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1.
Nat Commun ; 12(1): 1616, 2021 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-33712620

RESUMO

The polyketide natural product reveromycin A (RM-A) exhibits antifungal, anticancer, anti-bone metastasis, anti-periodontitis and anti-osteoporosis activities by selectively inhibiting eukaryotic cytoplasmic isoleucyl-tRNA synthetase (IleRS). Herein, a co-crystal structure suggests that the RM-A molecule occupies the substrate tRNAIle binding site of Saccharomyces cerevisiae IleRS (ScIleRS), by partially mimicking the binding of tRNAIle. RM-A binding is facilitated by the copurified intermediate product isoleucyl-adenylate (Ile-AMP). The binding assays confirm that RM-A competes with tRNAIle while binding synergistically with L-isoleucine or intermediate analogue Ile-AMS to the aminoacylation pocket of ScIleRS. This study highlights that the vast tRNA binding site of the Rossmann-fold catalytic domain of class I aminoacyl-tRNA synthetases could be targeted by a small molecule. This finding will inform future rational drug design.


Assuntos
Sítios de Ligação/efeitos dos fármacos , Ligases/química , Ligases/efeitos dos fármacos , Piranos/antagonistas & inibidores , RNA de Transferência/efeitos dos fármacos , Compostos de Espiro/antagonistas & inibidores , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/efeitos dos fármacos , Isoleucina , Isoleucina-tRNA Ligase/química , Isoleucina-tRNA Ligase/efeitos dos fármacos , Ligantes , Modelos Moleculares , Osteoporose/tratamento farmacológico , RNA de Transferência/química , Saccharomyces cerevisiae
2.
Int J Mol Sci ; 22(4)2021 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-33578647

RESUMO

Aminoacyl-tRNA synthetases (aaRSs) catalyze the esterification of tRNA with a cognate amino acid and are essential enzymes in all three kingdoms of life. Due to their important role in the translation of the genetic code, aaRSs have been recognized as suitable targets for the development of small molecule anti-infectives. In this review, following a concise discussion of aaRS catalytic and proof-reading activities, the various inhibitory mechanisms of reported natural and synthetic aaRS inhibitors are discussed. Using the expanding repository of ligand-bound X-ray crystal structures, we classified these compounds based on their binding sites, focusing on their ability to compete with the association of one, or more of the canonical aaRS substrates. In parallel, we examined the determinants of species-selectivity and discuss potential resistance mechanisms of some of the inhibitor classes. Combined, this structural perspective highlights the opportunities for further exploration of the aaRS enzyme family as antimicrobial targets.


Assuntos
Aminoacil-tRNA Sintetases/antagonistas & inibidores , Anti-Infecciosos/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/metabolismo , Animais , Anti-Infecciosos/química , Sítios de Ligação/efeitos dos fármacos , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Terapia de Alvo Molecular
3.
Biochim Biophys Acta Mol Cell Res ; 1868(1): 118889, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33091505

RESUMO

Aminoacyl-tRNA synthetases (ARSs) are a family of evolutionarily conserved housekeeping enzymes used for protein synthesis that have pivotal roles in the ligation of tRNA with their cognate amino acids. Recent advances in the structural and functional studies of ARSs have revealed many previously unknown biological functions beyond the classical catalytic roles. Sensing the sufficiency of intracellular nutrients such as amino acids, ATP, and fatty acids is a crucial aspect for every living organism, and it is closely connected to the regulation of diverse cellular physiologies. Notably, among ARSs, leucyl-tRNA synthetase 1 (LARS1) has been identified to perform specifically as a leucine sensor upstream of the amino acid-sensing pathway and thus participates in the coordinated control of protein synthesis and autophagy for cell growth. In addition to LARS1, other types of ARSs are also likely involved in the sensing and signaling of their cognate amino acids inside cells. Collectively, this review focuses on the mechanisms of ARSs interacting within amino acid signaling and proposes the possible role of ARSs as general intracellular amino acid sensors.


Assuntos
Aminoácidos/genética , Aminoacil-tRNA Sintetases/genética , Leucina-tRNA Ligase/genética , Leucina/genética , Aminoácidos/química , Aminoacil-tRNA Sintetases/química , Humanos , Leucina/química , Leucina-tRNA Ligase/química , Biossíntese de Proteínas/genética , RNA de Transferência/genética , Transdução de Sinais/genética
4.
Nat Commun ; 11(1): 6340, 2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33311509

RESUMO

Self-regeneration is a fundamental function of all living systems. Here we demonstrate partial molecular self-regeneration in a synthetic cell. By implementing a minimal transcription-translation system within microfluidic reactors, the system is able to regenerate essential protein components from DNA templates and sustain synthesis activity for over a day. By quantitating genotype-phenotype relationships combined with computational modeling we find that minimizing resource competition and optimizing resource allocation are both critically important for achieving robust system function. With this understanding, we achieve simultaneous regeneration of multiple proteins by determining the required DNA ratios necessary for sustained self-regeneration. This work introduces a conceptual and experimental framework for the development of a self-replicating synthetic cell.


Assuntos
Células Artificiais/metabolismo , Regeneração/fisiologia , Aminoacil-tRNA Sintetases/metabolismo , Biologia Computacional , DNA , Replicação do DNA , Microfluídica/métodos , Biossíntese de Proteínas , Biologia Sintética/métodos
5.
Sheng Li Xue Bao ; 72(4): 523-531, 2020 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-32820315

RESUMO

The imbalance of protein metabolism is the major cause of skeletal muscle atrophy, and the decrease of protein synthesis directly leads to the occurrence and development of age-related sarcopenia. The canonical role of leucyl-tRNA synthetase (LeuRS) is ligating leucine to the cognate tRNA, and thus it plays a central role in genetic coding. With the further studies of LeuRS in recent years, LeuRS has been found to control protein homeostasis in aging skeletal muscle via its non-canonical role. In this paper, we reviewed the structure and biological features of aminoacyl-tRNA synthetase and LeuRS, and summarized the recent advances in studies on the effects of LeuRS in regulating aging skeletal muscle protein synthesis as an intracellular leucine sensor. Moreover, we also analyzed the potential role of LeuRS in activation of mammalian target of rapamycin complex 1 (mTORC1) signaling transduction pathway in response to anabolic stimuli such as exercise and amino acids ingestion. This paper may provide some new ideas for the prevention, diagnosis and treatment of age-related sarcopenia.


Assuntos
Aminoacil-tRNA Sintetases/genética , Leucina-tRNA Ligase/genética , Músculo Esquelético , Biossíntese de Proteínas
6.
Am J Hum Genet ; 107(2): 311-324, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32738225

RESUMO

Aminoacyl-tRNA synthetases (ARSs) are ubiquitous, ancient enzymes that charge amino acids to cognate tRNA molecules, the essential first step of protein translation. Here, we describe 32 individuals from 21 families, presenting with microcephaly, neurodevelopmental delay, seizures, peripheral neuropathy, and ataxia, with de novo heterozygous and bi-allelic mutations in asparaginyl-tRNA synthetase (NARS1). We demonstrate a reduction in NARS1 mRNA expression as well as in NARS1 enzyme levels and activity in both individual fibroblasts and induced neural progenitor cells (iNPCs). Molecular modeling of the recessive c.1633C>T (p.Arg545Cys) variant shows weaker spatial positioning and tRNA selectivity. We conclude that de novo and bi-allelic mutations in NARS1 are a significant cause of neurodevelopmental disease, where the mechanism for de novo variants could be toxic gain-of-function and for recessive variants, partial loss-of-function.


Assuntos
Aspartato-tRNA Ligase/genética , Mutação com Ganho de Função/genética , Mutação com Perda de Função/genética , Transtornos do Neurodesenvolvimento/genética , Aminoacil-RNA de Transferência/genética , Alelos , Aminoacil-tRNA Sintetases/genética , Linhagem Celular , Feminino , Predisposição Genética para Doença/genética , Humanos , Masculino , Linhagem , RNA de Transferência/genética , Células-Tronco/fisiologia
7.
Nucleic Acids Res ; 48(15): 8740-8754, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32644155

RESUMO

In mammalian cells, eight cytoplasmic aminoacyl-tRNA synthetases (AARS), and three non-synthetase proteins, reside in a large multi-tRNA synthetase complex (MSC). AARSs have critical roles in interpretation of the genetic code during protein synthesis, and in non-canonical functions unrelated to translation. Nonetheless, the structure and function of the MSC remain unclear. Partial or complete crystal structures of all MSC constituents have been reported; however, the structure of the holo-MSC has not been resolved. We have taken advantage of cross-linking mass spectrometry (XL-MS) and molecular docking to interrogate the three-dimensional architecture of the MSC in human HEK293T cells. The XL-MS approach uniquely provides structural information on flexibly appended domains, characteristic of nearly all MSC constituents. Using the MS-cleavable cross-linker, disuccinimidyl sulfoxide, inter-protein cross-links spanning all MSC constituents were observed, including cross-links between eight protein pairs not previously known to interact. Intra-protein cross-links defined new structural relationships between domains in several constituents. Unexpectedly, an asymmetric AARS distribution was observed featuring a clustering of tRNA anti-codon binding domains on one MSC face. Possibly, the non-uniform localization improves efficiency of delivery of charged tRNA's to an interacting ribosome during translation. In summary, we show a highly compact, 3D structural model of the human holo-MSC.


Assuntos
Aminoacil-tRNA Sintetases/ultraestrutura , Complexos Multiproteicos/ultraestrutura , Conformação de Ácido Nucleico , Conformação Proteica , Aminoacil-tRNA Sintetases/genética , Células HEK293 , Humanos , Espectrometria de Massas , Conformação Molecular , Simulação de Acoplamento Molecular , Complexos Multiproteicos/genética , Ligação Proteica
8.
Zhongguo Dang Dai Er Ke Za Zhi ; 22(6): 595-601, 2020 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-32571458

RESUMO

OBJECTIVE: To study the clinical features of the diseases associated with aminoacyl-tRNA synthetases (ARS) deficiency. METHODS: A retrospective analysis was performed of the clinical and gene mutation data of 10 children who were diagnosed with ARS gene mutations, based on next-generation sequencing from January 2016 to October 2019. RESULTS: The age of onset ranged from 0 to 9 years among the 10 children. Convulsion was the most common initial symptom (7 children). Clinical manifestations included ataxia and normal or mildly retarded intellectual development (with or without epilepsy; n=4) and onset of epilepsy in childhood with developmental regression later (n=2). Some children experienced disease onset in the neonatal period and had severe epileptic encephalopathy, with myoclonus, generalized tonic-clonic seizure, and convulsive seizure (n=4); 3 had severe delayed development, 2 had feeding difficulty, and 1 had hearing impairment. Mutations were found in five genes: 3 had novel mutations in the AARS2 gene (c.331G>C, c.2682+5G>A, c.2164C>T, and c.761G>A), 2 had known mutations in the DARS2 gene (c.228-16C>A and c.536G>A), 1 had novel mutations in the CARS2 gene (c.1036C>T and c.323T>G), 1 had novel mutations in the RARS2 gene (c.1210A>G and c.622C>T), and 3 had novel mutations in the AARS gene (c.1901T>A, c.229C>T, c.244C>T, c.961G>C, c.2248C>T, and Chr16:70298860-70316687del). CONCLUSIONS: A high heterogeneity is observed in the clinical phenotypes of the diseases associated with the ARS deficiency. A total of 14 novel mutations in 5 genes are reported in this study, which enriches the clinical phenotypes and genotypes of the diseases associated with ARS deficiency.


Assuntos
Aminoacil-tRNA Sintetases/genética , Criança , Epilepsia , Humanos , Mutação , Fenótipo , Estudos Retrospectivos
9.
Sheng Wu Gong Cheng Xue Bao ; 36(5): 891-898, 2020 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-32567272

RESUMO

Unnatural amino acid orthogonal translation machinery can insert unnatural amino acids at desired sites of protein through stop codon by means of foreign orthogonal translation system composed of aminoacyl-tRNA synthetase and orthogonal tRNA genes. This new genetic engineering technology is not only a new tool for biochemical researches of proteins, but also an epoch-making technology for the development of new-type live viral vaccines. The mutated virus containing premature termination codon in genes necessary for replication can be propagated in transgenic cells harboring unnatural amino acid orthogonal translation machinery in media with corresponding unnatural amino acid, but it cannot replicate in conventional host cells. This replication-deficient virus is a new-type of live viral vaccine that possesses advantages of high efficacy of traditional attenuated vaccine and high safety of killed vaccine. This article reviews the application and prospect of unnatural amino acid orthogonal translation machinery in the development of novel replication-deficient virus vaccines.


Assuntos
Aminoácidos , Aminoacil-tRNA Sintetases , Engenharia Genética , Vacinas Virais , Aminoácidos/genética , Engenharia de Proteínas , RNA de Transferência
10.
Am J Physiol Lung Cell Mol Physiol ; 319(2): L369-L379, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32579851

RESUMO

Proper development of the respiratory bronchiole and alveolar epithelium proceeds through coordinated cross talk between the interface of epithelium and neighboring mesenchyme. Signals that facilitate and coordinate the cross talk as the bronchial forming canalicular stage transitions to construction of air-exchanging capillary-alveoli niche in the alveolar stage are poorly understood. Expressed within this decisive region, levels of aminoacyl-tRNA synthetase complex-interacting multifunctional protein 1 (AIMP1) inversely correlate with the maturation of the lung. The present study addresses the role of AIMP1 in lung development through the generation and characterization of Aimp1-/- mutant mice. Mating of Aimp1+/- produced offspring in expected Mendelian ratios throughout embryonic development. However, newborn Aimp1-/- pups exhibited neonatal lethality with mild cyanosis. Imaging both structure and ultrastructure of Aimp1-/- lungs showed disorganized bronchial epithelium, decreased type I but not type II cell differentiation, increased distal vessels, and disruption of E-cadherin deposition in cell-cell junctions. Supporting the in vivo findings of disrupted epithelial cell-cell junctions, in vitro biochemical experiments show that a portion of AIMP1 binds to phosphoinositides, the lipid anchor of proteins that have a fundamental role in both cellular membrane and actin cytoskeleton organization; a dramatic disruption in F-actin cytoskeleton was observed in Aimp1-/- mouse embryonic fibroblasts. Such observed structural defects may lead to disrupted cell-cell boundaries. Together, these results suggest a requirement of AIMP1 in epithelial cell differentiation in proper lung development.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Diferenciação Celular/fisiologia , Citocinas/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Pulmão/metabolismo , Pulmão/fisiologia , Actinas/metabolismo , Animais , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Feminino , Junções Intercelulares/metabolismo , Junções Intercelulares/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL
11.
PLoS One ; 15(6): e0234523, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32525903

RESUMO

OBJECTIVE: Surfactant protein D (SP-D) is considered a serum biomarker of various forms of interstitial lung disease (ILD). In this study, we examined the utility of SP-D as a predictive biomarker for mortality in patients with ILD associated with polymyositis/dermatomyositis (PM/DM) using large-scale multicentre cohort data. METHODS: We enrolled 381 patients with incident PM/DM-associated ILD in a multicentre retrospective cohort based on the availability of serum SP-D at the baseline. Demographic and clinical characteristics as well as the presence of autoantibodies to melanoma differentiation-associated gene 5 (MDA5) and aminoacyl tRNA synthetase were measured at the time of diagnosis, and follow-up survival data were collected prospectively. RESULTS: Seventy-eight patients died during the median observation period of 18 months, and the majority of patients died of ILD. The SP-D levels at baseline were significantly lower (P = 0.02) in a non-survivor subset than in a survivor subset among the entire enrolled patients. However, the SP-D levels were higher in the non-survivor subset than in the survivor subset based on the stratification by anti-MDA5-positive, anti-ARS-positive and, double-negativity, although there was an only statistically significant difference (P = 0.01) in the double-negative group. Surprisingly, the SP-D levels were within the upper limit of normal, 110 ng/mL, in 54 (87%) of 62 anti-MDA5-positive patients who died. In the double-negative group, the mortality rates were significantly higher (P = 0.002) in a subset with SP-D ≥127.6 ng/mL, the cut-off value for mortality calculated by the receiver operating characteristic curve, than the other subset. All of patients with SP-D <127.6 ng/mL survived. CONCLUSION: Serum SP-D levels behave differently among patients with stratified by anti-MDA5 antibody, anti-ARS antibody and both negativity in PM/DM-associated ILD. Its use in clinical practice should be applied with caution on the basis of the presence or absence of anti-MDA5 antibody or anti-ARS antibody.


Assuntos
Autoanticorpos/sangue , Dermatomiosite/complicações , Doenças Pulmonares Intersticiais/mortalidade , Proteína D Associada a Surfactante Pulmonar/sangue , Adulto , Idoso , Aminoacil-tRNA Sintetases/imunologia , Autoanticorpos/imunologia , Autoantígenos/imunologia , Biomarcadores/sangue , Dermatomiosite/sangue , Dermatomiosite/imunologia , Dermatomiosite/mortalidade , Estudos de Viabilidade , Feminino , Humanos , Helicase IFIH1 Induzida por Interferon/imunologia , Doenças Pulmonares Intersticiais/sangue , Doenças Pulmonares Intersticiais/etiologia , Doenças Pulmonares Intersticiais/imunologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Valores de Referência , Estudos Retrospectivos , Análise de Sobrevida
12.
Nucleic Acids Res ; 48(12): 6445-6457, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32484512

RESUMO

The accuracy in pairing tRNAs with correct amino acids by aminoacyl-tRNA synthetases (aaRSs) dictates the fidelity of translation. To ensure fidelity, multiple aaRSs developed editing functions that remove a wrong amino acid from tRNA before it reaches the ribosome. However, no specific mechanism within an aaRS is known to handle the scenario where a cognate amino acid is mischarged onto a wrong tRNA, as exemplified by AlaRS mischarging alanine to G4:U69-containing tRNAThr. Here, we report that the mischargeable G4:U69-containing tRNAThr are strictly conserved in vertebrates and are ubiquitously and abundantly expressed in mammalian cells and tissues. Although these tRNAs are efficiently mischarged, no corresponding Thr-to-Ala mistranslation is detectable. Mistranslation is prevented by a robust proofreading activity of ThrRS towards Ala-tRNAThr. Therefore, while wrong amino acids are corrected within an aaRS, a wrong tRNA is handled in trans by an aaRS cognate to the mischarged tRNA species. Interestingly, although Ala-tRNAThr mischarging is not known to occur in bacteria, Escherichia coli ThrRS also possesses robust cross-editing ability. We propose that the cross-editing activity of ThrRS is evolutionarily conserved and that this intrinsic activity allows G4:U69-containing tRNAThr to emerge and be preserved in vertebrates to have alternative functions without compromising translational fidelity.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Edição de RNA , RNA de Transferência/metabolismo , Alanina/genética , Animais , Evolução Molecular , Células HEK293 , Humanos , RNA de Transferência/genética , Treonina/genética , Vertebrados/genética
13.
Nucleic Acids Res ; 48(12): 6799-6810, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32484546

RESUMO

Structure and/or function of proteins are frequently affected by oxidative/nitrosative stress via posttranslational modifications. Aminoacyl-tRNA synthetases (aaRSs) constitute a class of ubiquitously expressed enzymes that control cellular protein homeostasis. Here, we found the activity of human mitochondrial (mt) threonyl-tRNA synthetase (hmtThrRS) is resistant to oxidative stress (H2O2) but profoundly sensitive to nitrosative stress (S-nitrosoglutathione, GSNO). Further study showed four Cys residues in hmtThrRS were modified by S-nitrosation upon GSNO treatment, and one residue was one of synthetic active sites. We analyzed the effect of modification at individual Cys residue on aminoacylation and editing activities of hmtThrRS in vitro and found that both activities were decreased. We further confirmed that S-nitrosation of mtThrRS could be readily detected in vivo in both human cells and various mouse tissues, and we systematically identified dozens of S-nitrosation-modified sites in most aaRSs, thus establishing both mitochondrial and cytoplasmic aaRS species with S-nitrosation ex vivo and in vivo, respectively. Interestingly, a decrease in the S-nitrosation modification level of mtThrRS was observed in a Huntington disease mouse model. Overall, our results establish, for the first time, a comprehensive S-nitrosation-modified aaRS network and a previously unknown mechanism on the basis of the inhibitory effect of S-nitrosation on hmtThrRS.


Assuntos
Mitocôndrias/genética , Nitrosação/genética , Estresse Nitrosativo/genética , Treonina-tRNA Ligase/genética , Aminoacil-tRNA Sintetases/genética , Aminoacilação/genética , Animais , Domínio Catalítico/efeitos dos fármacos , Células HeLa , Humanos , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/farmacologia , Cinética , Camundongos , Mitocôndrias/enzimologia , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/genética , Treonina-tRNA Ligase/química
14.
BMC Med Genet ; 21(1): 109, 2020 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-32423379

RESUMO

BACKGROUND: Perrault syndrome is a rare recessive and genetically heterogeneous disorder characterized by sensorineural hearing loss in males and females and gonadal dysgenesis in females. Mutations in seven different genes have been identified: HARS2, HSD17B4, CLLP, C10orf, ERAL1, TWNK and LARS2. To date, 19 variants have been reported in 18 individuals with LARS2-Perrault syndrome. CASE PRESENTATION: Here we describe the case of an 8-year-old girl with compound heterozygous missense mutations in the LARS2 gene. We identified two missense mutations [c.457A > C, p.(Asn153His) and c.1565C > A, p.(Thr522Asn)] and subsequent familial segregation showed that each parent had transmitted a mutation. CONCLUSIONS: These results have implications for genetic counseling and provide insight into the functional role of LARS2. This case highlights the importance of an early diagnosis. Systematic genetic screening of children with hearing loss allows the early identification of a Perrault syndrome in order to ensure specific endocrinological surveillance and management to prevent secondary complications. Clinical data are compared with the other cases reported in the literature.


Assuntos
Aminoacil-tRNA Sintetases/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Disgenesia Gonadal 46 XX/diagnóstico , Disgenesia Gonadal 46 XX/genética , Perda Auditiva Neurossensorial/diagnóstico , Perda Auditiva Neurossensorial/genética , Mutação , Alelos , Substituição de Aminoácidos , Biomarcadores , Criança , Gerenciamento Clínico , Feminino , Estudos de Associação Genética/métodos , Genótipo , Disgenesia Gonadal 46 XX/terapia , Perda Auditiva Neurossensorial/terapia , Humanos , Fenótipo
15.
Hum Genet ; 139(10): 1325-1343, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32399598

RESUMO

Perrault syndrome is a rare heterogeneous condition characterised by sensorineural hearing loss and premature ovarian insufficiency. Additional neuromuscular pathology is observed in some patients. There are six genes in which variants are known to cause Perrault syndrome; however, these explain only a minority of cases. We investigated the genetic cause of Perrault syndrome in seven affected individuals from five different families, successfully identifying the cause in four patients. This included previously reported and novel causative variants in known Perrault syndrome genes, CLPP and LARS2, involved in mitochondrial proteolysis and mitochondrial translation, respectively. For the first time, we show that pathogenic variants in PEX6 can present clinically as Perrault syndrome. PEX6 encodes a peroxisomal biogenesis factor, and we demonstrate evidence of peroxisomal dysfunction in patient serum. This study consolidates the clinical overlap between Perrault syndrome and peroxisomal disorders, and highlights the need to consider ovarian function in individuals with atypical/mild peroxisomal disorders. The remaining patients had variants in candidate genes such as TFAM, involved in mtDNA transcription, replication, and packaging, and GGPS1 involved in mevalonate/coenzyme Q10 biosynthesis and whose enzymatic product is required for mouse folliculogenesis. This genomic study highlights the diverse molecular landscape of this poorly understood syndrome.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Aminoacil-tRNA Sintetases/genética , Proteínas de Ligação a DNA/genética , Dimetilaliltranstransferase/genética , Endopeptidase Clp/genética , Farnesiltranstransferase/genética , Predisposição Genética para Doença , Geraniltranstransferase/genética , Disgenesia Gonadal 46 XX/genética , Perda Auditiva Neurossensorial/genética , Proteínas Mitocondriais/genética , Fatores de Transcrição/genética , Adolescente , Adulto , Sequência de Bases , Criança , DNA Mitocondrial/genética , Feminino , Expressão Gênica , Disgenesia Gonadal 46 XX/diagnóstico , Disgenesia Gonadal 46 XX/patologia , Perda Auditiva Neurossensorial/diagnóstico , Perda Auditiva Neurossensorial/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Ovário/metabolismo , Ovário/patologia , Linhagem , Peroxissomos/metabolismo , Peroxissomos/patologia
16.
RNA ; 26(8): 910-936, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32303649

RESUMO

The aminoacyl-tRNA synthetases are an essential and universally distributed family of enzymes that plays a critical role in protein synthesis, pairing tRNAs with their cognate amino acids for decoding mRNAs according to the genetic code. Synthetases help to ensure accurate translation of the genetic code by using both highly accurate cognate substrate recognition and stringent proofreading of noncognate products. While alterations in the quality control mechanisms of synthetases are generally detrimental to cellular viability, recent studies suggest that in some instances such changes facilitate adaption to stress conditions. Beyond their central role in translation, synthetases are also emerging as key players in an increasing number of other cellular processes, with far-reaching consequences in health and disease. The biochemical versatility of the synthetases has also proven pivotal in efforts to expand the genetic code, further emphasizing the wide-ranging roles of the aminoacyl-tRNA synthetase family in synthetic and natural biology.


Assuntos
Aminoacil-tRNA Sintetases/genética , RNA de Transferência/genética , Animais , Código Genético , Humanos , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , Aminoacilação de RNA de Transferência/genética
17.
Nat Biotechnol ; 38(8): 989-999, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32284585

RESUMO

A central challenge in expanding the genetic code of cells to incorporate noncanonical amino acids into proteins is the scalable discovery of aminoacyl-tRNA synthetase (aaRS)-tRNA pairs that are orthogonal in their aminoacylation specificity. Here we computationally identify candidate orthogonal tRNAs from millions of sequences and develop a rapid, scalable approach-named tRNA Extension (tREX)-to determine the in vivo aminoacylation status of tRNAs. Using tREX, we test 243 candidate tRNAs in Escherichia coli and identify 71 orthogonal tRNAs, covering 16 isoacceptor classes, and 23 functional orthogonal tRNA-cognate aaRS pairs. We discover five orthogonal pairs, including three highly active amber suppressors, and evolve new amino acid substrate specificities for two pairs. Finally, we use tREX to characterize a matrix of 64 orthogonal synthetase-orthogonal tRNA specificities. This work expands the number of orthogonal pairs available for genetic code expansion and provides a pipeline for the discovery of additional orthogonal pairs and a foundation for encoding the cellular synthesis of noncanonical biopolymers.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , RNA de Transferência/metabolismo , Sequência de Aminoácidos , Aminoacil-tRNA Sintetases/genética , Simulação por Computador , Escherichia coli , Regulação Bacteriana da Expressão Gênica , Proteínas de Fluorescência Verde , Ligação Proteica , Especificidade por Substrato
18.
Proc Natl Acad Sci U S A ; 117(16): 8900-8911, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32253314

RESUMO

Signaling pathways that sense amino acid abundance are integral to tissue homeostasis and cellular defense. Our laboratory has previously shown that halofuginone (HF) inhibits the prolyl-tRNA synthetase catalytic activity of glutamyl-prolyl-tRNA synthetase (EPRS), thereby activating the amino acid response (AAR). We now show that HF treatment selectively inhibits inflammatory responses in diverse cell types and that these therapeutic benefits occur in cells that lack GCN2, the signature effector of the AAR. Depletion of arginine, histidine, or lysine from cultured fibroblast-like synoviocytes recapitulates key aspects of HF treatment, without utilizing GCN2 or mammalian target of rapamycin complex 1 pathway signaling. Like HF, the threonyl-tRNA synthetase inhibitor borrelidin suppresses the induction of tissue remodeling and inflammatory mediators in cytokine-stimulated fibroblast-like synoviocytes without GCN2, but both aminoacyl-tRNA synthetase (aaRS) inhibitors are sensitive to the removal of GCN1. GCN1, an upstream component of the AAR pathway, binds to ribosomes and is required for GCN2 activation. These observations indicate that aaRS inhibitors, like HF, can modulate inflammatory response without the AAR/GCN2 signaling cassette, and that GCN1 has a role that is distinct from its activation of GCN2. We propose that GCN1 participates in a previously unrecognized amino acid sensor pathway that branches from the canonical AAR.


Assuntos
Aminoacil-tRNA Sintetases/antagonistas & inibidores , Anti-Inflamatórios/farmacologia , Artrite Reumatoide/tratamento farmacológico , Piperidinas/farmacologia , Quinazolinonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/metabolismo , Animais , Anti-Inflamatórios/uso terapêutico , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Artrite Reumatoide/cirurgia , Linhagem Celular , Fibroblastos , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana , Humanos , Pulmão/citologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Knockout , Piperidinas/uso terapêutico , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Quinazolinonas/uso terapêutico , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA-Seq , Transdução de Sinais/imunologia , Membrana Sinovial/citologia , Membrana Sinovial/patologia , Sinoviócitos , Transativadores/genética , Transativadores/metabolismo
19.
Sci China Life Sci ; 63(8): 1227-1239, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32189241

RESUMO

Aminoacyl-tRNA synthetases (aaRSs) are ubiquitously expressed, essential enzymes, synthesizing aminoacyl-tRNAs for protein synthesis. Functional defects of aaRSs frequently cause various human disorders. Human KARS encodes both cytosolic and mitochondrial lysyl-tRNA synthetases (LysRSs). Previously, two mutations (c.1129G>A and c.517T>C) were identified that led to hearing impairment; however, the underlying biochemical mechanism is unclear. In the present study, we found that the two mutations have no impact on the incorporation of LysRS into the multiple-synthetase complex in the cytosol, but affect the cytosolic LysRS level, its tertiary structure, and cytosolic tRNA aminoacylation in vitro. As for mitochondrial translation, the two mutations have little effect on the steady-state level, mitochondrial targeting, and tRNA binding affinity of mitochondrial LysRS. However, they exhibit striking differences in charging mitochondrial tRNALys, with the c.517T>C mutant being completely deficient in vitro and in vivo. We constructed two yeast genetic models, which are powerful tools to test the in vivo aminoacylation activity of KARS mutations at both the cytosolic and mitochondrial levels. Overall, our data provided biochemical insights into the potentially molecular pathological mechanism of KARS c.1129G>A and c.517T>C mutations and provided yeast genetic bases to investigate other KARS mutations in the future.


Assuntos
Aminoacilação/genética , Citoplasma/genética , Perda Auditiva/genética , Mitocôndrias/genética , RNA de Transferência de Lisina/metabolismo , Aminoacil-tRNA Sintetases/genética , Sequência de Bases , Domínio Catalítico , Regulação da Expressão Gênica , Perda Auditiva/metabolismo , Humanos , Modelos Moleculares , Mutação , Biossíntese de Proteínas , Conformação Proteica , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transfecção
20.
J Med Chem ; 63(8): 3908-3914, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32208684

RESUMO

Aminoacyl-tRNA synthetase interacting multifunctional proteins (AIMPs) have recently been considered novel therapeutic targets in several cancers. In this publication we report the development of novel 2-aminophenylpyrimidines as new AIMP2-DX2 inhibitors. In particular, aminophenylpyrimidine 3 not only exhibited promising in vitro and in vivo potency but also exerted selective inhibition of H460 and A549 cells and AIMP2-DX2 rather than WI-26 cells and AIMP2. Aminophenylpyrimidine 3 offers possible therapeutic potential in the treatment of lung cancer.


Assuntos
Aminoacil-tRNA Sintetases/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Células A549 , Aminoacil-tRNA Sintetases/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Nucleares/metabolismo , Pirimidinas/química , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
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