RESUMO
Retinoic acid inducible gene (RIG)-I-like receptors (RLRs), including RIG-I, melanoma differentiation associated-5 (MDA5), and laboratory of genetics and physiology 2 (LGP2), play pivotal roles in viral RNA sensing to initiate antiviral interferon (IFN) responses. We previously reported that an RNA-silencing regulator, transactivation response RNA-binding protein (TRBP), up-regulates MDA5/LGP2-mediated IFN responses through interaction with LGP2. Here, we aimed to investigate the mechanism underlying the TRBP-mediated up-regulation of IFN response. Data indicated that phosphomimetic TRBP showed a modest effect, whereas the nonphosphorylated form exhibited hyperactivity in enhancing Cardiovirus-triggered IFN responses. These results suggest that encephalomyocarditis virus (EMCV) attenuates the TRBP-mediated IFN response via TRBP phosphorylation, since EMCV infection activates the kinase responsible for TRBP phosphorylation for virus replication. Furthermore, we found that TRBP-mediated up-regulation of IFN response required the ATP hydrolysis and RNA binding of LGP2. TRBP enhanced RNA-dependent ATP hydrolysis by LGP2 but not that by RIG-I or MDA5. Nonphosphorylated TRBP exhibited higher levels of activity than phosphomimetic TRBP did, suggesting its possible involvement in the mechanism underlying the up-regulation of IFN response. TRBP activated the ATP hydrolysis of LGP2 and RIG-I, but not that of MDA5, in the absence of RNA. Collectively, we showed that TRBP differentially regulated RLR-mediated ATP hydrolysis. Further elucidation of the mechanism underlying the regulation of ATP hydrolysis leading to IFN response and self- and non-self-RNA discrimination could advance the development of effective therapeutic agents against autoimmune diseases.
Assuntos
Vírus da Encefalomiocardite , RNA Helicases , RNA Helicases/genética , Helicase IFIH1 Induzida por Interferon/genética , Helicase IFIH1 Induzida por Interferon/metabolismo , Ativação Transcricional , Hidrólise , Vírus da Encefalomiocardite/genética , Vírus da Encefalomiocardite/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA Viral/genética , Trifosfato de Adenosina , Imunidade InataRESUMO
A simultaneous analytical method based on LC-MS/MS was developed for the determination of flufenacet and its metabolites, [(4-fluorophenyl)(1-methylethyl) amino]oxo-acetic acid and [N-(4-fluorophenyl)-N-(1-methylethyl) acetamide]-2-sulfinylacetic acid, in agricultural products. The compounds were extracted from samples with methanol. The crude extracts were purified using Bond Elut C18 and InertSep GC/PSA, then determined by LC-MS/MS. The average recoveries (n=5) from 4 kinds of agricultural products (wheat, soybean, potato and tomato) spiked at the level of the MRLs or the uniform limits (0.01 µg/g) were 70.6-97.0%, and the relative standard deviations were less than 5%. The lower limits of quantitation of flufenacet and its metabolites were 0.01 µg/g.
