RESUMEN

Recent studies have given us a clue as to how modulations of both metabolic pathways and cyclins by the ubiquitin system influence cell cycle progression. Among these metabolic modulations, an aerobic glycolysis and glutaminolysis represent an initial step for metabolic machinery adaptation. The enzymes 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) and glutaminase-1 (GLS1) maintain a high abundance in glycolytic intermediates (for synthesis of non-essential amino acids, the use of ribose for the synthesis of nucleotides and hexosamine biosynthesis), as well as tricarboxylic acid cycle intermediates (replenishing the loss of mitochondrial citrate), respectively. On the one hand, regulation of these key metabolic enzymes by ubiquitin ligases anaphase-promoting complex/cyclosome (APC/C) and Skp1/cullin/F-box (SCF) has revealed the importance of anaplerosis by both glycolysis and glutaminolysis to overcome the restriction point of the G1 phase by maintaining high levels of glycolytic and glutaminolytic intermediates. On the other hand, only glutaminolytic intermediates are necessary to drive cell growth through the S and G2 phases of the cell cycle. It is interesting to appreciate how this reorganization of the metabolic machinery, which has been observed beyond cellular proliferation, is a crucial determinant of a cell's decision to proliferate. Here, we explore a unifying view of interactions between the ubiquitin system, metabolic activity, and cyclin-dependent kinase complexes activity during the cell cycle.

Asunto(s)

Ciclosoma-Complejo Promotor de la Anafase , Fase G1 , Glucosa/metabolismo , Glutamina/metabolismo , Fase S , Proteínas Ligasas SKP Cullina F-box/metabolismo , Glucólisis

RESUMEN

Anaphase-promoting complex/cyclosome (APC/C) is a key E3 ubiquitin ligase in cell division, which catalyses ubiquitination of cell-cycle regulators. Studying this complex could reveal important information regarding its application in cancer research and therapy. In this study, 4 synthesized small interfering RNAs (siRNAs) were transfected into HEK293T cells to suppress messenger RNA (mRNA) of Apc11; 2 of these reduced the amount of Apc11 mRNA by over 50%. Further experiments showed that rather than causing apoptosis, siRNA transfection led to cell-cycle distributions characterized by less time spent in G2/M phase and more time spent in G1 phase. This phenomenon was specifically induced by Apc11 silencing, as co-transfection of siRNA and an Apc11 plasmid could reverse this distribution bias. Our results suggested that siRNA targeted against Apc11 could hamper entry into G2/M phase. Current efforts are focused on elucidating the function and utility of the APC complex for clinical applications.

Asunto(s)

División Celular , Fase G2 , Técnicas de Silenciamiento del Gen , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ciclosoma-Complejo Promotor de la Anafase , Subunidad Apc11 del Ciclosoma-Complejo Promotor de la Anafase , Células HEK293 , Humanos , ARN Interferente Pequeño/metabolismo , Transfección

RESUMEN

Body measurement traits, influenced by genes and environmental factors, play numerous important roles in the value assessment of productivity and economy. There has been some indication that ANAPC13 influences adult height. We used PCR-SSCP and DNA sequencing technology to identify polymorphisms in the ANAPC13 gene. A polymorphism in intron 1 (A > G at base 17) was identified and an additional polymorphic site (C > T at base 42) was also uncovered, which accompanied the previous polymorphism in more than 98% of the subjects. The two novel polymorphisms in exon 1 were assayed and potential associations with body measurement traits were evaluated in 404 individuals. Three genotypes were detected in the study group, named AACC, AGCT and GGTT. Significant differences were observed between genotypes AACC and AGCT for body length, withers height, hip height, hip width, heart girth, pin bone width. However, no associations were found among any genotypes and chest depth. We conclude that polymorphisms and mutations in non-coding regions of the ANAPC13 gene significantly affect body measurement traits.

Asunto(s)

Pesos y Medidas Corporales , Bovinos/anatomía & histología , Bovinos/genética , Estudios de Asociación Genética , Polimorfismo de Nucleótido Simple/genética , Carácter Cuantitativo Heredable , Complejos de Ubiquitina-Proteína Ligasa/genética , Ciclosoma-Complejo Promotor de la Anafase , Animales , Secuencia de Bases , Distribución de Chi-Cuadrado , China , Exones/genética , Frecuencia de los Genes/genética , Sitios Genéticos/genética , Genotipo , Análisis de los Mínimos Cuadrados , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple/genética , Análisis de Secuencia de ADN

RESUMEN

BACKGROUND: The orderly progression through mitosis is regulated by the Anaphase-Promoting Complex (APC), a large multiprotein E3 ubiquitin ligase that targets key cell-cycle regulators for destruction by the 26 S proteasome. The APC is composed of at least 11 subunits and associates with additional regulatory activators during mitosis and interphase cycles. Despite extensive research on APC and activator functions in the cell cycle, only a few components have been functionally characterized in plants. RESULTS: Here, we describe an in-depth search for APC subunits and activator genes in the Arabidopsis, rice and poplar genomes. Also, searches in other genomes that are not completely sequenced were performed. Phylogenetic analyses indicate that some APC subunits and activator genes have experienced gene duplication events in plants, in contrast to animals. Expression patterns of paralog subunits and activators in rice could indicate that this duplication, rather than complete redundancy, could reflect initial specialization steps. The absence of subunit APC7 from the genome of some green algae species and as well as from early metazoan lineages, could mean that APC7 is not required for APC function in unicellular organisms and it may be a result of duplication of another tetratricopeptide (TPR) subunit. Analyses of TPR evolution suggest that duplications of subunits started from the central domains. CONCLUSIONS: The increased complexity of the APC gene structure, tied to the diversification of expression paths, suggests that land plants developed sophisticated mechanisms of APC regulation to cope with the sedentary life style and its associated environmental exposures.

Asunto(s)

Evolución Molecular , Proteínas de Plantas/genética , Plantas/genética , Complejos de Ubiquitina-Proteína Ligasa/genética , Secuencia de Aminoácidos , Ciclosoma-Complejo Promotor de la Anafase , Arabidopsis/genética , Secuencia de Bases , Chlorophyta/enzimología , Chlorophyta/genética , Mapeo Cromosómico , Cromosomas de las Plantas/genética , Regulación de la Expresión Génica de las Plantas , Variación Genética , Genoma de Planta/genética , Datos de Secuencia Molecular , Oryza/genética , Filogenia , Proteínas de Plantas/clasificación , Plantas/enzimología , Populus/genética , Subunidades de Proteína/clasificación , Subunidades de Proteína/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rhodophyta/enzimología , Rhodophyta/genética , Especificidad de la Especie , Sintenía , Complejos de Ubiquitina-Proteína Ligasa/clasificación

RESUMEN

Gastric cancer is the forth most frequent malignancy and the second most common cause of cancer death worldwide. DNA methylation is the most studied epigenetic alteration, occurring through a methyl radical addition to the cytosine base adjacent to guanine. Many tumor genes are inactivated by DNA methylation in gastric cancer. We evaluated the DNA methylation status of ANAPC1, CDKN2A and TP53 by methylation-specific PCR in 20 diffuse- and 26 intestinal-type gastric cancer samples and 20 normal gastric mucosa in individuals from Northern Brazil. All gastric cancer samples were advanced stage adenocarcinomas. Gastric samples were surgically obtained at the João de Barros Barreto University Hospital, State of Pará, and were stored at -80 degrees C before DNA extraction. Patients had never been submitted to chemotherapy or radiotherapy, nor did they have any other diagnosed cancer. None of the gastric cancer samples presented methylated DNA sequences for ANAPC1 and TP53. CDKN2A methylation was not detected in any normal gastric mucosa; however, the CDKN2A promoter was methylated in 30.4% of gastric cancer samples, with 35% methylation in diffuse-type and 26.9% in intestinal-type cancers. CDKN2A methylation was associated with the carcinogenesis process for ~30% diffuse-type and intestinal-type compared to non-neoplastic samples. Thus, ANAPC1 and TP53 methylation was probably not implicated in gastric carcinogenesis in our samples. CDKN2A can be implicated in the carcinogenesis process of only a subset of gastric neoplasias.

Asunto(s)

Adenocarcinoma/genética , Metilación de ADN/genética , Genes p16 , Genes p53 , Neoplasias Gástricas/genética , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Adulto , Anciano , Ciclosoma-Complejo Promotor de la Anafase , Subunidad Apc1 del Ciclosoma-Complejo Promotor de la Anafase , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología

RESUMEN

Sister-chromatid separation and exit from mitosis require ubiquitin-mediated proteolysis of cell cycle regulators such as cyclin B and securin. The specificity of the reaction is controlled by an ubiquitin-ligase multiprotein complex known as APC (Anaphase Promoting Complex). Comparison of the coding sequences of Arabidopsis genes with the Genbank database reveals extensive homology of the predicted ORFs with the corresponding proteins of other eukaryotes, indicating that the APC is well conserved in plants. However, different from other eukaryotes, the Arabidopsis genes have some particular characteristics, such as the presence of two copies of the CDC27 gene. Furthermore, expression analyses of the AtAPC genes disclose complex profiles that differ, depending on the tissue examined. In actively dividing cell suspensions there is a direct correspondence between the rates of proliferation and mRNA levels from the AtAPC components. On the other hand, in plant organs, dark-grown seedlings and during leaf growth, this correlation is lost and the AtAPC genes are highly expressed in tissues with low overall cell division. Moreover, expression patterns diverge between the subunit genes, raising the possibility that there could be more than one form of the APC, which would execute distinct functions during plant development. The results suggest that an important layer of regulation of APC/C in plants could operate through subunit availability in specific tissues and/or cellular compartments.

Asunto(s)

Proteínas de Arabidopsis/genética , Arabidopsis/enzimología , Regulación de la Expresión Génica de las Plantas , Complejos de Ubiquitina-Proteína Ligasa/genética , Secuencia de Aminoácidos , Ciclosoma-Complejo Promotor de la Anafase , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Ciclo Celular , Regulación Enzimológica de la Expresión Génica , Datos de Secuencia Molecular , Hojas de la Planta/crecimiento & desarrollo , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Empalme del ARN , ARN Mensajero/metabolismo , Distribución Tisular , Complejos de Ubiquitina-Proteína Ligasa/química , Complejos de Ubiquitina-Proteína Ligasa/metabolismo

RESUMEN

The Mre11-Rad50-Nbs1 protein complex has emerged as a central component in the human cellular DNA damage response, and recent observations suggest that these proteins are at least partially responsible for the linking of DNA damage detection to DNA repair and cell cycle checkpoint functions. We have identified Aspergillus nidulans sldI1444D mutant in a screen for dynein synthetic lethals. The sldI(RAD50) gene was cloned by complementation of the sporulation deficiency phenotype of this mutant. A transversion G-->C at the position 2509 (Ala-692-Pro amino acid change) in the sldI1444D mutant causes sensitivity to several DNA-damaging agents. The mutation sldI1 occurs at the CXXC hinge domain of Rad50. We have deleted part of the coiled-coil and few amino acids of the Rad50-Mre11 interaction region and assessed several phenotypic traits in this deletion strain. Besides sensitivity to a number of DNA-damaging agents, this deletion strain is also impaired in the DNA replication checkpoint response, and in ascospore viability. There is no delay of the S-phase when germlings of both sldI (RAD50) and mreA(MRE11) inactivation strains were exposed to the DNA damage caused by bleomycin. Transformation experiments and Southern blot analysis indicate homologous recombination is dependent on scaA(NBS1) function in the Mre11 complex. There are epistatic and synergistic interactions between sldI( RAD50) and bimE(APC1) at S-phase checkpoints and response to hydroxyurea and UV light. Our results suggest a possible novel feature of the Mre11 complex in A. nidulans, i.e. a relationship with bimE (APC1).

Asunto(s)

Aspergillus nidulans/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ciclosoma-Complejo Promotor de la Anafase , Subunidad Apc1 del Ciclosoma-Complejo Promotor de la Anafase , Aspergillus nidulans/efectos de los fármacos , Aspergillus nidulans/efectos de la radiación , Bleomicina/farmacología , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Daño del ADN/genética , Replicación del ADN/genética , Proteínas de Unión al ADN/genética , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Epistasis Genética , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Hidroxiurea/farmacología , Meiosis , Mutación , Subunidades de Proteína , Recombinación Genética , Fase S/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Schizosaccharomyces pombe/genética , Homología de Secuencia de Aminoácido , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Rayos Ultravioleta