RESUMO
The human adenovirus 5 (HAdV-5) E1A protein has a well defined canonical nuclear localization signal (NLS) located at its C-terminus. We used a genetic assay in the yeast Saccharomyces cerevisiae to demonstrate that the canonical NLS is present and functional in the E1A proteins of each of the six HAdV species. This assay also detects a previously described non-canonical NLS within conserved region 3 and a novel active NLS within the N-terminal/conserved region 1 portion of HAdV-5 E1A. These activities were also present in the E1A proteins of each of the other five HAdV species. These results demonstrate that, despite substantial differences in primary sequence, HAdV E1A proteins are remarkably consistent in that they contain one canonical and two non-canonical NLSs. By utilizing independent mechanisms, these multiple NLSs ensure nuclear localization of E1A in the infected cell.
Assuntos
Proteínas E1A de Adenovirus/genética , Adenovírus Humanos/genética , Sinais de Localização Nuclear , Humanos , Saccharomyces cerevisiae/genéticaRESUMO
We developed a quantitative method to measure the activity of cyclin-dependent kinases (Cdks) by western blotting, without radioisotopes. We prepared a recombinant protein substrate based upon the natural Cdk1 substrate, PP1Cα. By combining this substrate in a western blot method using fluorochrome based antibodies and phospho-imager analysis, we measured the Km of ATP binding to Cdk1 to be 3.5 µM. We then measured Cdk1 activity in cell extracts from interphase or mitotic cells, and demonstrated that previously identified Cdk inhibitors could be detected by this assay. Our data show that we have a safe, reliable assay to identify Cdk1 inhibitors and measure Cdk1 activity.
Assuntos
Western Blotting/métodos , Proteína Quinase CDC2/análise , Proteína Fosfatase 1/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Sequência de Aminoácidos , Western Blotting/normas , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Escherichia coli/genética , Expressão Gênica , Células HT29 , Humanos , Células MCF-7 , Mitose/genética , Dados de Sequência Molecular , Fosforilação , Proteína Fosfatase 1/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
The biochemical pathways that lead cells to mitotic catastrophe are not well understood. To identify these pathways, we have taken an approach of treating cells with a novel genotoxic compound and characterizing whether cells enter mitotic catastrophe or not. S23906 is a novel acronycine derivative that forms adducts with the N2 residue of guanine in the minor groove of the DNA helix and destabilizes base pairing to cause helix opening. We observed, in HeLa and HT-29 cells, that S23906 induced gamma-H2AX and activated checkpoint kinase 1, as did bleomycin, camptothecin, and cisplatin, when tested under equi-toxic conditions. S23906 also induced cyclin E1 protein, although this activity was not required for cytotoxicity because knock down of cyclin E1 by RNA interference did not affect the number of dead cells after treatment. Cyclin B1 levels first decreased and then increased after treatment with S23906. Cyclin B1 was associated with Cdk1 kinase activity, which correlated with an increase in the number of mitotic cells. By 32 h after treatment, at least 20% of the cells entered mitotic catastrophe as determined by microscopy. Suppression of the DNA checkpoint response by co-treatment with caffeine increased the number of cells in mitosis. These results suggest that mitotic catastrophe is one of the cellular responses to S23906 and that mitotic catastrophe may be a common cellular response to many different types of DNA damage.
Assuntos
Acronina/análogos & derivados , DNA/metabolismo , Mitose/efeitos dos fármacos , Acronina/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Northern Blotting , Proteína Quinase CDC2/metabolismo , Cafeína/farmacologia , Ciclina B1/metabolismo , Ciclina E/antagonistas & inibidores , Ciclina E/genética , Ciclina E/metabolismo , Imunofluorescência , Células HT29 , Células HeLa , Humanos , Proteínas Oncogênicas/antagonistas & inibidores , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , RNA Interferente Pequeno/farmacologiaRESUMO
BACKGROUND: Nuclear import of proteins is typically mediated by their physical interaction with soluble cytosolic receptor proteins via a nuclear localization signal (NLS). A simple genetic assay to detect active NLSs based on their function in the yeast Saccharomyces cerevisiae has been previously described. In that system, a chimera consisting of a modified bacterial LexA DNA binding domain and the transcriptional activation domain of the yeast Gal4 protein is fused to a candidate NLS. A functional NLS will redirect the chimeric fusion to the yeast cell nucleus and activate transcription of a reporter gene. RESULTS: We have reengineered this nuclear import system to expand its utility and tested it using known NLS sequences from adenovirus E1A. Firstly, the vector has been reconstructed to reduce the level of chimera expression. Secondly, an irrelevant "stuffer" sequence from the E. coli maltose binding protein was used to increase the size of the chimera above the passive diffusion limit of the nuclear pore complex. The improved vector also contains an expanded multiple cloning site and a hemagglutinin epitope tag to allow confirmation of expression. CONCLUSION: The alterations in expression level and composition of the fusions used in this nuclear import system greatly reduce background activity in beta-galactosidase assays, improving sensitivity and allowing more quantitative analysis of NLS bearing sequences.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Proteínas E1A de Adenovirus/química , Técnicas Genéticas , Proteínas Mutantes Quiméricas/metabolismo , Sinais Direcionadores de Proteínas/fisiologia , Proteínas E1A de Adenovirus/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência Consenso , Proteínas de Ligação a DNA , Proteínas de Escherichia coli/genética , Genes Reporter , Genes Sintéticos , Vetores Genéticos/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/análise , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Carioferinas/fisiologia , Dados de Sequência Molecular , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/genética , Proteínas Periplásmicas de Ligação/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Transformação GenéticaRESUMO
Investigations of storage lipid synthesis in developing flaxseed (Linum usitatissimum) provide useful information for designing strategies to enhance the oil content and nutritional value of this crop. Lipid content and changes in the FA composition during seed development were examined in two cultivars of flax (AC Emerson and Vimy). The oil content on a dry weight basis increased steadily until about 20 d after flowering (DAF). The proportion of alpha-linolenic acid (alpha-18:3, 18:3cisDelta9,12,15) in TAG increased during seed development in both cultivars while the proportions of linoleic acid (18:2cisDelta9,12) and saturated FA decreased. The developmental and substrate specificity characteristics of microsomal DAG acyltransferase (DGAT, EC 2.3.1.20) and lysophosphatidic acid acyltransferase (LPAAT, EC 2.3.1.51) were examined using cultivar AC Emerson. The maximal acyltransferase specific activities occurred in the range of 8-14 DAF, during rapid lipid accumulation on a per seed basis. Acyl-CoA of EPA (20:5cisDelta5,8,11,14,17) or DHA (22:6 cis4,7,10,13,16,19) were included in the specificity studies. DGAT displayed enhanced specificity for alpha-18:3-CoA, whereas the preferred substrate of [PAAT was 18:2-CoA. Both enzymes could use EPA- or DHA-CoA to varying extents. Developing flax embryos were able to take up and incorporate these nutritional FA into TAG and other intermediates in the TAG-formation pathway. This study suggests that if the appropriate acyl-CoA-dependent desaturation/elongation pathways are introduced and efficiently expressed in flax, this may lead to the conversion of alpha-18:3-CoA into EPA-CoA, thereby providing an activated substrate for TAG formation.
