Ly49P recognition of cytomegalovirus-infected cells expressing H2-Dk and CMV-encoded m04 correlates with the NK cell antiviral response

Natural killer (NK) cells are crucial in resistance to certain viral infections, but the mechanisms used to recognize infected cells remain largely unknown. Here, we show that the activating Ly49P receptor recognizes cells infected with mouse cytomegalovirus (MCMV) by a process that requires the presence of H2-Dk and the MCMV m04 protein. Using H2 chimeras between H2-Db and -Dk, we demonstrate that the H2-Dk peptide-binding platform is required for Ly49P recognition. We identified m04 as a viral component necessary for recognition using a panel of MCMV-deletion mutant viruses and complementation of m04-deletion mutant (Δm04) virus infection. MA/My mice, which express Ly49P and H2-Dk, are resistant to MCMV; however, infection with Δm04 MCMV abrogates resistance. Depletion of NK cells in MA/My mice abrogates their resistance to wild-type MCMV infection, but does not significantly affect viral titers in mice infected with Δm04 virus, implicating NK cells in host protection through m04-dependent recognition. These findings reveal a novel mechanism of major histocompatability complex class I–restricted recognition of virally infected cells by an activating NK cell receptor

Natural killer cell receptors and their MHC ligand interactions in innate resistance to mouse cytomegalovirus

Le premier but de mon projet de doctorat a ete la caracterisation moleculaire de l'interface entre les recepteurs activateurs des cellules Natural Killer (NK) et de leurs ligands exprimes dans les cellules infectees ainsi que l'implication de cette interaction sur la reponse a l'infection par le cytomegalovirus (MCMV).J'ai tire un avantage de la variation naturelle au sein des membres lies aux recepteurs Ly49C ainsi que de la disponibilite des structures cristallines des Ly49 afin de comprendre les determinants moleculaires des interactions Ly49H-m157 et egalement identifier les residus des acides amines qui permettent de discriminer entre les recepteurs qui se lient et ceux qui ne se lient pas a m157. Mes decouvertes suggerent que le "site 2" du contact entre le CMH de classe I et Ly49 n'est visiblement pas implique dans la liaison avec m157. Au contraire, les residus localises au niveau de l'interface homodimere-recepteur seraient probablement critiques a la reconnaissance fonctionnelle de la glycoproteine m157. Notre approche fonctionnelle et de modelisation tridimensionnelle suggerent que l'architecture du dimere Ly49H est cruciale pour l'accessibilite de m157 mais non pour les molecules de CMH de classe I et relient la variabilite dans l'homodimerisation des Ly49 a la reconnaissance directe des produits pathogeniques.Un autre mecanisme de la reponse de l'hote contre MCMV provient de l'etude de la souche de souris MA/My, laquelle, malgre l'absence du gene Ly49h ainsi que la proteine pour laquelle il code, y est hautement resistant. Des etudes anterieures ont demontre qu'une interaction epistatique entre un gene issu du groupe des genes Ly49 sur le chromosome 6 et le CMH (H2) sur le chromosome 17 est associee avec la resistance au virus. Utilisant une methode de co-culture de cellules reportrices NFAT-GFP exprimant les recepteurs activateurs Ly49 et de fibroblastes primaires infectes, j'ai montre que le recepteur activateur Ly49P des cellules NK reconnait specifiquement les cellules infectees par MCMV et que cette reconnaissance depend de la presence de l'haplotype H2k. Ce signal etait bloque par l'utilisation des anticorps anti-H2-D k mais non par anti-H2-Kk. Ces resultats indiquent l'existence d'un nouveau mecanisme des cellules NK implique dans la resistance au MCMV, lequel depend de l'interaction fonctionnelle entre le recepteur Ly49P et la molecule du MHC de classe I, H2-Dk, dans les cellules infectees par MCMV. Comme contribution directe de ce travail, nous avons demontre que la resistance chez MA/My est au moins partiellement dependante des interactions entre le recepteur Ly49P et la molecule H2-Dk modifiee par le virus dans les cellules infectees. Comme MCMV regule negativement l'expression des molecules du CMH de classe I, j'ai confirme la presence de H2-Dk dans les cellules infectees par l'utilisation d'un virus MCMV recombinant portant un gene rapporteur GFP. En permutant la plateforme peptidique de liaison, les domaines transmembranaires et intracellulaires entre les molecules H2-Db et H2-D k, j'ai demontre que la plateforme peptidique de liaison est critique pour la reconnaissance des cellules infectees. Par le criblage d'un panel de mutants MCMV portant des genes impliques dans l'evasion immune, j'ai demontre que l'infection de fibroblastes par le MCMV depourvu du gene m04 (Deltam04) abolit totalement l'activation de Ly49P. (Abstract shortened by UMI.

Syntéza a cholinesterázová inhibiční aktivita analogů solasodinu se sedmičlenným F kruhem

Solasodine analogues containing a seven-membered F ring with a nitrogen atom placed at position 22a were prepared from diosgenin or tigogenin in a four-step synthesis comprising of the simultaneous opening of the F-ring and introduction of cyanide in position 22 alpha, activation of the 26-hydroxyl group as mesylate, nitrile reduction, and N-cyclization. Solasodine, six obtained 22a(N)-homo analogues, as well as four 26a-homosolasodine derivatives and their open-chain precursors (13 in total) were tested as potential inhibitors of acetyland butyryl-cholinesterases and showed activity at micromolar concentrations. The structure-activity relationship study revealed that activities against studied esterases are affected by the structure of E/F rings and the substitution pattern of ring A. The most potent compound 8 acted as non-competitive inhibitors and exerted IC50 = 8.51 mu M and 7.05 mu M for eeAChE and eqBChE, respectively. Molecular docking studies revealed the hydrogen bond interaction of 8 with S293 of AChE; further rings are stabilized via hydrophobic interaction (ring A) or interaction with Y341 and W286 (rings B and C). Biological experiments showed no neurotoxicity of differentiated SH-SY5Y cells. More importantly, results from neuroprotective assay based on glutamate-induced cytotoxicity revealed that most derivatives had the ability to increase the viability of differentiated SH-SY5Y cells in comparison to galantamine and lipoic acid assayed as standards. The newly synthesized solasodine analogues are able to inhibit and to bind cholinesterases in noncompetitive mode of inhibition and exhibited neuroprotection potential of differentiated neuroblastoma cells after Glu-induced toxicity.Analogy solasodinu obsahující sedmičlenný kruh F s atomem dusíku umístěným v poloze 22a byly připraveny z diosgeninu nebo tigogeninu čtyřstupňovou syntézou zahrnující současné otevření F-kruhu a zavedení kyanidu do polohy 22 alfa, aktivaci 26-hydroxylová skupina jako mesylát, redukce nitrilu a N-cyklizace. Solasodin, šest získaných 22a(N)-homo analogů, stejně jako čtyři deriváty 26a-homosolasodinu a jejich prekurzory s otevřeným řetězcem (celkem 13) byly testovány jako potenciální inhibitory acetyl a butyryl-cholinesteráz a vykazovaly aktivitu při mikromolárních koncentracích. Studie vztahu struktura-aktivita odhalila, že aktivity proti studovaným esterázám jsou ovlivněny strukturou E/F kruhů a substitučním vzorem kruhu A. Nejúčinnější sloučenina 8 působila jako nekompetitivní inhibitory a vykazovala IC50 = 8,51 uM a 7,05 mu M pro eeACHE a eqBChE, v daném pořadí. Studie molekulárního dokování odhalily interakci vodíkové vazby 8 s S293 z AChE; další kruhy jsou stabilizovány hydrofobní interakcí (kruh A) nebo interakcí s Y341 a W286 (kruhy B a C). Biologické experimenty neprokázaly žádnou neurotoxicitu diferencovaných buněk SH-SY5Y. Ještě důležitější je, že výsledky neuroprotektivního testu založeného na cytotoxicitě indukované glutamátem odhalily, že většina derivátů měla schopnost zvýšit životaschopnost diferencovaných buněk SH-SY5Y ve srovnání s galantaminem a kyselinou lipoovou testovanými jako standardy. Nově syntetizované analogy solasodinu jsou schopny inhibovat a vázat cholinesterázy nekompetitivním způsobem inhibice a vykazují neuroprotektivní potenciál diferencovaných neuroblastomových buněk po toxicitě indukované Glu

Ly49P recognition of cytomegalovirus-infected cells expressing H2-Dk and CMV-encoded m04 correlates with the NK cell antiviral response.

Natural killer (NK) cells are crucial in resistance to certain viral infections, but the mechanisms used to recognize infected cells remain largely unknown. Here, we show that the activating Ly49P receptor recognizes cells infected with mouse cytomegalovirus (MCMV) by a process that requires the presence of H2-D(k) and the MCMV m04 protein. Using H2 chimeras between H2-D(b) and -D(k), we demonstrate that the H2-D(k) peptide-binding platform is required for Ly49P recognition. We identified m04 as a viral component necessary for recognition using a panel of MCMV-deletion mutant viruses and complementation of m04-deletion mutant (Deltam04) virus infection. MA/My mice, which express Ly49P and H2-D(k), are resistant to MCMV; however, infection with Deltam04 MCMV abrogates resistance. Depletion of NK cells in MA/My mice abrogates their resistance to wild-type MCMV infection, but does not significantly affect viral titers in mice infected with Deltam04 virus, implicating NK cells in host protection through m04-dependent recognition. These findings reveal a novel mechanism of major histocompatibility complex class I-restricted recognition of virally infected cells by an activating NK cell receptor

Sequestosome-1/p62 Is the Key Intracellular Target of Innate Defense Regulator Peptide*

Innate defense regulator-1 (IDR-1) is a synthetic peptide with no antimicrobial activity that enhances microbial infection control while suppressing inflammation. Previously, the effects of IDR-1 were postulated to impact several regulatory pathways including mitogen-activated protein kinase (MAPK) p38 and CCAAT-enhancer-binding protein, but how this was mediated was unknown. Using a combined stable isotope labeling by amino acids in cell culture-proteomics methodology, we identified the cytoplasmic scaffold protein p62 as the molecular target of IDR-1. Direct IDR-1 binding to p62 was confirmed by several biochemical binding experiments, and the p62 ZZ-type zinc finger domain was identified as the IDR-1 binding site. Co-immunoprecipitation analysis of p62 molecular complexes demonstrated that IDR-1 enhanced the tumor necrosis factor α-induced p62 receptor-interacting protein 1 (RIP1) complex formation but did not affect tumor necrosis factor α-induced p62-protein kinase ζ complex formation. In addition, IDR-1 induced p38 MAPK activity in a p62-dependent manner and increased CCAAT-enhancer-binding protein β activity, whereas NF-κB activity was unaffected. Collectively, these results demonstrate that IDR-1 binding to p62 specifically affects protein-protein interactions and subsequent downstream events. Our results implicate p62 in the molecular mechanisms governing innate immunity and identify p62 as a potential therapeutic target in both infectious and inflammatory diseases