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1.
Nat Commun ; 12(1): 5429, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34521822

RESUMO

Bacillus subtilis is a model gram-positive bacterium, commonly used to explore questions across bacterial cell biology and for industrial uses. To enable greater understanding and control of proteins in B. subtilis, here we report broad and efficient genetic code expansion in B. subtilis by incorporating 20 distinct non-standard amino acids within proteins using 3 different families of genetic code expansion systems and two choices of codons. We use these systems to achieve click-labelling, photo-crosslinking, and translational titration. These tools allow us to demonstrate differences between E. coli and B. subtilis stop codon suppression, validate a predicted protein-protein binding interface, and begin to interrogate properties underlying bacterial cytokinesis by precisely modulating cell division dynamics in vivo. We expect that the establishment of this simple and easily accessible chemical biology system in B. subtilis will help uncover an abundance of biological insights and aid genetic code expansion in other organisms.


Assuntos
Aminoácidos/genética , Aminoacil-tRNA Sintetases/genética , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Código Genético , Aminoácidos/química , Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/classificação , Aminoacil-tRNA Sintetases/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Códon , Citocinese/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Genoma Bacteriano , Ligação Proteica , Biossíntese de Proteínas , Mapeamento de Interação de Proteínas , RNA de Transferência/genética , RNA de Transferência/metabolismo
2.
Nat Commun ; 12(1): 5706, 2021 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-34588441

RESUMO

Genetic code expansion technologies supplement the natural codon repertoire with assignable variants in vivo, but are often limited by heterologous translational components and low suppression efficiencies. Here, we explore engineered Escherichia coli tRNAs supporting quadruplet codon translation by first developing a library-cross-library selection to nominate quadruplet codon-anticodon pairs. We extend our findings using a phage-assisted continuous evolution strategy for quadruplet-decoding tRNA evolution (qtRNA-PACE) that improved quadruplet codon translation efficiencies up to 80-fold. Evolved qtRNAs appear to maintain codon-anticodon base pairing, are typically aminoacylated by their cognate tRNA synthetases, and enable processive translation of adjacent quadruplet codons. Using these components, we showcase the multiplexed decoding of up to four unique quadruplet codons by their corresponding qtRNAs in a single reporter. Cumulatively, our findings highlight how E. coli tRNAs can be engineered, evolved, and combined to decode quadruplet codons, portending future developments towards an exclusively quadruplet codon translation system.


Assuntos
Anticódon/metabolismo , Códon/metabolismo , Evolução Molecular Direcionada , Escherichia coli/genética , RNA de Transferência/genética , Aminoácidos/genética , Aminoacil-tRNA Sintetases/metabolismo , Clonagem Molecular , Escherichia coli/enzimologia , Proteínas de Escherichia coli/biossíntese , Biossíntese de Proteínas , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA de Transferência/metabolismo
3.
J Mol Evol ; 89(7): 484-493, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34254168

RESUMO

During the endosymbiotic evolution of mitochondria, the genes for aminoacyl-tRNA synthetases were transferred to the ancestral nucleus. A further reduction of mitochondrial function resulted in mitochondrion-related organisms (MRO) with a loss of the organelle genome. The fate of the now redundant ancestral mitochondrial aminoacyl-tRNA synthetase genes is uncertain. The derived protein sequence for arginyl-tRNA synthetase from thirty mitosomal organisms have been classified as originating from the ancestral nuclear or mitochondrial gene and compared to the identity element at position 20 of the cognate tRNA that distinguishes the two enzyme forms. The evolutionary choice between loss and retention of the ancestral mitochondrial gene for arginyl-tRNA synthetase reflects the coevolution of arginyl-tRNA synthetase and tRNA identity elements.


Assuntos
Aminoacil-tRNA Sintetases , Arginina-tRNA Ligase , Sequência de Aminoácidos , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Arginina-tRNA Ligase/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , RNA de Transferência
4.
J Phys Chem B ; 125(28): 7651-7661, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34242030

RESUMO

Aminoacyl-tRNA synthetases (aaRSs), a family of ubiquitous and essential enzymes, can bind target tRNAs and catalyze the aminoacylation reaction in genetic code translation. In this work, we explore the dynamic properties and allosteric communication of human mitochondrial phenylalanyl-tRNA synthetase (hmPheRS) in free and bound states to understand the mechanisms of its tRNAPhe recognition and allostery using molecular dynamics simulations combined with the torsional mutual information-based network model. Our results reveal that hmPheRS's residue mobility and inter-residue motional coupling are significantly enhanced by tRNAPhe binding, and there occurs a strong allosteric communication which is critical for the aminoacylation reaction, suggesting the vital role of tRNAPhe binding in the enzyme's function. The identified signaling pathways mainly make the connections between the anticodon binding domain (ABD) and catalytic domain (CAD), as well as within the CAD composed of many functional fragments and active sites, revealing the co-regulation role of them to act coordinately and achieve hmPheRS's aminoacylation function. Besides, several key residues along the communication pathways are identified to be involved in mediating the coordinated coupling between anticodon recognition at the ABD and activation process at the CAD, showing their pivotal role in the allosteric network, which are well consistent with the experimental observation. This study sheds light on the allosteric communication mechanism in hmPheRS and can provide important information for the structure-based drug design targeting aaRSs.


Assuntos
Aminoacil-tRNA Sintetases , Fenilalanina-tRNA Ligase , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Anticódon/genética , Domínio Catalítico , Humanos , Mitocôndrias/metabolismo , Fenilalanina-tRNA Ligase/metabolismo
5.
Chem Biol Drug Des ; 98(3): 421-434, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33993612

RESUMO

Drug-resistant Mycobacterium tuberculosis poses a great threat to public health and remains one of the red-flag tagged infectious diseases, with the tendency of comorbidity with other disease conditions such as HIV/AIDS. This perhaps is responsible for redoubling of effort in tuberculosis research and continuous change in patient management to optimize the drug therapy. Aminoacyl-tRNA synthetases are essential enzymes in M. tuberculosis that catalyse the transfer of a particular amino acid to its corresponding specific tRNA to form an aminoacyl-tRNA. These enzymes are believed to be novel antibacterial, antifungal and antiparasitic drug targets because of their role in the process of protein translation. Therefore, their existence as a compliment of M. tuberculosis has attracted a lot of research interest with the aim of curbing the scourge and provide the most effective drug in the treatment of tuberculosis. This leads to the discovery of a pool of aminoacyl-tRNA synthetases with their essential inhibitors. This review seeks to articulate the current advances in the development of new TB drugs exhibiting novelty in their mode of action with specific emphasis on aminoacyl-tRNA synthetases as drug targets.


Assuntos
Aminoacil-tRNA Sintetases/antagonistas & inibidores , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/química , Mycobacterium tuberculosis/enzimologia , Aminoacil-tRNA Sintetases/metabolismo , Antituberculosos/química , Antituberculosos/metabolismo , Antituberculosos/farmacologia , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Farmacorresistência Bacteriana/efeitos dos fármacos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis/efeitos dos fármacos
6.
Transcription ; 12(1): 28-53, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-34000965

RESUMO

Diverse models have been advanced for the evolution of the genetic code. Here, models for tRNA, aminoacyl-tRNA synthetase (aaRS) and genetic code evolution were combined with an understanding of EF-Tu suppression of tRNA 3rd anticodon position wobbling. The result is a highly detailed scheme that describes the placements of all amino acids in the standard genetic code. The model describes evolution of 6-, 4-, 3-, 2- and 1-codon sectors. Innovation in column 3 of the code is explained. Wobbling and code degeneracy are explained. Separate distribution of serine sectors between columns 2 and 4 of the code is described. We conclude that very little chaos contributed to evolution of the genetic code and that the pattern of evolution of aaRS enzymes describes a history of the evolution of the code. A model is proposed to describe the biological selection for the earliest evolution of the code and for protocell evolution.


Assuntos
Aminoacil-tRNA Sintetases/genética , RNA de Transferência/genética , Aminoacil-tRNA Sintetases/metabolismo , Evolução Molecular , Código Genético
7.
Int J Mol Sci ; 22(9)2021 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-33926074

RESUMO

In most eukaryotes, mitochondrial protein synthesis is essential for oxidative phosphorylation (OXPHOS) as some subunits of the respiratory chain complexes are encoded by the mitochondrial DNA (mtDNA). Mutations affecting the mitochondrial translation apparatus have been identified as a major cause of mitochondrial diseases. These mutations include either heteroplasmic mtDNA mutations in genes encoding for the mitochondrial rRNA (mtrRNA) and tRNAs (mttRNAs) or mutations in nuclear genes encoding ribosomal proteins, initiation, elongation and termination factors, tRNA-modifying enzymes, and aminoacyl-tRNA synthetases (mtARSs). Aminoacyl-tRNA synthetases (ARSs) catalyze the attachment of specific amino acids to their cognate tRNAs. Differently from most mttRNAs, which are encoded by mitochondrial genome, mtARSs are encoded by nuclear genes and then imported into the mitochondria after translation in the cytosol. Due to the extensive use of next-generation sequencing (NGS), an increasing number of mtARSs variants associated with large clinical heterogeneity have been identified in recent years. Being most of these variants private or sporadic, it is crucial to assess their causative role in the disease by functional analysis in model systems. This review will focus on the contributions of the yeast Saccharomyces cerevisiae in the functional validation of mutations found in mtARSs genes associated with human disorders.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Aminoacil-tRNA Sintetases/fisiologia , Mitocôndrias/metabolismo , Citosol/metabolismo , DNA Mitocondrial/genética , Humanos , Mitocôndrias/fisiologia , Doenças Mitocondriais/genética , Doenças Mitocondriais/fisiopatologia , Mutação , Fosforilação Oxidativa , Biossíntese de Proteínas , Processamento Pós-Transcricional do RNA , RNA de Transferência/genética , Saccharomyces cerevisiae/metabolismo
8.
Bioorg Chem ; 110: 104806, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33799176

RESUMO

Aminoacyl-tRNA synthetases (aaRSs) are crucial for the correct assembly of amino acids to cognate tRNA to maintain the fidelity of proteosynthesis. AaRSs have become a hot target in antimicrobial research. Three aaRS inhibitors are already in clinical practice; antibacterial mupirocin inhibits the synthetic site of isoleucyl-tRNA synthetase, antifungal tavaborole inhibits the editing site of leucyl-tRNA synthetase, and antiprotozoal halofuginone inhibits proline-tRNA synthetase. According to the World Health Organization, tuberculosis globally remains the leading cause of death from a single infectious agent. The rising incidence of multidrug-resistant tuberculosis is alarming and urges the search for new antimycobacterial compounds, preferably with yet unexploited mechanism of action. In this literature review, we have covered the up-to-date state in the field of inhibitors of mycobacterial aaRSs. The most studied aaRS in mycobacteria is LeuRS with at least four structural types of inhibitors, followed by TyrRS and AspRS. Inhibitors of MetRS, LysRS, and PheRS were addressed in a single significant study each. In many cases, the enzyme inhibition activity translated into micromolar or submicromolar inhibition of growth of mycobacteria. The most promising aaRS inhibitor as an antimycobacterial compound is GSK656 (compound 8), the only aaRS inhibitor in clinical trials (Phase IIa) for systemic use against tuberculosis. GSK656 is orally available and shares the oxaborole tRNA-trapping mechanism of action with antifungal tavaborole.


Assuntos
Aminoacil-tRNA Sintetases/antagonistas & inibidores , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Aminoacil-tRNA Sintetases/metabolismo , Antibacterianos/síntese química , Antibacterianos/química , Bactérias/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-Atividade
9.
Angew Chem Int Ed Engl ; 60(27): 14811-14816, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-33871147

RESUMO

The catechol group of 3,4-dihydroxyphenylalanine (L-DOPA) derived from L-tyrosine oxidation is a key post-translational modification (PTM) in many protein biomaterials and has potential as a bioorthogonal handle for precision protein conjugation applications such as antibody-drug conjugates. Despite this potential, indiscriminate enzymatic modification of exposed tyrosine residues or complete replacement of tyrosine using auxotrophic hosts remains the preferred method of introducing the catechol moiety into proteins, which precludes many protein engineering applications. We have developed new orthogonal translation machinery to site-specifically incorporate L-DOPA into recombinant proteins and a new fluorescent biosensor to selectively monitor L-DOPA incorporation in vivo. We show simultaneous biosynthesis and incorporation of L-DOPA and apply this translation machinery to engineer a novel metalloprotein containing a DOPA-Fe chromophore.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Di-Hidroxifenilalanina/metabolismo , Aminoacil-tRNA Sintetases/química , Di-Hidroxifenilalanina/química , Modelos Moleculares , Estrutura Molecular
10.
Biomolecules ; 11(2)2021 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-33670192

RESUMO

Bioenergetics, genetic coding, and catalysis are all difficult to imagine emerging without pre-existing historical context. That context is often posed as a "Chicken and Egg" problem; its resolution is concisely described by de Grasse Tyson: "The egg was laid by a bird that was not a chicken". The concision and generality of that answer furnish no details-only an appropriate framework from which to examine detailed paradigms that might illuminate paradoxes underlying these three life-defining biomolecular processes. We examine experimental aspects here of five examples that all conform to the same paradigm. In each example, a paradox is resolved by coupling "if, and only if" conditions for reciprocal transitions between levels, such that the consequent of the first test is the antecedent for the second. Each condition thus restricts fluxes through, or "gates" the other. Reciprocally-coupled gating, in which two gated processes constrain one another, is self-referential, hence maps onto the formal structure of "strange loops". That mapping uncovers two different kinds of forces that may help unite the axioms underlying three phenomena that distinguish biology from chemistry. As a physical analog for Gödel's logic, biomolecular strange-loops provide a natural metaphor around which to organize a large body of experimental data, linking biology to information, free energy, and the second law of thermodynamics.


Assuntos
Metabolismo Energético , Código Genético , Trifosfato de Adenosina/metabolismo , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Evolução Biológica , Catálise , Biologia Computacional , Termodinâmica
11.
Sci Rep ; 11(1): 7162, 2021 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-33785838

RESUMO

Antibiotic resistance is a major problem of tuberculosis treatment. This provides the stimulus for the search of novel molecular targets and approaches to reduce or forestall resistance emergence in Mycobacterium tuberculosis. Earlier, we discovered a novel small-molecular inhibitor among 3-phenyl-5-(1-phenyl-1H-[1,2,3]triazol-4-yl)-[1,2,4]oxadiazoles targeting simultaneously two enzymes-mycobacterial leucyl-tRNA synthetase (LeuRS) and methionyl-tRNA synthetase (MetRS), which are promising molecular targets for antibiotic development. Unfortunately, the identified inhibitor does not reveal antibacterial activity toward M. tuberculosis. This study aims to develop novel aminoacyl-tRNA synthetase inhibitors among this chemical class with antibacterial activity toward resistant strains of M. tuberculosis. We performed molecular docking of the library of 3-phenyl-5-(1-phenyl-1H-[1,2,3]triazol-4-yl)-[1,2,4]oxadiazole derivatives and selected 41 compounds for investigation of their inhibitory activity toward MetRS and LeuRS in aminoacylation assay and antibacterial activity toward M. tuberculosis strains using microdilution assay. In vitro screening resulted in 10 compounds active against MetRS and 3 compounds active against LeuRS. Structure-related relationships (SAR) were established. The antibacterial screening revealed 4 compounds active toward M. tuberculosis mono-resistant strains in the range of concentrations 2-20 mg/L. Among these compounds, only one compound 27 has significant enzyme inhibitory activity toward mycobacterial MetRS (IC50 = 148.5 µM). The MIC for this compound toward M. tuberculosis H37Rv strain is 12.5 µM. This compound is not cytotoxic to human HEK293 and HepG2 cell lines. Therefore, 3-phenyl-5-(1-phenyl-1H-[1,2,3]triazol-4-yl)-[1,2,4]oxadiazole derivatives can be used for further chemical optimization and biological research to find non-toxic antituberculosis agents with a novel mechanism of action.


Assuntos
Aminoacil-tRNA Sintetases/antagonistas & inibidores , Antituberculosos/farmacologia , Proteínas Fúngicas/antagonistas & inibidores , Oxidiazóis/farmacologia , Tuberculose/tratamento farmacológico , Aminoacil-tRNA Sintetases/metabolismo , Antituberculosos/química , Antituberculosos/uso terapêutico , Proteínas de Ciclo Celular , Descoberta de Drogas , Farmacorresistência Bacteriana , Proteínas Fúngicas/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Oxidiazóis/química , Oxidiazóis/uso terapêutico , Tuberculose/microbiologia , Proteínas Supressoras de Tumor
12.
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
13.
Biochemistry ; 60(7): 489-493, 2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33560840

RESUMO

The ability to engineer the substrate specificity of natural aminoacyl-tRNA synthetase/tRNA pairs facilitates the site-specific incorporation of noncanonical amino acids (ncAAs) into proteins. The Methanocaldococcus jannaschii-derived tyrosyl-tRNA synthetase (MjTyrRS)/tRNA pair has been engineered to incorporate numerous ncAAs into protein expressed in bacteria. However, it cannot be used in eukaryotic cells due to cross-reactivity with its host counterparts. The Escherichia coli-derived tyrosyl-tRNA synthetase (EcTyrRS)/tRNA pair offers a suitable alternative to this end, but a much smaller subset of ncAAs have been genetically encoded using this pair. Here we report that this discrepancy, at least partly, stems from the structural robustness of EcTyrRS being lower than that of MjTyrRS. We show that the thermostability of engineered TyrRS mutants is generally significantly lower than those of their wild-type counterparts. Derived from a thermophilic archaeon, MjTyrRS is a remarkably sturdy protein and tolerates extensive active site engineering without a catastrophic loss of stability at physiological temperature. In contrast, EcTyrRS exhibits significantly lower thermostability, rendering some of its engineered mutants insufficiently stable at physiological temperature. Our observations identify the structural robustness of an aaRS as an important factor that significantly influences how extensively it can be engineered. To overcome this limitation, we have further developed chimeras between EcTyrRS and its homologue from a thermophilic bacterium, which offer an optimal balance between thermostability and activity. We show that the chimeric bacterial TyrRSs show enhanced tolerance for destabilizing active site mutations, providing a potentially more engineerable platform for genetic code expansion.


Assuntos
Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/metabolismo , Engenharia de Proteínas/métodos , Aminoácidos/genética , Aminoacil-tRNA Sintetases/genética , Domínio Catalítico/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Código Genético/genética , RNA de Transferência/metabolismo , Especificidade por Substrato/genética , Tirosina-tRNA Ligase/química , Tirosina-tRNA Ligase/genética , Tirosina-tRNA Ligase/metabolismo
14.
J Am Chem Soc ; 143(2): 1133-1143, 2021 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-33399460

RESUMO

Trimethylsilyl (TMS) groups present outstanding NMR probes of biological macromolecules as they produce intense singlets in 1H NMR spectra near 0 ppm, where few other proton resonances occur. We report a system for genetic encoding of N6-(((trimethylsilyl)methoxy)carbonyl)-l-lysine (TMSK) for site-specific incorporation into proteins. The system is based on pyrrolysyl-tRNA synthetase mutants, which deliver proteins with high yield and purity in vivo and in cell-free protein synthesis. As the TMS signal can readily be identified in 1D 1H NMR spectra of high-molecular weight systems without the need of isotopic labeling, TMSK delivers an excellent site-specific NMR probe for the study of protein structure and function, which is both inexpensive and convenient. We demonstrate the utility of TMSK to detect ligand binding, measure the rate of conformational change, and assess protein dimerization by paramagnetic relaxation enhancement. In addition, we present a system for dual incorporation of two different unnatural amino acids (TMSK and O-tert-butyl-tyrosine) in the same protein in quantities sufficient for NMR spectroscopy. Close proximity of the TMS and tert-butyl groups was readily detected by nuclear Overhauser effects.


Assuntos
Aminoacil-tRNA Sintetases/química , Lisina/química , Ressonância Magnética Nuclear Biomolecular , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Ligantes , Lisina/análogos & derivados , Lisina/genética , Modelos Moleculares , Estrutura Molecular , Peso Molecular , Mutação , Ligação Proteica
15.
Angew Chem Int Ed Engl ; 60(8): 3934-3939, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33063327

RESUMO

Polypeptides generated from proteolytic processing of protein precursors, or proteolytic proteoforms, play an important role in diverse biological functions and diseases. However, their often-small size and intricate post-translational biogenesis preclude the use of simple genetic tagging in their cellular studies. Herein, we develop a labeling strategy for this class of proteoforms, based on residue-specific genetic code expansion labeling with a molecular beacon design. We demonstrate the utility of such a design by creating a molecular beacon reporter to detect amyloid-ß peptides, known to be involved in the pathogenesis of Alzheimer's disease, as they are produced from amyloid precursor protein (APP) along the endocytic pathway of living cells.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Lisina/análogos & derivados , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Peptídeos beta-Amiloides/química , Precursor de Proteína beta-Amiloide/genética , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Código Genético , Células HEK293 , Humanos , Lisina/química , Lisina/metabolismo , Methanosarcina/enzimologia , Microscopia de Fluorescência , Mutagênese Sítio-Dirigida , Processamento de Proteína Pós-Traducional
16.
Trends Biotechnol ; 39(5): 460-473, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-32896440

RESUMO

The genetic code is the manual that cells use to incorporate amino acids into proteins. It is possible to artificially expand this manual through cellular, molecular, and chemical manipulations to improve protein functionality. Strategies for in vivo genetic code expansion are under the same functional constraints as natural protein synthesis. Here, we review the approaches used to incorporate noncanonical amino acids (ncAAs) into designer proteins through the manipulation of the translation machinery and draw parallels between these methods and natural adaptations that improve translation in extant organisms. Following this logic, we propose new nature-inspired tactics to improve genetic code expansion (GCE) in synthetic organisms.


Assuntos
Aminoácidos , Aminoacil-tRNA Sintetases , Código Genético , Proteínas , Aminoácidos/genética , Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Código Genético/genética , Biossíntese de Proteínas , Proteínas/química , RNA de Transferência/metabolismo , Biologia Sintética/tendências
17.
Nucleic Acids Res ; 49(7): 3603-3616, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33341895

RESUMO

During mRNA translation, tRNAs are charged by aminoacyl-tRNA synthetases and subsequently used by ribosomes. A multi-enzyme aminoacyl-tRNA synthetase complex (MSC) has been proposed to increase protein synthesis efficiency by passing charged tRNAs to ribosomes. An alternative function is that the MSC repurposes specific synthetases that are released from the MSC upon cues for functions independent of translation. To explore this, we generated mammalian cells in which arginyl-tRNA synthetase and/or glutaminyl-tRNA synthetase were absent from the MSC. Protein synthesis, under a variety of stress conditions, was unchanged. Most strikingly, levels of charged tRNAArg and tRNAGln remained unchanged and no ribosome pausing was observed at codons for arginine and glutamine. Thus, increasing or regulating protein synthesis efficiency is not dependent on arginyl-tRNA synthetase and glutaminyl-tRNA synthetase in the MSC. Alternatively, and consistent with previously reported ex-translational roles requiring changes in synthetase cellular localizations, our manipulations of the MSC visibly changed localization.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Biossíntese de Proteínas , RNA de Transferência de Arginina/metabolismo , RNA de Transferência de Glutamina/metabolismo , Ribossomos/metabolismo , Animais , Fibroblastos , Células HEK293 , Humanos , Camundongos
18.
Biosens Bioelectron ; 172: 112783, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33157411

RESUMO

Whole-cell amino acid biosensors can sense the concentrations of certain amino acids and output easily detectable signals, which are important for construction of microbial producers. However, many reported biosensors have poor specificity because they also sense non-target amino acids. Besides, biosensors for many amino acids are still unavailable. In this study, we proposed a new strategy for constructing whole-cell biosensors based on aminoacyl-tRNA synthetases (aaRSs), which take the advantage of their universality and intrinsically specific binding ability to corresponding amino acids. Taking isoleucine biosensor as an example, we first mutated the isoleucyl-tRNA synthetase in Escherichia coli to dramatically decrease its affinity to isoleucine. The engineered cells specifically sensed isoleucine and output isoleucine dose-dependent cell growth as an easily detectable signal. To further expand the sensing range, an isoleucine exporter was overexpressed to enhance excretion of intracellular isoleucine. Since cells equipped with the optimized whole-cell biosensor showed accelerated growth when cells produced higher concentrations of isoleucine, the biosensor was successfully applied in high-throughput selection of isoleucine overproducers from random mutation libraries. This work demonstrates the feasibility of engineering aaRSs to construct a new kind of whole-cell biosensors for amino acids. Considering all twenty proteinogenic and many non-canonical amino acids have their specific aaRSs, this strategy should be useful for developing biosensors for various amino acids.


Assuntos
Aminoacil-tRNA Sintetases , Técnicas Biossensoriais , Aminoácidos , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Isoleucina/genética , Isoleucina-tRNA Ligase/metabolismo , Cinética
19.
Amino Acids ; 53(1): 89-96, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33331978

RESUMO

Genetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair. Here, we investigated whether beneficial effect in unnatural amino acid incorporation caused by N-terminal mutations in PylRS of one species is transferable to PylRS of another species. It was shown that conserved mutations on the N-terminal domain of MmPylRS improved the unnatural amino acid incorporation efficiency up to five folds. As MbPylRS shares high sequence identity to MmPylRS, and the two homologs are often used interchangeably, we examined incorporation of five unnatural amino acids by four MbPylRS variants at two temperatures. Our results indicate that the beneficial N-terminal mutations in MmPylRS did not improve unnatural amino acid incorporation efficiency by MbPylRS. Knowledge from this work contributes to our understanding of PylRS homologs which are needed to improve the technique of genetic code expansion in the future.


Assuntos
Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/genética , Aminoácidos/química , Aminoacil-tRNA Sintetases/metabolismo , Código Genético , Methanosarcina/enzimologia , Methanosarcina/genética , Estrutura Molecular , Mutação , Biossíntese de Proteínas , RNA de Transferência/genética , RNA de Transferência/metabolismo , Temperatura
20.
Genes (Basel) ; 11(11)2020 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-33266490

RESUMO

Aminoacyl-tRNA synthetases (aaRSs) are key enzymes in the mRNA translation machinery, yet they possess numerous non-canonical functions developed during the evolution of complex organisms. The aaRSs and aaRS-interacting multi-functional proteins (AIMPs) are continually being implicated in tumorigenesis, but these connections are often limited in scope, focusing on specific aaRSs in distinct cancer subtypes. Here, we analyze publicly available genomic and transcriptomic data on human cytoplasmic and mitochondrial aaRSs across many cancer types. As high-throughput technologies have improved exponentially, large-scale projects have systematically quantified genetic alteration and expression from thousands of cancer patient samples. One such project is the Cancer Genome Atlas (TCGA), which processed over 20,000 primary cancer and matched normal samples from 33 cancer types. The wealth of knowledge provided from this undertaking has streamlined the identification of cancer drivers and suppressors. We examined aaRS expression data produced by the TCGA project and combined this with patient survival data to recognize trends in aaRSs' impact on cancer both molecularly and prognostically. We further compared these trends to an established tumor suppressor and a proto-oncogene. We observed apparent upregulation of many tRNA synthetase genes with aggressive cancer types, yet, at the individual gene level, some aaRSs resemble a tumor suppressor while others show similarities to an oncogene. This study provides an unbiased, overarching perspective on the relationship of aaRSs with cancers and identifies certain aaRS family members as promising therapeutic targets or potential leads for developing biological therapy for cancer.


Assuntos
Aminoacil-tRNA Sintetases/genética , Mutação , Neoplasias/enzimologia , Neoplasias/mortalidade , Aminoacil-tRNA Sintetases/metabolismo , Bases de Dados de Proteínas , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Variação Genética , Humanos , Neoplasias/genética , Proteômica/métodos , Análise de Sobrevida
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