RESUMEN
Adenosine to inosine editing at the wobble position allows decoding of multiple codons by a single tRNA. This reaction is catalyzed by adenosine deaminases acting on tRNA (ADATs) and is essential for viability. In bacteria, the anticodon-specific enzyme is a homodimer that recognizes a single tRNA substrate (tRNA(Arg)(ACG)) and can efficiently deaminate short anticodon stem-loop mimics of this tRNA in vitro. The eukaryal enzyme is composed of two nonidentical subunits, ADAT2 and ADAT3, which upon heterodimerization, recognize seven to eight different tRNAs as substrates, depending on the organism, and require a full-length tRNA for activity. Although crystallographic data have provided clues to why the bacterial deaminase can utilize short substrates, residues that provide substrate binding and recognition with the eukaryotic enzymes are not currently known. In the present study, we have used a combination of mutagenesis, binding studies, and kinetic analysis to explore the contribution of individual residues in Trypanosoma brucei ADAT2 (TbADAT2) to tRNA recognition. We show that deletion of the last 10 amino acids at the C terminus of TbADAT2 abolishes tRNA binding. In addition, single alanine replacements of a string of positively charged amino acids (KRKRK) lead to binding defects that correlate with losses in enzyme activity. This region, which we have termed the KR-domain, provides a first glance at key residues involved in tRNA binding by eukaryotic tRNA editing deaminases.
Asunto(s)
Adenosina Desaminasa/química , Adenosina Desaminasa/metabolismo , Dominios y Motivos de Interacción de Proteínas/fisiología , Edición de ARN , ARN de Transferencia/metabolismo , Trypanosoma brucei brucei/enzimología , Trypanosoma brucei brucei/genética , Adenosina Desaminasa/genética , Secuencia de Aminoácidos , Activación Enzimática/genética , Cinética , Datos de Secuencia Molecular , Unión Proteica/genética , Unión Proteica/fisiología , Dominios y Motivos de Interacción de Proteínas/genética , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína/genética , Estructura Terciaria de Proteína/fisiología , Edición de ARN/fisiología , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Trypanosoma brucei brucei/metabolismo , Estudios de Validación como AsuntoRESUMEN
Antisense oligonucleotides (ASO) containing 2'-O-methoxyethyl ribose (2'-MOE) modifications have been shown to possess both excellent pharmacokinetic properties and robust pharmacological activity in several animal models of human disease. 2'-MOE ASOs are generally well tolerated, displaying minimal to mild proinflammatory effect at doses far exceeding therapeutic doses. Although the vast majority of 2'-MOE ASOs are safe and well tolerated, a small subset of ASOs inducing acute inflammation in mice has been identified. The mechanism for these findings is not clear at this point, but the effects are clearly sequence-specific. One of those ASOs, ISIS 147420, causes a severe inflammatory response atypical of this class of oligonucleotides characterized by induction in interferon-ß (IFN-ß) at 48 h followed by acute transaminitis and extensive hepatocyte apoptosis and necrosis at 72 h. A large number of interferon-stimulated genes were significantly up-regulated in liver as early as 24 h. We speculated that a specific sequence motif might cause ISIS 147420 to be mistaken for viral RNA or DNA, thus triggering an acute innate immune response. ISIS 147420 toxicity was independent of Toll-like receptors, because there was no decrease in IFN-ß in Toll/interleukin-1 receptor-domain-containing adapter-inducing IFN-ß or Myd88-deficient mice. The involvement of cytosolic retinoic acid-inducible gene (RIG)-I-like pattern recognition receptors was also investigated. Pretreatment of mice with melanoma differentiation-associated gene 5 (MDA5) and IFN-ß promoter stimulator-1 ASOs, but not RIG-I or laboratory of genetics and physiology 2 (LGP2) ASOs, prevented the increase in IFN-ß and alanine aminotransferase induced by ISIS 147420. These results revealed a novel mechanism of oligonucleotide-mediated toxicity requiring both MDA5 and IPS-1 and resulting in the activation of the innate immune response.
Asunto(s)
ARN Helicasas DEAD-box/inmunología , ADN/inmunología , Inmunidad Innata/inmunología , Interferón Tipo I/inmunología , Oligonucleótidos Antisentido/inmunología , Ribosa/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/inmunología , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Alanina Transaminasa/genética , Alanina Transaminasa/inmunología , Alanina Transaminasa/metabolismo , Animales , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , ADN/genética , ADN/metabolismo , Hepatocitos/inmunología , Hepatocitos/metabolismo , Inmunidad Innata/genética , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Helicasa Inducida por Interferón IFIH1 , Interferón beta/genética , Interferón beta/inmunología , Interferón beta/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/inmunología , Factor 88 de Diferenciación Mieloide/metabolismo , Oligonucleótidos Antisentido/genética , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/inmunología , Receptores de Interleucina-1/metabolismo , Ribosa/genética , Ribosa/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología , Receptores Toll-Like/genética , Receptores Toll-Like/inmunología , Receptores Toll-Like/metabolismoRESUMEN
Adenosine-to-inosine editing in the anticodon of tRNAs is essential for viability. Enzymes mediating tRNA adenosine deamination in bacteria and yeast contain cytidine deaminase-conserved motifs, suggesting an evolutionary link between the two reactions. In trypanosomatids, tRNAs undergo both cytidine-to-uridine and adenosine-to-inosine editing, but the relationship between the two reactions is unclear. Here we show that down-regulation of the Trypanosoma brucei tRNA-editing enzyme by RNAi leads to a reduction in both C-to-U and A-to-I editing of tRNA in vivo. Surprisingly, in vitro, this enzyme can mediate A-to-I editing of tRNA and C-to-U deamination of ssDNA but not both in either substrate. The ability to use both DNA and RNA provides a model for a multispecificity editing enzyme. Notably, the ability of a single enzyme to perform two different deamination reactions also suggests that this enzyme still maintains specificities that would have been found in the ancestor deaminase, providing a first line of evidence for the evolution of editing deaminases.
Asunto(s)
Adenosina Desaminasa/fisiología , Citidina Desaminasa/fisiología , Edición de ARN , Adenosina/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular , Citidina/metabolismo , Desaminación , Inosina/metabolismo , Datos de Secuencia Molecular , Interferencia de ARN , Uridina/metabolismoRESUMEN
Previously, tubulin has been purified from Leishmania amazonensis and used to identify novel molecules with selective antimitotic activity. However, L. amazonensis is pathogenic and requires a relatively expensive medium for large-scale cultivation. Herein, the purification and characterization of tubulin from the non-pathogenic Leishmania tarentolae is reported, together with the sequence of alpha- and beta-tubulin from this organism. This protein was purified by sonication, diethylaminoethyl-Sepharose chromatography, and one assembly disassembly cycle in 1% overall recovery based on total cellular protein. Leishmania tarentolae tubulin was indistinguishable from the corresponding L. amazonensis protein in terms of binding affinity for dinitroaniline sulfanilamides and sensitivity to assembly inhibition by these compounds. The amino acid sequences derived from the L. tarentolae alpha- and beta-tubulin genes were 99.6 and 99.4% identical to the corresponding amino acid sequences from the Leishmania major Friedlin strain. These results indicate that tubulin from L. tarentolae is suitable for use in drug screening.
Asunto(s)
Leishmania/química , Tubulina (Proteína)/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Cromatografía en Agarosa , Dinitrobencenos/química , Dinitrobencenos/farmacología , Electroforesis en Gel de Poliacrilamida , Leishmania/efectos de los fármacos , Leishmania/genética , Ligandos , Datos de Secuencia Molecular , Alineación de Secuencia , Sonicación , Sulfanilamidas/química , Sulfanilamidas/farmacología , Tubulina (Proteína)/química , Tubulina (Proteína)/efectos de los fármacos , Tubulina (Proteína)/genética , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologíaRESUMEN
Editing of tRNAs is widespread in nature and either changes the decoding properties or restores the folding of a tRNA. Unlike the phylogenetically disperse adenosine (A) to inosine (I) editing, cytosine (C) to uridine (U) editing has only been previously described in organellar tRNAs. We have shown that cytoplasmic tRNA(Thr)(AGU) undergoes two distinct editing events in the anticodon loop: C to U and A to I. In vivo, every inosine-containing tRNA(Thr) is also C to U edited at position 32. In vitro, C to U editing stimulates conversion of A to I at the wobble base. Although the in vivo and in vitro requirements differ, in both cases, the C to U change plays a key role in A to I editing. Due to an unusual abundance of A34-containing tRNAs, our results also suggest that the unedited and edited tRNAs are functional, each dedicated to decoding a specific threonine codon. C to U editing of cytoplasmic tRNA expands the editing repertoire in eukaryotic cells, and when coupled to A to I changes, leads to an interrelation between editing sites.