Phosphoinositide 3-Kinase Regulates Glycolysis through Mobilization of Aldolase from the Actin Cytoskeleton.

The phosphoinositide 3-kinase (PI3K) pathway regulates multiple steps in glucose metabolism and also cytoskeletal functions, such as cell movement and attachment. Here, we show that PI3K directly coordinates glycolysis with cytoskeletal dynamics in an AKT-independent manner. Growth factors or insulin stimulate the PI3K-dependent activation of Rac, leading to disruption of the actin cytoskeleton, release of filamentous actin-bound aldolase A, and an increase in aldolase activity. Consistently, PI3K inhibitors, but not AKT, SGK, or mTOR inhibitors, cause a significant decrease in glycolysis at the step catalyzed by aldolase, while activating PIK3CA mutations have the opposite effect. These results point toward a master regulatory function of PI3K that integrates an epithelial cell's metabolism and its form, shape, and function, coordinating glycolysis with the energy-intensive dynamics of actin remodeling.

Beyond Glycolysis: Aldolase A Is a Novel Effector in Reelin-Mediated Dendritic Development.

Reelin, a secreted glycoprotein, plays a crucial role in guiding neocortical neuronal migration, dendritic outgrowth and arborization, and synaptic plasticity in the adult brain. Reelin primarily operates through the canonical lipoprotein receptors apolipoprotein E receptor 2 (Apoer2) and very low-density lipoprotein receptor (Vldlr). Reelin also engages with noncanonical receptors and unidentified coreceptors; however, the effects of which are less understood. Using high-throughput tandem mass tag (TMT) liquid chromatography tandem mass spectrometry (LC-MS/MS)-based proteomics and gene set enrichment analysis (GSEA), we identified both shared and unique intracellular pathways activated by Reelin through its canonical and noncanonical signaling in primary murine neurons of either sex during dendritic growth and arborization. We observed pathway cross talk related to regulation of cytoskeleton, neuron projection development, protein transport, and actin filament-based process. We also found enriched gene sets exclusively by the noncanonical Reelin pathway including protein translation, mRNA metabolic process, and ribonucleoprotein complex biogenesis suggesting Reelin fine-tunes neuronal structure through distinct signaling pathways. A key discovery is the identification of aldolase A, a glycolytic enzyme and actin-binding protein, as a novel effector of Reelin signaling. Reelin induced de novo translation and mobilization of aldolase A from the actin cytoskeleton. We demonstrated that aldolase A is necessary for Reelin-mediated dendrite growth and arborization in primary murine neurons and mouse brain cortical neurons. Interestingly, the function of aldolase A in dendrite development is independent of its known role in glycolysis. Altogether, our findings provide new insights into the Reelin-dependent signaling pathways and effector proteins that are crucial for dendritic development.

Restricted glycolysis is a primary cause of the reduced growth rate of zinc-deficient yeast cells.

Zinc is required for many critical processes, including intermediary metabolism. In Saccharomyces cerevisiae, the Zap1 activator regulates the transcription of ∼80 genes in response to Zn supply. Some Zap1-regulated genes are Zn transporters that maintain Zn homeostasis, while others mediate adaptive responses that enhance fitness. One adaptive response gene encodes the 2-cysteine peroxiredoxin Tsa1, which is critical to Zn-deficient (ZnD) growth. Depending on its redox state, Tsa1 can function as a peroxidase, a protein chaperone, or a regulatory redox sensor. In a screen for possible Tsa1 regulatory targets, we identified a mutation (cdc19S492A) that partially suppressed the tsa1Δ growth defect. The cdc19S492A mutation reduced activity of its protein product, pyruvate kinase isozyme 1 (Pyk1), implicating Tsa1 in adapting glycolysis to ZnD conditions. Glycolysis requires activity of the Zn-dependent enzyme fructose-bisphosphate aldolase 1, which was substantially decreased in ZnD cells. We hypothesized that in ZnD tsa1Δ cells, the loss of a compensatory Tsa1 regulatory function causes depletion of glycolytic intermediates and restricts dependent amino acid synthesis pathways, and that the decreased activity of Pyk1S492A counteracted this depletion by slowing the irreversible conversion of phosphoenolpyruvate to pyruvate. In support of this model, supplementing ZnD tsa1Δ cells with aromatic amino acids improved their growth. Phosphoenolpyruvate supplementation, in contrast, had a much greater effect on growth rate of WT and tsa1Δ ZnD cells, indicating that inefficient glycolysis is a major factor limiting yeast growth. Surprisingly however, this restriction was not primarily due to low fructose-bisphosphate aldolase 1 activity, but instead occurs earlier in glycolysis.

Mining cholesterol genes from thousands of mouse livers identifies aldolase C as a regulator of cholesterol biosynthesis.

The availability of genome-wide transcriptomic and proteomic datasets is ever-increasing and often not used beyond initial publication. Here, we applied module-based coexpression network analysis to a comprehensive catalog of 35 mouse genome-wide liver expression datasets (encompassing more than 3800 mice) with the goal of identifying and validating unknown genes involved in cholesterol metabolism. From these 35 datasets, we identified a conserved module of genes enriched with cholesterol biosynthetic genes. Using a systematic approach across the 35 datasets, we identified three genes (Rdh11, Echdc1, and Aldoc) with no known role in cholesterol metabolism. We then performed functional validation studies and show that each gene is capable of regulating cholesterol metabolism. For the glycolytic gene, Aldoc, we demonstrate that it contributes to de novo cholesterol biosynthesis and regulates cholesterol and triglyceride levels in mice. As Aldoc is located within a genome-wide significant genome-wide association studies locus for human plasma cholesterol levels, our studies establish Aldoc as a causal gene within this locus. Through our work, we develop a framework for leveraging mouse genome-wide liver datasets for identifying and validating genes involved in cholesterol metabolism.

ZNF692 drives malignant development of hepatocellular carcinoma cells by promoting ALDOA-dependent glycolysis.

Hepatocellular carcinoma (HCC) is one of the malignancies with the worst prognosis worldwide, in the occurrence and development of which glycolysis plays a central role. This study uncovered a mechanism by which ZNF692 regulates ALDOA-dependent glycolysis in HCC cells. RT-qPCR and western blotting were used to detect the expression of ZNF692, KAT5, and ALDOA in HCC cell lines and a normal liver cell line. The influences of transfection-induced alterations in the expression of ZNF692, KAT5, and ALDOA on the functions of HepG2 cells were detected by performing MTT, flow cytometry, Transwell, cell scratch, and colony formation assays, and the levels of glucose and lactate were determined using assay kits. ChIP and luciferase reporter assays were conducted to validate the binding of ZNF692 to the KAT5 promoter, and co-IP assays to detect the interaction between KAT5 and ALDOA and the acetylation of ALDOA. ZNF692, KAT5, and ALDOA were highly expressed in human HCC samples and cell lines, and their expression levels were positively correlated in HCC. ZNF692, ALDOA, or KAT5 knockdown inhibited glycolysis, proliferation, invasion, and migration and promoted apoptosis in HepG2 cells. ZNF692 bound to the KAT5 promoter and promoted its activity. ALDOA acetylation levels were elevated in HCC cell lines. KAT5 bound to ALDOA and catalyzed ALDOA acetylation. ALDOA or KAT5 overexpression in the same time of ZNF692 knockdown, compared to ZNF692 knockdown only, stimulated glycolysis, proliferation, invasion, and migration and reduced apoptosis in HepG2 cells. ZNF692 promotes the acetylation modification and protein expression of ALDOA by catalyzing KAT5 transcription, thereby accelerating glycolysis to drive HCC cell development.

Fructose-1, 6-Bisphosphate Aldolase B Suppresses Glycolysis and Tumor Progression of Gastric Cancer.

OBJECTIVE: Gastric cancer (GC) is believed to be one of the most common digestive tract malignant tumors. However, mounting evidence indicates a link between the glycolysis and tumorigenesis, including gastric cancer. METHODS: Our research identified 5508 differently expressed mRNAs in gastric cancer. Then, the genes highly associated with tumorigenesis were identified through weighted correlation network analysis (WGCNA). Bioinformatics analysis observed that these hub genes were significantly linked to the regulation of cell cycle, drug metabolism, and glycolysis. Among these hub genes, there is a critical gene involved in glycolysis regulation, namely fructose-bisphosphate B (ALDOB). RESULTS: Analysis based on The Cancer Genome Atlas (TCGA) and three Gene Expression Omnibus (GEO) datasets revealed that ALDOB was significantly downregulated in GC compared with normal tissues. In addition, cell viability assay confirmed that ALDOB acted as a tumor suppressor. Finally, drug sensitivity analysis revealed that ALDOB increased the sensitivity of gastric cancer cells to most antitumor drugs, especially talazoparib, XAV939, and FTI-277. Our results showed that the expression of ALDOB was significantly lower in GC tissues than in normal tissues. And ALDOB significantly inhibited proliferation and migration, delayed glycolysis in GC cells. Consequently, our study suggests that ALDOB may be a potential target for the clinical treatment of gastric cancer.

Mesocellular Silica Foam as Immobilization Carrier for Production of Statin Precursors.

The employment of 2-deoxyribose-5-phosphate aldolase (DERA) stands as a prevalent biocatalytic route for synthesizing statin side chains. The main problem with this pathway is the low stability of the enzyme. In this study, mesocellular silica foam (MCF) with different pore sizes was used as a carrier for the covalent immobilization of DERA. Different functionalizing and activating agents were tested and kinetic modeling was subsequently performed. The use of succinic anhydride as an activating agent resulted in an enzyme hyperactivation of approx. 140%, and the stability almost doubled compared to that of the free enzyme. It was also shown that the pore size of MCF has a decisive influence on the stability of the DERA enzyme.

Disruption of Erythritol Catabolism via the Deletion of Fructose-Bisphosphate Aldolase (Fba) and Transaldolase (Tal) as a Strategy to Improve the Brucella Rev1 Vaccine.

Brucellosis is a bacterial zoonosis caused by the genus Brucella, which mainly affects domestic animals. In these natural hosts, brucellae display a tropism towards the reproductive organs, such as the placenta, replicating in high numbers and leading to placentitis and abortion, an ability also exerted by the B. melitensis live-attenuated Rev1 strain, the only vaccine available for ovine brucellosis. It is broadly accepted that this tropism is mediated, at least in part, by the presence of certain preferred nutrients in the placenta, particularly erythritol, a polyol that is ultimately incorporated into the Brucella central carbon metabolism via two reactions dependent on transaldolase (Tal) or fructose-bisphosphate aldolase (Fba). In the light of these remarks, we propose that blocking the incorporation of erythritol into the central carbon metabolism of Rev1 by deleting the genes encoding Tal and Fba may impair the ability of the vaccine to proliferate massively in the placenta. Therefore, a Rev1ΔfbaΔtal double mutant was generated and confirmed to be unable to use erythritol. This mutant exhibited a reduced intracellular fitness both in BeWo trophoblasts and THP-1 macrophages. In the murine model, Rev1ΔfbaΔtal provided comparable protection to the Rev1 reference vaccine while inducing fewer adverse reproductive events in pregnant animals. Altogether, these results postulate the Rev1ΔfbaΔtal mutant as a reproductively safer Rev1-derived vaccine candidate to be studied in the natural host.

Genome-Wide Characterization of Fructose 1,6-Bisphosphate Aldolase Genes and Expression Profile Reveals Their Regulatory Role in Abiotic Stress in Cucumber.

The fructose-1,6-bisphosphate aldolase (FBA) gene family exists in higher plants, with the genes of this family playing significant roles in plant growth and development, as well as response to abiotic stresses. However, systematic reports on the FBA gene family and its functions in cucumber are lacking. In this study, we identified five cucumber FBA genes, named CsFBA1-5, that are distributed randomly across chromosomes. Phylogenetic analyses involving these cucumber FBAs, alongside eight Arabidopsis FBA proteins and eight tomato FBA proteins, were conducted to assess their homology. The CsFBAs were grouped into two clades. We also analyzed the physicochemical properties, motif composition, and gene structure of the cucumber FBAs. This analysis highlighted differences in the physicochemical properties and revealed highly conserved domains within the CsFBA family. Additionally, to explore the evolutionary relationships of the CsFBA family further, we constructed comparative syntenic maps with Arabidopsis and tomato, which showed high homology but only one segmental duplication event within the cucumber genome. Expression profiles indicated that the CsFBA gene family is responsive to various abiotic stresses, including low temperature, heat, and salt. Taken together, the results of this study provide a theoretical foundation for understanding the evolution of and future research into the functional characterization of cucumber FBA genes during plant growth and development.

LIPH contributes to glycolytic phenotype in pancreatic ductal adenocarcinoma by activating LPA/LPAR axis and maintaining ALDOA stability.

BACKGROUND: LIPH, a membrane-associated phosphatidic acid-selective phospholipase A1a, can produce LPA (Lysophosphatidic acid) from PA (Phosphatidic acid) on the outer leaflet of the plasma membrane. It is well known that LIPH dysfunction contributes to lipid metabolism disorder. Previous study shows that LIPH was found to be a potential gene related to poor prognosis with pancreatic ductal adenocarcinoma (PDAC). However, the biological functions of LIPH in PDAC remain unclear. METHODS: Cell viability assays were used to evaluate whether LIPH affected cell proliferation. RNA sequencing and immunoprecipitation showed that LIPH participates in tumor glycolysis by stimulating LPA/LPAR axis and maintaining aldolase A (ALDOA) stability in the cytosol. Subcutaneous, orthotopic xenograft models and patient-derived xenograft PDAC model were used to evaluate a newly developed Gemcitabine-based therapy. RESULTS: LIPH was significantly upregulated in PDAC and was related to later pathological stage and poor prognosis. LIPH downregulation in PDAC cells inhibited colony formation and proliferation. Mechanistically, LIPH triggered PI3K/AKT/HIF1A signaling via LPA/LPAR axis. LIPH also promoted glycolysis and de novo synthesis of glycerolipids by maintaining ALDOA stability in the cytosol. Xenograft models show that PDAC with high LIPH expression levels was sensitive to gemcitabine/ki16425/aldometanib therapy without causing discernible side effects. CONCLUSION: LIPH directly bridges PDAC cells and tumor microenvironment to facilitate aberrant aerobic glycolysis via activating LPA/LPAR axis and maintaining ALDOA stability, which provides an actionable gemcitabine-based combination therapy with limited side effects.

Loss of hepatic aldolase B activates Akt and promotes hepatocellular carcinogenesis by destabilizing the Aldob/Akt/PP2A protein complex.

Loss of hepatic fructose-1, 6-bisphosphate aldolase B (Aldob) leads to a paradoxical up-regulation of glucose metabolism to favor hepatocellular carcinogenesis (HCC), but the upstream signaling events remain poorly defined. Akt is highly activated in HCC, and targeting Akt is being explored as a potential therapy for HCC. Herein, we demonstrate that Aldob suppresses Akt activity and tumor growth through a protein complex containing Aldob, Akt, and protein phosphatase 2A (PP2A), leading to inhibition of cell viability, cell cycle progression, glucose uptake, and metabolism. Interestingly, Aldob directly interacts with phosphorylated Akt (p-Akt) and promotes the recruitment of PP2A to dephosphorylate p-Akt, and this scaffolding effect of Aldob is independent of its enzymatic activity. Loss of Aldob or disruption of Aldob/Akt interaction in Aldob R304A mutant restores Akt activity and tumor-promoting effects. Consistently, Aldob and p-Akt expression are inversely correlated in human HCC tissues, and Aldob down-regulation coupled with p-Akt up-regulation predicts a poor prognosis for HCC. We have further discovered that Akt inhibition or a specific small-molecule activator of PP2A (SMAP) efficiently attenuates HCC tumorigenesis in xenograft mouse models. Our work reveals a novel nonenzymatic role of Aldob in negative regulation of Akt activation, suggesting that directly inhibiting Akt activity or through reactivating PP2A may be a potential therapeutic approach for HCC treatment.

Fructose-1,6-bisphosphate and aldolase mediate glucose sensing by AMPK.

The major energy source for most cells is glucose, from which ATP is generated via glycolysis and/or oxidative metabolism. Glucose deprivation activates AMP-activated protein kinase (AMPK), but it is unclear whether this activation occurs solely via changes in AMP or ADP, the classical activators of AMPK. Here, we describe an AMP/ADP-independent mechanism that triggers AMPK activation by sensing the absence of fructose-1,6-bisphosphate (FBP), with AMPK being progressively activated as extracellular glucose and intracellular FBP decrease. When unoccupied by FBP, aldolases promote the formation of a lysosomal complex containing at least v-ATPase, ragulator, axin, liver kinase B1 (LKB1) and AMPK, which has previously been shown to be required for AMPK activation. Knockdown of aldolases activates AMPK even in cells with abundant glucose, whereas the catalysis-defective D34S aldolase mutant, which still binds FBP, blocks AMPK activation. Cell-free reconstitution assays show that addition of FBP disrupts the association of axin and LKB1 with v-ATPase and ragulator. Importantly, in some cell types AMP/ATP and ADP/ATP ratios remain unchanged during acute glucose starvation, and intact AMP-binding sites on AMPK are not required for AMPK activation. These results establish that aldolase, as well as being a glycolytic enzyme, is a sensor of glucose availability that regulates AMPK.

Escherichia coli BL21(DE3) optimized deletion mutant as the host for whole-cell biotransformation of N­acetyl­D­neuraminic acid.

N­Acetyl­D­neuraminic acid (Neu5Ac) is the crucial compound for the chemical synthesis of antiflu medicine Zanamivir. Chemoenzymatic synthesis of Neu5Ac involves N-acetyl-D-glucosamine 2-epimerase (AGE)-catalyzed epimerization of N-acetyl-D-glucosamine (GlcNAc) to N-acetyl-D-mannosamine (ManNAc), and aldolase-catalyzed condensation between ManNAc and pyruvate. Host optimization plays an important role in the whole-cell biotransformation of value-added compounds. In this study, via single-plasmid biotransformation system, we showed that the AGE gene BT0453, cloned from human gut microorganism Bacteroides thetaiotaomicron VPI-5482, showed the highest biotransformation yield among the AGE genes tested; and there is no clear Neu5Ac yield difference between the BT0453 coupled with one aldolase coding nanA gene and two nanA genes. Next, Escherichia coli chromosomal genes involved in substrate degradation, product exportation and pH change were deleted via recombineering and CRISPR/Cas9. With the final E. coli BL21(DE3) ΔnanA Δnag ΔpoxB as host, a significant 16.5% yield improvement was obtained. Furthermore, precursor (pyruvate) feeding resulted in 3.2% yield improvement, reaching 66.8% molar biotransformation. The result highlights the importance of host optimization, and set the stage for further metabolic engineering of whole-cell biotransformation of Neu5Ac.

The Network Basis for the Structural Thermostability and the Functional Thermoactivity of Aldolase B.

Thermostability is important for the thermoactivity of proteins including enzymes. However, it is still challenging to pinpoint the specific structural factors for different temperature thresholds to initiate their specific structural and functional perturbations. Here, graph theory was used to investigate how the temperature-dependent noncovalent interactions as identified in the structures of aldolase B and its prevalent A149P mutant could form a systematic fluidic grid-like mesh network with topological grids to regulate the structural thermostability and the functional thermoactivity upon cyclization against decyclization in an extended range of a subunit. The results showed that the biggest grid may determine the melting temperature thresholds for the changes in their secondary and tertiary structures and specific catalytic activities. Further, a highly conserved thermostable grid may serve as an anchor to secure the flexible active site to achieve the specific thermoactivity. Finally, higher grid-based systematic thermal instability may disfavor the thermoactivity. Thus, this computational study may provide critical clues for the structural thermostability and the functional thermoactivity of proteins including enzymes.

The metalloprotein YhcH is an anomerase providing N-acetylneuraminate aldolase with the open form of its substrate.

N-acetylneuraminate (Neu5Ac), an abundant sugar present in glycans in vertebrates and some bacteria, can be used as an energy source by several prokaryotes, including Escherichia coli. In solution, more than 99% of Neu5Ac is in cyclic form (≈92% beta-anomer and ≈7% alpha-anomer), whereas <0.5% is in the open form. The aldolase that initiates Neu5Ac metabolism in E. coli, NanA, has been reported to act on the alpha-anomer. Surprisingly, when we performed this reaction at pH 6 to minimize spontaneous anomerization, we found NanA and its human homolog NPL preferentially metabolize the open form of this substrate. We tested whether the E. coli Neu5Ac anomerase NanM could promote turnover, finding it stimulated the utilization of both beta and alpha-anomers by NanA in vitro. However, NanM is localized in the periplasmic space and cannot facilitate Neu5Ac metabolism by NanA in the cytoplasm in vivo. We discovered that YhcH, a cytoplasmic protein encoded by many Neu5Ac catabolic operons and belonging to a protein family of unknown function (DUF386), also facilitated Neu5Ac utilization by NanA and NPL and displayed Neu5Ac anomerase activity in vitro. YhcH contains Zn, and its accelerating effect on the aldolase reaction was inhibited by metal chelators. Remarkably, several transition metals accelerated Neu5Ac anomerization in the absence of enzyme. Experiments with E. coli mutants indicated that YhcH expression provides a selective advantage for growth on Neu5Ac. In conclusion, YhcH plays the unprecedented role of providing an aldolase with the preferred unstable open form of its substrate.

Recombinant Snail Sialic Acid Aldolase is Promiscuous towards Aliphatic Aldehydes.

Aldolases are enzymes that reversibly catalyze the cleavage of carbon-carbon bonds. Here we describe a recombinant sialic acid aldolase originating from the freshwater snail Biomphalaria glabrata (sNPL), and compare its substrate spectrum with a sialic acid aldolase originating from chicken (chNPL). In contrast to vertebrate animals which can synthesize, degrade, and incorporate sialic acids on glycoconjugate ubiquitously, snails (as all mollusks) cannot synthesize sialic acids endogenously, and therefore the biological function and substrate scope of sNPL ought to differ significantly from vertebrate sialic aldolases such as chNPL. sNPL was active towards a series of sialic acid derivatives but was in contrast to chNPL unable to catalyze the cleavage of N-acetylneuraminic acid into N-acetylmannosamine and pyruvate. Interestingly, chNPL and sNPL showed contrasting C4(R)/(S) diastereoselectivity towards the substrates d-mannose and d-galactose in the presence of pyruvate. In addition, sNPL was able to synthesize a series of 4-hydroxy-2-oxoates using the corresponding aliphatic aldehyde substrates in the presence of pyruvate, which could be not achieved by chNPL.

Fructose-1,6-Bisphosphate Aldolase B Depletion Promotes Hepatocellular Carcinogenesis Through Activating Insulin Receptor Signaling and Lipogenesis.

BACKGROUND AND AIMS: Insulin receptor (IR) transduces cell surface signal through phosphoinositide 3-kinase (PI3K)-AKT pathways or translocates to the nucleus and binds to the promoters to regulate genes associated with insulin actions, including de novo lipogenesis (DNL). Chronic activation of IR signaling drives malignant transformation, but the underlying mechanisms remain poorly defined. Down-regulation of fructose-1,6-bisphosphate aldolase (ALDO) B in hepatocellular carcinoma (HCC) is correlated with poor prognosis. We aim to study whether and how ALDOB is involved in IR signaling in HCC. APPROACH AND RESULTS: Global or liver-specific ALDOB knockout (L-ALDOB-/- ) mice were used in N-diethylnitrosamine (DEN)-induced HCC models, whereas restoration of ALDOB expression was achieved in L-ALDOB-/- mice by adeno-associated virus (AAV). 13 C6 -glucose was employed in metabolic flux analysis to track the de novo fatty acid synthesis from glucose, and nontargeted lipidomics and targeted fatty acid analysis using mass spectrometry were performed. We found that ALDOB physically interacts with IR and attenuates IR signaling through down-regulating PI3K-AKT pathways and suppressing IR nuclear translocation. ALDOB depletion or disruption of IR/ALDOB interaction in ALDOB mutants promotes DNL and tumorigenesis, which is significantly attenuated with ALDOB restoration in L-ALDOB-/- mice. Notably, attenuated IR/ALDOB interaction in ALDOB-R46A mutant exhibits more significant tumorigenesis than releasing ALDOB/AKT interaction in ALDOB-R43A, whereas knockdown IR sufficiently diminishes tumor-promoting effects in both mutants. Furthermore, inhibiting phosphorylated AKT or fatty acid synthase significantly attenuates HCC in L-ALDOB-/- mice. Consistently, ALDOB down-regulation is correlated with up-regulation of IR signaling and DNL in human HCC tumor tissues. CONCLUSIONS: Our study reports a mechanism by which loss of ALDOB activates IR signaling primarily through releasing IR/ALDOB interaction to promote DNL and HCC, highlighting a potential therapeutic strategy in HCC.

Loss-of-Function Genetic Screening Identifies Aldolase A as an Essential Driver for Liver Cancer Cell Growth Under Hypoxia.

BACKGROUND AND AIMS: Hypoxia is a common feature of the tumor microenvironment (TME), which promotes tumor progression, metastasis, and therapeutic drug resistance through a myriad of cell activities in tumor and stroma cells. While targeting hypoxic TME is emerging as a promising strategy for treating solid tumors, preclinical development of this approach is lacking in the study of HCC. APPROACH AND RESULTS: From a genome-wide CRISPR/CRISPR-associated 9 gene knockout screening, we identified aldolase A (ALDOA), a key enzyme in glycolysis and gluconeogenesis, as an essential driver for HCC cell growth under hypoxia. Knockdown of ALDOA in HCC cells leads to lactate depletion and consequently inhibits tumor growth. Supplementation with lactate partly rescues the inhibitory effects mediated by ALDOA knockdown. Upon hypoxia, ALDOA is induced by hypoxia-inducible factor-1α and fat mass and obesity-associated protein-mediated N6 -methyladenosine modification through transcriptional and posttranscriptional regulation, respectively. Analysis of The Cancer Genome Atlas shows that elevated levels of ALDOA are significantly correlated with poor prognosis of patients with HCC. In a screen of Food and Drug Administration-approved drugs based on structured hierarchical virtual platforms, we identified the sulfamonomethoxine derivative compound 5 (cpd-5) as a potential inhibitor to target ALDOA, evidenced by the antitumor activity of cpd-5 in preclinical patient-derived xenograft models of HCC. CONCLUSIONS: Our work identifies ALDOA as an essential driver for HCC cell growth under hypoxia, and we demonstrate that inhibition of ALDOA in the hypoxic TME is a promising therapeutic strategy for treating HCC.

Acetylation of fructose-bisphosphate aldolase-mediated glycolysis is essential for Bombyx mori nucleopolyhedrovirus infection.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a baculovirus that infects silkworms, and its interaction with silkworm has been considered an important model in the field of insect virology. Accumulating evidence indicates that most viruses promote glycolytic metabolism in host cells to favor infection. However, similar reports are lacking in insects, especially in the area of post-translational modifications of proteins. In this study, we found that BmNPV infection induced the acetylation of fructose-bisphosphate aldolase (ALDO) on lysine 42 (K42) to promote its enzyme activity. To explore the underlying mechanisms, site-directed mutagenesis of deacetylated mimic (K/R) was performed. The results demonstrated that K42 acetylation promoted viral proliferation by exacerbating the glycolytic flux induced by BmNPV infection, which resulted in increased ATP, glucose uptake and lactate accumulation. Inhibiting glycolysis with 2-deoxygucose (2DG) revealed that glycolysis was essential for optimal BmNPV infection. Finally, we showed that BmNPV-infected cells enhanced the transcription of glycolysis-related genes, including Glut1, Hk2 and Ldh. In parallel, K42 acetylation of ALDO also promoted the expression of these genes. Therefore, acetylation of ALDO could be considered a regulator of BmNPV-induced glycolysis. These finding provide insights into the interaction between silkworm and BmNPV.

Assessing the ceRNA hypothesis with quantitative measurements of miRNA and target abundance.

Recent studies have reported that competitive endogenous RNAs (ceRNAs) can act as sponges for a microRNA (miRNA) through their binding sites and that changes in ceRNA abundances from individual genes can modulate the activity of miRNAs. Consideration of this hypothesis would benefit from knowing the quantitative relationship between a miRNA and its endogenous target sites. Here, we altered intracellular target site abundance through expression of an miR-122 target in hepatocytes and livers and analyzed the effects on miR-122 target genes. Target repression was released in a threshold-like manner at high target site abundance (≥1.5 × 10(5) added target sites per cell), and this threshold was insensitive to the effective levels of the miRNA. Furthermore, in response to extreme metabolic liver disease models, global target site abundance of hepatocytes did not change sufficiently to affect miRNA-mediated repression. Thus, modulation of miRNA target abundance is unlikely to cause significant effects on gene expression and metabolism through a ceRNA effect.