Domain 2 of Hepatitis C Virus Protein NS5A Activates Glucokinase and Induces Lipogenesis in Hepatocytes

Hepatitis C virus (HCV) relies on cellular lipid metabolism for its replication, and actively modulates lipogenesis and lipid trafficking in infected hepatocytes. This translates into an intracellular accumulation of triglycerides leading to liver steatosis, cirrhosis and hepatocellular carcinoma, which are hallmarks of HCV pathogenesis. While the interaction of HCV with hepatocyte metabolic pathways is patent, how viral proteins are able to redirect central carbon metabolism towards lipogenesis is unclear. Here, we report that the HCV protein NS5A activates the glucokinase (GCK) isoenzyme of hexokinases through its D2 domain (NS5A-D2). GCK is the first rate-limiting enzyme of glycolysis in normal hepatocytes whose expression is replaced by the hexokinase 2 (HK2) isoenzyme in hepatocellular carcinoma cell lines. We took advantage of a unique cellular model specifically engineered to re-express GCK instead of HK2 in the Huh7 cell line to evaluate the consequences of NS5A-D2 expression on central carbon and lipid metabolism. NS5A-D2 increased glucose consumption but decreased glycogen storage. This was accompanied by an altered mitochondrial respiration, an accumulation of intracellular triglycerides and an increased production of very-low density lipoproteins. Altogether, our results show that NS5A-D2 can reprogram central carbon metabolism towards a more energetic and glycolytic phenotype compatible with HCV needs for replication.

Kinetic Modeling of Saccharomyces cerevisiae Central Carbon Metabolism: Achievements, Limitations, and Opportunities

Central carbon metabolism comprises the metabolic pathways in the cell that process nutrients into energy, building blocks and byproducts. To unravel the regulation of this network upon glucose perturbation, several metabolic models have been developed for the microorganism Saccharomyces cerevisiae. These dynamic representations have focused on glycolysis and answered multiple research questions, but no commonly applicable model has been presented. This review systematically evaluates the literature to describe the current advances, limitations, and opportunities. Different kinetic models have unraveled key kinetic glycolytic mechanisms. Nevertheless, some uncertainties regarding model topology and parameter values still limit the application to specific cases. Progressive improvements in experimental measurement technologies as well as advances in computational tools create new opportunities to further extend the model scale. Notably, models need to be made more complex to consider the multiple layers of glycolytic regulation and external physiological variables regulating the bioprocess, opening new possibilities for extrapolation and validation. Finally, the onset of new data representative of individual cells will cause these models to evolve from depicting an average cell in an industrial fermenter, to characterizing the heterogeneity of the population, opening new and unseen possibilities for industrial fermentation improvement.

Integrated Bioinformatics Analysis for Identification of the Hub Genes Linked with Prognosis of Ovarian Cancer Patients

Background. One of the most usual gynecological state of tumor is ovarian cancer and is a major reason of gynecological tumor-related global mortality rate. There have been multiple risk elements related to ovarian cancer like the background of past cases associated with breast cancer or ovarian cancer, or excessive body weight issues, case history of smoking, and untimely menstruation or menopause. Because of unclear expressions, more than 70% of the ovarian cancer patient cases are determined during the early stage. Material and Methods. GSE38666, GSE40595, and GSE66957 were the three microarray datasets which were analyzed using GEO2R for screening the differentially expressed genes. GO, Kyoto Encyclopedia of Genes, and protein expression studies were performed for analysis of hub genes. Then, survival analysis was performed for all the hub genes. Results. From the dataset, a total of 199 differentially expressed genes (DEGs) were identified. Through the KEGG pathway study, it was noted that the DEGs are mainly linked with the AGE-RAGE signaling pathway, central carbon metabolism, and human papillomavirus infection. The survival analysis showed 4 highly expressed hub genes COL4A1, SDC1, CDKN2A, and TOP2A which correlated with overall survival in ovarian cancer patients. Moreover, the expression of the 4 hub genes was validated by the GEPIA database and the Human Protein Atlas. Conclusion. The results have shown that all 4 hub genes were found to be upregulated in ovarian cancer tissues which predict poor prognosis in patients with ovarian cancer.

Liquid chromatography-mass spectrometry method for isomer separation and detection of sugars, phophorylated sugars and organic acids v1

This standard operating procedure is used to achieve effective separation of a wide range of polar metabolites found in central carbon metabolism via a hybrid liquid chromatographic method (ion-exchange chromatography and hydrophilic interaction liquid chromatography (HILIC)) using an Intrada Organic Acid column (Imtakt) coupled with triple quadrupole mass spectrometry. This method gives improved resolution while showing enhanced sensitivity for the detection of low abundance phosphorylated sugars compared with standard HILIC methods.

Emergence of growth and dormancy from a kinetic model of the Escherichia coli central carbon metabolism

Physiological states of bacterial cells exhibit a wide spectrum of timescale. Under nutrient-rich conditions, most of the cells in an isogenic bacterial population grow at certain rates, while a small subpopulation sometimes stays in a dormant state where the growth rates slow down by orders of magnitude. For revealing the origins of such heterogeneity of timescales, we studied the kinetic model of Escherichia coli central carbon metabolism including the dynamics of the energy currency molecules. We found that the model robustly exhibits both the growing- and the dormant state. In order to unveil the mechanism of distinct behaviours, we developed a recursive method to simplify the model without changing the qualitative feature of the dynamics. Analytical and numerical studies of the 2-variable minimal model revealed the necessary conditions for the distinct behaviour, namely, the depletion of energy due to the futile cycle and its non-uniform impact on the kinetics because of the coexistence of the energy currency-coupled and uncoupled reactions as well as branching of the network. The result is consistent with the experimental reports that the dormant cells commonly exhibit low ATP levels, and provides a possible explanation for the appearance of dormant cells that causes antibiotic persistence.

Purine Auxotrophic Starvation Evokes Phenotype Similar to Stationary Phase Cells in Budding Yeast

Purine auxotrophy is an abundant trait among eukaryotic parasites and a typical marker for many budding yeast strains. Supplementation with an additional purine source (such as adenine) is necessary to cultivate these strains. If not supplied in adequate amounts, purine starvation sets in. We explored purine starvation effects in a model organism, a budding yeast Saccharomyces cerevisiae ade8 knockout, at the level of cellular morphology, central carbon metabolism, and global transcriptome. We observed that purine-starved cells stopped their cycle in G1/G0 state and accumulated trehalose, and the intracellular concentration of AXP decreased, but adenylate charge remained stable. Cells became tolerant to severe environmental stresses. Intracellular RNA concentration decreased, and massive downregulation of ribosomal biosynthesis genes occurred. We proved that the expression of new proteins during purine starvation is critical for cells to attain stress tolerance phenotype Msn2/4p targets are upregulated in purine-starved cells when compared to cells cultivated in purine-rich media. The overall transcriptomic response to purine starvation resembles that of stationary phase cells. Our results demonstrate that the induction of a strong stress resistance phenotype in budding yeast can be caused not only by natural starvation, but also starvation for metabolic intermediates, such as purines.

Carbon flux through photosynthesis and central carbon metabolism show distinct patterns between algae, C3 and C4 plants

Photosynthesis-related pathways are regarded as a promising avenue for crop improvement. Whilst empirical studies have shown that photosynthetic efficiency is higher in microalgae than in C3 or C4 crops, the underlying reasons remain unclear. Using a tailor-made microfluidics labelling system to supply 13CO2 at steady state, we investigated in vivo labelling kinetics in intermediates of the Calvin Benson cycle and sugar, starch, organic acid and amino acid synthesis pathways, and in protein and lipids, in Chlamydomonas reinhardtii, Chlorella sorokiniana and Chlorella ohadii, which is the fastest growing green alga on record. We estimated flux patterns in these algae and compared them with published and new data from C3 and C4 plants. Our analyses identify distinct flux patterns supporting faster growth in photosynthetic cells, with some of the algae exhibiting faster ribulose 1,5-bisphosphate regeneration and increased fluxes through the lower glycolysis and anaplerotic pathways towards the tricarboxylic acid cycle, amino acid synthesis and lipid synthesis than in higher plants.

The Antibiotic Fosfomycin Mimics the Effects of the Intermediate Metabolites Phosphoenolpyruvate and Glyceraldehyde-3-Phosphate on the Stenotrophomonas maltophilia Transcriptome

Stenotrophomonas maltophilia is an opportunistic pathogen with an environmental origin, which presents a characteristically low susceptibility to antibiotics and is capable of acquiring increased levels of resistance to antimicrobials. Among these, fosfomycin resistance seems particularly intriguing; resistance to this antibiotic is generally due to the activity of fosfomycin-inactivating enzymes, or to defects in the expression or the activity of fosfomycin transporters. In contrast, we previously described that the cause of fosfomycin resistance in S. maltophilia was the inactivation of enzymes belonging to its central carbon metabolism. To go one step further, here we studied the effects of fosfomycin on the transcriptome of S. maltophilia compared to those of phosphoenolpyruvate—its structural homolog—and glyceraldehyde-3-phosphate—an intermediate metabolite of the mutated route in fosfomycin-resistant mutants—. Our results show that transcriptomic changes present a large degree of overlap, including the activation of the cell-wall-stress stimulon. These results indicate that fosfomycin activity and resistance are interlinked with bacterial metabolism. Furthermore, we found that the studied compounds inhibit the expression of the smeYZ efflux pump, which confers intrinsic resistance to aminoglycosides. This is the first description of efflux pump inhibitors that can be used as antibiotic adjuvants to counteract antibiotic resistance in S. maltophilia.

Evolutionary footprints of a cold relic in a rapidly warming world

With accelerating global warming, understanding the evolutionary dynamics of plant adaptation to environmental change is increasingly urgent. Here we reveal the enigmatic history of the genus Cochlearia (Brassicaceae), a Pleistocene relic that originated from a drought-adapted Mediterranean sister genus during the Miocene. Cochlearia rapidly diversified and adapted to circum-Arctic regions and other cold-characterized habitat types during the Pleistocene. This sudden change in ecological preferences was accompanied by a highly complex, reticulate polyploid evolution, which was apparently triggered by the impact of repeated Pleistocene glaciation cycles. Our results illustrate that two early diversified arctic-alpine diploid gene pools contributed differently to the evolution of this young polyploid genus now captured in a cold-adapted niche. Metabolomics revealed central carbon metabolism responses to cold in diverse species and ecotypes, likely due to continuous connections to cold habitats that may have facilitated widespread adaptation to alpine and subalpine habitats, and which we speculate were coopted from existing drought adaptations. Given the growing scientific interest in adaptive evolution of temperature-related traits, our results provide much-needed taxonomic and phylogenomic resolution of a model system as well as first insights into the origins of its adaptation to cold.