Fructose Diphosphate Research Articles

A novel variant of the Calvin\u2013Benson cycle bypassing fructose bisphosphate

The Calvin–Benson cycle (CB cycle) is quantitatively the most important metabolic pathway for CO2 fixation. In the canonical CB cycle, fructose 6-phosphate (F6P), fructose 1,6-bisphosphate (FBP), sedoheptulose 7-phosphate (S7P), and sedoheptulose 1,7-bisphosphate (SBP) appear as essential intermediates, where F6P is formed from FBP by the fructose 1,6-bisphosphatase (FBPase) reaction, and S7P is formed from SBP by the sedoheptulose 1,7-bisphosphatase (SBPase) reaction. Although the involvement of SBP and SBPase in the canonical CB cycle is consistent with the reported dependency of photosynthetic carbon metabolism on SBPase, the involvement of FBP and FBPase is not completely consistent with the reported FBP- or FBPase-related findings such as, although with a diminished growth rate, an Arabidopsis mutant lacking FBPase grew photoautotrophically in soil. Here, we show a novel variant of the CB cycle involving SBP, SBPase, and transaldolase, but neither FBP nor FBPase. This novel variant, named the S7P-removing transaldolase variant, bypasses FBP. This variant explains the FBP- or FBPase-related findings more easily than the canonical CB cycle as well as the dependency of photosynthetic carbon metabolism on SBPase and further suggests that co-overexpression of SBPase and transaldolase can be a strategy for enhancing photosynthetic carbon metabolism, which is important for the global environment.

Fructose-1,6-bisphosphate prevents pregnancy loss by inducing decidual COX-2+ macrophage differentiation.

Decidualization is an intricate biological process in which extensive remodeling of the endometrium occurs to support the development of an implanting blastocyst. However, the immunometabolic mechanisms underlying this process are still largely unknown. We found that the decidualization process is accompanied by the accumulation of fructose-1,6-bisphosphate (FBP). The combination of FBP with pyruvate kinase M stimulated IL-27 secretion by endometrial stromal cells in an ERK/c-FOS–dependent manner. IL-27 induced decidual COX-2+ M2-like macrophage differentiation, which promotes decidualization, trophoblast invasion, and maternal-fetal tolerance. Transfer of Ptgs2+/COX-2+ macrophages prevented fetal loss in Il27ra-deleted pregnant mice. FBP levels were low in plasma and decidual tissues of patients with unexplained recurrent spontaneous abortion. In therapeutic studies, FBP supplementation significantly improved embryo loss by up-regulation of IL-27–induced COX-2+ macrophage differentiation in a mouse model of spontaneous abortion. These findings collectively provide a scientific basis for a potential therapeutic strategy to prevent pregnancy loss.

Longitudinal Plasma Proteomics Analysis Reveals Novel Candidate Biomarkers in Acute COVID-19.

The host response to COVID-19 pathophysiology over the first few days of infection remains largely unclear, especially the mechanisms in the blood compartment. We report on a longitudinal proteomic analysis of acute-phase COVID-19 patients, for which we used blood plasma, multiple reaction monitoring with internal standards, and data-independent acquisition. We measured samples on admission for 49 patients, of which 21 had additional samples on days 2, 4, 7, and 14 after admission. We also measured 30 externally obtained samples from healthy individuals for comparison at baseline. The 31 proteins differentiated in abundance between acute COVID-19 patients and healthy controls belonged to acute inflammatory response, complement activation, regulation of inflammatory response, and regulation of protein activation cascade. The longitudinal analysis showed distinct profiles revealing increased levels of multiple lipid-associated functions, a rapid decrease followed by recovery for complement activation, humoral immune response, and acute inflammatory response-related proteins, and level fluctuation in the regulation of smooth muscle cell proliferation, secretory mechanisms, and platelet degranulation. Three proteins were differentiated between survivors and nonsurvivors. Finally, increased levels of fructose–bisphosphate aldolase B were determined in patients with exposure to angiotensin receptor blockers versus decreased levels in those exposed to angiotensin-converting enzyme inhibitors. Data are available via ProteomeXchange PXD029437.

Slow oscillations persist in pancreatic beta cells lacking phosphofructokinase M

Pulsatile insulin secretion by pancreatic beta cells is necessary for tight glucose control in the body. Glycolytic oscillations have been proposed as the mechanism for generating the electrical oscillations underlying pulsatile insulin secretion. The glycolytic enzyme 6-phosphofructokinase-1 (PFK) synthesizes fructose-1,6-bisphosphate (FBP) from fructose-6-phosphate. It has been proposed that the slow electrical and Ca2+ oscillations (periods of 3–5 min) observed in islets result from allosteric feedback activation of PFKM by FBP. Pancreatic beta cells express three PFK isozymes: PFKL, PFKM, and PFKP. A prior study of mice that were engineered to lack PFKM using a gene-trap strategy to delete Pfkm produced a mosaic reduction in global Pfkm expression, but the islets isolated from the mice still exhibited slow Ca2+ oscillations. However, these islets still expressed residual PFKM protein. Thus, to more fully test the hypothesis that beta cell PFKM is responsible for slow islet oscillations, we made a beta-cell-specific knockout mouse that completely lacked PFKM. While PFKM deletion resulted in subtle metabolic changes in vivo, islets that were isolated from these mice continued to exhibit slow oscillations in electrical activity, beta cell Ca2+ concentrations, and glycolysis, as measured using PKAR, an FBP reporter/biosensor. Furthermore, simulations obtained with a mathematical model of beta cell activity shows that slow oscillations can persist despite PFKM loss provided that one of the other PFK isoforms, such as PFKP, is present, even if its level of expression is unchanged. Thus, while we believe that PFKM may be the main regulator of slow oscillations in wild-type islets, PFKP can provide functional redundancy. Our model also suggests that PFKM likely dominates, in vivo, because it outcompetes PFKP with its higher FBP affinity and lower ATP affinity. We thus propose that isoform redundancy may rescue key physiological processes of the beta cell in the absence of certain critical genes.

Engineering a Xylose-Utilizing Synechococcus elongatus UTEX 2973 Chassis for 3-Hydroxypropionic Acid Biosynthesis under Photomixotrophic Conditions.

Photomixotrophic cultivation of cyanobacteria is considered a promising strategy to achieve both high cell density and product accumulation, since cyanobacteria can obtain carbon and energy sources from organic matter in addition to those obtained from CO2 and sunlight. Acetyl coenzyme A (acetyl-CoA) is a key precursor used for the biosynthesis of a wide variety of important value-added chemicals. However, the acetyl-CoA content in cyanobacteria is typically low under photomixotrophic conditions, which limits the productivity of the derived chemicals. In this study, a xylose utilization pathway from Escherichia coli was first engineered into fast-growing Synechococcus elongatus UTEX 2973 (hereafter Synechococcus 2973), enabling the xylose based photomixotrophy. Metabolomics analysis of the engineered strain showed that the utilization of xylose enhanced the carbon flow to the oxidative pentose phosphate (OPP) pathway, along with an increase in the intracellular abundance of metabolites such as fructose-6-phosphate (F6P), fructose-1,6-bisphosphate (FBP), ribose-5-phosphate (R5P), erythrose-4-phosphate (E4P), and glyceraldehyde-3-phosphate (G3P). Then, the native glycolytic pathway was rewired via heterologous phosphoketolase (Pkt) gene expression, combined with phosphofructokinase (Pfk) gene knockout and fructose-1,6-bisphosphatase (Fbp) gene overexpression, to drive more carbon flux from xylose to acetyl-CoA. Finally, a heterologous 3-hydroxypropionic acid (3-HP) biosynthetic pathway was introduced. The results showed that 3-HP biosynthesis was improved by up to approximately 4.1-fold (from 22.5 mg/L to 91.3 mg/L) compared with the engineered strain without a rewired metabolism under photomixotrophic conditions and up to approximately 14-fold compared with the strain under photoautotrophic conditions. Using 3-HP as a "proof-of-molecule", our results demonstrated that this strategy could be applied to improve the intracellular pool of acetyl-CoA for the photomixotrophic production of value-added chemicals that require acetyl-CoA as a precursor in a cyanobacterial chassis.

A glycolytic enzyme complex and creatine kinase locally and differentially regulate ATP-sensitive potassium (KATP) channels in pancreatic islet alpha and beta cells

The ATP-sensitive potassium channel (KATP) is a key regulator of membrane potential and islet hormone secretion. Here, using the inside-out excised patch configuration, we isolated the plasma membrane compartment containing KATP on β-cells and α-cells from humans and mice. The characteristic response of KATP to inhibition by ATP and activation by ADP was used to identify the presence of a glycolytic enzyme complex that is functionally coupled to KATP regulation. We found that the phosphofructokinase product fructose-1,6-bisphosphate (FBP), which oscillates in β-cells, can induce KATP closure by its metabolism through the glycolytic chain including aldolase, GAPDH, phosphoglycerate kinase, phosphoglycerate mutase, enolase and finally pyruvate kinase.

Synthesis of hydrophobically modified alginate and hydrophobically modified gelatin containing cubic phase for pH- and salt-responsive release of fructose diphosphate

Synthesis of hydrophobically modified alginate and hydrophobically modified gelatin containing cubic phase for pH- and salt-responsive release of fructose diphosphate

PFKP Activation Ameliorates Foot Process Fusion in Podocytes in Diabetic Kidney Disease.

BackgroundGlycolysis dysfunction is an important pathogenesis of podocyte injury in diabetic kidney disease (DKD). Foot process fusion of podocytes and increased albuminuria are markers of early DKD. Moreover, cytoskeletal remodeling has been found to be involved in the foot process fusion of podocytes. However, the connections between cytoskeletal remodeling and alterations of glycolysis in podocytes in DKD have not been clarified.MethodsmRNA sequencing of glomeruli obtained from db/db and db/m mice with albuminuria was performed to analyze the expression profiling of genes in glucose metabolism. Expressions of phosphofructokinase platelet type (PFKP) in the glomeruli of DKD patients were detected. Clotrimazole (CTZ) was used to explore the renal effects of PFKP inhibition in diabetic mice. Using Pfkp siRNA or recombinant plasmid to manipulate PFKP expression, the effects of PFKP on high glucose (HG) induced podocyte damage were assessed in vitro. The levels of fructose-1,6-bisphosphate (FBP) were measured. Targeted metabolomics was performed to observe the alterations of the metabolites in glucose metabolism after HG stimulation. Furthermore, aldolase type b (Aldob) siRNA or recombinant plasmid were applied to evaluate the influence of FBP level alteration on podocytes. FBP was directly added to podocyte culture media. Db/db mice were treated with FBP to investigate its effects on their kidney.ResultsmRNA sequencing showed that glycolysis enzyme genes were altered, characterized by upregulation of upstream genes (Hk1, and Pfkp) and down-regulation of downstream genes of glycolysis (Pkm, and Ldha). Moreover, the expression of PFKP was increased in glomeruli of DKD patients. The CTZ group presented more severe renal damage. In vitro, the Pfkp siRNA group and ALDOB overexpression group showed much more induced cytoskeletal remodeling in podocytes, while overexpression of PFKP and suppression of ALDOB in vitro rescued podocytes from cytoskeletal remodeling through regulation of FBP levels and inhibition of the RhoA/ROCK1 pathway. Furthermore, targeted metabolomics showed FBP level was significantly increased in HG group compared with the control group. Exogenous FBP addition reduced podocyte cytoskeletal remodeling and renal damage of db/db mice.ConclusionsThese findings provide evidence that PFKP may be a potential target for podocyte injury in DN and provide a rationale for applying podocyte glycolysis enhancing agents in patients with DKD.

The effects of fructose diphosphate on routine coagulation tests in vitro

To evaluate the effects of fructose diphosphate (FDP) on routine coagulation tests in vitro, we added FDP into the mixed normal plasma to obtain the final concentration of 0, 1, 2, 3, 4, 5, 6, 10, 15, 20, 25, 30 and 35 mg/mL of drug. Prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen (FBG) and thrombin time (TT) of samples were analyzed with blood coagulation analyzers from four different manufacturers(Sysmex, Stago, SEKISUI and Werfen) and their corresponding reagents, respectively. Before the experiment, we also observed whether there were significant differences in coagulation test results of different lots of reagents produced by each manufacturer. At the same time as the four routine clotting tests, the Sysmex blood coagulation analyzer and its proprietary analysis software were used to detect the change of maximum platelet aggregation rate in platelet-rich plasma after adding FDP (0, 1, 2, 3, 4, 5 and 6 mg/mL). The results of PT, aPTT and TT showed a FDP (0–35 mg/mL) concentration-dependent increase and a FBG concentration-dependent decrease. The degree of change (increase or decrease) varied depending on the assay system, with PT and aPTT being more affected by the Sysmex blood coagulation testing instrument reagent system and less affected by CEKISUI, TT less affected by CEKISUI and more affected by Stago, and FBG less affected by Stago and more affected by Sysmex. The results of PT, aPTT and TT were statistically positively correlated with their FDP concentrations, while FBG was negatively correlated. The correlation coefficients between FDP and the coagulation testing systems of Sysmex, Stago, Werfen and SEKISUI were 0.975, 0.988, 0.967, 0.986 for PT, and 0.993, 0.989, 0.990 and 0.962 for aPTT, 0.994, 0.960, 0.977 and 0.982 for TT, − 0.990, − 0.983, − 0.989 and − 0.954 for FBG, respectively. Different concentrations of FDP (0, 1, 2, 3, 4, 5 and 6 mg/mL) had different effects on the maximum aggregation rate of platelet induced by the agonists of adenosine diphosphate (ADP, 5 µmol/L), arachidonic acid (Ara, 1 mmol/L), collagen (Col, 2.5 µg/mL) and epinephrine (Epi,10 µmol/L), but the overall downward trend was consistent, that is, with the increase of FDP concentration, the platelet aggregation rate decreased significantly. Our experimental study demonstrated a possible effect of FDP on the assays of coagulation and Platelet aggregation, which may arise because the drug interferes with the coagulation and platelet aggregation detection system, or it may affect our in vivo coagulation system and Platelet aggregation function, the real mechanism of which remains to be further verified and studied.

Stoichiometric Conversion of Maltose for Biomanufacturing by In Vitro Synthetic Enzymatic Biosystems.

Maltose is a natural α-(1,4)-linked disaccharide with wide applications in food industries and microbial fermentation. However, maltose has scarcely been used for in vitro biosynthesis, possibly because its phosphorylation by maltose phosphorylase (MP) yields β-glucose 1-phosphate (β-G1P) that cannot be utilized by α-phosphoglucomutase (α-PGM) commonly found in in vitro synthetic enzymatic biosystems previously constructed by our group. Herein, we designed an in vitro synthetic enzymatic reaction module comprised of MP, β-phosphoglucomutase (β-PGM), and polyphosphate glucokinase (PPGK) for the stoichiometric conversion of each maltose molecule to two glucose 6-phosphate (G6P) molecules. Based on this synthetic module, we further constructed two in vitro synthetic biosystems to produce bioelectricity and fructose 1,6-diphosphate (FDP), respectively. The 14-enzyme biobattery achieved a Faraday efficiency of 96.4% and a maximal power density of 0.6 mW/cm2, whereas the 5-enzyme in vitro FDP-producing biosystem yielded 187.0 mM FDP from 50 g/L (139 mM) maltose by adopting a fed-batch substrate feeding strategy. Our study not only suggests new application scenarios for maltose but also provides novel strategies for the high-efficient production of bioelectricity and value-added biochemicals.

Coxiella burnetii inhibits host immunity by a protein phosphatase adapted from glycolysis

Coxiella burnetii is a bacterial pathogen that replicates within host cells by establishing a membrane-bound niche called the Coxiella-containing vacuole. Biogenesis of this compartment requires effectors of its Dot/Icm type IV secretion system. A large cohort of such effectors has been identified, but the function of most of them remain elusive. Here, by a cell-based functional screening, we identified the effector Cbu0513 (designated as CinF) as an inhibitor of NF-κB signaling. CinF is highly similar to a fructose-1,6-bisphosphate (FBP) aldolase/phosphatase present in diverse bacteria. Further study reveals that unlike its ortholog from Sulfolobus tokodaii, CinF does not exhibit FBP phosphatase activity. Instead, it functions as a protein phosphatase that specifically dephosphorylates and stabilizes IκBα. The IκBα phosphatase activity is essential for the role of CinF in C. burnetii virulence. Our results establish that C. burnetii utilizes a protein adapted from sugar metabolism to subvert host immunity.

SRNA23, a novel small RNA, regulates to the pathogenesis of Streptococcus suis serotype 2

ABSTRACT Streptococcus suis serotype 2 (S. suis 2) is an important ubiquitous zoonotic pathogen. To date, regulatory factors and their implication in S. suis pathogenesis are not fully understood. Small non-coding RNAs (sRNAs) have been proven to function as important regulatory factors in bacterial pathogenesis and stress adaptation. Here, we identified a differentially downregulated S. suis 05ZYH33 sRNA after iron starvation by RNA-seq, which we named sRNA23. The presence of sRNA23 was further confirmed by RACE and Northern blot. Expression of sRNA23 was significantly altered under different environmental stresses such as nutritional starvation, osmotic pressure, oxidative stress, and lysozymal exposure. A sRNA23-deleted mutant exhibited relatively shorter streptococcal chains and weakened biofilm-forming ability. The mutation also resulted in decreased adherence of the S. suis 05ZYH33 to human laryngeal epidermoid carcinoma (HEp-2) cells, increased sensitivity to phagocytosis by RAW264.7 macrophages, and significantly reduced hemolytic activity. Furthermore, we observed that a sRNA23-deleted mutant had a low survival rate in pig whole blood and attenuated virulence in a mouse model. Moreover, based on RNA pull-down and electrophoretic mobility shift assay, we found that sRNA23 can directly bind to two proteins involved in adhesion and biofilm formation, namely, moonlighting protein FBA (fructose diphosphate aldolase) and rplB (50S ribosomal protein L2), respectively. Collectively, sRNA23 enhances S. suis 2 pathogenicity and the binding between sRNA23 and FBA/rplB might play an essential role in the adherence and biofilm-forming ability of S. suis 2. Abbreviation sRNA: small noncoding RNA; FBA: fructose diphosphate aldolase; rplB: 50S ribosomal protein L2; RACE: rapid amplification of cDNA ends; EMSA: electrophoretic mobility shift assay; THB: Todd-Hewitt broth; FBS: fetal bovine serum; BIP: 2,2ʹ-Bipyridine

Transcriptome Analysis Reveals Catabolite Control Protein A Regulatory Mechanisms Underlying Glucose-Excess or -Limited Conditions in a Ruminal Bacterium, Streptococcus bovis.

Ruminants may suffer from rumen acidosis when fed with high-concentrate diets due to the higher proliferation and overproduction of lactate by Streptococcus bovis. The catabolite control protein A (CcpA) regulates the transcription of lactate dehydrogenase (ldh) and pyruvate formate-lyase (pfl) in S. bovis, but its role in response to different carbon concentrations remains unclear. To characterize the regulatory mechanisms of CcpA in S. bovis S1 at different levels of carbon, herein, we analyzed the transcriptomic and physiological characteristics of S. bovis S1 and its ccpA mutant strain grown in glucose-excess and glucose-limited conditions. A reduced growth rate and a shift in fermentation pattern from homofermentation to heterofermentation were observed under glucose-limited condition as compared to glucose-excess condition, in S. bovis S1. Additionally, the inactivation of ccpA significantly affected the growth and end metabolites in both conditions. For the glycolytic intermediate, fructose 1,6-bisphosphate (FBP), the concentration significantly reduced at lower glucose conditions; its concentration decreased significantly in the ccpA mutant strain. Transcriptomic results showed that about 46% of the total genes were differentially transcribed between the wild-type strain and ccpA mutant strain grown in glucose-excess conditions; while only 12% genes were differentially transcribed in glucose-limited conditions. Different glucose concentrations led to the differential expression of 38% genes in the wild-type strain, while only half of these were differentially expressed in the ccpA-knockout strain. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that the substrate glucose concentration significantly affected the gene expression in histidine metabolism, nitrogen metabolism, and some carbohydrate metabolism pathways. The deletion of ccpA affected several genes involved in carbohydrate metabolism, such as glycolysis, pyruvate metabolism, fructose and mannose metabolism, as well as in fatty acid biosynthesis pathways in bacteria grown in glucose-excess conditions; this effect was attenuated under glucose-limited conditions. Overall, these findings provide new information on gene transcription and metabolic mechanisms associated with substrate glucose concentration and validate the important role of CcpA in the regulation of carbon metabolism in S. bovis S1 at differential glucose availability.

New Insights to Regulation of Fructose-1,6-bisphosphatase during Anoxia in Red-Eared Slider, Trachemys scripta elegans.

The red-eared slider (Trachemys scripta elegans) undergoes numerous changes to its physiological and metabolic processes to survive without oxygen. During anoxic conditions, its metabolic rate drops drastically to minimize energy requirements. The alterations in the central metabolic pathways are often accomplished by the regulation of key enzymes. The regulation of one such enzyme, fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11), was characterized in the present study during anoxia in liver. FBPase is a crucial enzyme of gluconeogenesis. The FBPase was purified from liver tissue in both control and anoxic conditions and subsequently assayed to determine the kinetic parameters of the enzyme. The study revealed the relative degree of post-translational modifications in the FBPase from control and anoxic turtles. Further, this study demonstrated a significant decrease in the maximal activity in anoxic FBPase and decreased sensitivity to its substrate Fructose-1,6-bisphosphate (FBP) when compared to the control. Immunoblotting demonstrated increased threonine phosphorylation (~1.4-fold) in the anoxic FBPase. Taken together, these results suggest that the phosphorylation of liver FBPase is an important step in suppressing FBPase activity, ultimately leading to the inhibition of gluconeogenesis in the liver of the red-eared slider during anaerobic conditions.

The Role of B-Cells and Antibodies against Candida Vaccine Antigens in Invasive Candidiasis.

Systemic candidiasis is an invasive fungal infection caused by members of the genus Candida. The recent emergence of antifungal drug resistance and increased incidences of infections caused by non-albicans Candida species merit the need for developing immune therapies against Candida infections. Although the role of cellular immune responses in anti-Candida immunity is well established, less is known about the role of humoral immunity against systemic candidiasis. This review summarizes currently available information on humoral immune responses induced by several promising Candida vaccine candidates, which have been identified in the past few decades. The protective antibody and B-cell responses generated by polysaccharide antigens such as mannan, β-glucan, and laminarin, as well as protein antigens like agglutinin-like sequence gene (Als3), secreted aspartyl proteinase (Sap2), heat shock protein (Hsp90), hyphally-regulated protein (Hyr1), hyphal wall protein (Hwp1), enolase (Eno), phospholipase (PLB), pyruvate kinase (Pk), fructose bisphosphate aldolase (Fba1), superoxide dismutase gene (Sod5) and malate dehydrogenase (Mdh1), are outlined. As per studies reviewed, antibodies induced in response to leading Candida vaccine candidates contribute to protection against systemic candidiasis by utilizing a variety of mechanisms such as opsonization, complement fixation, neutralization, biofilm inhibition, direct candidacidal activity, etc. The contributions of B-cells in controlling fungal infections are also discussed. Promising results using anti-Candida monoclonal antibodies for passive antibody therapy reinforces the need for developing antibody-based therapeutics including anti-idiotypic antibodies, single-chain variable fragments, peptide mimotopes, and antibody-derived peptides. Future research involving combinatorial immunotherapies using humanized monoclonal antibodies along with antifungal drugs/cytokines may prove beneficial for treating invasive fungal infections.

Fructose 1,6-bisphosphate is anticonvulsant and improves oxidative glucose metabolism within the hippocampus and liver in the chronic pilocarpine mouse epilepsy model

Glucose metabolism is altered in epilepsy, and this may contribute to seizure generation. Recent research has shown that metabolic therapies including the ketogenic diet and medium chain triglycerides can improve energy metabolism in the brain. Fructose 1,6-bisphosphate (F16BP) is an intermediate of glycolysis and when administered exogenously is anticonvulsant in several rodent seizure models and may alter glucose metabolism. Here, we showed that F16BP elevated the seizure threshold in the acute 6-Hz mouse seizure model and investigated if F16BP could restore impairments in glucose metabolism occurring in the chronic stage of the pilocarpine mouse model of epilepsy. Two weeks after the pilocarpine injections, mice that experienced status epilepticus (SE, “epileptic”) and did not experience SE (no SE, “nonepileptic”) were injected with vehicle (0.9% saline) or F16BP (1 g/kg in 0.9% saline) daily for 5 consecutive days. At 3 weeks, mice were injected with [U-13C6]-glucose and the % enrichment of 13C in key metabolites in addition to the total levels of each metabolite was measured in the hippocampal formation and liver. Fructose 1,6-bisphosphate increased total GABA in the hippocampal formation, regardless of whether mice had experienced SE. In the hippocampal formation, F16BP prevented reductions in the % 13C enrichment of citrate, succinate, malate, glutamate, GABA and aspartate that occurred in the chronic stage of the pilocarpine model. Interestingly, % 13C enrichment in glucose-derived metabolites was reduced in the liver in the chronic stage of the pilocarpine model. Fructose 1,6-bisphosphate was also beneficial in the liver, preventing reductions in % 13C enrichment of lactate and alanine that were associated with SE. This study confirmed that F16BP is anticonvulsant and can improve elements of glucose metabolism that are dysregulated in the chronic stage of the pilocarpine model, which may be due to reduction of spontaneous seizures. Our results highlight that F16BP may be therapeutically beneficial for epilepsies refractory to treatment.

Glucose limitation activates AMPK coupled SENP1-Sirt3 signalling in mitochondria for T cell memory development

Metabolic programming and mitochondrial dynamics along with T cell differentiation affect T cell fate and memory development; however, how to control metabolic reprogramming and mitochondrial dynamics in T cell memory development is unclear. Here, we provide evidence that the SUMO protease SENP1 promotes T cell memory development via Sirt3 deSUMOylation. SENP1-Sirt3 signalling augments the deacetylase activity of Sirt3, promoting both OXPHOS and mitochondrial fusion. Mechanistically, SENP1 activates Sirt3 deacetylase activity in T cell mitochondria, leading to reduction of the acetylation of mitochondrial metalloprotease YME1L1. Consequently, deacetylation of YME1L1 suppresses its activity on OPA1 cleavage to facilitate mitochondrial fusion, which results in T cell survival and promotes T cell memory development. We also show that the glycolytic intermediate fructose-1,6-bisphosphate (FBP) as a negative regulator suppresses AMPK-mediated activation of the SENP1-Sirt3 axis and reduces memory development. Moreover, glucose limitation reduces FBP production and activates AMPK during T cell memory development. These data show that glucose limitation activates AMPK and the subsequent SENP1-Sirt3 signalling for T cell memory development.

Abstract 2519: Targeting glucose metabolism in HER2+breast cancer

Abstract Primary and metastatic breast cancers are driven by oncogenic mutations to rely on glucose metabolism to support their rapid proliferation. Of these genetic changes, the increased activity of the human epidermal growth factor receptor 2 (HER2/ErbB2) is observed in a third of all breast tumors. HER2 has been shown to activate downstream effectors (e.g. Ras, HIF-1α) that stimulate glycolysis - in part by activation of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase family of enzymes (PFKFB1-4). The PFKFBs produce fructose-2,6-bisphosphate (F26BP) which activates a key rate-limiting glycolytic enzyme, 6-phosphofructo-1-kinase (PFK1). In preliminary studies, we found that the PFKFB4 family member was highly expressed in a series of patient-derived HER2+ breast tumors relative to normal breast with markedly higher expression in their matched metastatic lymph nodes. We next determined that silencing PFKFB4 markedly reduced F26BP, glycolysis and cell survival in HER2+ breast cancer cells and inhibited tumor growth in vivo. Through virtual screening, we have discovered a first-in-class small molecule inhibitor of PFKFB4, termed 5MPN, that selectively inhibits PFKFB4 activity with a resultant decrease in F26BP, glycolysis and cancer cell growth. 5MPN also caused a significant decrease in the invasion and migration of HER2+ breast cancer cells in vitro, importantly at concentrations well below levels that affect cell viability and marked suppression in the growth of established tumors, without systemic toxicity. In additional studies, we have found that the related PFKFB3 isoform is also expressed in HER+ breast tumors. Our previous work found that the PFKFB4 enzyme localizes to the cytoplasm (where PFK1 and the glycolytic pathway function) indicating a possible dominant role for PFKFB4 in glycolytic regulation. PFKFB3 however largely localized to the nucleus where it serves an important function in proliferation via activation of cyclin-dependent kinases. Based on the different roles played by these enzymes in cancer cells, we next evaluated the effects of simultaneous administration of 5MPN ± a clinical candidate PFKFB3 inhibitor on survival and proliferation in HER2+ breast cancer and found that the combination synergistically and significantly decreased viability and growth in several examined HER2+ breast cancer cell lines. Our data indicate that targeting PFKFB4 may be a viable therapeutic option against breast cancer and strongly support the further exploration of co-targeting PFKFB4 and PFKFB3 as a potential therapeutic strategy for the successful treatment of HER2+ breast cancer. Citation Format: Simone Chang, Jason Chesney, John O. Trent, Sucheta Telang. Targeting glucose metabolism in HER2+breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2519.

Synthetic conjugate peptide Fba-Met6 (MP12) induces complement-mediated resistance against disseminated Candida albicans

The fungal genus Candida includes common commensals of the human mucosal membranes, and the most prevalently isolated species, C. albicans, poses a threat of candidemia and disseminated infection associated with an unacceptably high mortality rate and an immense $4 billion burden (US) yearly. Nevertheless, the demand for a vaccine remains wholly unfulfilled and increasingly pressing. We developed a double-peptide construct that is feasible for use in humans with the intention of preventing morbid infection by targeting epitopes derived from fructose bisphosphate aldolase (Fba) and methionine synthase (Met6) which are expressed on the C. albicans cell surface. To test the applicability of the design, we vaccinated mice via the intramuscular (IM) route with the conjugate denoted Fba-Met6 MP12 and showed that the vaccine enhanced survival against a lethal challenge. Because overall endpoint IgG1 and IgG2a antibody titers were robust and these mouse subclasses are associated with protective functionality, we investigated the potential of Fba and Met6 specific antibodies to facilitate the well-defined anti-Candida response by complement, which opsonizes fungi for degradation by primary effectors. Notably, reductions in the fungal burdens and enhanced survival were both abrogated in MP12-vaccinated mice that were pre-challenge dosed with cobra venom factor (CVF), a complement depleting factor. Altogether, we demonstrated that complement is relevant to MP12-based protection against disseminated C. albicans, delineating that a novel, multivalent targeted vaccine against proteins on the surface of C. albicans can enhance the natural response to infection.

129-OR: Glycolytic Enzymes and Creatine Kinase Locally Regulate KATP Channel Activity in Both ß- and a-Cells

The ATP-sensitive potassium channel (KATP) is a key regulator of membrane potential and islet hormone secretion. In ß-cells, it has been previously showed that the local activity of pyruvate kinase (PK) can sufficiently raise ATP/ADP to induce KATP closure, thus providing a another route to trigger membrane depolarization parallel to mitochondrial oxidative phosphorylation. Here, using direct single-channel measurements of KATP in both ß-and the lesser studied α-cells, we provide evidence for a larger network of metabolic enzymes that operate in the vicinity of KATP channels to fine-tune their activity. We found that the phosphofructokinase product fructose-1,6-bisphosphate (FBP), which oscillates in ß-cells, can induce KATP closure by its metabolism through the glycolytic chain including aldolase, GAPDH, phosphoglycerate kinase, phosphoglycerate mutase, enolase and finally PK. Strikingly, while withholding the substrates at GAPDH, we observed that FBP can independently inhibit KATP channels by direct action. In addition, we further provide evidence that creatine kinase (CK) activity can either oppose or enhance KATP closure, depending on the reaction direction. Finally, we demonstrated that lactate has an activating effect on KATP. In summary, these data provide evidence for the direct regulation of KATP by two metabolites, FBP and lactate, and identify two local ATP/ADP controllers, the glycolytic complex and the creatine kinase system in both ß-cells and α-cells. Disclosure T. Ho: None. E. Potapenko: None. M. J. Merrins: None.