Physiological relevance of fructose 2,6-bisphosphate in the regulation of spinach leaf pyrophosphate:fructose 6-phosphate 1-phosphotransferase

A major problem in defining the physiological role of pyrophosphate:fructose 6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90) is the 1,000-fold discrepancy between the apparent affinity of PFP for its activator, fructose 2,6-bisphosphate (Fru-2,6-P₂), determined under optimum conditions in vitro and the estimated concentration of this signal metabolite in vivo. The aim of this study was to investigate the combined influence of metabolic intermediates and inorganic phosphate (Pi) on the activation of PFP by Fru-2,6-P₂. The enzyme was purified to near-homogeneity from leaves of spinach (Spinacia oleracea L.). Under optimal in vitro assay conditions, the activation constant (K _a) of spinach leaf PFP for Fru-2,6-P₂ in the glycolytic direction was 15.8 nM. However, in the presence of physiological concentrations of fructose 6-phosphate, inorganic pyrophosphate (PPi), 3-phosphoglycerate (3PGA), phosphoenolpyruvate (PEP), ATP and Pi the K _a of spinach leaf PFP for Fru-2,6-P₂ was up to 2000-fold greater than that measured in the optimised assay and V _max decreased by up to 62%. Similar effects were observed with PFP purified from potato (Solanum tuberosum L.) tubers. Cytosolic metabolites and Pi also influenced the response of PFP to activation by its substrate fructose 1,6-bisphosphate (Fru-1,6-P₂). When assayed under optimum conditions in the gluconeogenic direction, the K _a of spinach leaf PFP for Fru-1,6-P₂ was approximately 50 μM. Physiological concentrations of PPi, 3PGA, PEP, ATP and Pi increased K _a up to 25-fold, and decreased V _max by over 65%. From these results it was concluded that physiological concentrations of metabolites and Pi increase the K _a of PFP for Fru-2,6-P₂ to values approaching the concentration of the activator in vivo. Hence, measured changes in cytosolic Fru-2,6-P₂ levels could appreciably alter the activation state of PFP in vivo. Moreover, the same levels of metabolites increase the K _a of PFP for Fru-1,6-P₂ to an extent that activation of PFP by this compound is unlikely to be physiologically relevant. Received: 21 July 2000 / Accepted: 15 September 2000     

Down-regulation of pyrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) activity in sugarcane enhances sucrose accumulation in immature internodes

Pyrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) activity was successfully down-regulated in sugarcane using constitutively expressed antisense and untranslatable forms of the sugarcane PFP-β gene. In young internodal tissue activity was reduced by up to 70% while no residual activity could be detected in mature tissues. The transgenic plants showed no visible phenotype or significant differences in growth and development under greenhouse and field conditions. Sucrose concentrations were significantly increased in the immature internodes of the transgenic plants but not in the mature internodes. This contributed to an increase in the purity of the immature tissues, resembling an early ripening phenotype. Both the immature and mature internodes of the transgenic plants had significantly higher fibre contents. These findings suggest that PFP influences the ability of young, biosynthetically active sugarcane culm tissue to accumulate sucrose but that the equilibrium of the glycolytic intermediates, including the stored sucrose, is restored when ATP-dependent phosphofructokinase and the residual PFP activity is sufficient to sustain the required glycolytic flux as the tissue matures. Moreover, it suggests a role for PFP in glycolytic carbon flow, which could be rate limiting under conditions of high metabolic activity.     

Altered expression of pyrophosphate: Fructose-6-phosphate 1-phosphotransferase affects the growth of transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis> plants

Pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) catalyzes the reversible interconversion of fructose-6-phosphate and fructose-1,6-bisphosphate, a key step in the regulation of the metabolic flux toward glycolysis or gluconeogenesis. To examine the role of PFP in plant growth, we have generated transgenic Arabidopsis plants that either overexpress or repress Arabidopsis PFP sub-unit genes. The overexpressing lines displayed increased PFP activity and slightly faster growth relative to wild type plants, although their photosynthetic activities and the levels of metabolites appeared not to have significantly changed. In contrast, the RNAi lines showed significantly retarded growth in parallel with the reduced PFP activity. Analysis of photosynthetic activity revealed that the growth retardation phenotype of the RNAi lines was accompanied by the reduced rates of CO₂ assimilation. Microarray analysis of our transgenic plants further revealed that the altered expression of AtPFPβ affects the expression of several genes involved in diverse physiological processes. Our current data thus suggest that PFP is important in carbohydrate metabolism and other cellular processes. These authors contributed equally to this study.     

Transgenic tobacco plants with strongly decreased expression of pyrophosphate: Fructose-6-phosphate 1-phosphotransferase do not differ significantly from wild type in photosynthate partitioning,plant growth or their ability to cope with limiting phosphate,limiting nitrogen and suboptimal temperatures

Transformation of tobacco with the potato gene encoding the subunit of pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) in the antisense orientation under the control of the constitutive CaMV 35S promoter, followed by selfing and crossing of the transformants, generated a line of tobacco (5–37) with up to an 85% reduction in PFP activity in the shoot. Transformants containing a sense construct (4-40-91) contained only 1–3% of wild-type PFP, presumably due to co-suppression. Rates of photosynthesis and partitioning between sucrose and starch in source leaves were identical in 4-40-91 transformants and the wild type. In the dark in sink leaves of 4-40-91 transformants, levels of hexose phosphates were up to 50% higher, glycerate-3-phosphate 30% lower and fructose-2,6-bisphosphate threefold higher than in the wild type; inorganic pyrophosphate, pyruvate and the ATP/ADP ratio were unaltered. Low -PFP and wild-type plants did not differ significantly in their rate of growth at 25° C and 200 mol quanta · m^–2 · s^–1 on full nutrient medium. Growth on limiting phosphate and limiting nitrogen was inhibited identically in the wild type and transformants, and transformants adjusted their shoot/root ratio in an identical manner to the wild type. Differences in fructose-2,6-bisphosphate and glycolytic metabolites between the wild type and transformants were no larger in these suboptimal nutrient conditions, than in optimal conditions. Growth of the wild type and 4-40-91 transformants was inhibited identically at 12° C compared to 25° C. Differences in fructose-2,6-bisphosphate were smaller when the genotypes were compared at 12° C than at 25° C. We conclude that PFP does not play an essential role in photosynthate partitioning in source leaves. During respiratory metabolism in sink leaves it catalyzes a net glycolytic flux, as in potato tubers. However, tobacco seedlings are able to compensate for a large decrease in expression of PFP without loss of growth, or the ability to cope with suboptimal phosphate, nitrogen or temperature.Abbreviations F2,6BP fructose-2,6-bisphosphate - F6P fructose-6-phosphate - G6P glucose-6-phosphate - PFK phosphofructokinase - PFP pyrophosphate-dependent fructose-6-phosphate 1-phosphotransferase - 3-PGA glycerate-3-phosphate - PPi inorganic pyrophosphate - PEP phosphoenolpyruvate This work was supported by the Bundesministerium für Forschung and Technologie (M.S, U.S.) and the Canadian Research Council (S.C., D.D). M.P. was supported by a Royal Society Fellowship.     

Fructose 2,6-bisphosphate in relation with the resumption of metabolic activity in slices of Jerusalem artichoke tubers

When slices of Jerusalem artichoke tubers were incubated at 25°C, their concentration in fructose 2,6-bisphosphate increased up to 250-fold within 2 h. Fructose 2,6-bisphosphate was also formed, although at a slower rate, in slices incubated at 0°C. Its formation could not be explained by an increase in the concentration of fructose 6-phosphate or of ATP either by an activation of phosphofructo-2-kinase. Pyrophosphate—fructose-6-phosphate 1-phosphotransferase was the only enzyme present in a tuber extract which was found to be sensitive to fructose 2,6-bisphosphate. An improved procedure for the assay of fructose 2,6-bisphosphate is also reported.     

蛇肌果糖1，6－二磷酸酯酶、果糖2，6－二磷酸、AMP的结合部位关系

腺苷－磷酸（ＡＭＰ）对４个快反应巯基被修饰的蛇肌果糖１,６－二磷酸酯酶活性的抑制作用增强,而该修饰的酶受果糖２,６－二磷酸的抑制脱敏。ＡＭＰ对酶抑制为半部位反应,酶受果糖２,６－二磷酸抑制的脱敏则表现为全部位反应。经枯草杆菌蛋白酶限制性酶解的果糖１,６－二磷酸酯酶的Ｋｉ（ＡＭＰ）增大１０倍,但受果糖２,６－二磷酸抑制的性质不变。经胰蛋白酶限制性酶解的果糖１,６－二磷酸酯酶的活性不再为ＡＭＰ抑制,但果糖２,６－二磷酸对该形式酶的抑制作用则明显增强,由于该酶失去受ＡＭＰ的抑制作用,因此ＡＭＰ促进果糖２,６－二磷酸抑制的性质亦随之丧失。据此提出在蛇肌果糖１,６－二磷酸酯酶中果糖２,６－二磷酸不是结合在ＡＭＰ结合部位上的看法。     

Transgenic potato plants with strongly decreased expression of pyrophosphate:fructose-6-phosphate phosphotransferase show no visible phenotype and only minor changes in metabolic fluxes in their tubers

Potato (Solanum tuberosum L.) plants were transformed with antisense constructs to the genes encoding the -and -subunits of pyrophosphate: fructose-6-phosphate phosphotransferase (PEP), their expression being driven by the constitutive CaMV 35S promotor. (i) In several independent transformant lines, PFP expression was decreased by 70–90% in growing tubers and by 88–99% in stored tubers. (ii) The plants did not show any visual phenotype, reduction of growth or decrease in total tuber yield. However, the tubers contained 20–40% less starch than the wild type. Sucrose levels were slightly increased in growing tubers, but not at other stages. The rates of accumulation of sucrose and free hexoses when tubers were stored at 4° C and the final amount accumulated were the same in antisense and wild-type tubers. (iii) Metabolites were investigated at four different stages in tuber life history; growing (sink) tubers, mature tubers, cold-sweetening tubers and sprouting (source) tubers. At all stages, compared to the wild type, antisense tubers contained slightly more hexose-phosphates, two- to threefold less glycerate-3-phosphate and phosphoenolpyruvate and up to four-to fivefold more fructose-2,6-bisphosphate. (iv) There was no accumulation or depletion of inorganic pyrophosphate (PPi), or of UDP-glucose relative to the hexose-phosphates. (v) The pyruvate content was unaltered or only marginally decreased, and the ATP/ADP ratio did not change. (vi) Labelling experiments on intact tubers did not reveal any significant decrease in the unidirectional rate of metabolism of [U-¹⁴C]sucrose to starch, organic acids or amino acids. Stored tubers with an extreme (90%) reduction of PFP showed a 25% decrease in the metabolism of [U¹⁴-C] sucrose. (vii) Metabolism (cycling) of [U-¹⁴C]glucose to surcrose increased 15-fold in discs from growing antisense tubers, compared with growing wild-type tubers. Resynthesis of sucrose was increased by 10–20% when discs from antisense and wild-type tubers stored at 4° C (cold sweetening) were compared. The conversion of [U-¹⁴C]glucose to starch was decreased by about 30% and 50%, respectively. (viii) The randomisation of [1-¹³C]glucose in the glucosyl and fructosyl moieties of sucrose was decreased from 13.8 and 15.7% in the wild type to 3.6 and 3.9% in an antisense transformant. Simultaneously, randomisation in glucosyl residues isolated from starch was reduced from 14.4 to 4.1%. (ix) These results provide evidence that PFP catalyses a readily reversible reaction in tubers, which is responsible for the recycling of label from triose-phosphates to hexose-phosphates, but with the net reaction in the glycolytic direction. The results do not support the notion that PFP is involved in regulating the cytosolic PPi concentration. They also demonstrate that PFP does not control the rate of glycolysis, and that tubers contain exessive capacity to phosphorylate fructose-6-phosphate. The decreased concentration of phosphoenolpyruvate and glycerate-3-phosphate compensates for the decrease of PFP protein by stimulating ATP-dependent phosphofructokinase, and by stimulating fructose-6-phosphate,2-kinase to increase the fructose-2,6-bisphosphate concentration and activate the residual PFP. The decreased starch accumulation is explained as an indirect effect, caused by the increased rate of resynthesis (cycling) of sucrose in the antisense tubers.Abbreviations Fru1,6bisP fructose-1,6-bisphosphate - Fru2,6bisP fructose-2,6-bisphosphate - Fru6P fructose-6-phosphate - Glc1P glucose-1-phosphate - Glc6P glucose-6-phosphate - NMR nuclear magnetic resonance - 3PGA glycerate-3-phosphate - PEP phosphoenolpyruvate - PEP pyrophosphate: fructose-6-phosphate phosphotransferase - PFK phosphofructokinase - UDPGlc UDP glucose - WT wild type This research was supported by the Bundesministerium for Forschung and Technology (M.S., U.S.), the Canadian Research Council (S.C., D.D.), the Agricultural and Food Research Council (R.V.) and Sandoz Agro Ltd. (M.H., M.S.).     

Pyrophosphate:fructose-6-phosphate 1-phosphotransferase from barley seedlings. Isolation, subunit composition and kinetic Characterization

Pyrophosphate:fructose-6-phosphate I-phosphotransferase (PFP: EC 2.7.1.90) was purified 260-fold from leaves of etiolated barley seedlings. The purified enzyme consisted of two subunits, with apparent molecular masses of 65 (α) and 60 (β) kDa. Polyclonal antibodies were raised against the denatured PFP protein eluted from an SDS-polyacrylamide gel. The antibodies recognized both denatured and native PFP. Western blots of crude extracts showed that the activity of PFP in barley leaves is correlated to the amount of PFP protein, and that both the α- and the β-subunits are present in near stoichiometric amounts in all investigated tissues. The apparent molecular mass of the boloenzyme. as determined by gel filtration chromatography, was dependent on the presence of pyrophosphate. In absence of pyrophosphate. barley PFP elutes as a heterotetramer whereas it elutes as a heterooctamer in the presence of 20 m M pyrophosphate. Pure PFP obtained by gel filtration chromatography in the presence of 20 m M pyropnosphaie reached a specific activity of 28 U mg⁻¹. Barley PFP was characterized with respect 10 kinetic properties in the forward direction (use of PP₁) and in the reverse direction (formation of PP₁). The affinity for the activator Fru-2.6-P₂: was very high, with an estimated K₃ of 2.8 n M when PFP activity was assayed in the forward direction.     

The subcellular location and characteristics of pyrophosphate-fructose-6-phosphate 1-phosphotransferase from suspension-cultured cells of soybean

The cytoplasm was identified as the probable location of pyrophosphate-fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.90) in suspension-cultured cells of soybean (Glycine max L.). The characteristics of the partially purified enzyme were investigated. The activity was strongly dependent on the presence of fructose 2,6-bisphosphate and this activator exerted its effects through a dramatic increase in the affinity of the enzyme for its substrates, fructose 6-phosphate and inorganic pyrophosphate. Saturation curves for all substrates were hyperbolic. The apparent molecular weight of the partially purified enzyme was 183000 by gel filtration chromatography and 128000 by sucrose-density-gradient centrifugation. The activation by fructose 2,6-bisphosphate was not accompanied by any measurable change in molecular weight. The possible role of this enzyme in the metabolism of non-photosynthetic sink tissues is discussed.Abbreviations PFP pyrophosphate-fructose-6-phosphate 1-phosphotransferase - P_i phosphate - PP_i pyrophosphate     

Activation of pyrophosphate:fructose-6-phosphate 1-phosphotransferase by fructose 2,6-bisphosphate stimulates conversion of hexose phosphates to triose phosphates but does not influence accumulation of carbohydrates in phosphate-deficient tobacco cells

The aim of this work was to investigate the contribution of fructose 2,6-bisphosphate to the regulation of carbohydrate metabolism under phosphate stress. The study exploited heterotrophic tobacco callus lines expressing a modified mammalian 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase that increased the fructose 2,6-bisphosphate content of the tissue. The phosphate status of two transgenic and one untransformed cell line was perturbed by incubation with 2-deoxyglucose, a phosphate-sequestering agent, and by growth of callus on phosphate-depleted media. 31P-NMR spectroscopy confirmed that both treatments decreased cellular levels of inorganic phosphate and phosphorylated metabolites. Despite large decreases in the amounts of phosphate esters, UDPglucose and adenylates in response to phosphate deficiency, the fructose 2,6-bisphosphate content of each line was unaffected by 2-deoxyglucose and increased during growth on phosphate-limited media. Short-term treatment of callus with 2-deoxyglucose had only minor effects on the carbohydrate status of each line, whereas long-term phosphate deficiency caused an increase in starch and a decrease in soluble sugar content in both transgenic and control lines. There were no consistent differences between the three callus lines in metabolism of [U-14C]glucose in response to incubation with 2-deoxyglucose. In contrast, there was a decrease in partitioning of label into glycolytic products (particularly organic acids) in untransformed callus during growth on phosphate-depleted medium. This decrease was greatly attenuated in the transgenic lines with increased fructose 2,6-bisphosphate content. This suggests that the conversion of hexose phosphates to triose phosphates is constrained under phosphate-deficient conditions, and that this restriction can be relieved by activation of pyrophosphate:fructose-6-phosphate 1-phosphotransferase. However, since the transgenic and control lines did not differ in the extent to which the carbohydrate content changed in response to growth on phosphate-depleted media, it is concluded that an increase in flux through pyrophosphate:fructose-6-phosphate 1-phosphotransferase is not a major component of the metabolic response of heterotrophic tobacco cells to phosphate deficiency.     

Glucose-6-phosphate dehydrogenase in barley roots: kinetic properties and localisation of the isoforms

Two different isoforms of glucose-6-phosphate dehydrogenase (Glc6PDH; EC 1.1.1.49) have been partially purified from barley (Hordeum vulgare L., cv. Alfeo) roots. The procedure included an ammonium sulfate step, Q-Sepharose and Reactive Blue agarose chromatography, and led to 60-fold and 150-fold purification for the two enzymes, respectively. The Glc6PDH 1 isoform accounts for 17% of total activity of the enzyme in roots, and is very sensitive to the effects of NADP⁺/NADPH ratio and dithiothreitol; the Glc6PDH 2 isoform is less affected by reducing power and represents 83% of the total activity. The isoforms showed distinct pH optima, isoelectric points, K _m for glucose-6-phosphate and a different electrophoretic mobility. The kinetic properties for the two enzymes were affected by ATP and metabolites. Both enzymes are inhibited to different extents by ATP when magnesium is omitted from the assay mixture, whereas the addition of ATP-Mg²⁺ had no effect on Glc6PDH activities. The Glc6PDH isoforms are usually present in the plastids and cytosol of plant cells. To verify the intracellular locations of the enzymes purified from barley roots, Glc6PDH was purified from isolated barley root plastids; this isoform showed kinetic parameters coincident with those found for Glc6PDH 1, suggesting a plastid location; the enzyme purified from the soluble fraction had kinetic parameters resembling those of Glc6PDH 2, confirming that this isoform is present in the cytosol of barley roots. Received: 21 June 2000 / Accepted: 28 July 2000     

Subunit interactions in pig-kidney fructose-1,6-bisphosphatase: Binding of substrate induces a second class of site with lowered affinity and catalytic activity

Background

Fructose-1,6-bisphosphatase, a major enzyme of gluconeogenesis, is inhibited by AMP, Fru-2,6-P₂ and by high concentrations of its substrate Fru-1,6-P₂. The mechanism that produces substrate inhibition continues to be obscure.

Methods

Four types of experiments were used to shed light on this: (1) kinetic measurements over a very wide range of substrate concentrations, subjected to detailed statistical analysis; (2) fluorescence studies of mutants in which phenylalanine residues were replaced by tryptophan; (3) effect of Fru-2,6-P₂ and Fru-1,6-P₂ on the exchange of subunits between wild-type and Glu-tagged oligomers; and (4) kinetic studies of hybrid forms of the enzyme containing subunits mutated at the active site residue tyrosine-244.

Results

The kinetic experiments with the wild-type enzyme indicate that the binding of Fru-1,6-P₂ induces the appearance of catalytic sites with lower affinity for substrate and lower catalytic activity. Binding of substrate to the high-affinity sites, but not to the low-affinity sites, enhances the fluorescence emission of the Phe219Trp mutant; the inhibitor, Fru-2,6-P₂, competes with the substrate for the high-affinity sites. Binding of substrate to the low-affinity sites acts as a “stapler” that prevents dissociation of the tetramer and hence exchange of subunits, and results in substrate inhibition.

Conclusions

Binding of the first substrate molecule, in one dimer of the enzyme, produces a conformational change at the other dimer, reducing the substrate affinity and catalytic activity of its subunits.

General significance

Mimics of the substrate inhibition of fructose-1,6-bisphosphatase may provide a future option for combatting both postprandial and fasting hyperglycemia.     

Brain energy metabolism in glutamate-receptor activation and excitotoxicity: Role for APC/C-Cdh1 in the balance glycolysis/pentose phosphate pathway

Recent advances in the field of brain energy metabolism strongly suggest that glutamate receptor-mediated neurotransmission is coupled with molecular signals that switch-on glucose utilization pathways to meet the high energetic requirements of neurons. Failure to adequately coordinate energy supply for neurotransmission ultimately results in a positive amplifying loop of receptor over-activation leading to neuronal death, a process known as excitotoxicity. In this review, we revisited current concepts in excitotoxic mechanisms, their involvement in energy substrate utilization, and the signaling pathways that coordinate both processes. In particular, we have focused on the novel role played by the E3 ubiquitin ligase, anaphase-promoting complex/cyclosome (APC/C)-Cdh1, in cell metabolism. Our laboratory identified 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) –a key glycolytic-promoting enzyme– as an APC/C-Cdh1 substrate. Interestingly, APC/C-Cdh1 activity is inhibited by over-activation of glutamate receptors through a Ca²⁺-mediated mechanism. Furthermore, by inhibiting APC/C-Cdh1 activity, glutamate-receptors activation promotes PFKFB3 stabilization, leading to increased glycolysis and decreased pentose-phosphate pathway activity. This causes a loss in neuronal ability to regenerate glutathione, triggering oxidative stress and delayed excitotoxicity. Further investigation is critical to identify novel molecules responsible for the coupling of energy metabolism with glutamatergic neurotransmission and excitotoxicity, as well as to help developing new therapeutic strategies against neurodegeneration.     

葡萄糖6-磷酸脱氢酶调控细胞周期蛋白E1和细胞周期蛋白依赖性激酶2促进细胞增殖及其在肾透明细胞癌中的预后价值

肾透明细胞癌(clear cell renal cell carcinoma,ccRCC)是一种转移率高、预后差的细胞代谢性疾病,对其有效诊疗及预后分子标志物的研究十分重要。葡萄糖6-磷酸脱氢酶(glucose 6-phosphatedehydrogenase, G6PD)在ccRCC中高表达,并提示患者不良预后,其促进ccRCC细胞增殖的分子机制有待进一步揭示。本研究发现,降低G6PD可抑制细胞周期G₁/S期转化并显著抑制ccRCC细胞增殖。G6PD可在细胞水平调控G₁/S期转化及增殖相关因子Cyclin D1,CDK4,CDK6,Cyclin E1和CDK2基因表达。TCGA数据库分析结果表明,ccRCC 中Cyclin D1,Cyclin E1 和 CDK2的mRNA 水平显著升高,而CDK4表达无明显差异,CDK6表达却显著降低。相关性分析结果显示,G6PD与Cyclin D1呈显著负相关(P<0.0001),G6PD与CDK4,CDK6之间无显著相关性(P>0.05),G6PD与Cyclin E1(P<0.0001)以及CDK2(P<0.05)显著正相关。进一步免疫组化检测结果表明,Cyclin E1和 CDK2在ccRCC肿瘤组织中表达显著升高。生存预后分析结果显示,Cyclin D1高表达提示ccRCC患者整体预后更为良好,CDK4和CDK6表达水平在ccRCC患者总生存率预测中无意义;而Cyclin E1和CDK2高表达均可提示ccRCC患者预后不良。进一步细胞水平检测发现,Cyclin E1、CDK2表达降低可显著逆转G6PD促进ccRCC细胞增殖的能力。综上,与增殖相关因子Cyclin D1,CDK4和CDK6相比,G6PD有可能通过促进Cyclin E1和CDK2表达升高而发挥促进 ccRCC肿瘤细胞增殖的作用,并且这3者的异常高表达有望成为ccRCC患者不良预后的独立生存预测因素。     

Expression of sucrose: fructan 6-fructosyltransferase (6-SFT) and myo -inositol 1-phosphate synthase (MIPS) genes in barley (Hordeum vulgare) leaves

Fructan is an important class of non-structural carbohydrates present in cool-season grasses. Sucrose: fructan 6-fructosyltransferase (6-SFT, EC 2.4.1.10), one of the enzymes thought to be involved in grass fructan biosynthesis, catalyzes the initiation and extension of 2,6-linked fructans.Myo-inositol is a central component in several metabolic pathways in higher plants.Myo-inositol 1-phosphate synthase (MIPS) (EC 5.5.1.4), the first enzyme in inositolde novo biosynthesis, catalyzes the formation ofmyo-inositol 1-phosphate (MIP) from glucose-6-phosphate. The expression of 6-SFT and MIPS genes is compared in barley (Hordeum vulgare L.) leaves under various conditions. In cool temperature treatments, both 6-SFT and MIPS mRNAs accumulate within two days and then decline after four days. Under warm temperatures and continuous illumination, the amount of 6-SFT and MIPS mRNA gradually accumulated in detached leaves and increased significantly by 8 h. In contrast, we observed no significant changes over time in attached (control) leaves. Treating detached leaves with glucose or sucrose in the dark resulted in accumulations of both 6-SFT and MIPS mRNA. Homologous expression patterns for 6-SFT and MIPS genes suggest that they may be similarly regulated in barley leaves. Although sucrose and glucose may play important roles in the expression of 6-SFT and MIPS genes, regulation likely involves multiple factors.     

Glutamine-Fructose-6-Phosphate Transaminase 2 (GFPT2) Is Upregulated in Breast Epithelial–Mesenchymal Transition and Responds to Oxidative Stress

Breast cancer cells that have undergone partial epithelial–mesenchymal transition (EMT) are believed to be more invasive than cells that have completed EMT. To study metabolic reprogramming in different mesenchymal states, we analyzed protein expression following EMT in the breast epithelial cell model D492 with single-shot LFQ supported by a SILAC proteomics approach. The D492 EMT cell model contains three cell lines: the epithelial D492 cells, the mesenchymal D492M cells, and a partial mesenchymal, tumorigenic variant of D492 that overexpresses the oncogene HER2. The analysis classified the D492 and D492M cells as basal-like and D492HER2 as claudin-low. Comparative analysis of D492 and D492M to tumorigenic D492HER2 differentiated metabolic markers of migration from those of invasion. Glutamine-fructose-6-phosphate transaminase 2 (GFPT2) was one of the top dysregulated enzymes in D492HER2. Gene expression analysis of the cancer genome atlas showed that GFPT2 expression was a characteristic of claudin-low breast cancer. siRNA-mediated knockdown of GFPT2 influenced the EMT marker vimentin and both cell growth and invasion in vitro and was accompanied by lowered metabolic flux through the hexosamine biosynthesis pathway (HBP). Knockdown of GFPT2 decreased cystathionine and sulfide:quinone oxidoreductase (SQOR) in the transsulfuration pathway that regulates H₂S production and mitochondrial homeostasis. Moreover, GFPT2 was within the regulation network of insulin and EGF, and its expression was regulated by reduced glutathione (GSH) and suppressed by the oxidative stress regulator GSK3-β. Our results demonstrate that GFPT2 controls growth and invasion in the D492 EMT model, is a marker for oxidative stress, and associated with poor prognosis in claudin-low breast cancer.