RESUMEN
The p53 tumor suppressor protein, known to be critically important in several processes including cell-cycle arrest and apoptosis, is highly regulated by multiple mechanisms, most certifiably the Murine Double Minute 2-Murine Double Minute X (MDM2-MDMX) heterodimer. The role of MDM2-MDMX in cell-cycle regulation through inhibition of p53 has been well established. Here we report that in cells either lacking p53 or expressing certain tumor-derived mutant forms of p53, loss of endogenous MDM2 or MDMX, or inhibition of E3 ligase activity of the heterocomplex, causes cell-cycle arrest. This arrest is correlated with a reduction in E2F1, E2F3, and p73 levels. Remarkably, direct ablation of endogenous p73 produces a similar effect on the cell cycle and the expression of certain E2F family members at both protein and messenger RNA levels. These data suggest that MDM2 and MDMX, working at least in part as a heterocomplex, may play a p53-independent role in maintaining cell-cycle progression by promoting the activity of E2F family members as well as p73, making them a potential target of interest in cancers lacking wild-type p53.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteína Tumoral p73/metabolismo , Animales , Apoptosis , Ciclo Celular/fisiología , Puntos de Control del Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Factor de Transcripción E2F1/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteína Tumoral p73/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Most well studied as proteins that restrain the p53 tumor suppressor protein, MDM2 and MDMX have rich lives outside of their relationship to p53. There is much to learn about how these two proteins are regulated and how they can function in cells that lack p53. Regulation of MDM2 and MDMX, which takes place at the level of transcription, post-transcription, and protein modification, can be very intricate and is context-dependent. Equally complex are the myriad roles that these two proteins play in cells that lack wild-type p53; while many of these independent outcomes are consistent with oncogenic transformation, in some settings their functions could also be tumor suppressive. Since numerous small molecules that affect MDM2 and MDMX have been developed for therapeutic outcomes, most if not all designed to prevent their restraint of p53, it will be essential to understand how these diverse molecules might affect the p53-independent activities of MDM2 and MDMX.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Animales , Humanos , Regiones Promotoras Genéticas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Correction for 'Thioamide quenching of intrinsic protein fluorescence' by Jacob M. Goldberg et al., Chem. Commun., 2012, 48, 1550-1552.
RESUMEN
MDM2 and MDMX, negative regulators of the tumor suppressor p53, can work separately and as a heteromeric complex to restrain p53's functions. MDM2 also has pro-oncogenic roles in cells, tissues, and animals that are independent of p53. There is less information available about p53-independent roles of MDMX or the MDM2-MDMX complex. We found that MDM2 and MDMX facilitate ferroptosis in cells with or without p53. Using small molecules, RNA interference reagents, and mutant forms of MDMX, we found that MDM2 and MDMX, likely working in part as a complex, normally facilitate ferroptotic death. We observed that MDM2 and MDMX alter the lipid profile of cells to favor ferroptosis. Inhibition of MDM2 or MDMX leads to increased levels of FSP1 protein and a consequent increase in the levels of coenzyme Q10, an endogenous lipophilic antioxidant. This suggests that MDM2 and MDMX normally prevent cells from mounting an adequate defense against lipid peroxidation and thereby promote ferroptosis. Moreover, we found that PPARα activity is essential for MDM2 and MDMX to promote ferroptosis, suggesting that the MDM2-MDMX complex regulates lipids through altering PPARα activity. These findings reveal the complexity of cellular responses to MDM2 and MDMX and suggest that MDM2-MDMX inhibition might be useful for preventing degenerative diseases involving ferroptosis. Furthermore, they suggest that MDM2/MDMX amplification may predict sensitivity of some cancers to ferroptosis inducers.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ferroptosis/genética , Metabolismo de los Lípidos/genética , PPAR alfa/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/fisiopatología , Proteínas de Ciclo Celular/genética , Glioblastoma/fisiopatología , Células HCT116 , Humanos , Mutación , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-mdm2/genética , Interferencia de ARN , Ratas , Proteína p53 Supresora de Tumor/metabolismo , Ubiquinona/análogos & derivados , Ubiquinona/metabolismoRESUMEN
Angiogenesis is regulated by complex interactions between endothelial cells and support cells of the vascular microenvironment, such as tissue myeloid cells and vascular mural cells. Multicellular interactions during angiogenesis are difficult to study in animals and challenging in a reductive setting. We incorporated stromal cells into an established bead-based capillary sprouting assay to develop assays that faithfully reproduce major steps of vessel sprouting and maturation. We observed that macrophages enhance angiogenesis, increasing the number and length of endothelial sprouts, a property we have dubbed "angiotrophism." We found that polarizing macrophages toward a pro-inflammatory profile further increased their angiotrophic stimulation of vessel sprouting, and this increase was dependent on macrophage Notch signaling. To study endothelial/pericyte interactions, we added vascular pericytes directly to the bead-bound endothelial monolayer. These pericytes formed close associations with the endothelial sprouts, causing increased sprout number and vessel caliber. We found that Jagged1 expression and Notch signaling are essential for the growth of both endothelial cells and pericytes and may function in their interaction. We observed that combining endothelial cells with both macrophages and pericytes in the same sprouting assay has multiplicative effects on sprouting. These results significantly improve bead-capillary sprouting assays and provide an enhanced method for modeling interactions between the endothelium and the vascular microenvironment. Achieving this in a reductive in vitro setting represents a significant step toward a better understanding of the cellular elements that contribute to the formation of mature vasculature.
Asunto(s)
Comunicación Celular , Microambiente Celular , Células Endoteliales de la Vena Umbilical Humana/citología , Macrófagos/citología , Modelos Biológicos , Neovascularización Fisiológica , Pericitos/citología , Receptores Notch/metabolismo , Animales , Línea Celular , Polaridad Celular , Supervivencia Celular , Técnicas de Cocultivo , Técnicas de Silenciamiento del Gen , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Inflamación/patología , Mediadores de Inflamación/metabolismo , Macrófagos/metabolismo , Ratones , Células Mieloides/citología , Células Mieloides/metabolismo , Pericitos/metabolismo , Proteínas Serrate-Jagged/metabolismo , Transducción de SeñalRESUMEN
Thioamides quench tryptophan and tyrosine fluorescence in a distance-dependent manner and thus can be used to monitor the binding of thioamide-containing peptides to proteins. Since thioamide analogs of the natural amino acids can be synthetically incorporated into peptides, they can function as minimally-perturbing probes of protein/peptide interactions.
Asunto(s)
Fluorescencia , Proteínas/química , Tioamidas/química , Modelos Moleculares , Péptidos/química , Triptófano/química , Tirosina/químicaRESUMEN
MicroRNAs (miRNAs) control tissue development, but their mechanism of regulation is not well understood. We used a gene complementation strategy combined with microarray screening to identify miRNAs involved in the formation of erythroid (red blood) cells. Two conserved miRNAs, miR 144 and miR 451, emerged as direct targets of the critical hematopoietic transcription factor GATA-1. In vivo, GATA-1 binds a distal upstream regulatory element to activate RNA polymerase II-mediated transcription of a single common precursor RNA (pri-miRNA) encoding both mature miRNAs. Zebrafish embryos depleted of miR 451 by using antisense morpholinos form erythroid precursors, but their development into mature circulating red blood cells is strongly and specifically impaired. These results reveal a miRNA locus that is required for erythropoiesis and uncover a new regulatory axis through which GATA-1 controls this process.