José V. Gimeno-Alcañiz

AMP-activated Protein Kinase Phosphorylates R5/PTG, the Glycogen Targeting Subunit of the R5/PTG-Protein Phosphatase 1 Holoenzyme, and Accelerates Its Down-regulation by the Laforin-Malin Complex

R5/PTG is one of the glycogen targeting subunits of type 1 protein phosphatase, a master regulator of glycogen synthesis. R5/PTG recruits the phosphatase to the places where glycogen synthesis occurs, allowing the activation of glycogen synthase and the inactivation of glycogen phosphorylase, thus increasing glycogen synthesis and decreasing its degradation. In this report, we show that the activity of R5/PTG is regulated by AMP-activated protein kinase (AMPK). We demonstrate that AMPK interacts physically with R5/PTG and modifies its basal phosphorylation status. We have also mapped the major phosphorylation sites of R5/PTG by mass spectrometry analysis, observing that phosphorylation of Ser-8 and Ser-268 increased upon activation of AMPK. We have recently described that the activity of R5/PTG is down-regulated by the laforin-malin complex, composed of a dual specificity phosphatase (laforin) and an E3-ubiquitin ligase (malin). We now demonstrate that phosphorylation of R5/PTG at Ser-8 by AMPK accelerates its laforin/malin-dependent ubiquitination and subsequent proteasomal degradation, which results in a decrease of its glycogenic activity. Thus, our results define a novel role of AMPK in glycogen homeostasis.

Study of the First Hours of Microvinification by the Use of Osmotic Stress-response Genes as Probes

When yeast cells are inoculated into grape must for vinification they find stress conditions because of osmolarity, which is due to very high sugar concentration, and pH lower than 4. In this work an analysis of the expression of three osmotic stress induced genes (GPD1, HSP12 and HSP104) under microvinification conditions is shown as a way to probe those stress situations and the regulatory mechanisms that control them. The results indicate that during the first hours of microvinification there is an increase in the GPDI mRNA levels with a maximum about one hour after inoculation, and a decrease in the amount of HSP12 and HSP104 mRNAs, although with differences between them. The RNA steady-state levels of all the genes considered, and in some cases the microvinification progress are significantly affected by the composition of the must (pH, nature of the osmotic agent and carbon source). These results point out the importance of the control of these parameters and the yeast molecular response during the first hours of vinification for an accurate winemaking process.

Performance of industrial strains of Saccharomyces cerevisae during wine fermentation is affected by manipulation strategies based on sporulation.

Genetic manipulation of industrial wine yeast strains has become an essential tool for both the study of the molecular mechanisms underlaying their physiology and the improvement of their fermentative properties. The construction of null mutants for any gene in these usually diploid strains, by using a procedure based on sporulation of a heterozygote lacking one copy of the gene of interest, has been tested as an alternative to the tedious work of sequential disruption of the complete set of copies. Our results indicate that most of the homozygotes resulting from sporulation of wine yeast strains are defective in glucose consumption under microvinification conditions in synthetic must and produce stuck fermentations. These kinds of defects are observed even for strains derived from sporulation of wild type. Alteration of genomic features of wine strains by sporulation is responsible for these defects.

Response to oxidative stress caused by H2O2 in Saccharomyces cerevisiae mutants deficient in trehalase genes

Abstract. The role of trehalose as cell protector against oxidative stress induced by H2O2 has been studied in Saccharomyces cerevisiae mutants in which the two trehalase genes ATH1 and NTH1 are deleted. The addition of low H2O2 concentrations to proliferating cultures of either strain did not harm cell viability and induced a marked activity to Nth1p, but with no significant level of trehalose accumulation. This pattern was reversed after a more severe H2O2 treatment that caused drastic cell killing. The most severe phenotype corresponded to the Δnth1 mutant. Under these conditions, the increase in Nth1p was abolished and a three-fold rise in trehalose content was recorded concomitant with activation of the trehalose synthase complex. The behavior of the double-disruptant Δath1Δnth1 mutant was identical to that of wild-type cells, although in exponential cultures Ath1p activity was virtually undetectable upon exposure to H2O2. Furthermore, these strains displayed an adaptive response to oxidative stress that was independent of intracellular trehalose synthesis. Our data strongly suggest that trehalose storage in budding yeasts is not an essential protectant in cell defense against oxidative challenge.

Trivalent arsenic species induce changes in expression and levels of proinflammatory cytokines in intestinal epithelial cells.

Chronic arsenic (As) toxicity in humans has been documented in many countries where exposure mostly occurs through drinking water. The As immunotoxic effects have been demonstrated in animal models as well as in humans. The studies of the immunotoxicity of As have centered on organs related to immune response or target organs, with few data being available at intestinal level. The present study has evaluated the changes in the expression and release of cytokines in Caco-2 cells, widely used as an intestinal epithelial model. Differentiated cells were exposed to 1 μM of As(III), 0.1 μM of monomethylarsonous acid [MMA(III)] and 1 μM of dimethylarsinous acid [DMA(III)] during 2, 4, 6 and 24 h. Additionally, the effect of As coexposure with lipopolysaccharide (LPS, 10 ng/mL) has been evaluated. The results show trivalent species to induce increases in the expression and release of the proinflammatory cytokines tumor necrosis factor alpha (TNFα), IL6, IL8 - the magnitude and time of response being different for each As species. The response of greatest magnitude corresponds to DMA(III), followed by As(III), while MMA(III) generates a limited response. Furthermore, the presence of LPS in the co-exposed cells could affect the expression and secretion of cytokines compared with individual exposure to arsenicals, especially for As(III)/LPS and DMA(III)/LPS.

Acid trehalase is involved in intracellular trehalose mobilization during postdiauxic growth and severe saline stress in Saccharomyces cerevisiae

The role of the acid trehalase encoded by the ATH1 gene in the yeast Saccharomyces cerevisiae is still unclear. In this work, we investigated the regulation of ATH1 transcription and found a clear involvement of the protein kinase Hog1p in the induction of this gene under severe stress conditions, such as high salt. We also detected changes in the acid trehalase activity and trehalose levels, indicating a role of the acid trehalase in intracellular trehalose mobilization. Finally, the growth analysis for different mutants in neutral and acid trehalases after high salt stress implicates acid trehalase activity in saline stress resistance.

Differential pattern of trehalose accumulation in wine yeast strains during the microvinification process

Trehalose accumulation in wine yeast strains growing under microvinification conditions was determined and compared to that obtained under laboratory conditions. Industrial strains accumulate 10-fold more trehalose than laboratory strains. Contrary to batch-culture growth, under microvinification conditions trehalose accumulation is not consequence of glucose exhaustion. Physiological relevance of trehalose during the process of wine making and their use for potential improvements of alcoholic fermentation are discussed.