RESUMEN
Uterine carcinosarcoma is an aggressive variant of endometrial carcinoma characterized by unusual histologic features including discrete malignant epithelial and mesenchymal components (carcinoma and sarcoma). Recent studies have confirmed a monoclonal origin, and comprehensive genomic characterizations have identified mutations such as Tp53 and Pten However, the biological origins and specific combination of driver events underpinning uterine carcinosarcoma have remained mysterious. Here, we explored the role of the tumor suppressor Fbxw7 in endometrial cancer through defined genetic model systems. Inactivation of Fbxw7 and Pten resulted in the formation of precancerous lesions (endometrioid intraepithelial neoplasia) and well-differentiated endometrioid adenocarcinomas. Surprisingly, all adenocarcinomas eventually developed into definitive uterine carcinosarcomas with carcinomatous and sarcomatous elements including heterologous differentiation, yielding a faithful genetically engineered model of this cancer type. Genomic analysis showed that most tumors spontaneously acquired Trp53 mutations, pointing to a triad of pathways (p53, PI3K, and Fbxw7) as the critical combination underpinning uterine carcinosarcoma, and to Fbxw7 as a key driver of this enigmatic endometrial cancer type. Lineage tracing provided formal genetic proof that the uterine carcinosarcoma cell of origin is an endometrial epithelial cell that subsequently undergoes a prominent epithelial-mesenchymal transition underlying the attainment of a highly invasive phenotype specifically driven by Fbxw7.
Asunto(s)
Neoplasias Endometriales/metabolismo , Transición Epitelial-Mesenquimal/fisiología , Proteína 7 que Contiene Repeticiones F-Box-WD/genética , Proteína 7 que Contiene Repeticiones F-Box-WD/metabolismo , Neoplasias Uterinas/metabolismo , Animales , Movimiento Celular , Proliferación Celular , Neoplasias Endometriales/genética , Neoplasias Endometriales/patología , Endometrio/patología , Células Epiteliales , Femenino , Humanos , Ratones , Mutación , Fosfohidrolasa PTEN/metabolismo , Fenotipo , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias Uterinas/genética , Neoplasias Uterinas/patologíaRESUMEN
Non-LTR retrotransposons are an important class of mobile elements that insert into host DNA by target-primed reverse transcription (TPRT). Non-LTR retrotransposons must bind to their mRNA, recognize and cleave their target DNA, and perform TPRT at the site of DNA cleavage. As DNA binding and cleavage are such central parts of the integration reaction, a better understanding of the endonuclease encoded by non-LTR retrotransposons is needed. This paper explores the R2 endonuclease domain from Bombyx mori using in vitro studies and in silico modeling. Mutations in conserved sequences located across the putative PD-(D/E)XK endonuclease domain reduced DNA cleavage, DNA binding and TPRT. A mutation at the beginning of the first α-helix of the modeled endonuclease obliterated DNA cleavage and greatly reduced DNA binding. It also reduced TPRT when tested on pre-cleaved DNA substrates. The catalytic K was located to a non-canonical position within the second α-helix. A mutation located after the fourth ß-strand reduced DNA binding and cleavage. The motifs that showed impaired activity form an extensive basic region. The R2 biochemical and structural data are compared and contrasted with that of two other well characterized PD-(D/E)XK endonucleases, restriction endonucleases and archaeal Holliday junction resolvases.
Asunto(s)
Endodesoxirribonucleasas/química , Retroelementos , Secuencia de Aminoácidos , Animales , Bombyx/enzimología , Secuencia Conservada , ADN/metabolismo , División del ADN , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Modelos Moleculares , Mutación , Estructura Secundaria de Proteína , Transcripción Reversa , Alineación de SecuenciaRESUMEN
Branched-chain amino acid (BCAA) metabolism fulfills numerous physiological roles and can be harnessed to produce valuable chemicals. However, the lack of eukaryotic biosensors specific for BCAA-derived products has limited the ability to develop high-throughput screens for strain engineering and metabolic studies. Here, we harness the transcriptional regulator Leu3p from Saccharomyces cerevisiae to develop a genetically encoded biosensor for BCAA metabolism. In one configuration, we use the biosensor to monitor yeast production of isobutanol, an alcohol derived from valine degradation. Small modifications allow us to redeploy Leu3p in another biosensor configuration that monitors production of the leucine-derived alcohol, isopentanol. These biosensor configurations are effective at isolating high-producing strains and identifying enzymes with enhanced activity from screens for branched-chain higher alcohol (BCHA) biosynthesis in mitochondria as well as cytosol. Furthermore, this biosensor has the potential to assist in metabolic studies involving BCAA pathways, and offers a blueprint to develop biosensors for other products derived from BCAA metabolism.