Transcriptional response of Saccharomyces cerevisiae to potassium starvation.

BACKGROUND: Ion homeostasis is essential for every cell and aberrant cation homeostasis is related to diseases like Alzheimer's disease and epilepsy. The mechanisms responsible for cation homeostasis are only partly understood. The yeast Saccharomyces cerevisiae is an excellent organism to study fundamental aspects of cation homeostasis. In this study we investigated the transcriptional response of this yeast to potassium starvation by using Serial Analysis of Gene Expression (SAGE)-tag sequencing. RESULTS: Comparison of transcript levels in cells grown for 60 min in media without potassium with those in cells grown under standard potassium concentrations showed that the mRNA levels of 105 genes were significantly (P < 0.01) up-regulated more than 2.0-fold during potassium starvation and the mRNA levels of 172 genes significantly down-regulated. These genes belong to several functional categories. Genes involved in stress response including HSP30, YRO2 and TPO2 and phosphate metabolism including PHO84, PHO5 and SPL2 were highly up-regulated. Analysis of the promoter of PHO84 encoding a high affinity phosphate transporter, revealed that increased PHO84 RNA levels are caused by both increased Pho4-dependent transcription and decreased RNA turnover. In the latter process antisense transcription may be involved. Many genes involved in cell cycle control, and to a lesser extent genes involved in amino acid transport, were strongly down-regulated. CONCLUSIONS: Our study showed that yeast cells respond to potassium starvation in a complex way and reveals a direct link between potassium homeostasis and phosphate metabolism.

Activating transcription factor 4 mediates up-regulation of alanine aminotransferase 2 gene expression under metabolic stress.

Alanine aminotransferase (ALT) provides a molecular link between carbohydrate and amino acid metabolism. In humans, two ALT isoforms have been characterized: ALT1, cytosolic, and ALT2, mitochondrial. To gain insight into the transcriptional regulation of the ALT2 gene, we cloned and characterized the human ALT2 promoter. 5'-deletion analysis of ALT2 promoter in transiently transfected HepG2 cells and site-directed mutagenesis allowed us to identify ATF4 as a new factor involved in the transcriptional regulation of ALT2 expression. Quantitative RT-PCR assays showed that the metabolic stressors histidinol and tunicamycin increased ATF4 levels and up-regulated ALT2 in HepG2 and Huh7 cells. Consistently, knock-down of ATF4 decreased ALT2 mRNA levels in HepG2 and Huh-7 cells. Moreover, ATF4 silencing prevented the activating effect of histidinol and tunicamycin on ATF4 and ALT2 expression. Our findings point to ALT2 as an enzyme involved in the metabolic adaptation of the cell to stress.

Hepatocyte nuclear factor 4α transactivates the mitochondrial alanine aminotransferase gene in the kidney of Sparus aurata.

Alanine aminotransferase (ALT) plays an important role in amino acid metabolism and gluconeogenesis. The preference of carnivorous fish for protein amino acids instead of carbohydrates as a source of energy lead us to study the transcriptional regulation of the mitochondrial ALT (mALT) gene and to characterize the enzyme kinetics and modulation of mALT expression in the kidney of gilthead sea bream (Sparus aurata) under different nutritional and hormonal conditions. 5'-Deletion analysis of mALT promoter in transiently transfected HEK293 cells, site-directed mutagenesis and electrophoretic mobility shift assays allowed us to identify HNF4α as a new factor involved in the transcriptional regulation of mALT expression. Quantitative RT-PCR assays showed that starvation and the administration of streptozotocin (STZ) decreased HNF4α levels in the kidney of S. aurata, leading to the downregulation of mALT transcription. Analysis of the tissue distribution showed that kidney, liver, and intestine were the tissues with higher mALT and HNF4α expression. Kinetic analysis indicates that mALT enzyme is more efficient in catalyzing the conversion of L: -alanine to pyruvate than the reverse reaction. From these results, we conclude that HNF4α transactivates the mALT promoter and that the low levels of mALT expression found in the kidney of starved and STZ-treated fish result from a decreased expression of HNF4α. Our findings suggest that the mALT isoenzyme plays a major role in oxidazing dietary amino acids, and points to ALT as a target for a biotechnological action to spare protein and optimize the use of dietary nutrients for fish culture.

Algal photosynthesis as the primary driver for a sustainable development in energy, feed, and food production.

High oil prices and global warming that accompany the use of fossil fuels are an incentive to find alternative forms of energy supply. Photosynthetic biofuel production represents one of these since for this, one uses renewable resources. Sunlight is used for the conversion of water and CO2 into biomass. Two strategies are used in parallel: plant-based production via sugar fermentation into ethanol and biodiesel production through transesterification. Both, however, exacerbate other problems, including regional nutrient balancing and the world's food supply, and suffer from the modest efficiency of photosynthesis. Maximizing the efficiency of natural and engineered photosynthesis is therefore of utmost importance. Algal photosynthesis is the system of choice for this particularly for energy applications. Complete conversion of CO2 into biomass is not necessary for this. Innovative methods of synthetic biology allow one to combine photosynthetic and fermentative metabolism via the so-called Photanol approach to form biofuel directly from Calvin cycle intermediates through use of the naturally transformable cyanobacterium Synechocystis sp. PCC 6803. Beyond providing transport energy and chemical feedstocks, photosynthesis will continue to be used for food and feed applications. Also for this application, arguments of efficiency will become more and more important as the size of the world population continues to increase. Photosynthetic cells can be used for food applications in various innovative forms, e.g., as a substitute for the fish proteins in the diet supplied to carnivorous fish or perhaps--after acid hydrolysis--as a complex, animal-free serum for growth of mammalian cells in vitro.

Transactivation of cytosolic alanine aminotransferase gene promoter by p300 and c-Myb.

Alanine aminotransferase (Alt) provides a molecular link between carbohydrate and amino acid metabolism. In the cell context, the predominant Alt isozyme is located in the cytosol. To gain insight into the transcriptional regulation of the cytosolic alt gene (calt), we cloned and characterized the calt promoter from gilthead sea bream (Sparus aurata). Transient transfection of sea bass larvae cells with deleted calt promoter constructs and electrophoretic mobility shift assays allowed us to identify p300 and c-Myb as new factors in the transcriptional regulation of calt expression. Transfection studies carried out with an acetylase-deficient mutant p300 (p300DY) revealed that the acetyltransferase activity of p300 is essential for the p300-mediated transcriptional activation of S. aurata calt. We had previously found up-regulation of liver cAlt2, an alternatively spliced isoform of calt, under gluconeogenic conditions and in streptozotocin (STZ)-treated S. aurata. Quantitative RT-PCR assays showed that increased p300 and c-Myb mRNA levels in the liver of starved S. aurata contribute to enhancing the transcription of cAlt2. Consistently, the administration of insulin decreased both p300 and c-Myb expression. The mRNA levels of p300 and c-Myb were also analyzed in the liver of STZ-induced diabetic S. aurata. Treatment with STZ increased the expression of p300, whereas it decreased c-Myb. Our findings suggest an involvement of p300 and c-Myb in up-regulation of cAlt2 in the liver of S. aurata under starvation. In addition, these results provide evidence for a role of p300 in diabetes.

A novel alternatively spliced transcript of cytosolic alanine aminotransferase gene associated with enhanced gluconeogenesis in liver of Sparus aurata.

Increased alanine aminotransferase (ALT) activity is associated with insulin resistance and the development of type 2 diabetes. The aim of this study was to characterize the modulation of cytosolic ALT expression in liver of gilthead sea bream (Sparus aurata) under conditions associated with increased gluconeogenesis and in streptozotocin (STZ)-treated fish. RT- and RACE-PCR assays allowed us to isolate a novel ALT isozyme (cALT2) generated from alternative splicing of cALT gene in S. aurata. HEK293 cells transfected with constructs expressing cALT2 as a C-terminal fusion with the enhanced green fluorescent protein allowed us to demonstrate that cALT2 is cytosolic. To unravel the molecular functions of cALT1 and cALT2 in liver of S. aurata, we examined tissue distribution, kinetic characterization of piscine cALT isozymes expressed in Saccharomyces cerevisiae, and regulation of hepatic cALT1 and cALT2 expression in various metabolic conditions. Kinetic analysis indicates that cALT2 is more efficient in catalysing the conversion of l-alanine to pyruvate than cALT1. Starvation increased cALT2 expression and decreased cALT1 mRNA in liver. Opposite effects were found in regularly fed fish at postprandial time 4-8h, and 6h after treatment with glucose or insulin. From these results we conclude that increased cALT2 expression occurred in liver under gluconeogenic conditions, while cALT1 was predominant during postprandial utilization of dietary nutrients. Since up-regulation of hepatic cALT2 expression occurred in STZ-induced diabetic S. aurata, increased hepatic cALT2 expression may be a promising marker in the prognosis of diabetes.

Sterol regulatory element binding protein-1a transactivates 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene promoter.

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) catalyzes the synthesis and degradation of fructose-2,6-bisphosphate, a key modulator of glycolysis-gluconeogenesis. To gain insight into the molecular mechanism behind hormonal and nutritional regulation of PFKFB expression, we have cloned and characterized the proximal promoter region of the liver isoform of PFKFB (PFKFB1) from gilthead sea bream (Sparus aurata). Transient transfection of HepG2 cells with deleted gene promoter constructs and electrophoretic mobility shift assays allowed us to identify a sterol regulatory element (SRE) to which SRE binding protein-1a (SREBP-1a) binds and transactivates PFKFB1 gene transcription. Mutating the SRE box abolished SREBP-1a binding and transactivation. The in vivo binding of SREBP-1a to the SRE box in the S. aurata PFKFB1 promoter was confirmed by chromatin immunoprecipitation assays. There is a great deal of evidence for a postprandial rise of PFKB1 mRNA levels in fish and rats. Consistently, starved-to-fed transition and treatment with glucose or insulin increased SREBP-1 immunodetectable levels, SREBP-1 association to PFKFB1 promoter, and PFKFB1 mRNA levels in the piscine liver. Our findings demonstrate involvement of SREBP-1a in the transcriptional activation of PFKFB1, and we conclude that SREBP-1a may exert a key role mediating postprandial activation of PFKFB1 transcription.