely playing a role in Be-presentation and sensitization

The effects of patient characteristics on ADHD diagnosis and treatment: a factorial study of family physicians

<p>Abstract</p> <p>Background</p> <p>Attention Deficit Hyperactivity Disorder (ADHD) is a costly and prevalent disorder in the U.S., especially among youth. However, significant disparities in diagnosis and treatment appear to be predicted by the race and insurance status of patients.</p> <p>Methods</p> <p>This study employed a web-based factorial survey with four ADHD cases derived from an ADHD clinic, two diagnosed with ADHD in actual evaluation, and two not. Randomized measures included race and insurance status of the patients. Participants N = (187) included clinician members of regional and national practice-based research networks and the U.S. clinical membership of the Society of Teachers of Family Medicine. The main outcomes were decisions to 1) diagnose and 2) treat the cases, based upon the information presented, analyzed via binary logistic regression of the randomized factors and case indicators on diagnosis and treatment.</p> <p>Results</p> <p>ADHD-positive cases were 8 times more likely to be diagnosed and 12 times more likely to be treated, and the male ADHD positive case was more likely to be diagnosed and treated than the female ADHD positive case. Uninsured cases were significantly more likely to be treated overall, but male cases that were uninsured were about half as likely to be diagnosed and treated with ADHD. Additionally, African-American race appears to increase the likelihood of medicinal treatment for ADHD and being both African-American and uninsured appears to cut the odds of medicinal treatment in half, but not significantly.</p> <p>Conclusions</p> <p>Family physicians were competent at discerning between near-threshold ADHD-negative and ADHD positive cases. However, insurance status and race, as well as gender, appear to affect the likelihood of diagnosis and treatment for ADHD in Family Medicine settings.</p

Crystal structure of phospholipase A2 from Indian cobra reveals a trimeric association.

Phospholipase A2 (PLA2) from Indian cobra venom (Naja naja naja) was crystallized from ethanol in space group P4(3)2(1)2 in the presence of Ca2+. The x-ray crystal structure was determined to 2.3-A resolution by molecular replacement techniques using a theoretical model constructed from homologous segments of the bovine pancreatic, porcine pancreatic, and rattlesnake venom crystal structures. The structure was refined to an R value of 0.174 for 17,542 reflections between 6.0- and 2.3-A resolution (F &gt; 2 sigma), including 148 water molecules. The 119-amino acid enzyme has an overall architecture strikingly similar to the other known PLA2 structures with regions implicated in catalysis showing the greatest structural conservation. Unexpectedly, three monomers were found to occupy the asymmetric unit and are oriented with their catalytic sites facing the pseudo-threefold axis with approximately 15% of the solvent accessible surface of each monomer buried in trimer contacts. The majority of the interactions at the subunit interfaces are made by residues unique to PLA2 sequences from cobra and krait venoms. The possible relevance of this unique trimeric structure is considered

Crystal structure of phospholipase A2 from Indian cobra reveals a trimeric association.

Phospholipase A2 (PLA2) from Indian cobra venom (Naja naja naja) was crystallized from ethanol in space group P4(3)2(1)2 in the presence of Ca2+. The x-ray crystal structure was determined to 2.3-A resolution by molecular replacement techniques using a theoretical model constructed from homologous segments of the bovine pancreatic, porcine pancreatic, and rattlesnake venom crystal structures. The structure was refined to an R value of 0.174 for 17,542 reflections between 6.0- and 2.3-A resolution (F &gt; 2 sigma), including 148 water molecules. The 119-amino acid enzyme has an overall architecture strikingly similar to the other known PLA2 structures with regions implicated in catalysis showing the greatest structural conservation. Unexpectedly, three monomers were found to occupy the asymmetric unit and are oriented with their catalytic sites facing the pseudo-threefold axis with approximately 15% of the solvent accessible surface of each monomer buried in trimer contacts. The majority of the interactions at the subunit interfaces are made by residues unique to PLA2 sequences from cobra and krait venoms. The possible relevance of this unique trimeric structure is considered

The optimization of peptide cargo bound to MHC class I molecules by the peptide-loading complex

Major histocompatibility complex (MHC) class I complexes present peptides from both self and foreign intracellular proteins on the surface of most nucleated cells. The assembled heterotrimeric complexes consist of a polymorphic glycosylated heavy chain, non-polymorphic β2 microglobulin, and a peptide of typically nine amino acids in length. Assembly of the class I complexes occurs in the endoplasmic reticulum and is assisted by a number of chaperone molecules. A multimolecular unit termed the peptide-loading complex (PLC) is integral to this process. The PLC contains a peptide transporter (transporter associated with antigen processing), a thiooxido-reductase (ERp57), a glycoprotein chaperone (calreticulin), and tapasin, a class I-specific chaperone. We suggest that class I assembly involves a process of optimization where the peptide cargo of the complex is edited by the PLC. Furthermore, this selective peptide loading is biased toward peptides that have a longer off-rate from the assembled complex. We suggest that tapasin is the key chaperone that directs this action of the PLC with secondary contributions from calreticulin and possibly ERp57. We provide a framework model for how this may operate at the molecular level and draw parallels with the proposed mechanism of action of human leukocyte antigen-DM for MHC class II complex optimization

Human C-type Lectin Domain Family 4, Member C (CLEC4C/BDCA-2/CD303) is a receptor for asialo-galactosyl-oligosaccharides

Plasmacytoid dendritic cells are specialized in the production of type I interferon (type I IFN), which promotes antiviral and antitumor responses, as well as autoimmune disorders. Activation of type I IFN secretion depends on the pattern recognition receptors TLR7 and TLR9, which sense microbial RNA and DNA, respectively. Type I IFN production is modulated by several receptors, including the type II C-type lectin domain family 4, member C (CLEC4C). The natural ligand of CLEC4C is unknown. To identify it, here we probed a glycan array with a soluble form of the CLEC4C ectodomain. We found that CLEC4C recognizes complex type sugars with terminal galactose. Importantly, soluble CLEC4C bound peripheral blood leukocytes and tumor cells that express glycans with galactose residues at the non-reducing ends. The positive and negative modulation of galactose residues on cell membranes was paralleled by the regulation of type I IFN secretion by plasmacytoid dendritic cells in co-culture experiments in vitro. These results suggest that the modulation in the expression of non-sialylated oligosaccharides by invading pathogens or transformed cells may affect type I IFN response and immune surveillance