PCL-1, uma ciclina multifuncional envolvida na regulação do metabolismo do glicogênio, germinação, divisão celular e na resposta ao estresse por cálcio em Neurospora crassa
Carregando...
Data
2016-09-15
Autores
Candido, Thiago de Souza [UNESP]
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
O fungo Neurospora crassa tem sido amplamente usado como um organismo modelo para os aspectos fundamentais da biologia dos eucariotos. Neste trabalho, foi investigado o papel funcional de uma ciclina de N. crassa (PCL-1), codificada pela ORF NCU08772 e ortóloga a Pcl10 de Saccharomyces cerevisiae. Na levedura, a proteína Pcl10, em conjunto com a proteína quinase dependente de ciclina Pho85, fosforila a enzima glicogênio sintase, a enzima regulatória da síntese de glicogênio. A fosforilação resulta na inativação da enzima e, portanto, em diminuição do acúmulo de glicogênio. A linhagem pcl-1 de N. crassa apresentou um atraso na germinação dos conídios e um retardo na progressão do ciclo celular quando comparado com a linhagem selvagem, sugerindo que esta ciclina pode regular o desenvolvimento e a divisão celular. Além disto, a linhagem nocauteada acumulou níveis mais elevados de glicogênio que a linhagem selvagem indicando o papel na regulação do metabolismo deste carboidrato. A fosforilação da enzima glicogênio sintase de N. crassa (GSN) foi analisada na linhagem nocaute através de análises de atividade enzimática, e os resultados mostraram que a GSN apresentou baixo índice de fosforilação, portanto alta atividade durante o crescimento, um resultado que pode explicar o alto acúmulo de glicogênio observado. Este resultado foi confirmado por análise de 2D-PAGE seguida por western blot, utilizando anticorpos anti-GSN. GSN apresentou na linhagem mutante isoformas menos fosforiladas que a enzima presente na linhagem selvagem. As proteínas recombinantes PHO85, PCL-1 foram utilizadas em ensaios de fosforilação in vitro da enzima GSN e os resultados mostraram que a enzima GSN foi fosforilada pelo complexo PHO85/PCL-1 no sítio putativo Ser636. O papel da ciclina na resposta ao cálcio foi investigado, e os resultados mostraram que a linhagem pcl-1 mostrou uma resistência ao estresse provocado por altas concentrações de cálcio quando comparada com a linhagem selvagem. Análises de expressão gênica por RT-qPCR foram realizadas e a linhagem pcl-1 mostrou estar envolvida na regulação de genes do metabolismo do cálcio. Os resultados indicam que a proteína PCL1 de N. crassa pode ser uma ciclina multifuncional e pode estar envolvida na regulação de vários processos celulares essenciais dependendo do ambiente.
The fungus Neurospora crassa has been widely used a model organism for the fundamental aspects of eukaryotes biology. This work investigated the functional role of a N. crassa cyclin (PCL-1), encoded by the ORF NCU08772, and orthologous to the Saccharomyces cerevisiae Pcl10 cyclin. In yeast, Pcl10 protein, together with the Pho85 cyclin-dependent protein kinase, phosphorylates the glycogen synthase enzyme, the regulatory enzyme in glycogen synthesis. Phosphorylation results in enzyme inactivation and therefore in decreased glycogen accumulation. The N. crassa pcl-1 strain showed a delay in conidia germination and in the progression of the cell cycle compared to the wild-type strain, suggesting that the cyclin may regulate development and cell division. Furthermore, the mutant strain accumulated higher glycogen levels than wild-type strain indicating its role in the regulation of the carbohydrate metabolism. The phosphorylation rate of the N. crassa glycogen synthase (GSN) was analyzed in the mutant strain by enzymatic activity assays, and the results showed that GSN was less phosphorylated during growth; therefore, high activity, and this result may explain the high glycogen accumulation observed in the mutant strain. This result was confirmed by 2D-PAGE gels followed by western blot using anti-GSN antibodies. The GSN isoforms presented in the mutant strain were less phosphorylated than the enzyme present in the wild-type strain. The recombinant proteins PHO85 and PCL-1 were used in in vitro phosphorylation assays of GSN enzyme, and the results showed that the enzyme was phosphorylated by the PHO85/PCL-1 complex at the putative site Ser636. The role of the cyclin in the response to calcium was investigated, and the results showed that the pcl-1 strain is more resistant than the wild-type strain to the stress caused by high calcium concentration. Gene expression analysis by RTqPCR was performed to analyze genes involved in calcium metabolism, and the pcl-1 strain showed to regulate the expression of some calcium metabolism genes. The results indicate that the N. crassa PCL1 cyclin may be multifunctional and may be involved in the regulation of several cellular processes depending on the environment.
The fungus Neurospora crassa has been widely used a model organism for the fundamental aspects of eukaryotes biology. This work investigated the functional role of a N. crassa cyclin (PCL-1), encoded by the ORF NCU08772, and orthologous to the Saccharomyces cerevisiae Pcl10 cyclin. In yeast, Pcl10 protein, together with the Pho85 cyclin-dependent protein kinase, phosphorylates the glycogen synthase enzyme, the regulatory enzyme in glycogen synthesis. Phosphorylation results in enzyme inactivation and therefore in decreased glycogen accumulation. The N. crassa pcl-1 strain showed a delay in conidia germination and in the progression of the cell cycle compared to the wild-type strain, suggesting that the cyclin may regulate development and cell division. Furthermore, the mutant strain accumulated higher glycogen levels than wild-type strain indicating its role in the regulation of the carbohydrate metabolism. The phosphorylation rate of the N. crassa glycogen synthase (GSN) was analyzed in the mutant strain by enzymatic activity assays, and the results showed that GSN was less phosphorylated during growth; therefore, high activity, and this result may explain the high glycogen accumulation observed in the mutant strain. This result was confirmed by 2D-PAGE gels followed by western blot using anti-GSN antibodies. The GSN isoforms presented in the mutant strain were less phosphorylated than the enzyme present in the wild-type strain. The recombinant proteins PHO85 and PCL-1 were used in in vitro phosphorylation assays of GSN enzyme, and the results showed that the enzyme was phosphorylated by the PHO85/PCL-1 complex at the putative site Ser636. The role of the cyclin in the response to calcium was investigated, and the results showed that the pcl-1 strain is more resistant than the wild-type strain to the stress caused by high calcium concentration. Gene expression analysis by RTqPCR was performed to analyze genes involved in calcium metabolism, and the pcl-1 strain showed to regulate the expression of some calcium metabolism genes. The results indicate that the N. crassa PCL1 cyclin may be multifunctional and may be involved in the regulation of several cellular processes depending on the environment.
Descrição
Palavras-chave
Polissacarídeos, Fosforilação, Neurospora crassa, Ciclo celular, Glicogênio, Protein kinase, Cyclin, Glycogen, GSN, PHO85, PCL1