RESUMO
During exposure of yeast cells to low levels of hydrogen peroxide (H2 O2 ), the expression of several genes is regulated for cells to adapt to the surrounding oxidative environment. Such adaptation involves modification of plasma membrane lipid composition, reorganization of ergosterol-rich microdomains and altered gene expression of proteins involved in lipid and vesicle traffic, to decrease permeability to exogenous H2 O2 . Opi1p is a transcriptional repressor that is inactive when present at the nuclear membrane/endoplasmic reticulum, but represseses transcription of inositol upstream activating sequence (UASINO )-containing genes, many of which are involved in the synthesis of phospholipids and fatty acids, when it is translocated to the nucleus. We investigated whether H2 O2 in concentrations inducing adaptation regulates Opi1p function. We found that, in the presence of H2 O2 , GFP-Opi1p fusion protein translocates to the nucleus and, concomitantly, the expression of UASINO -containing genes is affected. We also investigated whether cysteine residues of Opi1p were implicated in the H2 O2 -mediated translocation of this protein to the nucleus and identified cysteine residue 159 as essential for this process. Our work shows that Opi1p is redox-regulated and establishes a new mechanism of gene regulation involving Opi1p, which is important for adaptation to H2 O2 in yeast cells. Copyright © 2017 John Wiley & Sons, Ltd.
Assuntos
Núcleo Celular/metabolismo , Retículo Endoplasmático/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Proteínas Repressoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Adaptação Biológica , Fatores de Transcrição Hélice-Alça-Hélice Básicos/efeitos dos fármacos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Ácidos Graxos/biossíntese , Peróxido de Hidrogênio/química , Concentração de Íons de Hidrogênio , Inositol/análise , Inositol/química , Microdomínios da Membrana/metabolismo , Proteínas de Transporte de Monossacarídeos/efeitos dos fármacos , Proteínas de Transporte de Monossacarídeos/genética , Mio-Inositol-1-Fosfato Sintase/efeitos dos fármacos , Mio-Inositol-1-Fosfato Sintase/genética , Oxirredução , Estresse Oxidativo , Permeabilidade , Fosfolipídeos/biossíntese , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/efeitos dos fármacosRESUMO
L-myo-inositol 1-phosphate synthase (MIPS; EC5.5.1.4) catalyses the rate-limiting step in inositol biosynthetic pathway, and is extremely widespread in living organisms including plants. Several plants possess multiple copies of MIPS gene(s) indicating a possibility of differential expression of each gene to perform distinct physiological functions. To explore this, two MIPS genes (CaMIPS1 and CaMIPS2) were isolated from a drought-tolerant plant chickpea. Both genes are extremely divergent in respect to their introns, at the same time retaining 85% identity to their exons and functionally complementing inositol auxotroph Schizosaccharomyces pombe. Expression analysis showed both genes were expressed in all organs except seed, where only CaMIPS2 transcript was detected. Under environmental stresses, only CaMIPS2 was induced whereas CaMIPS1 expression remained same, which could be explained by the divergence of their 5' upstream regulatory sequences. Remarkably, both gene products exhibited similar biochemical characteristics; however, CaMIPS2 retained higher activity than CaMIPS1 at a high temperature and salt concentration. Furthermore, functional expression of CaMIPS2 in S. pombe results better growth response than CaMIPS1 under stress environment. Taken together, our results suggest that CaMIPS1 and CaMIPS2 are differentially expressed in chickpea to play discrete though overlapping roles in plant; however CaMIPS2 is likely to be evolved through gene duplication to function under environmental stresses.
Assuntos
Cicer/genética , Mio-Inositol-1-Fosfato Sintase/genética , Mio-Inositol-1-Fosfato Sintase/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Cicer/metabolismo , Clonagem Molecular , Biologia Computacional , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Teste de Complementação Genética , Inositol/metabolismo , Dados de Sequência Molecular , Mio-Inositol-1-Fosfato Sintase/efeitos dos fármacos , Mio-Inositol-1-Fosfato Sintase/isolamento & purificação , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , RNA de Plantas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Cloreto de Sódio/farmacologia , TemperaturaRESUMO
Bipolar affective disorder is a chronic, severe, debilitating illness affecting 1-2% of the population. Valproate, along with lithium and carbamazepine, are the only drugs for which long-term efficacy has been established. However, these drugs are ineffective for, and not well tolerated by, a large number of patients and are also associated with teratogenicity and reproductive defects. Therefore, there is a substantial need to develop more effective anti-bipolar drugs. We have previously shown that valproate, like lithium, decreases intracellular inositol, which supports the inositol depletion hypothesis. We employed inositol depletion in yeast as a screening tool to identify potential new anti-bipolar medications. We show here that hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, ethylhexanoate, and methyloctanoate decrease intracellular inositol levels and increase the expression of INO1, the gene encoding myo-inositol-3-phosphate synthase (MIPS). Similar to valproate, these inositol-depleting carboxylic acids inhibited MIPS indirectly. A correlation was shown between cell growth inhibition and the increase in INO1 expression by the carboxylic acids, factors that were reversed in the presence of inositol. Inositol depletion in yeast may be exploited as an easy and inexpensive screening test for potential new inositol depleting anti-bipolar drugs.
Assuntos
Anticonvulsivantes/efeitos adversos , Antimaníacos/efeitos adversos , Transtorno Bipolar/tratamento farmacológico , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Ácidos Carboxílicos/efeitos adversos , Inositol/deficiência , Inositol/metabolismo , Carbonato de Lítio/efeitos adversos , Ácido Valproico/efeitos adversos , Leveduras/fisiologia , Bioensaio , Proliferação de Células/efeitos dos fármacos , Doença Crônica , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Mio-Inositol-1-Fosfato Sintase/efeitos dos fármacos , Mio-Inositol-1-Fosfato Sintase/genética , Proteínas de Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Lithium and valproate are the prototypic mood stabilizers and have diverse structures and targets. Both drugs influence inositol metabolism. Lithium inhibits IMPase and valproate inhibits MIP synthase. This study shows that MIP synthase inhibition does not replicate or augment the effects of lithium in the inositol sensitive pilocarpine-induced seizures model. This lack of effects may stem from the low contribution of de-novo synthesis to cellular inositol supply or to the inhibition of the de-novo synthesis by lithium itself.