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PCL-1, a multifunctional cyclin involved in regulation of glycogen metabolism, germination, cell division and response to stress by calcium in Neurospora crassa

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Author(s):
Thiago de Souza Candido
Total Authors: 1
Document type: Doctoral Thesis
Press: Araraquara. 2016-10-04.
Institution: Universidade Estadual Paulista (Unesp). Instituto de Química. Araraquara
Defense date:
Advisor: Maria Célia Bertolini
Abstract

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. (AU)

FAPESP's process: 12/08652-4 - Protein kinases and regulation of glycogen metabolism in Neurospora crassa. Identification and functional and biochemical characterization
Grantee:Thiago de Souza Candido
Support Opportunities: Scholarships in Brazil - Doctorate