Mónica Montero-Lomelí

Gene expression analysis during dengue virus infection in HepG2 cells reveals virus control of innate immune response.

OBJECTIVES Liver damage occurs during Dengue Virus infection and constitutes a characteristic of severe forms of the disease. The present study was focused on the modulation of gene expression in a human hepatic cell lineage, HepG2, in response to Dengue Virus infection. METHODS The global gene expression changes in HepG2 cells after 6, 24 and 48h of infection with Dengue Virus were investigated using a new tool of microarray data analysis and real-time PCR. RESULTS HepG2 cells infected with Dengue Virus showed alterations in several signaling pathways involved in innate immune response. The analysis of pattern recognition pathways genes demonstrated that TLR3, TLR8, RIG-I and MDA5 mRNAs were up-regulated during Dengue Virus infection along with an increase in the expression of the type I interferon, IFN-beta and pro-inflammatory cytokines IL-6, IL-8 and RANTES genes. CONCLUSIONS Our results suggest that innate immune pathways are involved in the recognition of Dengue Virus by HepG2 cells. These observations may contribute to the understanding of the inflammatory responses induced by Dengue Virus-hepatocytes interaction during dengue diseases.

Regulation of monovalent ion homeostasis and pH by the Ser-Thr protein phosphatase SIT4 in Saccharomyces cerevisiae.

A gene, SIT4, was identified as corresponding to a serine/threonine protein phosphatase and when overexpressed confers lithium tolerance in galactose medium to the budding yeast Saccharomyces cerevisiae. This gene has been previously identified as a regulator of the cell cycle and involved in nitrogen sensing. It is shown that the transcription levels ofSIT4 are induced by low concentrations of Li+in a time-dependent manner. Na+ and K+ at high concentrations, but not sorbitol, also induce transcription. As a response to Na+ or Li+stress, yeast cells lower the intracellular K+ content. This effect is enhanced in cells overexpressing SIT4, which also increase 86Rb efflux after the addition of Na+ or Li+ to the extracellular medium. Another feature of SIT4-overexpressing cells is that they maintain a more alkaline pH of 6.64 compared with 6.17 in the wild type cells. It has been proposed that the main pathway of salt tolerance in yeast is mediated by a P-type ATPase, encoded by PMR2A/ENA1. However, our results show that in a sit4 strain, expression of ENA1 is still induced by monovalent cations, and overexpression of SIT4 does not alter the amount ofENA1 transcript. These results show that SIT4acts in a parallel pathway not involving induction of transcription ofENA1 and suggest a novel function for SIT4 in response to salt stress.

A New Fluorescence-Based Method Identifies Protein Phosphatases Regulating Lipid Droplet Metabolism

In virtually every cell, neutral lipids are stored in cytoplasmic structures called lipid droplets (LDs) and also referred to as lipid bodies or lipid particles. We developed a rapid high-throughput assay based on the recovery of quenched BODIPY-fluorescence that allows to quantify lipid droplets. The method was validated by monitoring lipid droplet turnover during growth of a yeast culture and by screening a group of strains deleted in genes known to be involved in lipid metabolism. In both tests, the fluorimetric assay showed high sensitivity and good agreement with previously reported data using microscopy. We used this method for high-throughput identification of protein phosphatases involved in lipid droplet metabolism. From 65 yeast knockout strains encoding protein phosphatases and its regulatory subunits, 13 strains revealed to have abnormal levels of lipid droplets, 10 of them having high lipid droplet content. Strains deleted for type I protein phosphatases and related regulators (ppz2, gac1, bni4), type 2A phosphatase and its related regulator (pph21 and sap185), type 2C protein phosphatases (ptc1, ptc4, ptc7) and dual phosphatases (pps1, msg5) were catalogued as high-lipid droplet content strains. Only reg1, a targeting subunit of the type 1 phosphatase Glc7p, and members of the nutrient-sensitive TOR pathway (sit4 and the regulatory subunit sap190) were catalogued as low-lipid droplet content strains, which were studied further. We show that Snf1, the homologue of the mammalian AMP-activated kinase, is constitutively phosphorylated (hyperactive) in sit4 and sap190 strains leading to a reduction of acetyl-CoA carboxylase activity. In conclusion, our fast and highly sensitive method permitted us to catalogue protein phosphatases involved in the regulation of LD metabolism and present evidence indicating that the TOR pathway and the SNF1/AMPK pathway are connected through the Sit4p-Sap190p pair in the control of lipid droplet biogenesis.

TORC1 Inhibition Induces Lipid Droplet Replenishment in Yeast

ABSTRACT Lipid droplets (LDs) are intracellular structures that regulate neutral lipid homeostasis. In mammals, LD synthesis is inhibited by rapamycin, a known inhibitor of the mTORC1 pathway. In Saccharomyces cerevisiae, LD dynamics are modulated by the growth phase; however, the regulatory pathways involved are unknown. Therefore, we decided to study the role of the TORC1 pathway on LD metabolism in S. cerevisiae. Interestingly, rapamycin treatment resulted in a fast LD replenishment and growth inhibition. The discovery that osmotic stress (1 M sorbitol) also induced LD synthesis but not growth inhibition suggested that the induction of LDs in yeast is not a secondary response to reduced growth. The induction of LDs by rapamycin was due to increased triacylglycerol but not sterol ester synthesis. Induction was dependent on the TOR downstream effectors, the PP2A-related phosphatase Sit4p and the regulatory protein Tap42p. The TORC1-controlled transcriptional activators Gln3p, Gat1p, Rtg1p, and Rtg3p, but not Msn2p and Msn4p, were required for full induction of LDs by rapamycin. Furthermore, we show that the deletion of Gln3p and Gat1p transcription factors, which are activated in response to nitrogen availability, led to abnormal LD dynamics. These results reveal that the TORC1 pathway is involved in neutral lipid homeostasis in yeast.

Probiotic Saccharomyces cerevisiae strains as biotherapeutic tools: is there room for improvement?

The probiotic yeast Saccharomyces cerevisiae var boulardii is widely used as a low cost and efficient adjuvant against gastrointestinal tract disorders such as inflammatory bowel disease and treatment of several types of diarrhea, both in humans and animals. S. boulardii exerts its protective mechanisms by binding and neutralizing enteric pathogens or their toxins, by reducing inflammation and by inducing the secretion of sIgA. Although several S. cerevisiae strains have proven probiotic potential in both humans and animals, only S. boulardii is currently licensed for use in humans. Recently, some researchers started using S. boulardii as heterologous protein expression systems. Combined with their probiotic activity, the use of these strains as prophylactic and therapeutic proteins carriers might result in a positive combined effort to fight specific diseases. Here, we provide an overview of the current use of S. cerevisiae strains as probiotics and their mechanisms of action. We also discuss their potential to produce molecules with biotherapeutic application and the advantages and hurdles of this approach. Finally, we suggest future directions and alternatives for which the combined effort of specific immunomodulatory effects of probiotic S. cerevisiae strains and ability to express desired foreign genes would find a practical application.

The unfolded protein response has a protective role in yeast models of classic galactosemia.

Classic galactosemia is a human autosomal recessive disorder caused by mutations in the GALT gene (GAL7 in yeast), which encodes the enzyme galactose-1-phosphate uridyltransferase. Here we show that the unfolded protein response pathway is triggered by galactose in two yeast models of galactosemia: lithium-treated cells and the gal7Δ mutant. The synthesis of galactose-1-phosphate is essential to trigger the unfolded protein response under these conditions because the deletion of the galactokinase-encoding gene GAL1 completely abolishes unfolded protein response activation and galactose toxicity. Impairment of the unfolded protein response in both yeast models makes cells even more sensitive to galactose, unmasking its cytotoxic effect. These results indicate that endoplasmic reticulum stress is induced under galactosemic conditions and underscores the importance of the unfolded protein response pathway to cellular adaptation in these models of classic galactosemia.

H+/K+ exchange in reconstituted yeast plasma membrane vesicles.

The activity of a putative H+/K+ exchange system in the plasma membrane of yeast was studied following the alkalinization of the interior of vesicles prepared with lecithin and yeast plasma membrane containing pyranine entrapped inside. The fluorescence of pyranine was used as an indicator of the internal pH of the vesicles. The addition of monovalent cations produced an increase of the internal pH, probably due to the activity of an exchange system, allowing H+ to leave the vesicle in an exchange for the cation added. The system showed partial selectivity towards K+ against other monovalent cations, and it was inhibited by amiloride. The activity of this system required the presence of the yeast plasma membrane in the vesicles, and it did not produce important changes of the membrane potential of the vesicles. The exchange depended partially on the relative values of the internal and the external pH of the vesicles. The system shows low affinity for the cations, and appears to be different from the mitochondrial H+/K+ exchange system, which is non-selective toward the different monovalent cations. This system could be involved in the regulation of the internal pH of the cells when they accumulate high concentrations of K+.

Pyruvate decarboxylase activity is regulated by the Ser/Thr protein phosphatase Sit4p in the yeast Saccharomyces cerevisiae

Deletion of SIT4 phosphatase decreased the pyruvate decarboxylase activity, which is essential for directing the glucose flux to ethanol production. Concomitantly, a reduction in the fermentative capacity was observed. As pyruvate decarboxylase expression was not altered, its post-translational phosphorylation was studied. Immunoblot analyses using anti-phosphoserine antibodies against the affinity-purified Pdc1p showed that Pdc1p is a phosphoenzyme. Dephosphorylation of Pdc1p by alkaline phosphatase inhibited activity by 50%. Moreover, phosphorylation of Pdc1p was dependent on the growth phase, being hyperphosphorylated in the logarithmic phase, which showed to be dependent on the presence of SIT4. A comparison of the kinetic parameters of pyruvate decarboxylase in total protein extracts from WT yeast and the Δsit4 mutant revealed that the apparent K(m) values of the cofactor thiamin pyrophosphate (TPP) were 81 and 205 μM, respectively, with V(max) values of 0.294 and 0.173 μmol mg⁻¹ min⁻¹, respectively. Treatment of the purified enzyme with alkaline phosphatase increased the K(m) for TPP from 20 to 84 μM and for pyruvate from 2.3 to 4.6 mM, while the V(max) changed from 0.806 to 0.673 μmol mg⁻¹ min⁻¹. These results suggest that the Pdc1p phosphorylation dependent on SIT4 occurs at residues that change the apparent affinity for TPP and pyruvate.

Lithium‐mediated suppression of morphogenesis and growth in Candida albicans

Hyphal development in Candida albicans contributes to virulence, and inhibition of filamentation is a target for the development of antifungal agents. Lithium is known to impair Saccharomyces cerevisiae growth in galactose-containing media by inhibition of phosphoglucomutase, which is essential for galactose metabolism. Lithium-mediated phosphoglucomutase inhibition is reverted by Mg(2+). In this study we have assessed the effect of lithium upon C. albicans and found that growth is inhibited preferentially in galactose-containing media. No accumulation of glucose-1-phosphate or galactose-1-phosphate was detected when yeasts were grown in the presence of galactose and 15 mM LiCl, though we observed that in vitro lithium-mediated phosphoglucomutase inhibition takes place with an IC(50) of 2 mM. Furthermore, growth inhibition by lithium was not reverted by Mg(2+). These results show that lithium-mediated inhibition of growth in a galactose-containing medium is not due to inhibition of galactose conversion to glucose-6-phosphate but is probably due to inhibition of a signaling pathway. Deletion of the Ser-Thr protein phosphatase SIT4 and treatment with rapamycin have been shown to inhibit filamentous differentiation. We observed that C. albicans filamentation was inhibited by lithium in solid medium containing either galactose as the sole carbon source or 10% fetal bovine serum. These results suggest that suppression of hyphal outgrowth by lithium could be related to inhibition of the target of rapamycin (TOR) pathway.

Dengue virus-induced regulation of the host cell translational machinery

Dengue virus (DV)-induced changes in the host cell protein synthesis machinery are not well understood. We investigated the transcriptional changes related to initiation of protein synthesis. The human hepatoma cell line, HepG2, was infected with DV serotype 2 for 1 h at a multiplicity of infection of one. RNA was extracted after 6, 24 and 48 h. Microarray results showed that 36.5% of the translation factors related to initiation of protein synthesis had significant differential expression (Z-score >or= +/-2.0). Confirmation was obtained by quantitative real-time reverse transcription-PCR. Of the genes involved in the activation of mRNA for cap-dependent translation (eIF4 factors), eIF4A, eIF4G1 and eIF4B were up-regulated while the negative regulator of translation eIF4E-BP3 was down-regulated. This activation was transient since at 24 h post-infection levels were not significantly different from control cells. However, at 48 h post-infection, eIF4A, eIF4E, eIF4G1, eIF4G3, eIF4B, and eIF4E-BP3 were down-regulated, suggesting that cap-dependent translation could be inhibited during the progression of infection. To test this hypothesis, phosphorylation of p70S6K and 4E-BP1, which induce cap-dependent protein synthesis, was assayed. Both proteins remained phosphorylated when assayed at 6 h after infection, while infection induced dephosphorylation of p70S6K and 4E-BP1 at 24 and 48 h of infection, respectively. Taken together, these results provide biological evidence suggesting that in HepG2 cells DV sustains activation of the cap-dependent machinery at early stages of infection, but progression of infection switches protein synthesis to a cap-independent process.