RESUMO
Rerouting the splicing machinery with steric-block oligonucleotides (ON) might lead to new therapeutic strategies in the treatment of diseases such as beta-thalassemia, Duchenne muscular dystrophy, or cancers. Interfering with splicing requires the sequence-specific and stable hybridization of RNase H-incompetent ON as peptide nucleic acids (PNA) or phosphorodiamidate morpholino oligomers (PMO). Unfortunately, these uncharged DNA mimics are poorly taken up by most cell types and conventional delivery strategies that rely on electrostatic interaction do not apply. Likewise, conjugation to cell penetrating peptides (CPPs) as Tat, Arg9, Lys8, or Pen leads to poor splicing correction efficiency at low concentration essentially because PNA- and PMO-CPP conjugates remain entrapped within endocytotic vesicles. Recently, we have designed an arginine-rich peptide (R-Ahx-R)4 (with Ahx for aminohexanoic acid) and an arginine-tailed Penetratin derivative which allow sequence-specific and efficient splicing correction at low concentration in the absence of endosomolytic agents. Both CPPs are undergoing structure-activity relationship studies for further optimization as steric-block ON delivery vectors.
Assuntos
Arginina/química , Oligonucleotídeos/química , Peptídeos/química , Peptídeos/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA , Animais , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Humanos , Peptídeos/administração & dosagem , Relação Estrutura-AtividadeRESUMO
Cationic CPPs (cell-penetrating peptides) have been used largely for intracellular delivery of low-molecular-mass drugs, biomolecules and particles. Most cationic CPPs bind to cell-associated glycosaminoglycans and are internalized by endocytosis, although the detailed mechanisms involved remain controversial. Sequestration and degradation in endocytic vesicles severely limits the efficiency of cytoplasmic and/or nuclear delivery of CPP-conjugated material. Re-routing the splicing machinery by using steric-block ON (oligonucleotide) analogues, such as PNAs (peptide nucleic acids) or PMOs (phosphorodiamidate morpholino oligomers), has consequently been inefficient when ONs are conjugated with standard CPPs such as Tat (transactivator of transcription), R(9) (nona-arginine), K(8) (octalysine) or penetratin in the absence of endosomolytic agents. New arginine-rich CPPs such as (R-Ahx-R)(4) (6-aminohexanoic acid-spaced oligo-arginine) or R(6) (hexa-arginine)-penetratin conjugated to PMO or PNA resulted in efficient splicing correction at non-cytotoxic doses in the absence of chloroquine. SAR (structure-activity relationship) analyses are underway to optimize these peptide delivery vectors and to understand their mechanisms of cellular internalization.
Assuntos
Sistemas de Liberação de Medicamentos , Oligonucleotídeos/metabolismo , Peptídeos/fisiologia , Sinais Direcionadores de Proteínas/fisiologia , Transporte Proteico/fisiologia , Animais , Humanos , Oligonucleotídeos/administração & dosagemRESUMO
The cellular uptake of PMOs (phosphorodiamidate morpholino oligomers) can be enhanced by their conjugation to arginine-rich CPPs (cell-penetrating peptides). Here, we discuss our recent findings regarding (R-Ahx-R)(4)AhxB (Ahx is 6-aminohexanoic acid and B is beta-alanine) CPP-PMO conjugates in DMD (Duchenne muscular dystrophy) and murine coronavirus research. An (R-Ahx-R)(4)AhxB-PMO conjugate was the most effective compound in inducing the correction of mutant dystrophin transcripts in myoblasts derived from a canine model of DMD. Similarly, normal levels of dystrophin expression were restored in the diaphragms of mdx mice, with treatment starting at the neonatal stage, and protein was still detecTable 22 weeks after the last dose of an (R-Ahx-R)(4)AhxB-PMO conjugate. Effects of length, linkage and carbohydrate modification of this CPP on the delivery of a PMO were investigated in a coronavirus mouse model. An (R-Ahx-R)(4)AhxB-PMO conjugate effectively inhibited viral replication, in comparison with other peptides conjugated to the same PMO. Shortening the CPP length, modifying it with a mannosylated serine moiety or replacing it with the R(9)F(2) CPP significantly decreased the efficacy of the resulting PPMO (CPP-PMO conjugate). We attribute the success of this CPP to its stability in serum and its capacity to transport PMO to RNA targets in a manner superior to that of poly-arginine CPPs.
Assuntos
Antivirais/farmacologia , Coronavirus/efeitos dos fármacos , Morfolinas/farmacologia , Distrofia Muscular de Duchenne/tratamento farmacológico , Peptídeos/uso terapêutico , Splicing de RNA/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Sistemas de Liberação de Medicamentos , Distrofina/biossíntese , Distrofina/genética , Humanos , Camundongos , Morfolinas/administração & dosagem , Distrofia Muscular de Duchenne/genética , Sinais Direcionadores de Proteínas/fisiologia , Transporte Proteico/fisiologia , Precursores de RNA/metabolismoRESUMO
Plasminogen activator inhibitor type I (PAI-1) plays a central role in metastatic behavior by increasing cells' migratory capacities as shown in several tumoral cell lines. Moreover, in vivo high expression of this factor helps tumoral growth, both by its role in extracellular matrix remodeling and by favoring angiogenesis. High levels of PAI-1 are correlated with bad prognosis in several cancers, particularly in breast cancer. The effect of PAI-1 upon angiogenesis is also involved in atherosclerosis, in which high levels of PAI-1 expression are observed. Breast carcinoma MDA MB 231 cells are known for both having important metastatic capacities and expressing high levels of PAI-1. We have demonstrated in these cells that the transfection of PAI-1 specific small interfering RNAs (siRNA) specifically inhibited the expression of this factor by 91%. We evaluated siRNA activity by determining PAI-1 mRNA level, as well as intracellular and extracellular PAI-1 protein by using RT Q-PCR, Western blot and ELISA analyses, respectively. Data confirmed inhibition at mRNA levels (primary aim of interference), intracellular protein, and secreted PAI-1, the latter being operative successfully in the cell microenvironment. The lipidic vector Delivery Liposomes System (DLS) used was adapted to siRNA delivery as observed by particle size distribution analysis, confocal microscopy and transfection into MDA MB 231, in the presence of serum. SiRNA activity was clearly detected at concentrations as low as 10 nM. Moreover, the low cytotoxicity of this vector makes it a good candidate for future in vivo siRNA delivery.
Assuntos
Neoplasias da Mama/genética , Inibidor 1 de Ativador de Plasminogênio/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Microquímica/métodos , Nanotecnologia/métodos , Inibidor 1 de Ativador de Plasminogênio/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
CPPs (cell-penetrating peptides) have given rise to much interest for the delivery of biomolecules such as peptides, proteins or ONs (oligonucleotides). CPPs and their conjugates were initially thought to translocate through the cell membrane by a non-endocytotic mechanism which has recently been re-evaluated. Basic-amino-acid-rich CPPs first interact with cell-surface proteoglycans before being internalized by endocytosis. Sequestration and degradation in endocytotic vesicles severely limits the cytoplasmic and nuclear delivery of the conjugated biomolecules. Accordingly, splicing correction by CPP-conjugated steric-block ON analogues is inefficient in the absence of endosomolytic agents. New arginine-rich CPPs allowing efficient splicing correction by conjugated PNAs (peptide nucleic acids) or PMO (phosphorodiamidate morpholino oligomer) steric blockers in the absence of endosomolytic agents have recently been defined in our group and are currently being characterized. They offer promising leads for the development of efficient cellular delivery vectors for therapeutic steric-block ON analogues.
Assuntos
Ácidos Nucleicos/administração & dosagem , Oligonucleotídeos/química , Peptídeos/química , Processamento Alternativo , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Sistemas de Liberação de Medicamentos , Técnicas de Transferência de Genes , Terapia Genética/métodos , Genômica/métodos , Humanos , Modelos BiológicosRESUMO
During the last decade several peptides have been extensively studied for their ability to translocate across the plasma membrane. These peptides have been called "cell penetrating peptides" (CPP) or "protein transduction domains" (PTD). These peptides also promote the cellular uptake of various cargo molecules. Their mechanism of cellular entry appeared very intriguing since most publications in the field highlighted an energy-independent process. Indeed, cellular uptake of these peptides was still observed by fluorescence microscopy at low temperature or in the presence of several drugs known to inhibit active transport. In addition, internalization was reported to be much faster than known endocytic processes. However the involvement of a specific cellular component responsible for this uptake process appeared unlikely following intensive structure activity relationship studies using a wide panel of Tat analogues. Several reports about a possible artefactual redistribution of CPPs, and their associated cargos, during the cell fixation step commonly used for fluorescence microscopy have recently emerged in the literature. Moreover strong ionic interactions of CPPs with the cell surface also led to an overestimation of the recorded cell-associated fluorescent signal. It now seems well established that arginine-rich peptides are internalized by an energy dependent process involving endocytosis. Whatever the case, however, an increasing number of data indicate that the conjugation of non-permeant molecules to these CPPs allows their cellular uptake and leads to the expected biological responses, thus pointing to the interest of this delivery strategy. However, initial structure activity relationship studies of these CPPs will have to be reconsidered and the relative potency of each peptide (and their analogues) to vectorize the cargos to their most appropriate subcellular compartment will require careful re-evaluation.
Assuntos
Produtos do Gene tat/metabolismo , Peptídeos/metabolismo , Animais , Arginina/química , Arginina/metabolismo , Proteínas de Transporte/química , Permeabilidade da Membrana Celular , Endocitose/fisiologia , Fixadores/metabolismo , Produtos do Gene tat/genética , HIV-1/metabolismo , Humanos , Peptídeos/genética , Transporte Proteico , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Fixação de Tecidos , Tripsina/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
Interferon alpha (IFNalpha) belongs to a cytokine family that exhibits antiviral properties, immuno-modulating effects, and antiproliferative activity on normal and neoplasic cells in vitro and in vivo. IFNalpha exerts antitumor action by inducing direct cytotoxicity against tumor cells. This toxicity is at least partly due to induction of apoptosis. Although the molecular basis of the inhibition of cell growth by IFNalpha is only partially understood, there is a direct correlation between the sensitivity of cells to the antiproliferative action of IFNalpha and the down-regulation of their mitochondrial mRNAs. Here, we studied the role of the 2-5A/RNase L system and its inhibitor RLI in this regulation of the mitochondrial mRNAs by IFNalpha. We found that a fraction of cellular RNase L and RLI is localized in the mitochondria. Thus, we down-regulated RNase L activity in human H9 cells by stably transfecting (i) RNase L antisense cDNA or (ii) RLI sense cDNA constructions. In contrast to control cells, no post-transcriptional down-regulation of mitochondrial mRNAs and no cell growth inhibition were observed after IFNalpha treatment in these transfectants. These results demonstrate that IFNalpha exerts its antiproliferative effect on H9 cells at least in part via the degradation of mitochondrial mRNAs by RNase L.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Nucleotídeos de Adenina/metabolismo , Chaperoninas , Endorribonucleases/metabolismo , Interferon-alfa/farmacologia , Mitocôndrias/genética , Oligorribonucleotídeos/metabolismo , Proteínas/metabolismo , RNA Mensageiro/genética , Sequência de Bases , Divisão Celular/efeitos dos fármacos , Primers do DNA , Endorribonucleases/antagonistas & inibidores , Humanos , RNA Mensageiro/metabolismo , Células Tumorais CultivadasRESUMO
Dinucleoside phosphates that harbor phosphate groups transiently blocked (caged) by o-nitrobenzyl or o-nitroveratryl residues were synthesized. It was shown that the conditions of the UV-induced deprotection largely depend on the nature of the protective group. The phosphotriesters obtained were resistant toward snake venom phosphodiesterase and nucleases of the cellular extract. The synthesis of the dinucleoside phosphates containing a photolabile group preceded the incorporation of the modified blocks into extended oligonucleotides by the phosphoramidite method.
Assuntos
Fosfatos de Dinucleosídeos/química , Fosfatos de Dinucleosídeos/síntese química , Oligonucleotídeos/química , Oligonucleotídeos/síntese química , Marcadores de Fotoafinidade , Raios UltravioletaRESUMO
PURPOSE: Recent studies have revealed abnormalities in the ribonuclease L pathway in peripheral blood mononuclear cells of patients with the chronic fatigue syndrome. We conducted a blinded study to detect possible differences in the distribution of 2-5A binding proteins in the cells of patients with chronic fatigue syndrome and controls. PATIENTS AND METHODS: We studied 57 patients with chronic fatigue syndrome and 53 control subjects (28 healthy subjects and 25 patients with depression or fibromyalgia). A radioactive probe was used to label 2-5A binding proteins in unfractionated peripheral blood mononuclear cell extracts and to compare their distribution in the three groups. RESULTS: A 37 kDa 2-5A binding polypeptide was found in 50 (88%) of the 57 patients with chronic fatigue syndrome compared with 15 (28%) of the 53 controls (P < 0.01). When present, the amount of 37 kDa protein was very low in the control groups. When expressed as the ratio of the 37 kDa protein to the 80 kDa protein, 41 (72%) of the 57 patients with chronic fatigue syndrome had a ratio > 0.05, compared with 3 (11%) of the 28 healthy subjects and none of the patients with fibromyalgia or depression. CONCLUSION: The presence of a 37 kDa 2-5A binding protein in extracts of peripheral blood mononuclear cells may distinguish patients with chronic fatigue syndrome from healthy subjects and those suffering from other diseases.
Assuntos
Nucleotídeos de Adenina/sangue , Síndrome de Fadiga Crônica/sangue , Oligorribonucleotídeos/sangue , Adulto , Biomarcadores/sangue , Estudos de Casos e Controles , Depressão/sangue , Diagnóstico Diferencial , Síndrome de Fadiga Crônica/diagnóstico , Feminino , Fibromialgia/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Monócitos/metabolismoRESUMO
The potential pharmacologic benefits of using peptide nucleic acid (PNA) as an antisense agent are tempered by its incapacity to activate RNase H. The mixed backbone oligonucleotide (ON) (or gapmer) approach, in which a short internal window of RNAse H-competent residues is embedded within an RNase H-incompetent ON has not been applied previously to PNA because PNA and DNA hybridize to RNA with very different helical structures, creating structural perturbations at the two PNA-DNA junctions. It is demonstrated here for the first time that a short internal phosphodiester window within a PNA is sufficient to evoke the RNase H-dependent cleavage of a targeted RNA and to abrogate translation elongation in a well-characterized in vitro assay.
Assuntos
Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos/farmacologia , Ácidos Nucleicos Peptídicos/farmacologia , RNA Mensageiro/metabolismo , Ribonuclease H/metabolismo , Animais , Catálise , Sistema Livre de Células , DNA/química , Relação Dose-Resposta a Droga , Escherichia coli/enzimologia , Marcação de Genes , Oligonucleotídeos Antissenso/química , Ácidos Nucleicos Peptídicos/química , Polímeros/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , Coelhos , Ribonuclease H/efeitos dos fármacos , Ribonuclease H/genéticaAssuntos
Oligodesoxirribonucleotídeos Antissenso/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Vírus da Estomatite Vesicular Indiana/genética , Animais , Linhagem Celular , Sistema Livre de Células , Dactinomicina/farmacologia , Células L , Estágios do Ciclo de Vida , Camundongos , Modelos Genéticos , RNA Mensageiro/genética , Coelhos , Reticulócitos/metabolismo , Vírus da Estomatite Vesicular Indiana/fisiologia , Ensaio de Placa Viral , Replicação ViralRESUMO
Oligonucleotides (ODNs) conjugated to rhodamin (Rh) and 4-[(N-2-chloroethyl-N-methyl)amino] benzylamine were used to investigate ODNs transport into keratinocytes. Affinity labeling of two proteins, 63 and 35 kDa, and the inhibition of the affinity labeling and ODNs uptake by the cells in the presence of nucleic acids, polyanions and trypsin suggest, that the proteins are involved in transport of nucleic acids in keratinocytes.
Assuntos
Queratinócitos/metabolismo , Oligonucleotídeos/metabolismo , Autorradiografia , Linhagem Celular , Humanos , Compostos de Mostarda/química , Oligonucleotídeos/química , Rodaminas/químicaRESUMO
Inadequate cellular compartmentalisation of plasmid DNA and antisense oligodeoxynucleotides (ODNs) is generally considered as a major limitation in their use. In this study, an approach combining in situ visual-isation of rhodamine-labelled ODNs and affinity modification of proteins by radiolabelled-alkylating ODN derivatives has been used to investigate the uptake of ODNs into keratinocytes. We confirm here that unmodified ODNs are efficiently taken up and accumulate in cell nuclei in primary keratinocytes as well as in HaCaT and A431 keratinocyte cell lines. Uptake is fast, irreversible, saturable and not significantly altered by incubation at low temperature. Affinity modification studies in keratinocyte cell lines has revealed two high-affinity, cell-specific interactions between ODNs and proteins of 61-63 kDa and 35 kDa. Trypsin pre-treatment of A431 cells and pre-incubation with polyanions, or with unlabelled nucleic acid competitors, inhibited the accumulation of rhodamine-labelled ODNs in nuclei as well as the affinity labelling of the 61-63 kDa doublet and 35 kDa ODN-binding proteins by reactive ODN derivatives. Finally, cell fractionation studies indicated that these ODN-binding proteins were essentially localised in the plasma membrane. Our results suggest that these ODN-binding proteins might be involved in the recognition and transport of ODNs into keratinocytes.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Queratinócitos/metabolismo , Proteínas de Membrana/metabolismo , Oligodesoxirribonucleotídeos Antissenso/metabolismo , Células Cultivadas , Corantes Fluorescentes/metabolismo , Células HeLa , Humanos , Rodaminas/metabolismo , Células Tumorais CultivadasRESUMO
The interferon-regulated 2-5A/RNase L pathway plays a major role in the antiviral and antiproliferative activities of these cytokines. Several viruses, however, have evolved strategies to escape the antiviral activity of the 2-5A/RNase L pathway. In this context, we have cloned a cDNA coding for the RNase L inhibitor (RLI), a protein that specifically inhibits RNase L and whose regulated expression in picornavirus-infected cells down regulates the activity of the 2-5A/RNase L pathway. We show here that RLI increases during the course of human immunodeficiency virus type 1 (HIV-1) infection, which may be related to the downregulation of RNase L activity that has been described to occur in HIV-infected cells. In order to establish a possible causal relationship between these observations, we have stably transfected H9 cells with RLI sense or antisense cDNA-expressing vectors. The overexpression of RLI causes a decrease in RNase L activity and a twofold enhancement of HIV production. This increase in HIV replication correlates with an increase in HIV RNA and proteins. In contrast, reduction of RLI levels in RLI antisense cDNA-expressing clones reverses the inhibition of RNase L activity associated with HIV multiplication and leads to a threefold decrease in the viral load. This anti-HIV activity correlated with a decrease in HIV RNA and proteins. These findings demonstrate that the level of RLI, via its modulation of RNase L activity, can severely impair HIV replication and suggest the involvement of RLI in the inhibition of the 2-5A/RNase L system observed during HIV infection.
Assuntos
Síndrome da Imunodeficiência Adquirida/metabolismo , Nucleotídeos de Adenina/antagonistas & inibidores , Endorribonucleases/antagonistas & inibidores , Inibidores Enzimáticos/metabolismo , HIV-1/fisiologia , Oligorribonucleotídeos/antagonistas & inibidores , Linhagem Celular , Regulação para Baixo , Endorribonucleases/metabolismo , Humanos , Transfecção , Replicação ViralRESUMO
Bis-peptide nucleic acid (bis-PNA) binding results in D-loop formation by strand displacement at complementary homopurine stretches in DNA duplexes. Transcription and replication in intact cells is mediated by multienzymatic complexes involving several proteins other than polymerases. The behaviour of the highly stable clamp structure formed by bis-PNAs has thus far been studied with respect to their capacity to arrest RNA polymerases. Little attention has been given to their recognition and processing by DNA helicases. In this report we have investigated the inhibitory effect of a bis-PNA on the DNA-helicase activity of the well characterized herpes simplex type I UL9 protein. Unwinding by UL9 of a synthetic substrate is significantly inhibited by a bis-PNA and the addition of the ICP8 protein, which increases UL9 processivity, does not relieve this inhibition.
Assuntos
DNA Helicases/antagonistas & inibidores , Proteínas de Ligação a DNA/antagonistas & inibidores , Herpesvirus Humano 1/enzimologia , Ácidos Nucleicos Peptídicos/farmacologia , Proteínas Virais/antagonistas & inibidores , DNA/metabolismo , DNA Helicases/genética , Replicação do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/farmacologia , Proteínas Recombinantes/farmacologia , Proteínas Virais/genética , Proteínas Virais/farmacologiaRESUMO
Antisense oligonucleotides (ON) allow the specific control of gene expression and phosphorothioate derivatives are currently being evaluated for possible clinical applications. Numerous second generation ON analogues with improved pharmacological properties have been described. Most of them, however, do not recruit RNase H, which is known to increase ON potency by eliciting the specific degradation of the target RNA. Silverman, Torrence and colleagues have conjugated 2,5A to natural antisense ON and demonstrated the preferential cleavage of a target RNA in cell-free and intact cell experiments. We have established for the first time that RNase H-incompetent ON, viz. alpha-anomeric ON analogues, can be converted into sequence-specific nucleases upon conjugation to 2,5A. The use of alpha-ON- and beta-ON-2,5A chimeras has allowed us to delineate the part played by RNase H and RNase L in target RNA degradation and translation arrest. Finally, the present studies have revealed limitations which are encountered in the choice of a suitable target for such ON-2,5A chimeras.
Assuntos
Oligonucleotídeos Antissenso , Ribonuclease H/metabolismo , Ribonuclease Pancreático/metabolismo , Animais , Linhagem Celular , Humanos , Camundongos , Modelos Biológicos , Hibridização de Ácido Nucleico , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Spodoptera , Células Tumorais CultivadasRESUMO
The interferon-(IFN)-inducible 2',5'-oligoadenylate (2-5A)/endoribonuclease L (RNase L) pathway plays a major role in the antiviral and antiproliferative effects of IFN. The 2-5A/RNase L pathway appears to be regulated by the cell-growth status or viral infection. Viruses, and picornaviruses in particular, have evolved strategies to escape the 2-5A/RNase L-pathway-associated antiviral activity. We have recently cloned a cDNA coding for RLI, a RNase-L-specific protein inhibitor. Its regulated expression by viral infection could provide a new strategy to modulate the 2-5A/RNase L pathway. Since RNase L had been shown to be down regulated upon encephalomyocarditis (EMCV) infection, we stably transfected HeLa cells with a RLI antisense cDNA expressing vector. Four independent clones named VAS1, VAS2, VAS3 and VAS4 and one clone transfected with the empty vector (VV) as control, were analyzed. The level of RLI was decreased by 20% for VAS1, 25% for VAS2, 75% for VAS3 and 50% for VAS4. The inactivation of RNase L observed during EMCV infection was decreased in these clones as compared to control HeLa cells. Here again the results vary between the four clones. The maximum inhibition of RNase L (90%) was observed in control cells and in VAS1 while 48% inhibition was observed in VAS4 and 25% in VAS3. The reversal in RNase L inhibition thus reflects closely the resulting RLI level, in keeping with a major role of RLI in EMCV-induced down regulation of 2-5A-binding activity of RNase L. Moreover, cells expressing a low level of RLI (VAS3 and VAS 4) are partially resistant to EMCV infection.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Infecções por Cardiovirus/enzimologia , Chaperoninas , Vírus da Encefalomiocardite , Endorribonucleases/antagonistas & inibidores , Inibidores Enzimáticos/metabolismo , Proteínas/antagonistas & inibidores , Anticorpos , Infecções por Cardiovirus/genética , Infecções por Cardiovirus/virologia , DNA Antissenso/genética , DNA Antissenso/farmacologia , Regulação para Baixo/efeitos dos fármacos , Vírus da Encefalomiocardite/isolamento & purificação , Endorribonucleases/metabolismo , Inibidores Enzimáticos/farmacologia , Células HeLa , Humanos , Proteínas/genética , Proteínas/metabolismo , TransfecçãoRESUMO
The 2-5A/RNase L system is one of the pathways induced by interferon (IFN). It plays a major role in the antiviral and antiproliferative activities of IFNs. Recently, we have shown that the activity of the RNase L could be inhibited by a proteic inhibitor, the RNase L Inhibitor (RLI). Human RLI (Hu-RLI) was cloned and characterized. We describe here the isolation and characterization of the cDNA encoding the murine RLI (Mu-RLI). Hu-RLI and Mu-RLI protein have 98% amino acid identity. Mu-RLI is functionally homologous to Hu-RLI, and all the structural features and amino acid sequence motifs of Hu-RLI are conserved in Mu-RLI. Moreover, reticulocyte lysate translated Mu-RLI protein is also able to inhibit 2-5A binding on 2-5A-dependent RNAse-L. Northern blot analysis revealed that Mu-RLI cDNA hybridizes with one mRNA of 3.5 kb except for the testis where two mRNA of 3.5 and 2.1 kb, respectively, are detected, suggesting a tissue-specific regulation.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Chaperoninas , Inibidores Enzimáticos/química , Biossíntese de Proteínas , Proteínas/química , Nucleotídeos de Adenina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Ligação Competitiva , Clonagem Molecular , Sequência Conservada , DNA Complementar , Inibidores Enzimáticos/farmacologia , Humanos , Masculino , Mathanococcus/genética , Camundongos , Dados de Sequência Molecular , Oligorribonucleotídeos/metabolismo , Especificidade de Órgãos , Proteínas/farmacologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
Antisense oligonucleotides (ONs) are designed to hybridize target mRNA in a sequence-specific manner and inhibit gene expression by preventing translation, either by activation of RNase H or steric blockage of the ribosome complex. Second-generation ONs, which possess greater binding affinity for target RNA relative to the isosequential phosphodiester (PO) ONs, have been developed and include, among others, peptide nucleic acids (PNA) and N3' P5' phosphoramidate oligonucleotides (npONs). In the present study, PNA and npON derivatives were targeted to the coding portion of the complementary mRNA of the N protein of the vesicular stomatitis virus (VSV) in order to evaluate their ability to arrest translation in an in vitro rabbit reticulocyte lysate system. High-affinity hybridization of ONs lacking RNase H activity was not sufficient to block translation in this test system. Only antisense ONs acting via an RNase H mechanism or by steric hindrance through covalent attachment (via transplatin modification) to the target mRNA were found to definitively arrest translation in this study.