Intracellular Citrate Research Articles

Abstract We087: The mitochondrial citrate carrier SLC25A1 is a regulator of metabolic reprogramming and morphogenesis in the developing heart

Background: Congenital heart defects (CHDs) are the most common type of birth defect, yet the etiology of most CHDs are unknown. A major genetic risk factor for CHD is 22q11.2 deletion syndrome (22q11.2DS), a multisystem chromosomal microdeletion disorder where ~75% of patients present with CHD. SLC25A1 is a gene found within the 22q11.2DS microdeletion region that encodes for the mitochondrial citrate carrier, an inner membrane transporter that regulates intracellular citrate compartmentalization. Through this role, SLC25A1 is thought to contribute to cytosolic acetyl-CoA availability, and processes like protein acetylation and the epigenetic regulation of gene expression. We have previously found that SLC25A1 is a central gene contributing to the neurodevelopmental presentation of 22q11.2DS and importantly, neuronal mitochondrial function. Because the heart, like the brain, is highly reliant on energy derived from mitochondria, and the developing heart must undergo metabolic maturation whereby metabolism is reprogrammed from primary reliance on glycolysis towards mitochondrial oxidative metabolism, we hypothesized that SLC25A1 plays a key role in the metabolic maturation that is required for proper cardiac development. Objective: Here, we studied the role of Slc25a1 in the developing heart to examine the link between mitochondria and cardiac morphogenesis. Methods and Results: By studying mice with the systemic knockout of SLC25A1, we discovered that Slc25a1 null embryos displayed impaired growth, cardiac malformations, mitochondrial ultrastructural defects and impaired mitochondrial oxygen consumption. Transcriptomics analyses of metabolism-related genes revealed that Slc25a1 deletion causes widespread alterations in metabolic gene expression. Further, metabolic modelling predicted that loss of SLC25A1 downregulates oxidative phosphorylation, while increasing reliance on glucose. Finally, we found that loss of SLC25A1 decreases cardiac H3K9 acetylation levels at promoter regions of dysregulated metabolic genes. Mechanistically, SLC25A1 may regulating mitochondrial function and metabolism through epigenetic control of gene expression to promote metabolic remodeling in the developing heart. Conclusions: Our work suggests that SLC25A1 as an an important mitochondrial regulator of gene expression in the developing heart. Importantly, it positions SLC25A1 as a novel mitochondrial regulator of ventricular morphogenesis as well as cardiac metabolic maturation.

Retraction Note: Intracellular citrate accumulation by oxidized ATM-mediated metabolism reprogramming via PFKP and CS enhances hypoxic breast cancer cell invasion and metastasis

Retraction Note: Intracellular citrate accumulation by oxidized ATM-mediated metabolism reprogramming via PFKP and CS enhances hypoxic breast cancer cell invasion and metastasis

Metabolomics of Escherichia coli for Disclosing Novel Metabolic Engineering Strategies for Enhancing Hydrogen and Ethanol Production.

The biological production of hydrogen is an appealing approach to mitigating the environmental problems caused by the diminishing supply of fossil fuels and the need for greener energy. Escherichia coli is one of the best-characterized microorganisms capable of consuming glycerol-a waste product of the biodiesel industry-and producing H2 and ethanol. However, the natural capacity of E. coli to generate these compounds is insufficient for commercial or industrial purposes. Metabolic engineering allows for the rewiring of the carbon source towards H2 production, although the strategies for achieving this aim are difficult to foresee. In this work, we use metabolomics platforms through GC-MS and FT-IR techniques to detect metabolic bottlenecks in the engineered ΔldhΔgndΔfrdBC::kan (M4) and ΔldhΔgndΔfrdBCΔtdcE::kan (M5) E. coli strains, previously reported as improved H2 and ethanol producers. In the M5 strain, increased intracellular citrate and malate were detected by GC-MS. These metabolites can be redirected towards acetyl-CoA and formate by the overexpression of the citrate lyase (CIT) enzyme and by co-overexpressing the anaplerotic human phosphoenol pyruvate carboxykinase (hPEPCK) or malic (MaeA) enzymes using inducible promoter vectors. These strategies enhanced specific H2 production by up to 1.25- and 1.49-fold, respectively, compared to the reference strains. Other parameters, such as ethanol and H2 yields, were also enhanced. However, these vectors may provoke metabolic burden in anaerobic conditions. Therefore, alternative strategies for a tighter control of protein expression should be addressed in order to avoid undesirable effects in the metabolic network.

The Influence of Zinc and Citrate on T Cell Activation and Differentiation in Mixed Lymphocyte Cultures.

Zinc is important for a balanced immune system, but the mechanisms are not yet fully elucidated. One possibility is an interaction of zinc with the tricarboxylic acid cycle (TCA), in which zinc inhibits the mitochondrial aconitase leading to an increase in intracellular citrate concentration as described for prostate cells. Therefore, the immune modulatory effects of zinc and citrate and their interaction in mixed lymphocyte cultures (MLC) are studied. After allogeneic (MLC) or superantigen stimulation, the interferon-γ (IFNγ) production is quantified by ELISA and T cell subpopulations are determined by Western Blot. Intracellular concentrations of citrate and zinc are measured. Zinc and citrate reduce the IFNγ expression and the pro-inflammatory T helper cells (Th) 1 and Th17 in MLC. While zinc increases regulatory T cells, citrate reduces them. After superantigen stimulation IFNγ production is decreased only by citrate but increased by zinc. Zinc does not affect citrate concentration, while citrate impairs zinc uptake. Thus, zinc and citrate independently regulate IFNy expression. These results may explain the immunosuppressive effect of blood products anticoagulated by citrate. In addition, high citrate consumption may lead to immunosuppressive effects, so upper limits for citrate should be established.

Wild-type IDH1 Knockout Leads to G0/G1 Arrest, Impairs Cancer Cell Proliferation, Altering Glycolysis, and the TCA Cycle in Colon Cancer.

The isocitrate dehydrogenase (IDH), which participates in the TCA cycle, is an important key enzyme in regulating cell metabolism. The effect of the metabolic IDH enzyme on cancer pathogenesis has recently been shown in different types of cancer. However, the role of wild-type (wt) IDH1 in the development of colon cancer is still unknown. Our study investigated the role of the IDH1 enzyme in key hallmarks of colon cancer using various methods such as wound healing, cell cycle, colony formation ability, invasion, and apoptosis analysis. Furthermore, cell metabolism was investigated by pyruvate analysis, dinitrosalicylic acid, and HPLC methods. In addition, CRISPR/Cas9 tool was utilized to knockout the IDH1 gene in colon adenocarcinoma cells (SW620). Further studies were performed in two isogenic IDH1 KO clones. Our findings in both clones suggest that IDH1 KO results in G0/G1 arrest, and reduces proliferation by approximately twofold compared to IDH1 WT cells. In addition, the invasion, migration, and colony formation abilities of IDH1 KO clones were significantly decreased accompanied by significant morphological changes. In the context of metabolism, intracellular glucose, pyruvate, αKG, and malate levels were decreased, while the intracellular citrate level was increased in IDH1 KO clones as compared to IDH1 WT cells. Our results reveal that wt IDH1 knockout leads to a decrease in the aggressive features of colon cancer cells. In conclusion, we reported that wt IDH1 has an effective role in colon cancer progression and could be a potential therapeutic target.

Quantification of Intracellular Citrate Concentrations with Genetically Encoded Biosensors.

Citrate is a central intracellular metabolite with roles in a variety of normal and aberrant biological processes. The methods for quantifying citrate concentration in cells can enable the study of the molecular mechanisms of citrate-related biological processes and diseases. Compared to existing analytical methods such as enzymatic assays and mass spectrometry, genetically encoded biosensors based on fluorescent proteins (FPs) offer the advantage of noninvasively tracking intracellular ion and small molecule dynamics with high spatial-temporal resolution. These biosensors are less toxic than chemosensors and can be targeted to specific organelles for subcellular imaging. Here we present a protocol for quantification of cytosolic and mitochondrial citrate in mammalian cells with recently reported genetically encoded biosensors for citrate.

Metabolic reprogramming in the OPA1-deficient cells.

OPA1, a dynamin-related GTPase mutated in autosomal dominant optic atrophy, is essential for the fusion of the inner mitochondrial membrane. Although OPA1 deficiency leads to impaired mitochondrial morphology, the role of OPA1 in central carbon metabolism remains unclear. Here, we aim to explore the functional role and metabolic mechanism of OPA1 in cell fitness beyond the control of mitochondrial fusion. We applied [U-13C]glucose and [U-13C]glutamine isotope tracing techniques to OPA1-knockout (OPA1-KO) mouse embryonic fibroblasts (MEFs) compared to OPA1 wild-type (OPA1-WT) controls. Furthermore, the resulting tracing data were integrated by metabolic flux analysis to understand the underlying metabolic mechanism through which OPA1 deficiency reprograms cellular metabolism. OPA1-deficient MEFs were depleted of intracellular citrate, which was consistent with the decreased oxygen consumption rate in these cells with mitochondrial fission that is not balanced by mitochondrial fusion. Whereas oxidative glucose metabolism was impaired, OPA1-deficient cells activated glutamine-dependent reductive carboxylation and subsequently relied on this reductive metabolism to produce cytosolic citrate as a predominant acetyl-CoA source for de novo fatty acid synthesis. Prevention of cytosolic glutamine reductive carboxylation by GSK321, an inhibitor of isocitrate dehydrogenase 1 (IDH1), largely repressed lipid synthesis and blocked cell proliferation in OPA1-deficient MEFs. Our data support that, when glucose oxidation failed to support lipogenesis and proliferation in cells with unbalanced mitochondrial fission, OPA1 deficiency stimulated metabolic anaplerosis into glutamine-dependent reductive carboxylation in an IDH1-mediated manner.

Phenobarbital induces SLC13A5 expression through activation of PXR but not CAR in human primary hepatocytes

Phenobarbital (PB), a widely used anti‐epileptic drug, is known to upregulate the expression of numerous drug‐metabolizing enzymes and transporters in the liver primarily via activation of the constitutive androstane receptor (CAR, NR1I3). The solute carrier family 13 member 5 (SLC13A5), a sodium‐coupled citrate transporter, plays an important role in intracellular citrate homeostasis that is associated with a number of metabolic syndromes and neurological disorders. Here, we show that PB markedly elevates the expression of SLC13A5 through a pregnane X receptor (PXR)‐dependent but CAR‐independent signaling pathway. In human primary hepatocytes, the mRNA and protein expression of SLC13A5 was robustly induced by PB treatment, while genetic knockdown or pharmacological inhibition of PXR significantly attenuated this induction. Utilizing genetically modified HepaRG cells, we found that PB induces SLC13A5 expression in both wild type (WT) and CAR‐knockout HepaRG cells, whereas such induction was fully abolished in the PXR‐knockout HepaRG cells. Mechanistically, we identified and functionally characterized three enhancer modules located upstream of the transcription start site or introns of the SLC13A5 gene that are associated with the regulation of PXR‐mediated SLC13A5 induction. Moreover, metformin, a deactivator of PXR, dramatically suppressed PB‐mediated induction of hepatic SLC13A5 as well as its activation of the SLC13A5 luciferase reporter activity via PXR. Collectively, these data reveal PB as a potent inducer of SLC13A5 through the activation of PXR but not CAR in human primary hepatocytes.

Increased Fatty Acid Synthesis and Catabolism Supports Metastatic Breast Cancer Cell Migration

Lipid accumulation is positively associated with breast cancer aggressiveness and poorer clinical outcomes. Despite this observation, mechanisms underlying the increase and role of stored lipid in breast cancer progression remain incompletely understood. The accumulation of triacylglycerol (TAG) is dependent on the balance of uptake, synthesis, and utilization of fatty acids (FAs). In the current study, we utilized non‐metastatic MCF10A‐ras and metastatic MCF10CA1a human breast cancer cells to determine differences in TAG storage and catabolism for sustaining migration—a critical step in the metastatic cascade. Our results demonstrate that MCF10CA1a cells have 90% more TAG than MCF10A‐ras (TAG Assay and Transmission Electron Microscopy), although no significant difference in 14C‐palmitate uptake. MCF10CA1a cells have greater FA synthesis as shown by greater incorporation of 13C‐acetate (60%), 13C‐glucose (65%), and 13C‐glutamine (50%) into palmitate compared to the MCF10A‐ras cells (LC‐MS/MS), as well as higher protein levels of FA synthase (FASN). Additionally, MCF10CA1a cells display greater flux of uniformly‐labeled 13C‐glucose and 13C‐glutamine to the FA synthesis precursor, citrate, and lower intracellular citrate pool size compared to MCF10A‐ras. In addition to increased FA anabolism, MCF10CA1a cells rely on FA oxidation (FAO) for cellular migration compared to MCF10A‐ras cells, as determined by inhibiting the rate‐limiting enzyme of FAO, carnitine palmitoyltransferase 1, with etomoxir (transwell and wound‐healing assays). Pretreatment with the FASN inhibitor TVB‐3166 (3 d) significantly reduced TAG levels and subsequent migration by 67% compared to vehicle‐treated cells. Similarly, pretreatment with inhibitors of the TAG‐synthesizing enzymes diacylglycerol O‐acyltransferase 1 and 2 reduced TAG accumulation and subsequent migration (75%) of MCF10CA1a cells compared to vehicle. Together, the data suggest that FA synthesis may contribute to TAG storage necessary to sustain breast cancer migration. To test this hypothesis, we inhibited the initial enzyme of the TAG lipolysis pathway, adipose triacylglycerol lipase (ATGL; inhibitor ATGListatin), and measured migration. Inhibition of ATGL alone reduced MCF10CA1a cell migration (20% decrease compared to vehicle), and addition of the FAO inhibitor (etomoxir) provided no further effect. Our study indicates that metastatic MCF10CA1a cells accumulate FAs by increasing de novo lipogenesis, store them as TAG, and that catabolism of these stores drives FAO‐dependent migration.

Phenobarbital Induces SLC13A5 Expression through Activation of PXR but Not CAR in Human Primary Hepatocytes.

Phenobarbital (PB), a widely used antiepileptic drug, is known to upregulate the expression of numerous drug-metabolizing enzymes and transporters in the liver primarily via activation of the constitutive androstane receptor (CAR, NR1I3). The solute carrier family 13 member 5 (SLC13A5), a sodium-coupled citrate transporter, plays an important role in intracellular citrate homeostasis that is associated with a number of metabolic syndromes and neurological disorders. Here, we show that PB markedly elevates the expression of SLC13A5 through a pregnane X receptor (PXR)-dependent but CAR-independent signaling pathway. In human primary hepatocytes, the mRNA and protein expression of SLC13A5 was robustly induced by PB treatment, while genetic knockdown or pharmacological inhibition of PXR significantly attenuated this induction. Utilizing genetically modified HepaRG cells, we found that PB induces SLC13A5 expression in both wild type and CAR-knockout HepaRG cells, whereas such induction was fully abolished in the PXR-knockout HepaRG cells. Mechanistically, we identified and functionally characterized three enhancer modules located upstream from the transcription start site or introns of the SLC13A5 gene that are associated with the regulation of PXR-mediated SLC13A5 induction. Moreover, metformin, a deactivator of PXR, dramatically suppressed PB-mediated induction of hepatic SLC13A5 as well as its activation of the SLC13A5 luciferase reporter activity via PXR. Collectively, these data reveal PB as a potent inducer of SLC13A5 through the activation of PXR but not CAR in human primary hepatocytes.

Bronchial Epithelial Cells Accumulate Citrate Intracellularly in Response to Pneumococcal Hydrogen Peroxide.

Community-acquired pneumonia is an infection of the lower respiratory tract caused by various viral and bacterial pathogens, including influenza A virus (IAV), Streptococcus pneumoniae, and Staphylococcus aureus. To understand the disease pathology, it is important to delineate host metabolic responses to an infection. In this study, metabolome profiling of mono- and coinfected human bronchial epithelial cells was performed. We show that IAV and S. aureus silently survive within the cells with almost negligible effects on the host metabolome. In contrast, S. pneumoniae significantly altered various host pathways such as glycolysis, tricarboxylic acid cycle, and amino acid metabolism. Intracellular citrate accumulation was the most prominent signature of pneumococcal infections and was directly attributed to the action of pneumococci-derived hydrogen peroxide. No coinfection specific metabolome signatures were observed.

Biochemical elucidation of citrate accumulation in Synechocystis sp. PCC 6803 via kinetic analysis of aconitase

A unicellular cyanobacterium Synechocystis sp. PCC 6803 possesses a unique tricarboxylic acid (TCA) cycle, wherein the intracellular citrate levels are approximately 1.5–10 times higher than the levels of other TCA cycle metabolite. Aconitase catalyses the reversible isomerisation of citrate and isocitrate. Herein, we biochemically analysed Synechocystis sp. PCC 6803 aconitase (SyAcnB), using citrate and isocitrate as the substrates. We observed that the activity of SyAcnB for citrate was highest at pH 7.7 and 45 °C and for isocitrate at pH 8.0 and 53 °C. The Km value of SyAcnB for citrate was higher than that for isocitrate under the same conditions. The Km value of SyAcnB for isocitrate was 3.6-fold higher than the reported Km values of isocitrate dehydrogenase for isocitrate. Therefore, we suggest that citrate accumulation depends on the enzyme kinetics of SyAcnB, and 2-oxoglutarate production depends on the chemical equilibrium in this cyanobacterium.

Possible role of corticosterone in proteolysis, glycolytic, and amino acid metabolism in primary cultured avian myotubes incubated at high-temperature conditions

Excess glucocorticoid secretion induces oxidative damage and muscle proteolysis and modulates glucose and lipid metabolism. It is known that the high-temperature (HT) treatment enhances corticosterone (CORT) secretion, muscle proteolysis, and mitochondrial reactive oxygen species (mtROS) generation in chickens. The present study investigated the co-effects of CORT on proteolysis and mtROS production, together with glucose, fatty acid, and amino acid metabolism in HT-treated cells. Myoblast cells were isolated from the major pectoralis muscle of five 0- or 1-day-old neonatal chicks and were precultured at 37°C/CO2 conditions for 48 h to reach subconfluent (80%–90%) conditions. Cells were then reseeded onto a 6- or 24-well microplate for the subsequent measurement, followed by the culture under a control temperature (37°C, control) or HT (41°C) conditions for 1 or 6 h. The HT-treated cells were cocultured with physiologically relevant concentrations of CORT (20 ng/mL in dimethyl sulfoxide). The HT treatment decreased cellular protein content (P < 0.05) and increased atrogin-1 mRNA levels and mtROS generation levels compared to the control group (P < 0.05), whereas HT/CORT co-treatment did not induce changes in either parameter. The mRNA level of glucose transporter-1 was decreased in HT-treated cells compared to that in normal cells (P < 0.05), and the decrease was increased in the CORT co-treatment (P < 0.05). While HT treatment did not alter pyruvate dehydrogenase kinase-4 mRNA level, the level was increased in the CORT co-treatment compared to the control and HT-treated cells (P < 0.05). Neither HT nor HT/CORT treatments altered the mRNA levels of fatty acid oxidation-related factors, carnitine palmitoyl transferase-1, and cluster of differentiation 36. The study conducted a metabolic analysis using gas chromatography-mass spectrometry. The results showed that HT/CORT-treated cells had decreased intracellular citrate and α-ketoglutarate levels (P < 0.05) and increased extracellular alanine and amino acid that have gluconeogenic properties, as well as increased aspartate, isoleucine, serine, methionine, and threonine levels (P < 0.05) compared to HT-treated cells. These results suggest that CORT may not affect proteolysis and mtROS production but can suppress pyruvate oxidation and promote alanine production in HT-treated chickens.

Silibinin down-regulates PD-L1 expression in nasopharyngeal carcinoma by interfering with tumor cell glycolytic metabolism

The upregulation of checkpoint inhibitor PD-L1 expression has recently been associated with nasopharyngeal carcinoma (NPC) resistance to therapy. The mechanism of induction of PD-L1 has also been linked to enhanced aerobic glycolysis promoted by HIF1-α dysregulation and LDH-A activity in cancer. Here, we investigated the effect of the anti-tumoral compound Silibinin on HIF-1α/LDH-A mediated cancer cell metabolism and PD-L1 expression in NPC. Our results demonstrate that exposure to Silibinin potently inhibits tumor growth and promotes a shift from aerobic glycolysis toward oxidative phosphorylation. The EBV + NPC cell line C666-1 and glycolytic human tumor explants treated with Silibinin displayed a reduction in LDH-A activity which consistently associated with a reduction in lactate levels. This effect was accompanied by an increase in intracellular citrate levels in C666-1 cells. Accordingly, expression of HIF-1α, a critical regulator of glycolysis, was down-regulated after treatment. This event associated with a down-regulation in PD-L1. Altogether, our results provide evidence that silibinin can alter PD-L1 expression by interfering with HIF-1α/LDH-A mediated cell metabolism in NPC. These results provide a new perspective for Silibinin use to overcome PD-L1 mediated NPC resistance to therapy.

Abstract 3798: Acetate supplementation eliminates hypoxia mediated resistance to differentiation therapy in neuroblastoma cells

Abstract Neuroblastoma is the most common extracranial solid tumor in childhood. Retinoic acid (RA) leads to cell cycle arrest and induces cell differentiation in neuroblastoma cells, thus it has been used to treat neuroblastoma as differentiating agent for decades. However, therapy resistance often occurs and the overall survival remains low. Hypoxia, a common metabolic stress existing in tumor microenvironment, increases tumor resistance to RA by promoting dedifferentiation of neuroblastoma cells. The aim of this study was to elucidate the mechanisms underlying hypoxia-mediated therapy resistance and develop therapeutic approach to improve the response to differentiation therapy. By RNA-Seq analysis, we identified a group of neuron-specific differentiation markers that induced by RA treatment, while the induction was diminished under hypoxia. Associated with the silencing of differentiation markers expression, a significant loss of histone acetylation of H3K9, H3K27 and total H3 was observed under hypoxia. Metabolomics analysis indicated hypoxia inhibited mitochondrial oxidative phosphorylation to decrease intracellular acetyl-CoA and citrate, which provides acetyl-CoA for histone acetyltransferase. Importantly, acetate supplementation restored cellular acetyl-CoA level, histone acetylation, and the expression of differentiation markers under hypoxia. Differentiation morphology was also restored by acetate supplementation under hypoxia. In conclusion, our finding suggested hypoxia contributed to the resistance to RA-induced differentiation by reprogramming cellular metabolism and inducing histone hypoacetylation. Acetate supplementation is an efficacious therapeutic approach to improve the sensitivity of hypoxic tumor to RA-based differentiation therapy. Citation Format: Yang Li, Yiren Zhou, John M. Maris, Amato J. Giaccia, Jiangbin Ye. Acetate supplementation eliminates hypoxia mediated resistance to differentiation therapy in neuroblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3798.

RETRACTED ARTICLE: Intracellular citrate accumulation by oxidized ATM-mediated metabolism reprogramming via PFKP and CS enhances hypoxic breast cancer cell invasion and metastasis

Citrate, a substance being related to de novo fatty acid synthesis and tricarboxylic acid (TCA) cycle, has a pivotal role in cell survival. However, the molecular mechanisms that regulate intracellular citrate in triple-negative breast cancer (TNBC), especially under hypoxic condition, remain poorly understood. Here we find that hypoxia (1% O2) induces DNA damage-independent ATM activation (oxidized ATM) and suppression of oxidized ATM reduces intracellular citrate via decreasing the levels of phosphofructokinase (PFKP) and citrate synthase (CS), two key glucose metabolism-associated enzymes. Mechanistically, PFKP is regulated by HIF1A at the translational level, whereas CS is of posttranscriptional regulation by UBR5-mediated ubiquitination. Interestingly, accumulation of citrate in cytoplasm or exogenous citrate significantly enhances cell migration, invasion, and metastasis of hypoxic TNBC cells in vitro and in mice xenografts. The underlying mechanism mainly involves citrate-stimulated activation of the AKT/ERK/MMP2/9 signaling axis. Our findings unravel a novel function of oxidized ATM in promoting migration, invasion, and metastasis of TNBC.

Identification and application of a growth-regulated promoter for improving l-valine production in Corynebacterium glutamicum

BackgroundPromoters are commonly used to regulate the expression of specific target genes or operons. Although a series of promoters have been developed in Corynebacterium glutamicum, more precise and unique expression patterns are needed that the current selection of promoters cannot produce. RNA-Seq technology is a powerful tool for helping us to screen out promoters with expected transcriptional strengths.ResultsThe promoter PCP_2836 of an aldehyde dehydrogenase coding gene from Corynebacterium glutamicum CP was identified via RNA-seq and RT-PCR as a growth-regulated promoter. Comparing with the strong constitutive promoter Ptuf, the transcriptional strength of PCP_2836 showed a significant decrease that from about 75 to 8% in the stationary phase. By replacing the native promoters of the aceE and gltA genes with PCP_2836 in the C. glutamicum ATCC 13032-derived l-valine-producing strain AN02, the relative transcriptional levels of the aceE and gltA genes decreased from 1.2 and 1.1 to 0.35 and 0.3, and the activity of their translation products decreased to 43% and 35%, respectively. After 28 h flask fermentation, the final cell density of the obtained strains, GRaceE and GRgltA, exhibited a 7–10% decrease. However, l-valine production increased by 23.9% and 27.3%, and the yield of substrate to product increased 43.8% and 62.5%, respectively. In addition, in the stationary phase, the intracellular citrate levels in GRaceE and GRgltA decreased to 27.0% and 33.6% of AN02, and their intracellular oxaloacetate levels increased to 2.7 and 3.0 times that of AN02, respectively.ConclusionsThe PCP_2836 promoter displayed a significant difference on its transcriptional strength in different cell growth phases. With using PCP_2836 to replace the native promoters of aceE and gltA genes, both the transcriptional levels of the aceE and gltA genes and the activity of their translation products demonstrated a significant decrease in the stationary phase. Thus, the availability of pyruvate was significantly increased for the synthesis of l-valine without any apparent irreversible negative impacts on cell growth. Use of this promoter can enhance the selectivity and control of gene expression and could serve as a useful research tool for metabolic engineering.

Structural and Biochemical Analysis of the Citrate-Responsive Mechanism of the Regulatory Domain of Catabolite Control Protein E from Staphylococcus aureus.

Catabolite control protein E (CcpE) is a LysR-type transcriptional regulator that positively regulates the transcription of the first two enzymes of the TCA cycle, namely, citZ and citB, by sensing accumulated intracellular citrate. CcpE comprises an N-terminal DNA-binding domain and a C-terminal regulatory domain (RD) and senses citrate with conserved arginine residues in the RD. Although the crystal structure of the apo SaCcpE-RD has been reported, the citrate-responsive and DNA-binding mechanisms by which CcpE regulates TCA activity remain unclear. Here, we report the crystal structure of the apo and citrate-bound SaCcpE-RDs. The SaCcpE-RD exhibits conformational changes between the two subdomains via hinge motion of the central β4 and β10 strands. The citrate molecule is located in a positively charged cavity between the two subdomains and interacts with the highly conserved Ser98, Leu100, Arg145, and Arg256 residues. Compared with that of the apo SaCcpE-RD, the distance between the two subdomains of the citrate-bound SaCcpE-RD is more than ∼3 Å due to the binding of the citrate molecule, and this form exhibits a closed structure. The SaCcpE-RD exhibits various citrate-binding-independent conformational changes at the contacting interface. The SaCcpE-RD prefers the dimeric state in solution, whereas the SaCcpE-FL prefers the tetrameric state. Our results provide insight into the molecular function of SaCcpE.

Extracellular Citrate Affects Critical Elements of Cancer Cell Metabolism and Supports Cancer Development In Vivo.

Glycolysis and fatty acid synthesis are highly active in cancer cells through cytosolic citrate metabolism, with intracellular citrate primarily derived from either glucose or glutamine via the tricarboxylic acid cycle. We show here that extracellular citrate is supplied to cancer cells through a plasma membrane-specific variant of the mitochondrial citrate transporter (pmCiC). Metabolomic analysis revealed that citrate uptake broadly affected cancer cell metabolism through citrate-dependent metabolic pathways. Treatment with gluconate specifically blocked pmCiC and decreased tumor growth in murine xenografts of human pancreatic cancer. This treatment altered metabolism within tumors, including fatty acid metabolism. High expression of pmCiC was associated with invasion and advanced tumor stage across many human cancers. These findings support the exploration of extracellular citrate transport as a novel potential target for cancer therapy.Significance: Uptake of extracellular citrate through pmCiC can be blocked with gluconate to reduce tumor growth and to alter metabolic characteristics of tumor tissue. Cancer Res; 78(10); 2513-23. ©2018 AACR.

Suppressed de novo lipogenesis by plasma membrane citrate transporter inhibitor promotes apoptosis in HepG2 cells.

Suppression of the expression or activities of enzymes that are involved in the synthesis of de novo lipogenesis (DNL) in cancer cells triggers cell death via apoptosis. The plasma membrane citrate transporter (PMCT) is the initial step that translocates citrate from blood circulation into the cytoplasm for de novo long‐chain fatty acids synthesis. This study investigated the antitumor effect of the PMCT inhibitor (PMCTi) in inducing apoptosis by inhibiting the DNL pathway in HepG2 cells. The present findings showed that PMCTi reduced cell viability and enhanced apoptosis through decreased intracellular citrate levels, which consequently caused inhibition of fatty acid and triacylglycerol productions. Thus, as a result of the reduction in fatty acid synthesis, the activity of carnitine palmitoyl transferase‐1 (CPT‐1) was suppressed. Decreased CPT‐1 activity also facilitated the disruption of mitochondrial membrane potential (ΔΨm) leading to stimulation of apoptosis. Surprisingly, primary human hepatocytes were not affected by PMCTi. Increased caspase‐8 activity as a consequence of reduction in fatty acid synthesis was also found to cause disruption of ΔΨm. In addition, apoptosis induction by PMCTi was associated with an enhanced reactive oxygen species generation. Taken together, we suggest that inhibition of the DNL pathway following reduction in citrate levels is an important regulator of apoptosis in HepG2 cells via suppression of CPT‐1 activity. Thus, targeting the DNL pathway mediating CPT‐1 activity by PMCTi may be a selective potential anticancer therapy.