RESUMEN
The leukodystrophies cause severe neurodevelopmental defects from birth and follow an incurable and progressive course that often leads to premature death. It has recently been reported that abnormalities in aminoacyl t-RNA synthetase (ARS) genes are linked to various unique leukodystrophies and leukoencephalopathies. Aminoacyl t-RNA synthetase proteins are fundamentally known as the first enzymes of translation, catalysing the conjugation of amino acids to cognate tRNAs for protein synthesis. It is known that certain aminoacyl t-RNA synthetase have multiple non-canonical roles in both transcription and translation, and their disruption results in varied and complicated phenotypes. We clinically and genetically studied seven patients (six male and one female; aged 2 to 12 years) from five unrelated families who all showed the same phenotypes of severe developmental delay or arrest (7/7), hypotonia (6/7), deafness (7/7) and inability to speak (6/7). The subjects further developed intractable epilepsy (7/7) and nystagmus (6/6) with increasing age. They demonstrated characteristic laboratory data, including increased lactate and/or pyruvate levels (7/7), and imaging findings (7/7), including calcification and abnormal signals in the white matter and pathological involvement (2/2) of the corticospinal tracts. Through whole-exome sequencing, we discovered genetic abnormalities in lysyl-tRNA synthetase (KARS). All patients harboured the variant [c.1786C>T, p.Leu596Phe] KARS isoform 1 ([c.1702C>T, p.Leu568Phe] of KARS isoform 2) either in the homozygous state or compound heterozygous state with the following KARS variants, [c.879+1G>A; c.1786C>T, p.Glu252_Glu293del; p.Leu596Phe] ([c.795+1G>A; c.1702C>T, p.Glu224_Glu255del; p.Leu568Phe]) and [c.650G>A; c.1786C>T, p.Gly217Asp; p.Leu596Phe] ([c.566G>A; c.1702C>T, p.Gly189Asp; p.Leu568Phe]). Moreover, similarly disrupted lysyl-tRNA synthetase (LysRS) proteins showed reduced enzymatic activities and abnormal CNSs in Xenopus embryos. Additionally, LysRS acts as a non-canonical inducer of the immune response and has transcriptional activity. We speculated that the complex functions of the abnormal LysRS proteins led to the severe phenotypes in our patients. These KARS pathological variants are novel, including the variant [c.1786C>T; p.Leu596Phe] (c.1702C>T; p.Leu568Phe) shared by all patients in the homozygous or compound-heterozygous state. This common position may play an important role in the development of severe progressive leukodystrophy. Further research is warranted to further elucidate this relationship and to investigate how specific mutated LysRS proteins function to understand the broad spectrum of KARS-related diseases.
Asunto(s)
Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/fisiopatología , Lisina-ARNt Ligasa/genética , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/fisiología , Animales , Niño , Preescolar , Modelos Animales de Enfermedad , Femenino , Homocigoto , Humanos , Leucoencefalopatías/genética , Lisina-ARNt Ligasa/fisiología , Masculino , Mutación , Linaje , Fenotipo , Secuenciación del Exoma , Xenopus laevisRESUMEN
In yeast, the import of tRNALys with CUU anticodon (tRK1) relies on a complex mechanism where interaction with enolase 2 (Eno2p) dictates a deep conformational change of the tRNA. This event is believed to mask the tRNA from the cytosolic translational machinery to re-direct it towards the mitochondria. Once near the mitochondrial outer membrane, the precursor of the mitochondrial lysyl-tRNA synthetase (preMsk1p) takes over enolase to carry the tRNA within the mitochondrial matrix, where it is supposed to participate in translation following correct refolding. Biochemical data presented in this report focus on the role of enolase. They show that despite the inability of Eno2p alone to form a complex with tRK1, mitochondrial import can be recapitulated in vitro using fractions of yeast extracts sharing either recombinant or endogenous yeast Eno2p as one of the main components. Taken together, our data suggest the existence of a protein complex containing Eno2p that is involved in RNA mitochondrial import.
Asunto(s)
Lisina-ARNt Ligasa/fisiología , Mitocondrias/metabolismo , Fosfopiruvato Hidratasa/fisiología , ARN de Transferencia de Lisina/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/ultraestructura , Transporte Biológico , Proteínas de Transporte de Catión/metabolismo , Mitocondrias/enzimología , Complejos Multiproteicos/química , Complejos Multiproteicos/fisiología , Fosfopiruvato Hidratasa/metabolismo , ARN de Transferencia/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologíaRESUMEN
Autoantibodies against various components of host are known to occur in leprosy. Nerve damage is the primary cause of disability associated with leprosy. The aim of this study was to detect the level of autoantibodies and lympho-proliferative response against myelin basic protein (MBP) in leprosy patients (LPs) and their correlation with clinical phenotypes of LPs. Further, probable role of molecular mimicry in nerve damage of LPs was investigated. We observed significantly high level of anti-MBP antibodies in LPs across the spectrum and a positive significant correlation between the level of anti-MBP antibodies and the number of nerves involved in LPs. We report here that 4 B cell epitopes of myelin A1 and Mycobacterium leprae proteins, 50S ribosomal L2 and lysyl tRNA synthetase are cross-reactive. Further, M. leprae sonicated antigen hyperimmunization was responsible for induction of autoantibody response in mice which could be adoptively transferred to naive mice. For the first time our findings suggest the role of molecular mimicry in nerve damage in leprosy.
Asunto(s)
Enfermedades Desmielinizantes/microbiología , Lepra/microbiología , Lisina-ARNt Ligasa/fisiología , Imitación Molecular/fisiología , Mycobacterium leprae/patogenicidad , Proteína Básica de Mielina/fisiología , Proteínas Ribosómicas/fisiología , Animales , Enfermedades Desmielinizantes/complicaciones , Enfermedades Desmielinizantes/etiología , Humanos , Lepra/complicaciones , Lepra/etiología , Ratones , Ratones Endogámicos BALB C/sangre , ConejosRESUMEN
Control of foodborne Salmonella within the farm-retail continuum is a complex issue since over 2500 serovars of Salmonella exist, the host range of Salmonella spp. varies greatly, and Salmonella is environmentally ubiquitous. To identify Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) genes important for pathogen survival, our research group previously screened a signature-tagged mutagenesis bank in an ex vivo swine stomach content assay. A mutation in the poxA gene, a member of the gene family encoding class-II aminoacyl-tRNA synthetases, decreased survival of Salmonella Typhimurium in the ex vivo swine stomach content assay. In the current study, complementation with a plasmid-encoded poxA gene restored survival of the poxA mutant to the level of the parental, wild-type strain. In vivo analysis of the poxA mutant in the natural porcine host revealed significantly reduced fecal shedding of Salmonella, decreased colonization of the tonsils, and decreased detection of the mutant strain in the cecal contents of the pigs at 7 days postinoculation (p < 0.05). Body temperature (fever) of the pigs inoculated with wild-type Salmonella Typhimurium was significantly higher than that of pigs inoculated with the poxA mutant (p < 0.05). Two-dimensional gel electrophoresis revealed characteristic differences in the protein profile of the poxA mutant relative to the wild-type strain, indicating that deletion of poxA in Salmonella Typhimurium exerts selective effects on translation and/or posttranslational modifications of mRNA species that are necessary for stress survival and colonization of the natural swine host.
Asunto(s)
Lisina-ARNt Ligasa/fisiología , Viabilidad Microbiana , Mutación , Biosíntesis de Proteínas , Salmonelosis Animal/microbiología , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidad , Animales , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Derrame de Bacterias , Traslocación Bacteriana , Ciego/microbiología , Línea Celular , Heces/microbiología , Femenino , Fiebre/etiología , Enfermedades Gastrointestinales/microbiología , Enfermedades Gastrointestinales/fisiopatología , Enfermedades Gastrointestinales/veterinaria , Genes Bacterianos , Humanos , Lisina-ARNt Ligasa/genética , Masculino , Tonsila Palatina/microbiología , Salmonelosis Animal/fisiopatología , Salmonella typhimurium/enzimología , Salmonella typhimurium/fisiología , Sus scrofaRESUMEN
Retroviruses rely on host RNA-binding proteins to modulate various steps in their replication. Previously several animal retroviruses were determined to mediate Dhx9/RNA helicase A (RHA) interaction with a 5' terminal post-transcriptional control element (PCE) for efficient translation. Herein PCE reporter assays determined HTLV-1 and HIV-1 RU5 confer orientation-dependent PCE activity. The effect of Dhx9/RHA down-regulation and rescue with siRNA-resistant RHA on expression of HIV-1(NL4-3) provirus determined that RHA is necessary for efficient HIV-1 RNA translation and requires ATPase-dependent helicase function. Quantitative analysis determined HIV-1 RNA steady-state and cytoplasmic accumulation were not reduced; rather the translational activity of viral RNA was reduced. Western blotting determined that RHA-deficient virions assemble with Lys-tRNA synthetase, exhibit processed reverse transcriptase and contain similar level of viral RNA, but they are poorly infectious on primary lymphocytes and HeLa cells. The results demonstrate RHA is an important host factor within the virus-producer cell and within the viral particle. The identification of RHA-dependent PCE activity in cellular junD RNA and in six of seven genera of Retroviridae suggests conservation of this translational control mechanism among vertebrates, and convergent evolution of Retroviridae to utilize this host mechanism.
Asunto(s)
ARN Helicasas DEAD-box/fisiología , VIH-1/genética , VIH-1/fisiología , Proteínas de Neoplasias/fisiología , Biosíntesis de Proteínas , Virión/fisiología , Secuencia de Bases , Línea Celular , Duplicado del Terminal Largo de VIH , Virus Linfotrópico T Tipo 1 Humano/genética , Humanos , Lisina-ARNt Ligasa/fisiología , Datos de Secuencia Molecular , ARN Viral/metabolismo , Secuencias Repetidas Terminales , Ensamble de Virus , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/biosíntesis , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
HIV-1 is a retrovirus that causes AIDS in humans. The RNA genome of the virus encodes a Gag polyprotein, which is further processed into matrix, capsid and nucleocapsid proteins. These proteins play a significant role at several steps in the viral life cycle. In addition, various stages of assembly, infection and replication of the virus involve necessary interactions with a large number of supplementary proteins/cofactors within the infected host cell. This minireview focuses on the proteomics of the capsid protein, its influence on the packaging of nonviral molecules into HIV-1 virions and the subsequent role of the molecules themselves. These interactions and their characterization present novel frontiers for the design and advancement of antiviral therapeutics.
Asunto(s)
Proteínas de la Cápside/fisiología , VIH-1/fisiología , Ensamble de Virus , Factores de Restricción Antivirales , Proteínas de la Cápside/química , Proteínas Portadoras/fisiología , Ciclofilina A/fisiología , VIH-1/química , Humanos , Lisina-ARNt Ligasa/fisiología , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína LigasasRESUMEN
Lysyl tRNA synthetase (LysRS) is an aminoacyl-tRNA synthetase (AaRS). This group of ancient proteins, known for their critical role in translation, was found in recent years to function in a variety of other roles. Besides its enzymatic activity in aminoacylation of tRNA, LysRS can produce dinucleotide diadenosine tetraphosphate (Ap(4)A). Intracellularly, it is found mainly in the cytoplasm as a part of a multisynthetase complex where it interacts with several proteins, most notably AIMP2. Besides its role in translation it has been demonstrated that LysRS can act as a cytokine-like molecule, secreted by cells and having distinct effects on macrophages. Moreover, LysRS can bind to the transcription factors USF2 and MITF and can influence their transcriptional activities following immunological stimulation of mast cells. In this review, we focus on the nontranslational functions of LysRS related to the immune system. We begin with a short discussion of "gene sharing," proceed to a description of its structural and enzymatic function and then describe some of the in vivo functions of this enzyme.
Asunto(s)
Sistema Inmunológico , Lisina-ARNt Ligasa/fisiología , Animales , Autoinmunidad , Humanos , Factor de Transcripción Asociado a Microftalmía/metabolismo , Unión Proteica , Factores Estimuladores hacia 5'/metabolismoRESUMEN
Lysyl-tRNA synthetase (LysRS) was found to produce diadenosine tetraphosphate (Ap(4)A) in vitro more than two decades ago. Here, we used LysRS silencing in mast cells in combination with transfected normal and mutated LysRS to demonstrate in vivo the critical role played by LysRS in the production of Ap(4)A in response to immunological challenge. Upon such challenge, LysRS was phosphorylated on serine 207 in a MAPK-dependent manner, released from the multisynthetase complex, and translocated into the nucleus. We previously demonstrated that LysRS forms a complex with MITF and its repressor Hint-1, which is released from the complex by its binding to Ap(4)A, enabling MITF to transcribe its target genes. Here, silencing LysRS led to reduced Ap(4)A production in immunologically activated cells, which resulted in a lower level of MITF inducible genes. Our data demonstrate that specific LysRS serine 207 phosphorylation regulates Ap(4)A production in immunologically stimulated mast cells, thus implying that LysRS is a key mediator in gene regulation.
Asunto(s)
Regulación de la Expresión Génica , Inmunidad Celular/genética , Lisina-ARNt Ligasa/fisiología , Transducción de Señal , Transporte Activo de Núcleo Celular , Animales , Línea Celular , Fosfatos de Dinucleósidos/biosíntesis , Humanos , Lisina-ARNt Ligasa/metabolismo , Sistema de Señalización de MAP Quinasas , Factor de Transcripción Asociado a Microftalmía/genética , Factor de Transcripción Asociado a Microftalmía/metabolismo , Fosforilación , Ratas , Serina/metabolismoRESUMEN
Aminoacyl-tRNA synthetases (aaRSs) are responsible for attaching amino acids to their cognate tRNAs during protein synthesis. In eukaryotes aaRSs are commonly found in multi-enzyme complexes, although the role of these complexes is still not completely clear. Associations between aaRSs have also been reported in archaea, including a complex between prolyl-(ProRS) and leucyl-tRNA synthetases (LeuRS) in Methanothermobacter thermautotrophicus that enhances tRNA(Pro) aminoacylation. Yeast two-hybrid screens suggested that lysyl-tRNA synthetase (LysRS) also associates with LeuRS in M. thermautotrophicus. Co-purification experiments confirmed that LeuRS, LysRS, and ProRS associate in cell-free extracts. LeuRS bound LysRS and ProRS with a comparable K(D) of about 0.3-0.9 microm, further supporting the formation of a stable multi-synthetase complex. The steady-state kinetics of aminoacylation by LysRS indicated that LeuRS specifically reduced the Km for tRNA(Lys) over 3-fold, with no additional change seen upon the addition of ProRS. No significant changes in aminoacylation by LeuRS or ProRS were observed upon the addition of LysRS. These findings, together with earlier data, indicate the existence of a functional complex of three aminoacyl-tRNA synthetases in archaea in which LeuRS improves the catalytic efficiency of tRNA aminoacylation by both LysRS and ProRS.
Asunto(s)
Aminoacil-ARNt Sintetasas/fisiología , Proteínas Arqueales/fisiología , Methanobacteriaceae/enzimología , Aminoacil-ARNt Sintetasas/aislamiento & purificación , Aminoacil-ARNt Sintetasas/metabolismo , Aminoacilación , Proteínas Arqueales/aislamiento & purificación , Proteínas Arqueales/metabolismo , Catálisis , Leucina-ARNt Ligasa/aislamiento & purificación , Leucina-ARNt Ligasa/metabolismo , Leucina-ARNt Ligasa/fisiología , Lisina-ARNt Ligasa/aislamiento & purificación , Lisina-ARNt Ligasa/metabolismo , Lisina-ARNt Ligasa/fisiología , Methanobacteriaceae/fisiología , Mapeo de Interacción de Proteínas , Transducción de Señal/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
In the post genome project era, it is well established that the human genome contains a smaller number of genes than expected. The complexity found in higher organisms can be explained if proteins are multifunctional. Indeed, recent studies are continuing to reveal proteins that are capable of a broad repertoire of functions. A good paradigm for multifunctionality can be found in the amino-acyl tRNA synthetases (aaRSs), an ancient conserved family of proteins. This unique family, which is comprised of 20 different enzymes, is well known for its participation in protein synthesis. Several studies have described numerous examples of these "housekeeping" proteins taking part in extensive critical cellular activities. In this review, we focus on a member of that family, lysyl-tRNA synthetase (LysRS), which has been shown to have a dual functionality. In addition to its contribution to the translation process, LysRS also takes part in the regulation of MITF and USF2 target genes. This phenomenon was first described in mast cells.
Asunto(s)
Lisina-ARNt Ligasa/fisiología , Mastocitos/fisiología , Biosíntesis de Proteínas , Transcripción Genética , Animales , Factor de Transcripción Asociado a Microftalmía/fisiología , Modelos Genéticos , Factores Estimuladores hacia 5'/fisiologíaRESUMEN
Accurate aminoacyl-tRNA synthesis is essential for faithful translation of the genetic code and consequently has been intensively studied for over three decades. Until recently, the study of aminoacyl-tRNA synthesis in archaea had received little attention. However, as in so many areas of molecular biology, the advent of archaeal genome sequencing has now drawn researchers to this field. Investigations with archaea have already led to the discovery of novel pathways and enzymes for the synthesis of numerous aminoacyl-tRNAs. The most surprising of these findings has been a transamidation pathway for the synthesis of asparaginyl-tRNA and a novel lysyl-tRNA synthetase. In addition, seryl- and phenylalanyl-tRNA synthetases that are only marginally related to known examples outside the archaea have been characterized, and the mechanism of cysteinyl-tRNA formation in Methanococcus jannaschii and Methanobacterium thermoautotrophicum is still unknown. These results have revealed completely unexpected levels of complexity and diversity, questioning the notion that aminoacyl-tRNA synthesis is one of the most conserved functions in gene expression. It has now become clear that the distribution of the various mechanisms of aminoacyl-tRNA synthesis in extant organisms has been determined by numerous gene transfer events, indicating that, while the process of protein biosynthesis is orthologous, its constituents are not.
Asunto(s)
Aminoacil-ARNt Sintetasas/fisiología , Archaea/enzimología , Proteínas Arqueales/fisiología , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Archaea/genética , Proteínas Arqueales/genética , Euryarchaeota/enzimología , Evolución Molecular , Regulación de la Expresión Génica Arqueal , Lisina-ARNt Ligasa/genética , Lisina-ARNt Ligasa/fisiología , Fenilalanina-ARNt Ligasa/genética , Fenilalanina-ARNt Ligasa/fisiología , Filogenia , ARN de Archaea/genética , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Selenocisteína/metabolismo , Serina-ARNt Ligasa/genética , Serina-ARNt Ligasa/fisiologíaRESUMEN
The complete nucleotide sequence of lysU, the gene for the heat-inducible lysyl-tRNA synthetase of Escherichia coli, was determined and compared with the published sequence of lysS (herC), the gene for the constitutive lysyl-tRNA synthetase. These unlinked genes were found to be identical over 72% of their lengths. The deduced amino acid sequences of the respective gene products, LysU and LysS, were identical over 85% and similar over 92% of their lengths. Accumulation of high levels of LysU during growth of strains carrying the wild-type allele of lysU on multicopy plasmids had no observable effect on growth or on the synthesis of LysS. A lysU deletion strain was constructed and was shown to grow normally at low temperature (28 degrees C) but poorly at 44 degrees C; the slow growth (45% of normal) at elevated temperature was fully reversed by plasmids bearing wild-type lysU. The implications of these findings for the existence of two aminoacyl-tRNA synthetases for lysine are discussed.