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1.
Hum Cell ; 35(4): 1126-1139, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35429326

RESUMO

Colon cancer cells rely on mitochondrial respiration as major source of energy for supporting their proliferation and invasion, thus promoting colon cancer malignancy and progression. In this study, we comprehensively investigated the prognostic significance of mitochondria-related genes in colon cancer and identified the hub genes that control colon cancer cell mitochondrial respiration and proliferation. We first systematically evaluated the prognostic significance of differentially expressed mitochondria-related genes in colon cancer specimens. Furthermore, a protein-protein interaction network was constructed to explore the hub genes. Eventually, five hub genes were identified, namely, POLG, FASTK, MRPS5, AARS2, and VARS2. Functional analyses showed that all these five hub genes are essential for maintaining mitochondrial respiration and proliferation of colon cancer cells. Mechanistic studies revealed the roles of these five hub genes in modulating mitochondrial DNA expression, that in turn influence mitochondrial respiration. In summary, our study demonstrated that POLG, FASTK, MRPS5, AARS2, and VARS2 may potentially serve as prognostic biomarkers and therapeutic targets for colon cancer.


Assuntos
Neoplasias do Colo , Proliferação de Células/genética , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Antígenos HLA , Humanos , Mitocôndrias/metabolismo , Proteínas Serina-Treonina Quinases , Respiração , Valina-tRNA Ligase/metabolismo
2.
J Biosci Bioeng ; 133(5): 436-443, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35216933

RESUMO

Isoleucyl-tRNA synthetase (IleRS), leucyl-tRNA synthetase (LeuRS), and valyl-tRNA synthetase (ValRS) are enzymes that have potential for the determination of l-isoleucine, l-leucine, and l-valine in food products and plasma. However, the disadvantages of these enzymes are their specificity and sensitivity. Here, we examined the substrate specificity of IleRS, LeuRS, and ValRS under various conditions of pyrophosphate amplification to improve their specificity and sensitivity. The amount of pyrophosphate produced in IleRS, LeuRS, and ValRS reactions was amplified after the addition of excess adenosine-5'-triphosphate and magnesium ions, and was approximately 9-, 8-, and 7-fold higher, respectively, for each of the initial l-amino acid substrates (50 µM). However, in addition to their target amino acids, IleRS, LeuRS, and ValRS also reacted with l-valine, l-lysine, and l-threonine, respectively. This substrate misrecognition was overcome by making the reaction pH more acidic and by increasing the magnesium ion concentration. The pyrophosphate amplification in IleRS, LeuRS, and ValRS reactions resulted in the production of p1, p4-di (adenosine) 5'-tetraphosphate. We also observed a strong positive correlation (R = 0.99) between the amount of pyrophosphate produced and the initial concentration of l-amino acid with 5 and 50 µM l-isoleucine, l-leucine, and l-valine. Our results suggest that amino acid assays using IleRS, LeuRS, and ValRS are promising methods to accurately measure l-valine, l-isoleucine, and l-leucine in food products and plasma.


Assuntos
Aminoacil-tRNA Sintetases , Leucina-tRNA Ligase , Adenosina/metabolismo , Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/metabolismo , Difosfatos , Escherichia coli/metabolismo , Isoleucina , Leucina/metabolismo , Leucina-tRNA Ligase/química , Leucina-tRNA Ligase/genética , Leucina-tRNA Ligase/metabolismo , Magnésio/metabolismo , RNA de Transferência , Especificidade por Substrato , Valina/metabolismo , Valina-tRNA Ligase/química , Valina-tRNA Ligase/genética , Valina-tRNA Ligase/metabolismo
3.
Nature ; 601(7893): 428-433, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34937946

RESUMO

Although deregulation of transfer RNA (tRNA) biogenesis promotes the translation of pro-tumorigenic mRNAs in cancers1,2, the mechanisms and consequences of tRNA deregulation in tumorigenesis are poorly understood. Here we use a CRISPR-Cas9 screen to focus on genes that have been implicated in tRNA biogenesis, and identify a mechanism by which altered valine tRNA biogenesis enhances mitochondrial bioenergetics in T cell acute lymphoblastic leukaemia (T-ALL). Expression of valine aminoacyl tRNA synthetase is transcriptionally upregulated by NOTCH1, a key oncogene in T-ALL, underlining a role for oncogenic transcriptional programs in coordinating tRNA supply and demand. Limiting valine bioavailability through restriction of dietary valine intake disrupted this balance in mice, resulting in decreased leukaemic burden and increased survival in vivo. Mechanistically, valine restriction reduced translation rates of mRNAs that encode subunits of mitochondrial complex I, leading to defective assembly of complex I and impaired oxidative phosphorylation. Finally, a genome-wide CRISPR-Cas9 loss-of-function screen in differential valine conditions identified several genes, including SLC7A5 and BCL2, whose genetic ablation or pharmacological inhibition synergized with valine restriction to reduce T-ALL growth. Our findings identify tRNA deregulation as a critical adaptation in the pathogenesis of T-ALL and provide a molecular basis for the use of dietary approaches to target tRNA biogenesis in blood malignancies.


Assuntos
Leucemia-Linfoma Linfoblástico de Células T Precursoras , Valina-tRNA Ligase , Valina , Animais , Disponibilidade Biológica , Sistemas CRISPR-Cas , Dieta , Complexo I de Transporte de Elétrons/genética , Transportador 1 de Aminoácidos Neutros Grandes , Camundongos , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , RNA de Transferência/genética , Valina/metabolismo , Valina-tRNA Ligase/metabolismo
4.
FEBS J ; 287(4): 800-813, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31486189

RESUMO

Isoleucyl-tRNA synthetase (IleRS) is a paradigm for understanding how specificity against smaller hydrophobic substrates evolved in both the synthetic and editing reactions. IleRS misactivates nonproteinogenic norvaline (Nva) and proteinogenic valine (Val), with a 200-fold lower efficiency than the cognate isoleucine (Ile). Translational errors are, however, prevented by IleRS hydrolytic editing. Nva and Val are both smaller than Ile by a single methylene group. How does the removal of one additional methylene group affects IleRS specificity? We found that the nonproteinogenic α-aminobutyrate (Abu) is activated 30-fold less efficiently than Nva and Val, indicating that the removal of the second methylene group comes with a lower penalty. As with Nva and Val, discrimination against Abu predominantly originated from a higher KM . To examine whether increased hydrophobicity could compensate for the loss of van der Waals interactions, we tested fluorinated Abu analogues. We found that fluorination further hampered activation by IleRS, and even more so by the evolutionary-related ValRS. This suggests that hydrophobicity is not a main driving force of substrate binding in these enzymes. Finally, a discrimination factor of 7100 suggests that IleRS is not expected to edit Abu. However, we found that the IleRS editing domain hydrolyzes Abu-tRNAIle with a rate of 40 s-1 and the introduction of fluorine did not slow down the hydrolysis. This raises interesting questions regarding the mechanism of specificity of the editing domain and its evolution. Understanding what shapes IleRS specificity is also of importance for reengineering translation to accommodate artificial substrates including fluorinated amino acids. ENZYMES: Isoleucyl-tRNA synthetase (EC 6.1.1.5), leucyl-tRNA synthetase (EC 6.1.1.4), valyl-tRNA synthetase (EC 6.1.1.9).


Assuntos
Aminobutiratos/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Isoleucina-tRNA Ligase/química , Leucina-tRNA Ligase/química , Valina-tRNA Ligase/química , Aminobutiratos/metabolismo , Sítios de Ligação , Clonagem Molecular , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Halogenação , Isoleucina-tRNA Ligase/genética , Isoleucina-tRNA Ligase/metabolismo , Cinética , Leucina-tRNA Ligase/genética , Leucina-tRNA Ligase/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Termodinâmica , Valina-tRNA Ligase/genética , Valina-tRNA Ligase/metabolismo
5.
Sci Adv ; 5(11): eaax7462, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31799395

RESUMO

Lysosomal storage disorders (LSDs) result from an enzyme deficiency within lysosomes. The systemic administration of the missing enzyme, however, is not effective in the case of LSDs with central nervous system (CNS)-involvement. Here, an enzyme delivery system based on the encapsulation of cross-linked enzyme aggregates (CLEAs) into poly-(lactide-co-glycolide) (PLGA) nanoparticles (NPs) functionalized with brain targeting peptides (Ang2, g7 or Tf2) is demonstrated for Krabbe disease, a neurodegenerative LSD caused by galactosylceramidase (GALC) deficiency. We first synthesize and characterize Ang2-, g7- and Tf2-targeted GALC CLEA NPs. We study NP cell trafficking and capability to reinstate enzymatic activity in vitro. Then, we successfully test our formulations in the Twitcher mouse. We report enzymatic activity measurements in the nervous system and in accumulation districts upon intraperitoneal injections, demonstrating activity recovery in the brain up to the unaffected mice level. Together, these results open new therapeutic perspectives for all LSDs with major CNS-involvement.


Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Terapia de Reposição de Enzimas/métodos , Galactosilceramidase/administração & dosagem , Leucodistrofia de Células Globoides/terapia , Nanopartículas/metabolismo , Animais , Encéfalo/metabolismo , Linhagem Celular , Galactosilceramidase/deficiência , Células HEK293 , Antígenos HLA/metabolismo , Humanos , Leucodistrofia de Células Globoides/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ribonuclease Pancreático/metabolismo , Valina-tRNA Ligase/metabolismo
6.
Org Biomol Chem ; 16(7): 1073-1078, 2018 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-29367962

RESUMO

The ability to incorporate non-canonical amino acids (ncAA) using translation offers researchers the ability to extend the functionality of proteins and peptides for many applications including synthetic biology, biophysical and structural studies, and discovery of novel ligands. Here we describe the high promiscuity of an editing-deficient valine-tRNA synthetase (ValRS T222P). Using this enzyme, we demonstrate ribosomal translation of 11 ncAAs including those with novel side chains, α,α-disubstitutions, and cyclic ß-amino acids.


Assuntos
Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Sequência de Aminoácidos , Aminoácidos/química , Biossíntese de Proteínas/genética , Engenharia de Proteínas , Valina-tRNA Ligase/metabolismo
7.
Hum Genomics ; 11(1): 28, 2017 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-29137650

RESUMO

BACKGROUND: Most mitochondrial and cytoplasmic aminoacyl-tRNA synthetases (aaRSs) are encoded by nuclear genes. Syndromic disorders resulting from mutation of aaRSs genes display significant phenotypic heterogeneity. We expand aaRSs-related phenotypes through characterization of the clinical and molecular basis of a novel autosomal-recessive syndrome manifesting severe mental retardation, ataxia, speech impairment, epilepsy, short stature, microcephaly, hypogonadism, and growth hormone deficiency. RESULTS: A G>A variant in exon 29 of VARS2 (c.3650G>A) (NM_006295) was identified in the index case. This homozygous variant was confirmed by Sanger sequencing and segregated with disease in the family studied. The c.3650G>A change results in alteration of arginine to histidine at residue 1217 (R1217H) of the mature protein and is predicted to be pathogenic. CONCLUSIONS: These findings contribute to a growing list of aaRSs disorders, broadens the spectrum of phenotypes attributable to VARS2 mutations, and provides new insight into genotype-phenotype correlations among the mitochondrial synthetase genes.


Assuntos
Epilepsia/genética , Antígenos HLA/genética , Hormônio do Crescimento Humano/deficiência , Hipogonadismo/genética , Deficiência Intelectual/genética , Valina-tRNA Ligase/genética , Estatura/genética , Mapeamento Cromossômico , Exoma , Feminino , Genes Recessivos , Transtornos do Crescimento/genética , Antígenos HLA/metabolismo , Hormônio do Crescimento Humano/genética , Humanos , Masculino , Linhagem , Gravidez , Síndrome , Valina-tRNA Ligase/metabolismo , Adulto Jovem
8.
Plant Physiol ; 170(4): 2110-23, 2016 04.
Artigo em Inglês | MEDLINE | ID: mdl-26839129

RESUMO

Chloroplasts and mitochondria contain their own genomes and transcriptional and translational systems. Establishing these genetic systems is essential for plant growth and development. Here we characterized a mutant form of a Val-tRNA synthetase (OsValRS2) from Oryza sativa that is targeted to both chloroplasts and mitochondria. A single base change in OsValRS2 caused virescent to albino phenotypes in seedlings and white panicles at heading. We therefore named this mutant white panicle 1 (wp1). Chlorophyll autofluorescence observations and transmission electron microscopy analyses indicated that wp1 mutants are defective in early chloroplast development. RNA-seq analysis revealed that expression of nuclear-encoded photosynthetic genes is significantly repressed, while expression of many chloroplast-encoded genes also changed significantly in wp1 mutants. Western-blot analyses of chloroplast-encoded proteins showed that chloroplast protein levels were reduced in wp1 mutants, although mRNA levels of some genes were higher in wp1 than in wild type. We found that wp1 was impaired in chloroplast ribosome biogenesis. Taken together, our results show that OsValRS2 plays an essential role in chloroplast development and regulating chloroplast ribosome biogenesis.


Assuntos
Cloroplastos/metabolismo , Biogênese de Organelas , Oryza/enzimologia , Proteínas de Plantas/metabolismo , Ribossomos/metabolismo , Valina-tRNA Ligase/metabolismo , Núcleo Celular/genética , Cloroplastos/ultraestrutura , Mapeamento Cromossômico , Clonagem Molecular , Fluorescência , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Mutação , Oryza/genética , Fenótipo , Fotossíntese , Proteínas de Plantas/genética , Biossíntese de Proteínas , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Plântula/metabolismo , Análise de Sequência de RNA , Frações Subcelulares/enzimologia , Valina-tRNA Ligase/genética
9.
Cell Rep ; 9(2): 476-83, 2014 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-25310979

RESUMO

Bacterial ribosomes stall on polyproline stretches and require the elongation factor P (EF-P) to relieve the arrest. Yet it remains unclear why evolution has favored the development of EF-P rather than selecting against the occurrence of polyproline stretches in proteins. We have discovered that only a single polyproline stretch is invariant across all domains of life, namely a proline triplet in ValS, the tRNA synthetase, that charges tRNA(Val) with valine. Here, we show that expression of ValS in vivo and in vitro requires EF-P and demonstrate that the proline triplet located in the active site of ValS is important for efficient charging of tRNA(Val) with valine and preventing formation of mischarged Thr-tRNA(Val) as well as efficient growth of E. coli in vivo. We suggest that the critical role of the proline triplet for ValS activity may explain why bacterial cells coevolved the EF-P rescue system.


Assuntos
Sequência Conservada , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Fatores de Alongamento de Peptídeos/genética , Peptídeos/genética , Valina-tRNA Ligase/genética , Sequência de Aminoácidos , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Evolução Molecular , Dados de Sequência Molecular , Mutação , Fatores de Alongamento de Peptídeos/química , Fatores de Alongamento de Peptídeos/metabolismo , Valina-tRNA Ligase/química , Valina-tRNA Ligase/metabolismo
10.
Hum Mutat ; 35(8): 983-9, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24827421

RESUMO

By way of whole-exome sequencing, we identified a homozygous missense mutation in VARS2 in one subject with microcephaly and epilepsy associated with isolated deficiency of the mitochondrial respiratory chain (MRC) complex I and compound heterozygous mutations in TARS2 in two siblings presenting with axial hypotonia and severe psychomotor delay associated with multiple MRC defects. The nucleotide variants segregated within the families, were absent in Single Nucleotide Polymorphism (SNP) databases and are predicted to be deleterious. The amount of VARS2 and TARS2 proteins and valyl-tRNA and threonyl-tRNA levels were decreased in samples of afflicted patients according to the genetic defect. Expression of the corresponding wild-type transcripts in immortalized mutant fibroblasts rescued the biochemical impairment of mitochondrial respiration and yeast modeling of the VARS2 mutation confirmed its pathogenic role. Taken together, these data demonstrate the role of the identified mutations for these mitochondriopathies. Our study reports the first mutations in the VARS2 and TARS2 genes, which encode two mitochondrial aminoacyl-tRNA synthetases, as causes of clinically distinct, early-onset mitochondrial encephalopathies.


Assuntos
Antígenos HLA/genética , Mitocôndrias/genética , Encefalomiopatias Mitocondriais/genética , Mutação , Treonina-tRNA Ligase/genética , Valina-tRNA Ligase/genética , Linhagem Celular , Criança , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Antígenos HLA/metabolismo , Heterozigoto , Homozigoto , Humanos , Lactente , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Encefalomiopatias Mitocondriais/enzimologia , Encefalomiopatias Mitocondriais/patologia , Polimorfismo Genético , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência de Treonina/genética , RNA de Transferência de Treonina/metabolismo , RNA de Transferência de Valina/genética , RNA de Transferência de Valina/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Treonina-tRNA Ligase/metabolismo , Valina-tRNA Ligase/metabolismo
11.
J Biol Chem ; 286(47): 41057-68, 2011 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-21965654

RESUMO

Four distinct aminoacyl-tRNA synthetases (aaRSs) found in some cyanobacterial species contain a novel protein domain that bears two putative transmembrane helices. This CAAD domain is present in glutamyl-, isoleucyl-, leucyl-, and valyl-tRNA synthetases, the latter of which has probably recruited the domain more than once during evolution. Deleting the CAAD domain from the valyl-tRNA synthetase of Anabaena sp. PCC 7120 did not significantly modify the catalytic properties of this enzyme, suggesting that it does not participate in its canonical tRNA-charging function. Multiple lines of evidence suggest that the function of the CAAD domain is structural, mediating the membrane anchorage of the enzyme, although membrane localization of aaRSs has not previously been described in any living organism. Synthetases containing the CAAD domain were localized in the intracytoplasmic thylakoid membranes of cyanobacteria and were largely absent from the plasma membrane. The CAAD domain was necessary and apparently sufficient for protein targeting to membranes. Moreover, localization of aaRSs in thylakoids was important under nitrogen limiting conditions. In Anabaena, a multicellular filamentous cyanobacterium often used as a model for prokaryotic cell differentiation, valyl-tRNA synthetase underwent subcellular relocation at the cell poles during heterocyst differentiation, a process also dependent on the CAAD domain.


Assuntos
Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/metabolismo , Membrana Celular/enzimologia , Sequência de Aminoácidos , Aminoacil-tRNA Sintetases/genética , Anabaena/citologia , Anabaena/enzimologia , Membrana Celular/metabolismo , Evolução Molecular , Espaço Intracelular/metabolismo , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Transporte Proteico , Valina-tRNA Ligase/metabolismo
12.
Mol Biol Evol ; 27(6): 1415-24, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20106903

RESUMO

Previous studies showed that VAS1 of Saccharomyces cerevisiae encodes both cytosolic and mitochondrial forms of valyl-tRNA synthetase (ValRS) through alternative initiation of translation. We show herein that except for Schizosaccharomyces pombe, all yeast species studied contained a single ValRS gene encoding both forms, and all of the mature protein forms deduced from those genes possessed an N-terminal appended domain (Ad) that was absent from their bacterial relatives. In contrast, S. pombe contained two distinct nuclear ValRS genes, one encoding the mitochondrial form and the other its cytosolic counterpart. Although the cytosolic form closely resembles other yeast ValRS sequences (approximately 60% identity), the mitochondrial form exhibits significant divergence from others (approximately 35% identity). Both genes are active and essential for the survival of the yeast. Most conspicuously, the mitochondrial form lacks the characteristic Ad. A phylogenetic analysis further suggested that both forms of S. pombe ValRS are of mitochondrial origin, and the mitochondrial form is ancestral to the cytoplasmic form.


Assuntos
Genes Essenciais , Genes Fúngicos , Mitocôndrias/genética , Schizosaccharomyces/enzimologia , Valina-tRNA Ligase/genética , Sequência de Aminoácidos , Aminoacilação , Citoplasma/metabolismo , Técnicas de Inativação de Genes , Teste de Complementação Genética , Histocitoquímica , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Alinhamento de Sequência , Valina-tRNA Ligase/metabolismo
13.
J Biol Chem ; 284(36): 23954-60, 2009 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-19574213

RESUMO

Previous studies showed that cytoplasmic and mitochondrial forms of yeast valyl-tRNA synthetase (ValRS) are specified by the VAS1 gene through alternative initiation of translation. Sequence comparison suggests that the yeast cytoplasmic (or mature mitochondrial) ValRS contains an N-terminal appendage that acts in cis as a nonspecific tRNA-binding domain (TRBD) and is absent from its bacterial relatives. We show here that Escherichia coli ValRS can substitute for the mitochondrial and cytoplasmic functions of VAS1 by fusion of a mitochondrial targeting signal and a TRBD, respectively. In addition, the bacterial ValRS gene can be converted into a dual functional yeast gene encoding both cytoplasmic and mitochondrial activities by fusion of a DNA sequence specifying both the mitochondrial targeting signal and TRBD. In vitro assays suggested that fusion of a nonspecific TRBD to the bacterial enzyme significantly enhanced its yeast tRNA-binding and aminoacylation activities. These results not only underscore the necessity of retaining a TRBD for functioning of a tRNA synthetase in yeast cytoplasm, but also provide insights into the evolution of tRNA synthetase genes.


Assuntos
Citoplasma/enzimologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Evolução Molecular , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Valina-tRNA Ligase/metabolismo , Citoplasma/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Iniciação Traducional da Cadeia Peptídica/fisiologia , Sinais Direcionadores de Proteínas/fisiologia , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Valina-tRNA Ligase/genética
14.
Biophys Chem ; 143(1-2): 34-43, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19398261

RESUMO

The main role of aminoacyl-tRNA synthetases (aaRSs) is to transfer the cognate amino acids to the 3'-end of their tRNA by strictly discriminating from non-cognate amino acids. Some aaRSs accomplish this via proofreading and editing mechanisms, among which valyl-tRNA synthetase (ValRS) hydrolyses the non-cognate amino acid, threonine. In ValRS, existence of pre-transfer editing process is still unclear, although crystal structure of editing site with pre-transfer substrate analog (Thr-AMS) was released. In the case of isoleucyl-tRNA synthetase (IleRS), editing mechanism is well studied and mutational analyses revealed the existence of post- and pre-transfer editing mechanisms. Our aim is to investigate the possibility of pre-transfer editing process by performing molecular dynamics (MD) simulation studies. Simulations were carried out for ValRS with pre-transfer substrates (Thr-AMP/Val-AMP) and post-transfer substrates (Thr-A76/Val-A76) to understand their binding pattern. Two important point mutation studies were performed to observe their effect on editing process. This study also intends to compare and contrast the pre-transfer editing with post-transfer editing of ValRS. Interestingly, the MD simulation results revealed that non-cognate substrates (Thr-AMP/Thr-A76) bind more strongly than the cognate substrates (Val-AMP/Val-A76) in both pre- and post-transfer editing respectively. The editing site mutations (Lys270Ala and Asp279Ala) severely affected the binding ability of pre-transfer substrate (Thr-AMP) by different ways. Even though pre- and post-transfer substrates bind to the same site, specific differences were observed which has led us to believe the existence of the pre-transfer editing process in ValRS.


Assuntos
Monofosfato de Adenosina/metabolismo , Thermus thermophilus/enzimologia , Treonina/metabolismo , Valina-tRNA Ligase/química , Valina-tRNA Ligase/metabolismo , Valina/metabolismo , Monofosfato de Adenosina/química , Ácido Aspártico/genética , Simulação por Computador , Lisina/genética , Modelos Moleculares , Mutação Puntual , Ligação Proteica , Conformação Proteica , RNA de Transferência/química , RNA de Transferência/metabolismo , Especificidade por Substrato , Treonina/química , Valina/química , Valina-tRNA Ligase/genética
15.
J Biol Chem ; 283(45): 30699-706, 2008 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-18755686

RESUMO

Previous studies showed that valyl-tRNA synthetase of Saccharomyces cerevisiae contains an N-terminal polypeptide extension of 97 residues, which is absent from its bacterial relatives, but is conserved in its mammalian homologues. We showed herein that this appended domain and its human counterpart are both nonspecific tRNA-binding domains (K(d) approximately 0.5 microm). Deletion of the appended domain from the yeast enzyme severely impaired its tRNA binding, aminoacylation, and complementation activities. This N-domain-deleted yeast valyl-tRNA synthetase mutant could be rescued by fusion of the equivalent domain from its human homologue. Moreover, fusion of the N-domain of the yeast enzyme or its human counterpart to Escherichia coli glutaminyl-tRNA synthetase enabled the otherwise "inactive" prokaryotic enzyme to function as a yeast enzyme in vivo. Different from the native yeast enzyme, which showed different affinities toward mixed tRNA populations, the fusion enzyme exhibited similar binding affinities for all yeast tRNAs. These results not only underscore the significance of nonspecific tRNA binding in aminoacylation, but also provide insights into the mechanism of the formation of aminoacyl-tRNAs.


Assuntos
RNA de Transferência/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Aminoacilação de RNA de Transferência/fisiologia , Valina-tRNA Ligase/metabolismo , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia , RNA de Transferência/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Valina-tRNA Ligase/genética
16.
Nucleic Acids Res ; 36(9): 3065-74, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18400783

RESUMO

Phenotypic diversity associated with pathogenic mutations of the human mitochondrial genome (mtDNA) has often been explained by unequal segregation of the mutated and wild-type genomes (heteroplasmy). However, this simple hypothesis cannot explain the tissue specificity of disorders caused by homoplasmic mtDNA mutations. We have previously associated a homoplasmic point mutation (1624C>T) in MTTV with a profound metabolic disorder that resulted in the neonatal deaths of numerous siblings. Affected tissues harboured a marked biochemical defect in components of the mitochondrial respiratory chain, presumably due to the extremely low (<1%) steady-state levels of mt-tRNA(Val). In primary myoblasts and transmitochondrial cybrids established from the proband (index case) and offspring, the marked respiratory deficiency was lost and steady-state levels of the mutated mt-tRNA(Val) were greater than in the biopsy material, but were still an order of magnitude lower than in control myoblasts. We present evidence that the generalized decrease in steady-state mt-tRNA(Val) observed in the homoplasmic 1624C>T-cell lines is caused by a rapid degradation of the deacylated form of the abnormal mt-tRNA(Val). By both establishing the identity of the human mitochondrial valyl-tRNA synthetase then inducing its overexpression in transmitochondrial cell lines, we have been able to partially restore steady-state levels of the mutated mt-tRNA(Val), consistent with an increased stability of the charged mt-tRNA. These data indicate that variations in the levels of VARS2L between tissue types and patients could underlie the difference in clinical presentation between individuals homoplasmic for the 1624C>T mutation.


Assuntos
Antígenos HLA/metabolismo , Miopatias Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mutação Puntual , RNA de Transferência de Valina/genética , RNA/genética , Valina-tRNA Ligase/metabolismo , Sequência de Bases , Linhagem Celular , Células Cultivadas , Humanos , Mitocôndrias/enzimologia , Dados de Sequência Molecular , RNA/química , RNA/metabolismo , Estabilidade de RNA , RNA Mitocondrial , RNA de Transferência de Valina/química , RNA de Transferência de Valina/metabolismo
17.
Chem Biol ; 13(10): 1091-100, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17052613

RESUMO

Aminoacyl-tRNA synthetases prevent mistranslation, or genetic code ambiguity, through specialized editing reactions. Mutations that disrupt editing in bacteria adversely affect cell growth and viability, and recent work in the mouse supports the idea that translational errors caused by an editing defect lead to a neurological disease-like phenotype. To further investigate the connection of mistranslation to cell pathology, we introduced an inducible transgene expressing an editing-deficient valyl-tRNA synthetase into mammalian cells. Introducing mistranslation precipitated a disruption of cell morphology and membrane blebbing, accompanied by activation of caspase-3, consistent with an apoptotic response. Addition of a noncanonical amino acid that is misactivated, but not cleared, by the editing-defective enzyme exacerbated these effects. A special ambiguity-detecting sensor provided direct readout of mistranslation in vivo, supporting the possibility that decreased translational fidelity could be associated with disease.


Assuntos
Muridae/genética , Edição de RNA , Transcrição Gênica , Valina-tRNA Ligase/genética , Animais , Apoptose/genética , Técnicas Biossensoriais , Caspase 3/metabolismo , Células Cultivadas , Camundongos , Modelos Moleculares , Células NIH 3T3 , Conformação Proteica , Transgenes , Valina-tRNA Ligase/metabolismo
18.
J Mol Biol ; 359(4): 901-12, 2006 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-16697013

RESUMO

In isoleucyl-tRNA synthetase (IleRS), the "editing" domain contributes to accurate aminoacylation by hydrolyzing the mis-synthesized intermediate, valyl-adenylate, in the "pre-transfer" editing mode and the incorrect final product, valyl-tRNA(Ile), in the "post-transfer" editing mode. In the present study, we determined the crystal structures of the Thermus thermophilus IleRS editing domain complexed with the substrate analogues in the pre and post-transfer modes, both at 1.7 A resolution. The active site accommodates the two analogues differently, with the valine side-chain rotated by about 120 degrees and the adenosine moiety oriented upside down. The substrate-binding pocket adjusts to the adenosine-monophosphate and adenosine moieties in the pre and post-transfer modes, respectively, by flipping the Trp227 side-chain by about 180 degrees . The substrate recognition mechanisms of IleRS are characterized by the active-site rearrangement between the two editing modes, and therefore differ from those of the homologous valyl and leucyl-tRNA synthetases from T.thermophilus, in which the post-transfer mode is predominant. Both modes of editing activities were reduced by replacements of Trp227 with Ala, Val, Leu, and His, but not by those with Phe and Tyr, indicating that the aromatic ring of Trp227 is important for the substrate recognition. In both editing modes, Thr233 and His319 recognize the substrate valine side-chain, regardless of the valine side-chain rotation, and reject the isoleucine side-chain. The T233A and H319A mutants have detectable editing activities against the cognate isoleucine.


Assuntos
Isoleucina-tRNA Ligase/química , Isoleucina-tRNA Ligase/metabolismo , Thermus thermophilus/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Histidina/metabolismo , Isoleucina/genética , Isoleucina/metabolismo , Isoleucina-tRNA Ligase/genética , Leucina-tRNA Ligase/química , Leucina-tRNA Ligase/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Estrutura Terciária de Proteína , Edição de RNA , Homologia de Sequência de Aminoácidos , Homologia Estrutural de Proteína , Triptofano/genética , Valina/metabolismo , Valina-tRNA Ligase/química , Valina-tRNA Ligase/metabolismo
19.
RNA ; 10(3): 493-503, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-14970394

RESUMO

To correct misactivation and misacylation errors, Escherichia coli valyl-tRNA synthetase (ValRS) catalyzes a tRNA(Val)-dependent editing reaction at a site distinct from its aminoacylation site. Here we examined the effects of replacing the conserved 3'-adenosine of tRNA(Val) with nucleoside analogs, to identify structural elements of the 3'-terminal nucleoside necessary for tRNA function at the aminoacylation and editing sites of ValRS. The results show that the exocyclic amino group (N6) is not essential: purine riboside-substituted tRNA(Val) is active in aminoacylation and in stimulating editing. Presence of an O6 substituent (guanosine, inosine, xanthosine) interferes with aminoacylation as well as posttransfer and total editing (pre- plus posttransfer editing). Because ValRS does not recognize substituents at the 6-position, these results suggest that an unprotonated N1, capable of acting as an H-bond acceptor, is an essential determinant for both the aminoacylation and editing reactions. Substituents at the 2-position of the purine ring, either a 2-amino group (2-aminopurine, 2,6-diaminopurine, guanosine, and 7-deazaguanosine) or a 2-keto group (xanthosine, isoguanosine), strongly inhibit both aminoacylation and editing. Although aminoacylation by ValRS is at the 2'-OH, substitution of the 3'-terminal adenosine of tRNA(Val) with 3'-deoxyadenosine reduces the efficiency of valine acceptance and of posttransfer editing, demonstrating that the 3'-terminal hydroxyl group contributes to tRNA recognition at both the aminoacylation and editing sites. Our results show a strong correlation between the amino acid accepting activity of tRNA and its ability to stimulate editing, suggesting misacylated tRNA is a transient intermediate in the editing reaction, and editing by ValRS requires a posttransfer step.


Assuntos
Escherichia coli/enzimologia , RNA de Transferência de Lisina/metabolismo , Valina-tRNA Ligase/metabolismo , Adenosina/análogos & derivados , Escherichia coli/metabolismo , Ligação de Hidrogênio , Edição de RNA/fisiologia
20.
RNA ; 9(1): 100-11, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12554880

RESUMO

The molecular interactions between valyl-tRNA synthetase (ValRS) and tRNA(Val), with the C34-A35-C36 anticodon, from Thermus thermophilus were studied by crystallographic analysis and structure-based mutagenesis. In the ValRS-bound structure of tRNA(Val), the successive A35-C36 residues (the major identity elements) of tRNA(Val) are base-stacked upon each other, and fit into a pocket on the alpha-helix bundle domain of ValRS. Hydrogen bonds are formed between ValRS and A35-C36 of tRNA(Val) in a base-specific manner. The C-terminal coiled-coil domain of ValRS interacts electrostatically with A20 and hydrophobically with the G19*C56 tertiary base pair. The loss of these interactions by the deletion of the coiled-coil domain of ValRS increased the K(M) value for tRNA(Val) 28-fold and decreased the k(cat) value 19-fold in the aminoacylation. The tRNA(Val) K(M) and k(cat) values were increased 21-fold and decreased 32-fold, respectively, by the disruption of the G18*U55 and G19*C56 tertiary base pairs, which associate the D- and T-loops for the formation of the L-shaped tRNA structure. Therefore, the coiled-coil domain of ValRS is likely to stabilize the L-shaped tRNA structure during the aminoacylation reaction.


Assuntos
RNA de Transferência de Valina/metabolismo , Valina-tRNA Ligase/metabolismo , Anticódon , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Conformação Proteica , Thermus thermophilus/enzimologia , Valina-tRNA Ligase/química
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