RESUMEN
Catalytic antibody 27C1 bears binding sites for both a substrate- and a functionalized small nonprotein component in the active site. We investigated the possibility of exploiting imine and enamine intermediates using a primary amine molecule into the active site of antibody 27C1. The antibody catalyzed ß-keto acid decarboxylation with a rate enhancement (kcat/Km/kuncat) of 140,000, as well as highly regioselective cross-aldol reactions of ketones and p-nitrobenzaldehyde. These studies provide new strategies for the generation of catalytic antibodies possessing binding sites for functionalized components.
Asunto(s)
Aldehídos/química , Aminas/química , Anticuerpos/metabolismo , Benzaldehídos/química , Sitios de Unión , Catálisis , Dominio Catalítico , Descarboxilación , Cetonas/química , Cinética , EstereoisomerismoRESUMEN
Antisense transcription is a widespread phenomenon in the mammalian genome and is believed to play a role in regulating gene expression. However, the exact functional significance of antisense transcription is largely unknown. Here, we show that natural antisense (AS) RNA is an important modulator of interferon-α1 (IFN-α1) mRNA levels. A ~4-kb, spliced IFN-α1 AS RNA targets a single-stranded region within a conserved secondary structure element of the IFN-α1 mRNA, an element which was previously reported to function as the nuclear export element. Following infection of human Namalwa lymphocytes with Sendai virus or infection of guinea pig 104C1 fetal fibroblasts with influenza virus A/PR/8/34, expression of IFN-α1 AS RNA becomes elevated. This elevated expression results in increased IFN-α1 mRNA stability because of the cytoplasmic (but not nuclear) interaction of the AS RNA with the mRNA at the single-stranded region. This results in increased IFN-α protein production. The silencing of IFN-α1 AS RNA by sense oligonucleotides or over-expression of antisense oligoribonucleotides, which were both designed from the target region, confirmed the critical role of the AS RNA in the post-transcriptional regulation of IFN-α1 mRNA levels. This AS RNA stabilization effect is caused by the prevention of the microRNA (miRNA)-induced destabilization of IFN-α1 mRNA due to masking of the miR-1270 binding site. This discovery not only reveals a regulatory pathway for controlling IFN-α1 gene expression during the host innate immune response against virus infection but also suggests a reason for the large number of overlapping complementary transcripts with previously unknown function.
Asunto(s)
Interferón-alfa/genética , ARN sin Sentido/genética , ARN Mensajero/genética , Animales , Linfocitos B/virología , Secuencia de Bases , Línea Celular , Fibroblastos/virología , Silenciador del Gen , Cobayas , Humanos , MicroARNs/metabolismo , Estabilidad del ARN , ARN sin Sentido/química , ARN sin Sentido/metabolismo , ARN Mensajero/química , ARN Mensajero/metabolismo , Infecciones por Respirovirus/genética , Infecciones por Respirovirus/metabolismo , Virus Sendai/aislamiento & purificación , Regulación hacia ArribaRESUMEN
Human immunodeficiency virus type 1 Rev (regulator of the expression of the virion) protein was shown to reduce the expression level of the co-transfected luciferase reporter gene (luc+) introduced to monitor transfection efficiency. We studied the mechanism of the inhibitory Rev effect. The effect, caused by nuclear retention of luc+ mRNA, was reversed if rev had a point mutation that makes its nuclear export signal (NES) unable to associate with cellular transport factors. The Rev NES receptor CRM1 (chromosome region maintenance 1)-specific inhibitor, leptomycin B, blocked luc+ mRNA export. This finding was also supported by the overexpression of delta CAN, another specific CRM1 inhibitor that caused inhibition of luciferase gene expression. Experiments involving tsBN2 cells, which have a temperature-sensitive RCC1 (regulator of chromosome condensation 1) allele, demonstrated that luc+ expression required generation of the GTP-bound form of RanGTPase (RanGTP) by RCC1. The constitutive transport element (CTE)-mediated nuclear export of luc+ mRNA was found to also depend upon RanGTP. Nuclear export of luc+ mRNA is thus suggested to involve CRM1 and RanGTP, which Rev employs to transport viral mRNA. The Rev effect is therefore considered to involve competition between two molecules for common transport factors.
Asunto(s)
Núcleo Celular/metabolismo , Carioferinas/metabolismo , Luciferasas de Luciérnaga/genética , Proteínas de Complejo Poro Nuclear/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteína de Unión al GTP ran/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Línea Celular Tumoral , Ácidos Grasos Insaturados/farmacología , Genes rev , VIH-1/genética , Células HeLa , Humanos , Luciferasas de Luciérnaga/metabolismo , Señales de Exportación Nuclear/efectos de los fármacos , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Unión al ARN/genética , Transfección , Proteína Exportina 1RESUMEN
Human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env)-mediated membrane fusion occurs as a sequence of events that is triggered by CD4 binding to the Env gp120 subunit. In this study, we analyzed the dynamics of Env-mediated membrane fusion at the single-cell level using fluorescent fusion proteins and confocal laser fluorescent microscopy. Either enhanced cyan or yellow fluorescent protein (CFP and YFP, respectively) was fused to the end of the cytoplasmic regions of the HIV-1 receptors (CD4 and CCR5) and Env proteins. Real-time imaging of membrane fusion mediated by these recombinant proteins revealed that the kinetics of fusion in our system was faster than that previously reported. Analysis of the receptor interaction by fluorescence resonance energy transfer (FRET) at the single-cell level demonstrated a tendency for oligomerization of CD4-CD4, but not of CD4-CCR5, in the absence of Env-expressing cells. However, when Env-expressing cells attached to the receptor cells, FRET produced by CD4-CCR5 interaction was increased; the FRET intensity began to decline before the formation of the fusion pore. These changes in FRET may represent the temporal association of these receptors, triggered by gp120 binding, and their dissociation during the formation of the fusion pore. In addition, the FRET analysis of receptor interactions in the presence of fusion inhibitors showed that not only inhibitors acting on CCR5 but also the gp41-derived peptide T-20 interfered with CD4-CCR5 interaction during fusion. These data suggest that T-20 could affect the formation of Env-receptors complexes during the membrane fusion.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Productos del Gen env/metabolismo , VIH-1/patogenicidad , Fusión de Membrana/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Antígenos CD4/genética , Antígenos CD4/metabolismo , Fusión Celular , Línea Celular , Colorantes Fluorescentes/metabolismo , Productos del Gen env/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , VIH-1/metabolismo , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Receptores CCR5/genética , Receptores CCR5/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
G protein-coupled receptor CCR5 is the main coreceptor for macrophage-tropic human immunodeficiency virus type 1 (HIV-1), and various small-molecule CCR5 antagonists are being developed to treat HIV-1 infection. It has been reported that such CCR5 antagonists, including TAK-779, bind to a putative binding pocket formed by transmembrane domains (TMs) 1, 2, 3 and 7 of CCR5, indicating the importance of the conformational changes of the TMs during virus entry. In this report, using a single-round infection assay with human CCR5 and its substitution mutants, we demonstrated that a new CCR5 antagonist, TAK-220, shares the putative interacting amino acid residues Asn252 and Leu255 in TM6 with TAK-779 but also requires the distinct residues Gly163 and Ile198 in TMs 4 and 5, respectively, for its inhibitory effect. We suggested that, together with molecular models of the interactions between the drugs and CCR5, the inhibitory activity of TAK-220 could involve direct interactions with amino acid residues in TMs 4, 5, and 6 in addition to those in the previously postulated binding pocket. The possible interaction of drugs with additional regions of the CCR5 molecule would help to develop a new small-molecule CCR5 antagonist.