Pisiferdiol restores the growth of a mutant yeast suffering from hyperactivated Ca 2+ signalling through calcineurin inhibition
ABSTRACT In the course of our screening program for a new inhibitor of the Ca(2+) signalling pathway using mutant yeast [Saccharomyces cerevisiae (zds1Δ erg3Δ pdr1Δ pdr3Δ)], a mouse PP2Cα activator, pisiferdiol, isolated from Chamaecyparis pisifera, was found to alleviate the Ca(2+) signal-mediated growth inhibition. Pisiferdiol showed growth inhibition activity against the mpk1Δ strain compared with the cnb1Δ strain and induced Li(+) sensitivity to the wild-type strain, indicating that it suppresses the calcineurin pathway in the yeast. However, the Li(+) sensitivity to ptc1Δ strain by pisiferdiol was diminished. Pisiferdiol showed growth restored activity in the zds1Δ strain without immunophilins Fkb1p or Cph1p, and in the pmc1Δ strain. It inhibited calcineurin-induced expression in the reporter gene assay and decreased the protein expression (Western blots) of calcineurin (Cnb1p) in addition to a decrease of Swe1p and phosphorylation of Cdc28p in the mutant yeast. These results showed that pisiferdiol could suppress indirectly the action of calcineurin and restored the growth inhibition of the mutant yeast through Ptc1p activation.
- [Show abstract] [Hide abstract]
ABSTRACT: The ubiquitin/proteasome system plays significant and important roles in the regulation of metabolism of various proteins. The dysfunction of this system is involved in several diseases, for example, cancer, neurogenic diseases and chronic inflammation. Therefore, the compounds, which regulate the ubiquitin/proteasome system, might be candidates for the development use as clinical drugs. The Saccharomyces cerevisiae mutant (rsp5(A401E)) has a single amino acid change, Ala401Glu, in the RSP5 gene, which encodes an essential E3 ubiquitin ligase, is hypersensitive to high-temperature stress. Here, we found that the immunosuppressants FK506 and cyclosporin A, both known as calcineurin inhibitors, complemented the high-temperature stress-induced growth defect of rsp5(A401E) strain. The defect of calcineurin pathway by disrupting the CNB1 and CRZ1 gene also partially complemented the high-temperature stress sensitivity of rsp5(A401E) cells. Thus, these results suggest that inhibition of the calcineurin pathway confers the tolerance to high-temperature stress on rsp5(A401E) cells. Furthermore, some diterpenoid compounds, which restore the growth of rsp5(A401E) cells, showed the activities of calcineurin inhibition and protein phosphatase 2C activation. These results indicate that calcineurin inhibitors suppress the high-temperature stress sensitivity of rsp5(A401E) cells and that analysis of their physiological function is effective for the screening of calcineurin inhibitors in yeast cells.FEMS Yeast Research 03/2014; 14(4):567-574. DOI:10.1111/1567-1364.12143