RESUMEN

ASC-2, a multifunctional coactivator, forms a steady-state complex, named ASCOM (for ASC-2 COMplex), that contains the histone H3-lysine-4 (H3K4)-methyltransferase MLL3 or its paralogue MLL4. Somewhat surprisingly, given prior indications of redundancy between MLL3 and MLL4, targeted inactivation of the MLL3 H3K4-methylation activity in mice is found to result in ureter epithelial tumors. Interestingly, this phenotype is exacerbated in a p53(+/-) background and the tumorigenic cells are heavily immunostained for gammaH2AX, indicating a contribution of MLL3 to the DNA damage response pathway through p53. Consistent with the in vivo observations, and the demonstration of a direct interaction between p53 and ASCOM, cell-based assays have revealed that ASCOM, through ASC-2 and MLL3/4, acts as a p53 coactivator and is required for H3K4-trimethyation and expression of endogenous p53-target genes in response to the DNA damaging agent doxorubicin. In support of redundant functions for MLL3 and MLL4 for some events, siRNA-mediated down-regulation of both MLL3 and MLL4 is required to suppress doxorubicin-inducible expression of several p53-target genes. Importantly, this study identifies a specific H3K4 methytransferase complex, ASCOM, as a physiologically relevant coactivator for p53 and implicates ASCOM in the p53 tumor suppression pathway in vivo.

Asunto(s)

N-Metiltransferasa de Histona-Lisina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Transformación Celular Neoplásica , Daño del ADN/genética , Reparación del ADN/genética , Activación Enzimática , N-Metiltransferasa de Histona-Lisina/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Transgénicos , Coactivadores de Receptor Nuclear , Proteína p53 Supresora de Tumor/genética , Neoplasias Ureterales/enzimología , Neoplasias Ureterales/genética , Neoplasias Ureterales/patología

RESUMEN

Activating signal cointegrator-2 (ASC-2), a transcriptional coactivator of multiple transcription factors that include the adipogenic factors peroxisome proliferator-activated receptor gamma (PPARgamma) and C/EBPalpha, is associated with histone H3-Lys-4-methyltransferase (H3K4MT) MLL3 or its paralogue MLL4 in a complex named ASCOM (ASC-2 complex). Indeed, ASC-2-null mouse embryonic fibroblasts (MEFs) have been demonstrated to be refractory to PPARgamma-stimulated adipogenesis and fail to express the PPARgamma-responsive adipogenic marker gene aP2. However, the specific roles for MLL3 and MLL4 in adipogenesis remain undefined. Here, we provide evidence that MLL3 plays crucial roles in adipogenesis. First, MLL3(Delta/Delta) mice expressing a H3K4MT-inactivated mutant of MLL3 have significantly less white fat. Second, MLL3(Delta/Delta) MEFs are mildly but consistently less responsive to inducers of adipogenesis than WT MEFs. Third, ASC-2, MLL3, and MLL4 are recruited to the PPARgamma-activated aP2 gene during adipogenesis, and PPARgamma is shown to interact directly with the purified ASCOM. Moreover, although H3K4 methylation of aP2 is readily induced in WT MEFs, it is not induced in ASC-2(-/-) MEFs and only partially induced in MLL3(Delta/Delta) MEFs. These results suggest that ASCOM-MLL3 and ASCOM-MLL4 likely function as crucial but redundant H3K4MT complexes for PPARgamma-dependent adipogenesis.

Asunto(s)

Adipogénesis/fisiología , N-Metiltransferasa de Histona-Lisina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Obesidad/fisiopatología , Adipocitos Blancos/enzimología , Tejido Adiposo Blanco/citología , Tejido Adiposo Blanco/metabolismo , Animales , Peso al Nacer , Grasas de la Dieta/farmacología , Femenino , N-Metiltransferasa de Histona-Lisina/genética , Masculino , Metilación , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Células 3T3 NIH , Coactivadores de Receptor Nuclear , Obesidad/genética , PPAR gamma/metabolismo , Fenotipo

RESUMEN

Baculoviral inhibitor of apoptosis repeat-containing (Birc)6 gene/BIRC6 (Bruce/APOLLON) encodes an inhibitor of apoptosis and a chimeric E2/E3 ubiquitin ligase in mammals. The physiological role of Bruce in antiapoptosis is unknown. Here, we show that deletion of the C-terminal half of Bruce, including the UBC domain, causes activation of caspases and apoptosis in the placenta and yolk sac, leading to embryonic lethality. This apoptosis is associated with up-regulation and nuclear localization of the tumor suppressor p53 and activation of mitochondrial apoptosis, which includes up-regulation of Bax, Bak, and Pidd, translocation of Bax and caspase-2 onto mitochondria, release of cytochrome c and apoptosis-inducing factor, and activation of caspase-9 and caspase-3. Mutant mouse embryonic fibroblasts are sensitive to multiple mitochondrial death stimuli but resistant to TNF. In addition, eliminating p53 by RNA interference rescues cell viability induced by Bruce ablation in human cell line H460. This viability preservation results from reduced expression of proapoptotic factors Bax, Bak, and Pidd and from prevention of activation of caspase-2, -9, and -3. The amount of second mitochondrial-derived activator of caspase and Omi does not change. We conclude that p53 is a downstream effector of Bruce, and, in response to loss of Bruce function, p53 activates Pidd/caspase-2 and Bax/Bak, leading to mitochondrial apoptosis.

Asunto(s)

Apoptosis , Desarrollo Embrionario/genética , Mitocondrias/metabolismo , Proteínas de Neoplasias/genética , Proteína p53 Supresora de Tumor/genética , Animales , Caspasa 2 , Caspasas/metabolismo , Pérdida del Embrión , Regulación del Desarrollo de la Expresión Génica , Homocigoto , Proteínas Inhibidoras de la Apoptosis , Proteínas de la Membrana/metabolismo , Ratones , Ratones Mutantes , Mutación , Proteínas de Neoplasias/fisiología , Placenta/patología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Saco Vitelino/patología , Proteína Destructora del Antagonista Homólogo bcl-2

RESUMEN

The inhibitor of apoptosis (IAP) proteins bind and inhibit caspases via their baculovirus IAP repeat domains. Some of these IAPs are capable of ubiquitinating themselves and their interacting proteins through the ubiquitin-protein isopeptide ligase activity of their RING domain. The Drosophila IAP antagonists Reaper, Hid, and Grim can accelerate the degradation of Drosophila IAP1 and some mammalian IAPs by promoting their ubiquitin-protein isopeptide ligase activity. Here we show that Smac/DIABLO, a mammalian functional homolog of Reaper/Hid/Grim, selectively causes the rapid degradation of c-IAP1 and c-IAP2 but not XIAP and Livin in HeLa cells, although it efficiently promotes the auto-ubiquitination of them all. Smac binding to c-IAP via its N-terminal IAP-binding motif is the prerequisite for this effect, which is further supported by the findings that Smac N-terminal peptide is sufficient to enhance c-IAP1 ubiquitination, and Smac no longer promotes the ubiquitination of mutant c-IAP1 lacking all three baculovirus IAP repeat domains. In addition, different IAPs require the same ubiquitin-conjugating enzymes UbcH5a and UbcH6 for their ubiquitination. Taken together, Smac may serve as a key molecule in vivo to selectively reduce the protein level of c-IAPs through the ubiquitin/proteasome pathway.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/fisiología , Proteínas de Unión a Hierro , Proteínas Mitocondriales/fisiología , Proteínas de Neoplasias/biosíntesis , Biosíntesis de Proteínas , Proteínas , Secuencias de Aminoácidos , Proteínas Reguladoras de la Apoptosis , Western Blotting , Proteínas Portadoras/metabolismo , Cisteína Endopeptidasas/metabolismo , Escherichia coli/metabolismo , Glutatión Transferasa/metabolismo , Células HeLa , Humanos , Proteínas Inhibidoras de la Apoptosis , Péptidos y Proteínas de Señalización Intracelular , Proteínas Mitocondriales/metabolismo , Complejos Multienzimáticos/metabolismo , Mutación , Plásmidos/metabolismo , Complejo de la Endopetidasa Proteasomal , Unión Proteica , Estructura Terciaria de Proteína , Transfección , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/química , Ubiquitina-Proteína Ligasas , Proteína Inhibidora de la Apoptosis Ligada a X

RESUMEN

Omi/HtrA2 is a mitochondrial serine protease that is released into the cytosol during apoptosis to antagonize inhibitors of apoptosis (IAPs) and contribute to caspase-independent cell death. Here, we demonstrate that Omi/HtrA2 directly cleaves various IAPs in vitro, and the cleavage efficiency is determined by its IAP-binding motif, AVPS. Cleavage of IAPs such as c-IAP1 substantially reduces its ability to inhibit and ubiquitylate caspases. In contrast to the stoichiometric anti-IAP activity by Smac/DIABLO, Omi/HtrA2 cleavage of c-IAP1 is catalytic and irreversible, thereby more efficiently inactivating IAPs and promoting caspase activity. Elimination of endogenous Omi by RNA interference abolishes c-IAP1 cleavage and desensitizes cells to apoptosis induced by TRAIL. In addition, overexpression of cleavage-site mutant c-IAP1 makes cells more resistant to TRAIL-induced caspase activation. This IAP cleavage by Omi is independent of caspase. Taken together, these results indicate that unlike Smac/DIABLO, Omi/HtrA2's catalytic cleavage of IAPs is a key mechanism for it to irreversibly inactivate IAPs and promote apoptosis.

Asunto(s)

Apoptosis/fisiología , Caspasas/metabolismo , Proteínas/metabolismo , Serina Endopeptidasas/metabolismo , Proteínas Reguladoras de la Apoptosis , Sitios de Unión , Proteínas Portadoras/metabolismo , Catálisis , Activación Enzimática/fisiología , Células HeLa , Serina Peptidasa A2 que Requiere Temperaturas Altas , Humanos , Proteínas Inhibidoras de la Apoptosis , Péptidos y Proteínas de Señalización Intracelular , Ligasas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Mitocondriales/metabolismo , Mutación , Estructura Terciaria de Proteína , Proteínas/genética , Serina Endopeptidasas/genética , Especificidad por Sustrato , Ligando Inductor de Apoptosis Relacionado con TNF , Factor de Necrosis Tumoral alfa/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas

RESUMEN

To reveal the possible role of the amino-terminal domain of G protein-coupled receptor kinases(GRKs)in receptor phosphorylation and/or modulation of its kinase activity, a truncated mutant of GRK-2 lacking the amino-terminal domain(deltaN-GRK2)was made. deltaN-GRK2 was expressed effectively in E.coli as a GST fusion protein and was purified by affinity chromatography on a GSH-Sepharose column. deltaN-GRK2 was then separated from GST tag by thrombin cleavage and recovered. Although deltaN-GRK2 had nearly identical activity with wild-type GRK-2 in phosphorylation of peptide substrate, it completely lost the ability to phosphorylate the light-activated receptor rhodopsin. Furthermore, deletion of the amino-terminal domain rendered GRK-2 unresponsive to the regulation of kinase activity by a truncated form of rhodopsin, (329)G-Rho(*) and beta gamma subunits of G protein. These results demonstrated that the amino-terminal domain was necessary to GRK2 for both the phosphorylation of receptor and the regulation of its kinase activity by the receptor. It was reasonable to postulate that this domain has little, if any effect on the catalytic domain of natural form of GRK2.

RESUMEN

Proteomics is a new research area of the post-genomic era that aims at the analysis and identification of the entire proteins present in the cell, tissue or organism, and of the functions and the linkages of these proteins. Protein-protein interactions are characteristic of cellular activities and of course an important part of proteomics. One of the effective techniques to detect protein-protein interaction is yeast two-hybrid system. This system was successfully employed to analyze the protein linkage map of the yeast mRNA splicing machinery, which boded well for its application on human proteome research.