The Emi2 Protein of Saccharomyces cerevisiae is a Hexokinase Expressed under Glucose Limitation

Hexokinases catalyze glucose phosphorylation at the first step in glycolysis in eukaryotes. In the budding yeast Saccharomyces cerevisiae , three enzymes for glucose phosphorylation have long been known: Hxk1, Hxk2, and Glk1. In this study, we focus on Emi2, a previously uncharacterized hexokinase-like protein of S. cerevisiae . Our data show that the recombinant Emi2 protein (rEmi2), expressed in Escherichia coli , possesses glucose-phosphorylating activity in the presence of ATP and Mg ²⁺ . It was also found that rEmi2 phosphorylates not only glucose but also fructose, mannose and glucosamine in vitro . In addition, we examined changes in the level of endogenous Emi2 protein in S. cerevisiae in the presence or absence of glucose and a non-fermentable carbon source. We found that the expression of Emi2 protein is tightly suppressed during proliferation in high glucose, while it is strongly upregulated in response to glucose limitation and the presence of a non-fermentable carbon source. Our data suggest that the expression of the endogenous Emi2 protein in S. cerevisiae is regulated under the control of Hxk2 in response to glucose availability in the environment.     

Effects of growth with ethanol on fermentation and membrane fluidity of Saccharomyces cerevisiae

Saccharomyces cerevisiae HSc was grown with ethanol at concentrations up to 10% (v/v). The immediate effects of additions of externally added ethanol on CO₂ production and O₂ consumption of washed organisms were studied by stopped-flow membrane inlet quadrupole mass spectrometry. Fermentative activities of organisms grown with ethanol (0–5% v/v) showed similar sensitivities to inhibition by ethanol, whereas those grown with 10% (v/v) ethanol had become protected and were markedly less sensitive. The fluidity of subcellular membrane fractions was measured by determination of the temperature dependence of the rotational order parameter of the spin label 5-doxyl stearic acid (free radical) by electron spin resonance. Mitochondria prepared from yeasts grown with 0, 7 and 9% (v/v) ethanol showed similar overall fluidity, although differences in temperature-dependent behaviour indicate altered lipid composition or lateral phase separations. On the other hand the microsomal fraction from organisms grown with 9% ethanol showed a remarkable increase in fluidity. These data suggest that the protective effects of growth with ethanol near the limit of tolerance on fermentative activities may arise from altered plasma membrane fluidity properties.     

Radiation-Induced Senescence Reprograms Secretory and Metabolic Pathways in Colon Cancer HCT-116 Cells

Understanding the global metabolic changes during the senescence of tumor cells can have implications for developing effective anti-cancer treatment strategies. Ionizing radiation (IR) was used to induce senescence in a human colon cancer cell line HCT-116 to examine secretome and metabolome profiles. Control proliferating and senescent cancer cells (SCC) exhibited distinct morphological differences and expression of senescent markers. Enhanced secretion of pro-inflammatory chemokines and IL-1, anti-inflammatory IL-27, and TGF-β1 was observed in SCC. Significantly reduced levels of VEGF-A indicated anti-angiogenic activities of SCC. Elevated levels of tissue inhibitors of matrix metalloproteinases from SCC support the maintenance of the extracellular matrix. Adenylate and guanylate energy charge levels and redox components NAD and NADP and glutathione were maintained at near optimal levels indicating the viability of SCC. Significant accumulation of pyruvate, lactate, and suppression of the TCA cycle in SCC indicated aerobic glycolysis as the predominant energy source for SCC. Levels of several key amino acids decreased significantly, suggesting augmented utilization for protein synthesis and for use as intermediates for energy metabolism in SCC. These observations may provide a better understanding of cellular senescence basic mechanisms in tumor tissues and provide opportunities to improve cancer treatment.     

Bioenergetic Alterations of Metabolic Redox Coenzymes as NADH,FAD and FMN by Means of Fluorescence Lifetime Imaging Techniques

Metabolic FLIM (fluorescence lifetime imaging) is used to image bioenergetic status in cells and tissue. Whereas an attribution of the fluorescence lifetime of coenzymes as an indicator for cell metabolism is mainly accepted, it is debated whether this is valid for the redox state of cells. In this regard, an innovative algorithm using the lifetime characteristics of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) to calculate the fluorescence lifetime induced redox ratio (FLIRR) has been reported so far. We extended the FLIRR approach and present new results, which includes FLIM data of the various enzymes, such as NAD(P)H, FAD, as well as flavin mononucleotide (FMN). Our algorithm uses a two-exponential fitting procedure for the NAD(P)H autofluorescence and a three-exponential fit of the flavin signal. By extending the FLIRR approach, we introduced FLIRR1 as protein-bound NAD(P)H related to protein-bound FAD, FLIRR2 as protein-bound NAD(P)H related to free (unbound) FAD and FLIRR3 as protein-bound NAD(P)H related to protein-bound FMN. We compared the significance of extended FLIRR to the metabolic index, defined as the ratio of protein-bound NAD(P)H to free NAD(P)H. The statistically significant difference for tumor and normal cells was found to be highest for FLIRR1.     

非线性规划在生物代谢仿真过程中的应用

随着系统生物学研究的不断深入,生物过程网络的规模逐步拓展,其模型的复杂程度也越来越高,数值积分等常规方法很难求解生物代谢网络这类大规模复杂动态系统,客观上需要新的分析方法去应对。针对典型的酿酒酵母糖酵解途径的动态模型,首先利用Matlab进行常规的数值积分求解,以此作为参照,提出将糖酵解的动态模型离散化后利用非线性规划解题器IPOPT进行动态仿真的思路,采用不同的离散步长,计算得到不同的仿真结果。计算结果显示,通过调整计算参数,IPOPT可得到与参照一致的仿真结果,这表明非线性规划可作为分析优化生物过程这类大规模复杂动态系统的新方法,为下一步进行模型参数估计、多目标动态优化等打下基础。     

Accumulation of triosephosphate isomerase, with sequence homology to Beta amyloid peptides, in vessel walls of the newborn piglet hippocampus

We investigated whether beta-amyloid (Abeta)-like immunoreactivity was seen in the brains of newborn piglets. The immunoreactivity for Abeta(1-42) and Abeta(1-40) proteins, but not Abeta precursor protein, was present in CD68-positive perivascular cells of the hippocampus and in parts of the meninges. It was colocalized with immunoreactivity for receptor for advanced glycation end product and tumor necrosis factor-alpha. The protein with a molecular mass of 27 kDa, which was recognized by the Abeta antibodies, was identified as triosephosphate isomerase (TPI) with sequence homology to Abeta peptides by N-terminal amino acid sequencing, mass fingerprint analysis using matrix-associated laser desorption/ionization mass spectrometry, and Western blotting. Western blotting assay also revealed that detectable expression of Abeta proteins were not seen in the piglet brains. These findings indicate that TPI with sequence homology to Abeta peptides accumulates in perivascular cells of the microglia/macrophage lineage located around arterial vessels of the newborn piglet hippocampus.     

Oncostatic-Cytoprotective Effect of Melatonin and Other Bioactive Molecules: A Common Target in Mitochondrial Respiration

For several years, oncostatic and antiproliferative properties, as well as thoses of cell death induction through 5-methoxy-N-acetiltryptamine or melatonin treatment, have been known. Paradoxically, its remarkable scavenger, cytoprotective and anti-apoptotic characteristics in neurodegeneration models, such as Alzheimer’s disease and Parkinson’s disease are known too. Analogous results have been confirmed by a large literature to be associated to the use of many other bioactive molecules such as resveratrol, tocopherol derivatives or vitamin E and others. It is interesting to note that the two opposite situations, namely the neoplastic pathology and the neurodegeneration, are characterized by deep alterations of the metabolome, of mitochondrial function and of oxygen consumption, so that the oncostatic and cytoprotective action can find a potential rationalization because of the different metabolic and mitochondrial situations, and in the effect that these molecules exercise on the mitochondrial function. In this review we discuss historical and general aspects of melatonin, relations between cancers and the metabolome and between neurodegeneration and the metabolome, and the possible effects of melatonin and of other bioactive molecules on metabolic and mitochondrial dynamics. Finally, we suggest a common general mechanism as responsible for the oncostatic/cytoprotective effect of melatonin and of other molecules examined.     

The WWOX/HIF1A Axis Downregulation Alters Glucose Metabolism and Predispose to Metabolic Disorders

Recent reports indicate that the hypoxia-induced factor (HIF1α) and the Warburg effect play an initiating role in glucotoxicity, which underlies disorders in metabolic diseases. WWOX has been identified as a HIF1α regulator. WWOX downregulation leads to an increased expression of HIF1α target genes encoding glucose transporters and glycolysis’ enzymes. It has been proven in the normoglycemic mice cells and in gestational diabetes patients. The aim of the study was to determine WWOX’s role in glucose metabolism regulation in hyperglycemia and hypoxia to confirm its importance in the development of metabolic disorders. For this purpose, the WWOX gene was silenced in human normal fibroblasts, and then cells were cultured under different sugar and oxygen levels. Thereafter, it was investigated how WWOX silencing alters the genes and proteins expression profile of glucose transporters and glycolysis pathway enzymes, and their activity. In normoxia normoglycemia, higher glycolysis genes expression, their activity, and the lactate concentration were observed in WWOX KO fibroblasts in comparison to control cells. In normoxia hyperglycemia, it was observed a decrease of insulin-dependent glucose uptake and a further increase of lactate. It likely intensifies hyperglycemia condition, which deepen the glucose toxic effect. Then, in hypoxia hyperglycemia, WWOX KO caused weaker glucose uptake and elevated lactate production. In conclusion, the WWOX/HIF1A axis downregulation alters glucose metabolism and probably predispose to metabolic disorders.     

Synapses: The Brain’s Energy-Demanding Sites

The brain is one of the most energy-consuming organs in the mammalian body, and synaptic transmission is one of the major contributors. To meet these energetic requirements, the brain primarily uses glucose, which can be metabolized through glycolysis and/or mitochondrial oxidative phosphorylation. The relevance of these two energy production pathways in fulfilling energy at presynaptic terminals has been the subject of recent studies. In this review, we dissect the balance of glycolysis and oxidative phosphorylation to meet synaptic energy demands in both resting and stimulation conditions. Besides ATP output needs, mitochondria at synapse are also important for calcium buffering and regulation of reactive oxygen species. These two mitochondrial-associated pathways, once hampered, impact negatively on neuronal homeostasis and synaptic activity. Therefore, as mitochondria assume a critical role in synaptic homeostasis, it is becoming evident that the synaptic mitochondria population possesses a distinct functional fingerprint compared to other brain mitochondria. Ultimately, dysregulation of synaptic bioenergetics through glycolytic and mitochondrial dysfunctions is increasingly implicated in neurodegenerative disorders, as one of the first hallmarks in several of these diseases are synaptic energy deficits, followed by synapse degeneration.