RESUMEN
Recent studies suggested an essential role for seryl-tRNA synthetase (SerRS) in vascular development. This role is specific to SerRS among all tRNA synthetases and is independent of its well-known aminoacylation function in protein synthesis. A unique nucleus-directing domain, added at the invertebrate-to-vertebrate transition, confers this novel non-translational activity of SerRS. Previous studies showed that SerRS, in some unknown way, controls VEGFA expression to prevent vascular over-expansion. Using in vitro, cell and animal experiments, we show here that SerRS intervenes by antagonizing c-Myc, the major transcription factor promoting VEGFA expression, through a tandem mechanism. First, by direct head-to-head competition, nuclear-localized SerRS blocks c-Myc from binding to the VEGFA promoter. Second, DNA-bound SerRS recruits the SIRT2 histone deacetylase to erase prior c-Myc-promoted histone acetylation. Thus, vertebrate SerRS and c-Myc is a pair of 'Yin-Yang' transcriptional regulator for proper development of a functional vasculature. Our results also discover an anti-angiogenic activity for SIRT2.DOI: http://dx.doi.org/10.7554/eLife.02349.001.
Asunto(s)
Proteínas Proto-Oncogénicas c-myc/genética , Serina-ARNt Ligasa/genética , Secuencia de Aminoácidos , Inductores de la Angiogénesis/farmacología , Animales , Línea Celular , Epigénesis Genética , Femenino , Silenciador del Gen , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Masculino , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Conformación Proteica , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Serina-ARNt Ligasa/farmacología , Sirtuina 2/genética , Sirtuina 2/farmacología , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Pez CebraRESUMEN
Like all other eukaryal cytosolic seryl-tRNA synthetase (SerRS) enzymes, Saccharomyces cerevisiae SerRS contains a C-terminal extension not found in the enzymes of eubacterial and archaeal origin. Overexpression of C-terminally truncated SerRS lacking the 20-amino acid appended domain (SerRSC20) is toxic to S. cerevisiae possibly because of altered substrate recognition. Compared to wild-type SerRS the truncated enzyme displays impaired tRNA-dependent serine recognition and is less stable. This suggests that the C-terminal peptide is important for the formation or maintenance of the enzyme structure optimal for substrate binding and catalysis.
Asunto(s)
ADN de Hongos/metabolismo , ARN de Transferencia de Serina/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Serina-ARNt Ligasa/farmacología , Secuencia de Aminoácidos , Estabilidad de Enzimas , Cinética , Datos de Secuencia Molecular , Conformación Proteica , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Serina-ARNt Ligasa/genética , Serina-ARNt Ligasa/metabolismo , Especificidad por SustratoRESUMEN
The activation process of N-hydroxy-4-aminoazobenzene (N-OH-AAB) dyes, proximate mutagenic or carcinogenic metabolites of AAB dyes, to the ultimate mutagenic or carcinogenic metabolites was studied by the use of an amino acid conjugation (aminoacylation) system catalyzed by yeast seryl-tRNA synthetase and [3H]ATP. A potent mutagen, N-hydroxy-3-methoxy-AAB (N-OH-3-MeO-AAB), as well as a non-mutagen, N-OH-2-MeO-AAB, were equally susceptible to N-O-serine conjugation. A weak mutagen, N-OH-AAB, and a moderate mutagen, N-OH-2,5-diMeO-AAB, were also susceptible to the aminoacylation, but to a lesser extent than the 2- or 3-methoxyl homologs. In contrast, N-hydroxy-N-methyl-4-aminoazobenzene and a moderate mutagen, N-OH-4'-MeO-AAB, were not susceptible to the aminoacylation. The ability of these N-OH-AAB dyes to bind with nucleic acid after serine conjugation was proportional to the susceptibility of the dyes to serine conjugation. Serine conjugates of N-OH-AAB dyes reacted with poly G, but not with poly A, poly C or poly U, suggesting that the azo dyes selectively bind with guanine base of nucleic acids. The susceptibility of N-OH-AAB dyes to aminoacylation was compared with the carcinogenic, mutagenic and unscheduled DNA synthesis-inducing activities of these and the mother AAB dyes.