RESUMEN

Gene regulatory grids (GRGs) encompass the space of all the possible transcription factor (TF)-target gene interactions that regulate gene expression, with gene regulatory networks (GRNs) representing a temporal and spatial manifestation of a portion of the GRG, essential for the specification of gene expression. Thus, understanding GRG architecture provides a valuable tool to explain how genes are expressed in an organism, an important aspect of synthetic biology and essential toward the development of the "in silico" cell. Progress has been made in some unicellular model systems (eg, yeast), but significant challenges remain in more complex multicellular organisms such as plants. Key to understanding the organization of GRGs is therefore identifying the genes that TFs bind to, and control. The application of sensitive and high-throughput methods to investigate genome-wide TF-target gene interactions is providing a wealth of information that can be linked to important agronomic traits. We describe here the methods and resources that have been developed to investigate the architecture of plant GRGs and GRNs. We also provide information regarding where to obtain clones or other resources necessary for synthetic biology or metabolic engineering.

Asunto(s)

Regulación de la Expresión Génica de las Plantas , Redes Reguladoras de Genes , Plantas/genética , Inmunoprecipitación de Cromatina/métodos , ADN de Plantas/genética , ADN de Plantas/metabolismo , Genes de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Técnicas del Sistema de Dos Híbridos

RESUMEN

αA-crystallin is a molecular chaperone and an antiapoptotic protein. This study investigated the mechanism of inhibition of apoptosis by human αA-crystallin and determined if the chaperone activity of αA-crystallin is required for the antiapoptotic function. αA-crystallin inhibited chemical-induced apoptosis in Chinese hamster ovary (CHO) cells and HeLa cells by inhibiting activation of caspase-3 and -9. In CHO cells, it inhibited apoptosis induced by the overexpression of human proapoptotic proteins, Bim and Bax. αA-crystallin inhibited doxorubicin-mediated activation of human procaspase-3 in CHO cells and it activated the PI3K/Akt cell survival pathway by promoting the phosphorylation of PDK1, Akt and phosphatase tensin homologue in HeLa cells. The phosphoinositide 3 kinase (PI3K) activity was increased by αA-crystallin overexpression but the protein content was unaltered. Downregulation of PI3K by the expression of a dominant-negative mutant or inhibition by LY294002 abrogated the ability of αA-crystallin to phosphorylate Akt. These antiapoptotic functions of αA-crystallin were enhanced in a mutant protein (R21A) that shows increased chaperone activity than the wild-type (Wt) protein. Interestingly, a mutant protein (R49A) that shows decreased chaperone activity was far weaker than the Wt protein in its antiapoptotic functions. Together, our study results show that αA-crystallin inhibits apoptosis by enhancing PI3K activity and inactivating phosphatase tensin homologue and that the antiapoptotic function is directly related to its chaperone activity.

Asunto(s)

Apoptosis/fisiología , Cristalinas/fisiología , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Células CHO , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Inhibidores de Caspasas , Cricetinae , Cricetulus , Cristalinas/genética , Citocromos c/metabolismo , Doxorrubicina/farmacología , Activación Enzimática/genética , Inhibidores Enzimáticos/metabolismo , Células HeLa , Humanos , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Proteína X Asociada a bcl-2/metabolismo

RESUMEN

Apigenin, a natural plant flavonoid with antiproliferative activity, is emerging as a promising compound for cancer prevention and therapy, but its mechanism of action remains unclear. High expression of the small heat-shock protein-27 (Hsp27) in leukemia contributes to the resistance of these cells to cancer treatments. Changes in Hsp27 phosphorylation have been associated with heat and metabolic stress, but its role in flavonoid anticancer activity has not been investigated. In this study, we examined the effect of apigenin in the regulation of Hsp27 on leukemia. We showed that apigenin does not affect Hsp27 expression but induces a bimodal phosphorylation on Ser78 and Ser82. The phosphorylation at early times was regulated by p38. At later times, Hsp27 phosphorylation was dependent on p38 activity and for some residues on PKCδ. Silencing of p38 expression reduced apigenin-induced phosphorylation on Ser15, Ser78, and Ser82, whereas silencing of PKCδ expression reduced the phosphorylation on Ser15 and Ser82 without affecting Ser78. In addition, we found that apigenin-induced PKCδ activity is mediated by p38. We also showed that the phosphorylation of Hsp27 significantly increased the susceptibility of leukemia cells to apigenin-induced apoptosis. Together, these results identify a complex signaling network regulating the cytotoxic effect of apigenin through Hsp27 phosphorylation.

Asunto(s)

Aminoácidos/metabolismo , Apigenina/farmacología , Apoptosis/efectos de los fármacos , Proteínas de Choque Térmico HSP27/metabolismo , Leucemia/patología , Línea Celular Tumoral , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Leucemia/enzimología , Modelos Biológicos , Fosforilación/efectos de los fármacos , Proteína Quinasa C-delta/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo

RESUMEN

The signaling pathways activated by the macrophage colony-stimulating factor (M-CSF) to promote survival of monocyte and macrophage lineage cells are not well established. In an effort to elucidate these pathways, we have used two cell types responsive to M-CSF: NIH 3T3 fibroblasts genetically engineered to express human M-CSF receptors (3T3-FMS cells) and human monocytes. M-CSF treatment induced M-CSF receptor tyrosine phosphorylation and recruitment of the p85 subunit of phosphatidylinositol 3-kinase (PI3K) to these receptors. These M-CSF receptor events correlated with activation of the serine/threonine kinase Akt. To clarify that PI3K products activate Akt in response to M-CSF, NIH 3T3 fibroblasts expressing mutant human M-CSF receptors (3T3-FMS(Y809F)) that fail to activate Ras in response to M-CSF also exhibit increased Akt kinase activity in response to M-CSF challenge. Furthermore, Akt appears to be the primary regulator of survival in 3T3-FMS cells, as transfection of genes encoding dominant-negative Akt isoforms into these fibroblasts blocked M-CSF-induced survival. In normal human monocytes, M-CSF increased the levels of tyrosine-phosphorylated proteins and induced Akt activation in a PI3K-dependent manner. The PI3K inhibitor LY294002 blocked M-CSF-mediated monocyte survival, an effect that was partially restored by caspase-9 inhibitors. These data suggest that M-CSF may induce cell survival through Akt-induced suppression of caspase-9 activation.

Asunto(s)

Supervivencia Celular/fisiología , Factor Estimulante de Colonias de Macrófagos/fisiología , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/metabolismo , Células 3T3 , Animales , Caspasa 9 , Caspasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Humanos , Ratones , Monocitos/efectos de los fármacos , Monocitos/enzimología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt

RESUMEN

Apoptosis is an important mechanism for regulating the numbers of monocytes and macrophages. Caspases (cysteine-aspartate-specific proteases) are key molecules in apoptosis and require proteolytic removal of prodomains for activity. Caspase-1 and caspase-3 have both been connected to apoptosis in other model systems. The present study attempted to delineate what role these caspases play in spontaneous monocyte apoptosis. In serum-free conditions, monocytes showed a commitment to apoptosis as early as 4 h in culture, as evidenced by caspase-3-like activity. Apoptosis, as defined by oligonucleosomal DNA fragmentation, was prevented by a generalized caspase inhibitor, z-VAD-FMK, and the more specific caspase inhibitor, z-DEVD-FMK. The caspase activity was specifically attributable to caspase-3 by the identification of cleavage of procaspase-3 to active forms by immunoblots and by cleavage of the fluorogenic substrate DEVD-AFC. In contrast, a caspase-1 family inhibitor, YVAD-CMK, did not protect monocytes from apoptosis, and the fluorogenic substrate YVAD-AFC failed to show an increase in activity in apoptotic monocytes. When cultured with LPS (1 microgram/ml), monocyte apoptosis was prevented, as was the activation of caspase-3. Unexpectedly, LPS did not change baseline caspase-1 activity. These findings link spontaneous monocyte apoptosis to the proteolytic activation of caspase-3.

Asunto(s)

Apoptosis/inmunología , Caspasa 1/fisiología , Caspasas/fisiología , Lipopolisacáridos/farmacología , Monocitos/citología , Monocitos/enzimología , Caspasa 3 , Inhibidores de Caspasas , Caspasas/sangre , Supervivencia Celular/inmunología , Activación Enzimática/inmunología , Humanos , Interleucina-1/metabolismo , Monocitos/inmunología , Precursores de Proteínas/metabolismo , Transducción de Señal/inmunología

RESUMEN

Caspases are cysteine proteases that mediate apoptosis by proteolysis of specific substrates. Although many caspase substrates have been identified, for most substrates the physiologic caspase(s) required for cleavage is unknown. The Bcl-2 protein, which inhibits apoptosis, is cleaved at Asp-34 by caspases during apoptosis and by recombinant caspase-3 in vitro. In the present study, we show that endogenous caspase-3 is a physiologic caspase for Bcl-2. Apoptotic extracts from 293 cells cleave Bcl-2 but not Bax, even though Bax is cleaved to an 18-kDa fragment in SK-NSH cells treated with ionizing radiation. In contrast to Bcl-2, cleavage of Bax was only partially blocked by caspase inhibitors. Inhibitor profiles indicate that Bax may be cleaved by more than one type of noncaspase protease. Immunodepletion of caspase-3 from 293 extracts abolished cleavage of Bcl-2 and caspase-7, whereas immunodepletion of caspase-7 had no effect on Bcl-2 cleavage. Furthermore, MCF-7 cells, which lack caspase-3 expression, do not cleave Bcl-2 following staurosporine-induced cell death. However, transient transfection of caspase-3 into MCF-7 cells restores Bcl-2 cleavage after staurosporine treatment. These results demonstrate that in these models of apoptosis, specific cleavage of Bcl-2 requires activation of caspase-3. When the pro-apoptotic caspase cleavage fragment of Bcl-2 is transfected into baby hamster kidney cells, it localizes to mitochondria and causes the release of cytochrome c into the cytosol. Therefore, caspase-3-dependent cleavage of Bcl-2 appears to promote further caspase activation as part of a positive feedback loop for executing the cell.

Asunto(s)

Apoptosis , Caspasas/metabolismo , Grupo Citocromo c/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Caspasa 3 , Cricetinae , Activación Enzimática , Células HL-60 , Humanos , Especificidad por Sustrato

Asunto(s)

Electroforesis en Gel Bidimensional/métodos , Endopeptidasas/metabolismo , Proteínas/aislamiento & purificación , Proteínas/metabolismo , Animales , Autorradiografía , Bovinos , Estudios de Evaluación como Asunto , Peso Molecular , Proteínas/química , Dodecil Sulfato de Sodio , Especificidad por Sustrato

RESUMEN

Saccharomyces cerevisiae mutations that cause a requirement for SSD1-v for viability were isolated, yielding one new gene, LAS1, and three previously identified genes, SIT4, BCK1/SLK1, and SMP3. Three of these genes, LAS1, SIT4, and BCK1/SLK1, encode proteins that have roles in bud formation or morphogenesis. LAS1 is essential and loss of LAS1 function causes the cells to arrest as 80% unbudded cells and 20% large budded cells that accumulate many vesicles at the mother-daughter neck. Overexpression of LAS1 results in extra cell surface projections in the mother cell, alterations in actin and SPA2 localization, and the accumulation of electron-dense structures along the periphery of both the mother cell and the bud. The nuclear localization of LAS1 suggests a role of LAS1 for regulating bud formation and morphogenesis via the expression of components that function directly in these processes.

Asunto(s)

Proteínas Fúngicas/fisiología , Genes Fúngicos , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Nucleares/fisiología , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/citología , Secuencia de Aminoácidos , Tamaño de la Célula , Pared Celular/metabolismo , Proteínas Fúngicas/genética , Datos de Secuencia Molecular , Morfogénesis , Proteínas Nucleares/genética , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/fisiología , Proteínas Quinasas/genética , Proteínas Quinasas/fisiología , Proteína Fosfatasa 2 , Proteínas Recombinantes de Fusión/biosíntesis , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo

RESUMEN

The Mu transposons of the Robertsons's Mutator transposable element system in maize are unusual in many respects, when compared to the other known plant transposon systems. The excision of these elements occurs late in somatic tissues and very rarely in the germ line. Unlike the other plant transposons, there is no experimental evidence directly linking Mu element excision and integration. We have analyzed the excision products generated by a Mu1 transposon inserted into the bronze 1 locus of maize. We find that the excision products or 'footprints' left by the Mu1 element resemble those of the other plant transposable elements, rather than those of the animal transposable element systems. We also find some novel types of footprints resembling recombinational events. We suggest that the Mu1 element can promote intrachromosomal crossovers and conversions near its site of insertion, and that this may be another mechanism by which transposons can accelerate the evolution of genomes.

Asunto(s)

Elementos Transponibles de ADN , Zea mays/genética , Secuencia de Bases , Clonación Molecular , Genes , Datos de Secuencia Molecular , Oligonucleótidos/química , Reacción en Cadena de la Polimerasa , Recombinación Genética