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1.
Nucleic Acids Res ; 52(D1): D476-D482, 2024 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-37986218

RESUMO

The incorporation of non-canonical amino acids (ncAAs) into proteins is a powerful technique used in various research fields. Genetic code expansion (GCE) is the most common way to achieve this: a specific codon is selected to be decoded by a dedicated tRNA orthogonal to the endogenous ones. In the past 30 years, great progress has been made to obtain novel tRNA synthetases (aaRSs) accepting a variety of ncAAs with distinct physicochemical properties, to develop robust in vitro assays or approaches for codon reassignment. This sparked the use of the technique, leading to the accumulation of publications, from which gathering all relevant information can appear daunting. Here we present iNClusive (https://non-canonical-aas.biologie.uni-freiburg.de/), a manually curated, extensive repository using standardized nomenclature that provides organized information on ncAAs successfully incorporated into target proteins as verified by mass spectrometry. Since we focused on tRNA synthetase-based tRNA loading, we provide the sequence of the tRNA and aaRS used for the incorporation. Derived from more than 687 peer-reviewed publications, it currently contains 2432 entries about 466 ncAAs, 569 protein targets, 500 aaRSs and 144 tRNAs. We foresee iNClusive will encourage more researchers to experiment with ncAA incorporation thus contributing to the further development of this exciting technique.


Assuntos
Sequência de Aminoácidos , Aminoácidos , Bases de Dados de Proteínas , Proteínas , Aminoácidos/química , Aminoácidos/metabolismo , Códon/genética , Código Genético , Proteínas/genética , RNA de Transferência/genética , RNA de Transferência/metabolismo , Internet
2.
ACS Synth Biol ; 11(10): 3529-3533, 2022 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-36180042

RESUMO

The optogenetic tool LEXY consists of the second light oxygen voltage (LOV) domain of Avena sativa phototropin 1 mutated to contain a nuclear export signal. It allows exporting from the nucleus with blue light proteins of interest (POIs) genetically fused to it. Mutations slowing the dark recovery rate of the LOV domain within LEXY were recently shown to allow for better depletion of some POIs from the nucleus in Drosophila embryos and for the usage of low light illumination regimes. We investigated these variants in mammalian cells and found they increase the cytoplasmic localization of the proteins we tested after illumination, but also during the dark phases, which corresponds to higher leakiness of the system. These data suggest that, when aiming to sequester into the nucleus a protein with a cytoplasmic function, the original LEXY is preferable. The iLEXY variants are, instead, advantageous when wanting to deplete the nucleus of the POI as much as possible.


Assuntos
Proteínas Nucleares , Fototropinas , Animais , Fototropinas/genética , Fototropinas/metabolismo , Proteínas Nucleares/metabolismo , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo , Sinais de Exportação Nuclear/genética , Luz , Avena/genética , Avena/metabolismo , Oxigênio/metabolismo , Mamíferos/metabolismo
3.
J Biochem ; 169(3): 273-286, 2021 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-33245128

RESUMO

The nucleus is a very complex organelle present in eukaryotic cells. Having the crucial task to safeguard, organize and manage the genetic information, it must tightly control its molecular constituents, its shape and its internal architecture at any given time. Despite our vast knowledge of nuclear cell biology, much is yet to be unravelled. For instance, only recently we came to appreciate the existence of a dynamic nuclear cytoskeleton made of actin filaments that regulates processes such as gene expression, DNA repair and nuclear expansion. This suggests further exciting discoveries ahead of us. Modern cell biologists embrace a new methodology relying on precise perturbations of cellular processes that require a reversible, highly spatially confinable, rapid, inexpensive and tunEable external stimulus: light. In this review, we discuss how optogenetics, the state-of-the-art technology that uses genetically encoded light-sensitive proteins to steer biological processes, can be adopted to specifically investigate nuclear cell biology.


Assuntos
Núcleo Celular/metabolismo , Optogenética/métodos , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Animais , Biologia Celular , Núcleo Celular/genética , Citoesqueleto/metabolismo , Regulação da Expressão Gênica , Humanos , Luz , Engenharia de Proteínas/métodos , Transporte Proteico
4.
Nucleic Acids Res ; 46(3): 1470-1485, 2018 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-29244160

RESUMO

In Pseudomonas aeruginosa the RNA chaperone Hfq and the catabolite repression control protein (Crc) act as post-transcriptional regulators during carbon catabolite repression (CCR). In this regard Crc is required for full-fledged Hfq-mediated translational repression of catabolic genes. RNAseq based transcriptome analyses revealed a significant overlap between the Crc and Hfq regulons, which in conjunction with genetic data supported a concerted action of both proteins. Biochemical and biophysical approaches further suggest that Crc and Hfq form an assembly in the presence of RNAs containing A-rich motifs, and that Crc interacts with both, Hfq and RNA. Through these interactions, Crc enhances the stability of Hfq/Crc/RNA complexes, which can explain its facilitating role in Hfq-mediated translational repression. Hence, these studies revealed for the first time insights into how an interacting protein can modulate Hfq function. Moreover, Crc is shown to interfere with binding of a regulatory RNA to Hfq, which bears implications for riboregulation. These results are discussed in terms of a working model, wherein Crc prioritizes the function of Hfq toward utilization of favored carbon sources.


Assuntos
Proteínas de Bactérias/genética , Repressão Catabólica , Fator Proteico 1 do Hospedeiro/genética , Biossíntese de Proteínas , Pseudomonas aeruginosa/genética , RNA Bacteriano/genética , Proteínas Repressoras/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Bordetella pertussis/genética , Bordetella pertussis/metabolismo , Metabolismo dos Carboidratos/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/química , Fator Proteico 1 do Hospedeiro/metabolismo , Cinética , Modelos Moleculares , Motivos de Nucleotídeos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Pseudomonas aeruginosa/metabolismo , RNA Bacteriano/química , RNA Bacteriano/metabolismo , Regulon , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Transcriptoma
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