RESUMEN
The tumor suppressor p53 and the DNA repair factor 53BP1 (p53 binding protein 1) regulate gene transcription and responses to genotoxic stresses. Upon DNA damage, p53 undergoes dimethylation at Lys382 (p53K382me2), and this posttranslational modification is recognized by 53BP1. The molecular mechanism of nonhistone methyl-lysine mark recognition remains unknown. Here we report a 1. 6-A-resolution crystal structure of the tandem Tudor domain of human 53BP1 bound to a p53K382me2 peptide. In the complex, dimethylated Lys382 is restrained by a set of hydrophobic and cation-pi interactions in a cage formed by four aromatic residues and an aspartate of 53BP1. The signature HKKme2 motif of p53, which defines specificity, is identified through a combination of NMR resonance perturbations, mutagenesis, measurements of binding affinities and docking simulations, and analysis of the crystal structures of 53BP1 bound to p53 peptides containing other dimethyl-lysine marks, p53K370me2 (p53 dimethylated at Lys370) and p53K372me2 (p53 dimethylated at Lys372). Binding of the 53BP1 Tudor domain to p53K382me2 may facilitate p53 accumulation at DNA damage sites and promote DNA repair as suggested by chromatin immunoprecipitation and DNA repair assays. Together, our data detail the molecular mechanism of p53-53BP1 association and provide the basis for deciphering the role of this interaction in the regulation of p53 and 53BP1 functions.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismo , Inmunoprecipitación de Cromatina , Cristalografía por Rayos X , Reparación del ADN , Humanos , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Estructura Cuaternaria de Proteína , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
Sequence-specific nucleated protein aggregation is closely linked to the pathogenesis of most neurodegenerative diseases and constitutes the molecular basis of prion formation. Here we report that fibrillar polyglutamine peptide aggregates can be internalized by mammalian cells in culture where they gain access to the cytosolic compartment and become co-sequestered in aggresomes together with components of the ubiquitin-proteasome system and cytoplasmic chaperones. Remarkably, these internalized fibrillar aggregates are able to selectively recruit soluble cytoplasmic proteins with which they share homologous but not heterologous amyloidogenic sequences, and to confer a heritable phenotype on cells expressing the homologous amyloidogenic protein from a chromosomal locus.
Asunto(s)
Amiloidosis/metabolismo , Citoplasma/metabolismo , Endocitosis/fisiología , Cuerpos de Inclusión/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Péptidos/metabolismo , Amiloide/biosíntesis , Amiloidosis/patología , Amiloidosis/fisiopatología , Comunicación Celular/fisiología , Línea Celular , Citoplasma/patología , Transmisión de Enfermedad Infecciosa , Humanos , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/fisiopatología , Enfermedades Neurodegenerativas/patología , Enfermedades Neurodegenerativas/fisiopatología , Neurofibrillas/metabolismo , Neurofibrillas/patología , Péptidos/toxicidad , Enfermedades por Prión/metabolismo , Enfermedades por Prión/fisiopatología , Complejo de la Endopetidasa Proteasomal/metabolismo , Complejo de la Endopetidasa Proteasomal/ultraestructura , Expansión de Repetición de Trinucleótido/genéticaRESUMEN
Modification of histone proteins by lysine methylation is a principal chromatin regulatory mechanism (Shi, Y., and Whetstine, J. R. (2007) Mol. Cell 25, 1-14). Recently, lysine methylation has been shown also to play a role in regulating non-histone proteins, including the tumor suppressor protein p53 (Huang, J., and Berger, S. L. (2008) Curr. Opin. Genet. Dev. 18, 152-158). Here, we identify a novel p53 species that is dimethylated at lysine 382 (p53K382me2) and show that the tandem Tudor domain of the DNA damage response mediator 53BP1 acts as an "effector" for this mark. We demonstrate that the 53BP1 tandem Tudor domain recognizes p53K382me2 with a selectivity relative to several other protein lysine methylation sites and saturation states. p53K382me2 levels increase with DNA damage, and recognition of this modification by 53BP1 facilitates an interaction between p53 and 53BP1. The generation of p53K382me2 promotes the accumulation of p53 protein that occurs upon DNA damage, and this increase in p53 levels requires 53BP1. Taken together, our study identifies a novel p53 modification, demonstrates a new effector function for the 53BP1 tandem Tudor domain, and provides insight into how DNA damage signals are transduced to stabilize p53.
Asunto(s)
Daño del ADN , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteína p53 Supresora de Tumor/química , Secuencia de Aminoácidos , ADN/química , Histonas/química , Humanos , Lisina/química , Metilación , Datos de Secuencia Molecular , Péptidos/química , Estructura Terciaria de Proteína , Transducción de Señal , Especificidad por Sustrato , Transfección , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
Reversible covalent methylation of lysine residues on histone proteins constitutes a principal molecular mechanism that links chromatin states to diverse biological outcomes. Recently, lysine methylation has been observed on nonhistone proteins, suggesting broad cellular roles for the enzymes generating and removing methyl moieties. Here we report that the lysine methyltransferase enzyme SET8/PR-Set7 regulates the tumor suppressor protein p53. We find that SET8 specifically monomethylates p53 at lysine 382 (p53K382me1). This methylation event robustly suppresses p53-mediated transcription activation of highly responsive target genes but has little influence on weak targets. Further, depletion of SET8 augments the proapoptotic and checkpoint activation functions of p53, and accordingly, SET8 expression is downregulated upon DNA damage. Together, our study identifies SET8 as a p53-modifying enzyme, identifies p53K382me1 as a regulatory posttranslational modification of p53, and begins to dissect how methylation may contribute to a dynamic posttranslational code that modulates distinct p53 functions.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/metabolismo , Lisina/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Secuencia de Aminoácidos , Anticuerpos/inmunología , Línea Celular Tumoral , Daño del ADN , N-Metiltransferasa de Histona-Lisina/deficiencia , Humanos , Metilación , Modelos Biológicos , Datos de Secuencia Molecular , Interferencia de ARN , Especificidad por Sustrato , Activación Transcripcional/genética , Proteína p53 Supresora de Tumor/químicaRESUMEN
The PHD finger motif is a signature chromatin-associated motif that is found throughout eukaryotic proteomes. Here we have determined the histone methyl-lysine binding activity of the PHD fingers present within the Saccharomyces cerevisiae proteome. We provide evidence on the genomic scale that PHD fingers constitute a general class of effector modules for histone H3 trimethylated at lysine 4 (H3K4me3) and histone H3 trimethylated at lysine 36 (H3K36me3). Structural modeling of PHD fingers demonstrates a conserved mechanism for recognizing the trimethyl moiety and provides insight into the molecular basis of affinity for the different methyl-histone ligands. Together, our study suggests that a common function for PHD fingers is to transduce methyl-lysine events and sheds light on how a single histone modification can be linked to multiple biological outcomes.
Asunto(s)
Histonas/metabolismo , Proteínas de Homeodominio , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas de Unión al ADN , Proteínas de Homeodominio/química , Proteínas de Homeodominio/metabolismo , Lisina , Metilación , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Proteoma , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
A chromogenic bioassay was utilized to determine the antithrombin activity of methylene chloride and methanol extracts prepared from 30 plants of central Florida. Extracts of Ardisia crenata, Tetrapanax papyriferus, Lagerstroemia indica, Callistemon lanceolatus, Antigonon Leptopus, Magnolia virginiana, and Myrica cerifera demonstrated activity of 80% or higher in this bioassay system.