Transforming growth factor ß1 upregulates 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase-4 expression in A549 and MCF-10A cells.

Transforming growth factor ß1 (TGFß1) induces a cellular process known as epithelial-mesenchymal transition (EMT) associated with metabolic reprogramming, including enhanced glycolysis. Given the involvement of 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFKFB) enzymes in glycolysis, we aimed to investigate whether TGFß1 regulates expressions of PFKFB genes and if PFKFBs are required for TGFß1-driven phenotypes. A549 and MCF-10A cell lines were used as TGFß1-driven EMT models. Messenger RNA expressions of PFKFB and EMT genes were determined by real-time quantitative polymerase chain reaction. A small interfering RNA approach was used to deplete PFKFB4 expression. A Matrigel invasion assay was conducted to assess the effect of PFKFB4 silencing on the TGFß1-enhanced invasion of A549 cells. F2,6BP levels were analyzed using an enzyme-coupled assay. Glucose and lactate concentrations were determined using colorimetric assays. TGFß1 robustly induced expression of the fourth isoform of PFKFBs, PFKFB4, in both cell lines. PFKFB4 depletion partially inhibits mesenchymal transdifferentiation caused by TGFß1 in A549 cells, as assessed by microscopy. Inductions of Snail in MCF-10A cells and Fibronectin in A549 cells and repressions of E-cadherin in both cell lines by TGFß1 are attenuated by PFKFB4 silencing. PFKFB4 silencing reduces F2,6BP and glycolytic activity, although TGFß1 alone does not affect these parameters. Finally, PFKFB4 depletion suppresses the TGFß1-driven invasion of A549 cells through Matrigel. Presented data suggest that TGFß1 induces the expression of PFKFB4 in A549 and MCF-10 cells, and PFKFB4 may be required for TGFß1-driven phenotypes such as EMT and invasion in these models.

Functional and structural characterization of an endo-ß-1,3-glucanase from Euglena gracilis.

Endo-ß-1,3-glucanases from several organisms have attracted much attention in recent years because of their capability for in vitro degrading ß-1,3-glucan as a critical step for both biofuels production and short-chain oligosaccharides synthesis. In this study, we biochemically characterized a putative endo-ß-1,3-glucanase (EgrGH64) belonging to the family GH64 from the single-cell protist Euglena gracilis. The gene coding for the enzyme was heterologously expressed in a prokaryotic expression system supplemented with 3% (v/v) ethanol to optimize the recombinant protein right folding. Thus, the produced enzyme was highly purified by immobilized-metal affinity and gel filtration chromatography. The enzymatic study demonstrated that EgrGH64 could hydrolyze laminarin (KM 23.5 mg ml-1,kcat 1.20 s-1) and also, but with less enzymatic efficiency, paramylon (KM 20.2 mg ml-1,kcat 0.23 ml mg-1 s-1). The major product of the hydrolysis of both substrates was laminaripentaose. The enzyme could also use ramified ß-glucan from the baker's yeast cell wall as a substrate (KM 2.10 mg ml-1, kcat 0.88 ml mg-1 s-1). This latter result, combined with interfacial kinetic analysis evidenced a protein's greater efficiency for the yeast polysaccharide, and a higher number of hydrolysis sites in the ß-1,3/ß-1,6-glucan. Concurrently, the enzyme efficiently inhibited the fungal growth when used at 1.0 mg/mL (15.4 µM). This study contributes to assigning a correct function and determining the enzymatic specificity of EgrGH64, which emerges as a relevant biotechnological tool for processing ß-glucans.

Simultaneous inhibition of PFKFB3 and GLS1 selectively kills KRAS-transformed pancreatic cells.

Activating mutations of the oncogenic KRAS in pancreatic ductal adenocarcinoma (PDAC) are associated with an aberrant metabolic phenotype that may be therapeutically exploited. Increased glutamine utilization via glutaminase-1 (GLS1) is one such feature of the activated KRAS signaling that is essential to cell survival and proliferation; however, metabolic plasticity of PDAC cells allow them to adapt to GLS1 inhibition via various mechanisms including activation of glycolysis, suggesting a requirement for combinatorial anti-metabolic approaches to combat PDAC. We investigated whether targeting the glycolytic regulator 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) in combination with GLS1 can selectively prevent the growth of KRAS-transformed cells. We show that KRAS-transformation of pancreatic duct cells robustly sensitizes them to the dual targeting of GLS1 and PFKFB3. We also report that this sensitivity is preserved in the PDAC cell line PANC-1 which harbors an activating KRAS mutation. We then demonstrate that GLS1 inhibition reduced fructose-2,6-bisphosphate levels, the product of PFKFB3, whereas PFKFB3 inhibition increased glutamine consumption, and these effects were augmented by the co-inhibition of GLS1 and PFKFB3, suggesting a reciprocal regulation between PFKFB3 and GLS1. In conclusion, this study identifies a novel mutant KRAS-induced metabolic vulnerability that may be targeted via combinatorial inhibition of GLS1 and PFKFB3 to suppress PDAC cell growth.

Elucidating carbohydrate metabolism in Euglena gracilis: Reverse genetics-based evaluation of genes coding for enzymes linked to paramylon accumulation.

Euglena gracilis is a eukaryotic single-celled and photosynthetic organism grouped under the kingdom Protista. This phytoflagellate can accumulate the carbon photoassimilate as a linear ß-1,3-glucan chain called paramylon. This storage polysaccharide can undergo degradation to provide glucose units to obtain ATP and reducing power both in aerobic and anaerobic growth conditions. Our group has recently characterized an essential enzyme for accumulating the polysaccharide, the UDP-glucose pyrophosphorylase (Biochimie vol 154, 2018, 176-186), which catalyzes the synthesis of UDP-glucose (the substrate for paramylon synthase). Additionally, the identification of nucleotide sequences coding for putative UDP-sugar pyrophosphorylases suggests the occurrence of an alternative source of UDP-glucose. In this study, we demonstrate the active involvement of both pyrophosphorylases in paramylon accumulation. Using techniques of single and combined knockdown of transcripts coding for these proteins, we evidenced a substantial decrease in the polysaccharide synthesis from 39 ± 7 µg/106 cells determined in the control at day 21st of growth. Thus, the paramylon accumulation in Euglena gracilis cells decreased by 60% and 30% after a single knockdown of the expression of genes coding for UDP-glucose pyrophosphorylase and UDP-sugar pyrophosphorylase, respectively. Besides, the combined knockdown of both genes resulted in a ca. 65% reduction in the level of the storage polysaccharide. Our findings indicate the existence of a physiological dependence between paramylon accumulation and the partitioning of sugar nucleotides into other metabolic routes, including the Leloir pathway's functionality in Euglena gracilis.

PFKFB2 regulates glycolysis and proliferation in pancreatic cancer cells.

Tumor cells increase glucose metabolism through glycolysis and pentose phosphate pathways to meet the bioenergetic and biosynthetic demands of rapid cell proliferation. The family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) are key regulators of glucose metabolism via their synthesis of fructose-2,6-bisphosphate (F2,6BP), a potent activator of glycolysis. Previous studies have reported the co-expression of PFKFB isozymes, as well as the mRNA splice variants of particular PFKFB isozymes, suggesting non-redundant functions. Majority of the evidence demonstrating a requirement for PFKFB activity in increased glycolysis and oncogenic properties in tumor cells comes from studies on PFKFB3 and PFKFB4 isozymes. In this study, we show that the PFKFB2 isozyme is expressed in tumor cell lines of various origin, overexpressed and localizes to the nucleus in pancreatic adenocarcinoma, relative to normal pancreatic tissue. We then demonstrate the differential intracellular localization of two PFKFB2 mRNA splice variants and that, when ectopically expressed, cytoplasmically localized mRNA splice variant causes a greater increase in F2,6BP which coincides with an increased glucose uptake, as compared with the mRNA splice variant localizing to the nucleus. We then show that PFKFB2 expression is required for steady-state F2,6BP levels, glycolytic activity, and proliferation of pancreatic adenocarcinoma cells. In conclusion, this study may provide a rationale for detailed investigation of PFKFB2's requirement for the glycolytic and oncogenic phenotype of pancreatic adenocarcinoma cells.

Heterologous expression and kinetic characterization of the α, ß and αß blend of the PPi-dependent phosphofructokinase from Citrus sinensis.

This work reports the molecular cloning and heterologous expression of the genes coding for α and ß subunits of pyrophosphate-dependent phosphofructokinase (PPi-PFK) from orange. When expressed individually, both recombinant subunits were produced as highly purified monomeric proteins able to phosphorylate fructose-6-phosphate at the expenses of PPi (specific activity of 0.075 and 0.017 units. mg-1 for α and ß subunits, respectively). On the other hand, co-expression rendered a α3ß3 hexamer with specific activity three orders of magnitude higher than the single subunits. All the conformations of the enzyme were characterized with respect to its kinetic properties and sensitivity to the regulator fructose-2,6-bisphosphate. A thorough review of current knowledge on the matter indicates that this is the first report of the recombinant production of active plant PPi-PFK and the characterization of its different conformations. This is a main contribution for future studies focused to better understand the enzyme properties and how it accomplishes its relevant role in plant metabolism.

Elucidating paramylon and other carbohydrate metabolism in Euglena gracilis: Kinetic characterization, structure and cellular localization of UDP-glucose pyrophosphorylase.

Many oligo and polysaccharides (including paramylon) are critical in the Euglena gracilis life-cycle and they are synthesized by glycosyl transferases using UDP-glucose as a substrate. Herein, we report the molecular cloning of a gene putatively coding for a UDP-glucose pyrophosphorylase (EgrUDP-GlcPPase) in E. gracilis. After heterologous expression of the gene in Escherichia coli, the recombinant enzyme was characterized structural and functionally. Highly purified EgrUDP-GlcPPase exhibited a monomeric structure, able to catalyze synthesis of UDP-glucose with a Vmax of 3350 U.mg-1. Glucose-1P and UTP were the preferred substrates, although the enzyme also used (with lower catalytic efficiency) TTP, galactose-1P and mannose-1P. Oxidation by hydrogen peroxide inactivated the enzyme, an effect reversed by reduction with dithiothreitol or thioredoxin. The redox process would involve sulfenic acid formation, since no pair of the 7 cysteine residues is close enough in the 3D structure of the protein to form a disulfide bridge. Electrophoresis studies suggest that, after oxidation, the enzyme arranges in many enzymatically inactive structural conformations; which were also detected in vivo. Finally, confocal fluorescence microscopy provided evidence for a cytosolic (mainly in the flagellum) localization of the enzyme.