Compensatory activity of the PC-ME1 metabolic axis underlies differential sensitivity to mitochondrial complex I inhibition.

Deficiencies in the electron transport chain (ETC) lead to mitochondrial diseases. While mutations are distributed across the organism, cell and tissue sensitivity to ETC disruption varies, and the molecular mechanisms underlying this variability remain poorly understood. Here we show that, upon ETC inhibition, a non-canonical tricarboxylic acid (TCA) cycle upregulates to maintain malate levels and concomitant production of NADPH. Our findings indicate that the adverse effects observed upon CI inhibition primarily stem from reduced NADPH levels, rather than ATP depletion. Furthermore, we find that Pyruvate carboxylase (PC) and ME1, the key mediators orchestrating this metabolic reprogramming, are selectively expressed in astrocytes compared to neurons and underlie their differential sensitivity to ETC inhibition. Augmenting ME1 levels in the brain alleviates neuroinflammation and corrects motor function and coordination in a preclinical mouse model of CI deficiency. These studies may explain why different brain cells vary in their sensitivity to ETC inhibition, which could impact mitochondrial disease management.

Toxicity and Metabolomic Dysfunction Invoked by Febrifugin, a Harmful Component of Edible Nut of Swietenia macrophylla.

Swietenia macrophylla fruit is a valuable and historically significant medicinal plant with anti-hypertension and anti-diabetes. We identified a toxic component, Febrifugin, from the edible part of the nut following zebrafish toxicity-guided isolation. Febrifugin is a mexicanolide-type limonoid compound. The toxic factor induced acute toxicity in zebrafish, including yolk sac edema and pericardial edema, reduced body length, decreased melanin deposition, and presented acute skeletal developmental issues. Further exploration of the acute toxicity mechanism through metabolomics revealed that Febrifugin caused significant changes in 13 metabolites in zebrafish larvae, which are involved in the pentose phosphate, tricarboxylic acid (TCA) cycle, and amino acid biosynthesis. The bioassay of oxidative stress capacity and qRT-PCR measurement showed that the compound significantly affected the h6pd gene in the pentose phosphate pathway and the mRNA expression of cs, idh3a, fh, and shda genes in the TCA cycle, leading to reactive oxygen species (ROS) accumulation and a notable decrease in glutathione (GSH) activity in zebrafish. These findings provide a basis for the rational use of S. macrophylla as a medicinal plant and raise awareness of the safety of medicinal plants.

TCA metabolism regulates DNA hypermethylation in LPS and Mycobacterium tuberculosis-induced immune tolerance.

Severe and chronic infections, including pneumonia, sepsis, and tuberculosis (TB), induce long-lasting epigenetic changes that are associated with an increase in all-cause postinfectious morbidity and mortality. Oncology studies identified metabolic drivers of the epigenetic landscape, with the tricarboxylic acid (TCA) cycle acting as a central hub. It is unknown if the TCA cycle also regulates epigenetics, specifically DNA methylation, after infection-induced immune tolerance. The following studies demonstrate that lipopolysaccharide and Mycobacterium tuberculosis induce changes in DNA methylation that are mediated by the TCA cycle. Infection-induced DNA hypermethylation is mitigated by inhibitors of cellular metabolism (rapamycin, everolimus, metformin) and the TCA cycle (isocitrate dehydrogenase inhibitors). Conversely, exogenous supplementation with TCA metabolites (succinate and itaconate) induces DNA hypermethylation and immune tolerance. Finally, TB patients who received everolimus have less DNA hypermethylation demonstrating proof of concept that metabolic manipulation can mitigate epigenetic scars.

Oxaloacetate anaplerosis differently contributes to pathogenicity in plant pathogenic fungi Fusarium graminearum and F. oxysporum.

Anaplerosis refers to enzymatic reactions or pathways replenishing metabolic intermediates in the tricarboxylic acid (TCA) cycle. Pyruvate carboxylase (PYC) plays an important anaplerotic role by catalyzing pyruvate carboxylation, forming oxaloacetate. Although PYC orthologs are well conserved in prokaryotes and eukaryotes, their pathobiological functions in filamentous pathogenic fungi have yet to be fully understood. Here, we delve into the molecular functions of the ortholog gene PYC1 in Fusarium graminearum and F. oxysporum, prominent fungal plant pathogens with distinct pathosystems, demonstrating variations in carbon metabolism for pathogenesis. Surprisingly, the PYC1 deletion mutant of F. oxysporum exhibited pleiotropic defects in hyphal growth, conidiation, and virulence, unlike F. graminearum, where PYC1 deletion did not significantly impact virulence. To further explore the species-specific effects of PYC1 deletion on pathogenicity, we conducted comprehensive metabolic profiling. Despite shared metabolic changes, distinct reprogramming in central carbon and nitrogen metabolism was identified. Specifically, alpha-ketoglutarate, a key link between the TCA cycle and amino acid metabolism, showed significant down-regulation exclusively in the PYC1 deletion mutant of F. oxysporum. The metabolic response associated with pathogenicity was notably characterized by S-methyl-5-thioadenosine and S-adenosyl-L-methionine. This research sheds light on how PYC1-mediated anaplerosis affects fungal metabolism and reveals species-specific variations, exemplified in F. graminearum and F. oxysporum.

Novel sampling and gas-phase derivatization strategy: Proof-of-concept by profiling ionic polar metabolites using gas chromatography-mass spectrometry.

Metabolomic research involves the comprehensive analysis of metabolites in biological samples and has many applications. Gas chromatography-mass spectrometry (GC-MS) is an established and widely used approach for metabolic profiling. However, sample preparation and metabolite derivatization are time-consuming, and derivatization options are limited. We propose gas-solid phase derivatization (GSPD) as a novel sampling and derivatization method that uses a silica monolith substrate and gaseous derivatization reagents for metabolomics using GC-MS. We developed a method to measure the organic acids and sugar phosphates responsible for glycolysis and the tricarboxylic acid (TCA) cycle. GSPD simplifies the sample preparation and can be applied to derivatization reactions that are difficult to perform in solution owing to solvent limitations. The developed method was applied to human plasma and tomato pulp and was shown to have a higher detection performance than the conventional method. This study provides a strategy to simplify sample preparation and expand derivatization options for GC-MS-based metabolomics.

Biofluorescence imaging-guided spatial metabolic tracing: In vivo tracking of metabolic activity in circulating tumor cell-mediated multi-organ metastases.

Circulating tumor cells (CTC) are considered metastatic precursors that are shed from the primary or metastatic deposits and navigate the bloodstream before undergoing extravasation to establish distant metastases. Metabolic reprogramming appears to be a hallmark of metastatic progression, yet current methods for evaluating metabolic heterogeneity within organ-specific metastases in vivo are limited. To overcome this challenge, we present Biofluorescence Imaging-Guided Spatial Metabolic Tracing (BIGSMT), a novel approach integrating in vivo biofluorescence imaging, stable isotope tracing, stain-free laser capture microdissection, and liquid chromatography-mass spectrometry. This innovative technology obviates the need for staining or intricate sample preparation, mitigating metabolite loss, and substantially enhances detection sensitivity and accuracy through chemical derivatization of polar metabolites in central carbon pathways. Application of BIGSMT to a preclinical CTC-mediated metastasis mouse model revealed significant heterogeneity in the in vivo carbon flux from glucose into glycolysis and the tricarboxylic acid (TCA) cycle across distinct metastatic sites. Our analysis indicates that carbon predominantly enters the TCA cycle through the enzymatic reaction catalyzed by pyruvate dehydrogenase. Thus, our spatially resolved BIGSMT technology provides fresh insights into the metabolic heterogeneity and evolution during melanoma CTC-mediated metastatic progression and points to novel therapeutic opportunities.

Isocitrate dehydrogenases 2-mediated dysfunctional metabolic reprogramming promotes intestinal cancer progression via regulating HIF-1A signaling pathway.

Changes in isocitrate dehydrogenases (IDH) lead to the production of the cancer-causing metabolite 2-hydroxyglutarate, making them a cause of cancer. However, the specific role of IDH in the progression of colon cancer is still not well understood. Our current study provides evidence that IDH2 is significantly increased in colorectal cancer (CRC) cells and actively promotes cell growth in vitro and the development of tumors in vivo. Inhibiting the activity of IDH2, either through genetic silencing or pharmacological inhibition, results in a significant increase in α-ketoglutarate (α-KG), indicating a decrease in the reductive citric acid cycle. The excessive accumulation of α-KG caused by the inactivation of IDH2 obstructs the generation of ATP in mitochondria and promotes the downregulation of HIF-1A, eventually inhibiting glycolysis. This dual metabolic impact results in a reduction in ATP levels and the suppression of tumor growth. Our study reveals a metabolic trait of colorectal cancer cells, which involves the active utilization of glutamine through reductive citric acid cycle metabolism. The data suggests that IDH2 plays a crucial role in this metabolic process and has the potential to be a valuable target for the advancement of treatments for colorectal cancer.

Tricarboxylic Acid Cycle Relationships with Non-Metabolic Processes: A Short Story with DNA Repair and Its Consequences on Cancer Therapy Resistance.

Metabolic changes involving the tricarboxylic acid (TCA) cycle have been linked to different non-metabolic cell processes. Among them, apart from cancer and immunity, emerges the DNA damage response (DDR) and specifically DNA damage repair. The oncometabolites succinate, fumarate and 2-hydroxyglutarate (2HG) increase reactive oxygen species levels and create pseudohypoxia conditions that induce DNA damage and/or inhibit DNA repair. Additionally, by influencing DDR modulation, they establish direct relationships with DNA repair on at least four different pathways. The AlkB pathway deals with the removal of N-alkylation DNA and RNA damage that is inhibited by fumarate and 2HG. The MGMT pathway acts in the removal of O-alkylation DNA damage, and it is inhibited by the silencing of the MGMT gene promoter by 2HG and succinate. The other two pathways deal with the repair of double-strand breaks (DSBs) but with opposite effects: the FH pathway, which uses fumarate to help with the repair of this damage, and the chromatin remodeling pathway, in which oncometabolites inhibit its repair by impairing the homologous recombination repair (HRR) system. Since oncometabolites inhibit DNA repair, their removal from tumor cells will not always generate a positive response in cancer therapy. In fact, their presence contributes to longer survival and/or sensitization against tumor therapy in some cancer patients.

Immune suppression by human thymus-derived effector Tregs relies on glucose/lactate-fueled fatty acid synthesis.

Regulatory T cells (Tregs) suppress pro-inflammatory conventional T cell (Tconv) responses. As lipids impact cell signaling and function, we compare the lipid composition of CD4+ thymus-derived (t)Tregs and Tconvs. Lipidomics reveal constitutive enrichment of neutral lipids in Tconvs and phospholipids in tTregs. TNFR2-co-stimulated effector tTregs and Tconvs are both glycolytic, but only in tTregs are glycolysis and the tricarboxylic acid (TCA) cycle linked to a boost in fatty acid (FA) synthesis (FAS), supported by relevant gene expression. FA chains in tTregs are longer and more unsaturated than in Tconvs. In contrast to Tconvs, tTregs effectively use either lactate or glucose for FAS and rely on this process for proliferation. FASN and SCD1, enzymes responsible for FAS and FA desaturation, prove essential for the ability of tTregs to suppress Tconvs. These data illuminate how effector tTregs can thrive in inflamed or cancerous tissues with limiting glucose but abundant lactate levels.

Hypoxia-induced downregulation of PGK1 crotonylation promotes tumorigenesis by coordinating glycolysis and the TCA cycle.

Protein post-translational modifications (PTMs) are crucial for cancer cells to adapt to hypoxia; however, the functional significance of lysine crotonylation (Kcr) in hypoxia remains unclear. Herein we report a quantitative proteomics analysis of global crotonylome under normoxia and hypoxia, and demonstrate 128 Kcr site alterations across 101 proteins in MDA-MB231 cells. Specifically, we observe a significant decrease in K131cr, K156cr and K220cr of phosphoglycerate kinase 1 (PGK1) upon hypoxia. Enoyl-CoA hydratase 1 (ECHS1) is upregulated and interacts with PGK1, leading to the downregulation of PGK1 Kcr under hypoxia. Abolishment of PGK1 Kcr promotes glycolysis and suppresses mitochondrial pyruvate metabolism by activating pyruvate dehydrogenase kinase 1 (PDHK1). A low PGK1 K131cr level is correlated with malignancy and poor prognosis of breast cancer. Our findings show that PGK1 Kcr is a signal in coordinating glycolysis and the tricarboxylic acid (TCA) cycle and may serve as a diagnostic indicator for breast cancer.

Metabolomic profiling of pheochromocytomas in dogs: Catecholamine phenotype and tricarboxylic acid cycle metabolites.

BACKGROUND: In humans with pheochromocytomas (PCCs), targeted metabolomics is used to determine the catecholamine phenotype or to uncover underlying pathogenic variants in tricarboxylic acid (TCA) cycle genes such as succinate dehydrogenase subunits (SDHx). HYPOTHESIS/OBJECTIVES: To analyze catecholamine contents and TCA cycle metabolites of PCCs and normal adrenals (NAs). ANIMALS: Ten healthy dogs, 21 dogs with PCC. METHODS: Prospective observational study. Dogs diagnosed with PCC based on histopathological and immunohistochemical confirmation were included. Tissue catecholamine contents and TCA metabolites in PCCs and NAs were measured by liquid chromatography with mass spectrometry or electrochemical detection. RESULTS: Compared to NAs, PCCs had significantly higher tissue proportion of norepinephrine (88% [median: range, 38%-98%] vs 14% [11%-26%]; P < .001), and significantly lower tissue proportion of epinephrine (12% [1%-62%] vs 86% [74%-89%]; P < .001). Pheochromocytomas exhibited significantly lower fumarate (0.4-fold; P < .001), and malate (0.5-fold; P = .008) contents than NAs. Citrate was significantly higher in PCCs than in NAs (1.6-fold; P = .015). One dog in the PCC group had an aberrant succinate : fumarate ratio that was 25-fold higher than in the other PCCs, suggesting an SDHx mutation. CONCLUSIONS AND CLINICAL IMPORTANCE: This study reveals a distinct catecholamine content and TCA cycle metabolite profile in PCCs. Metabolite profiling might be used to uncover underlying pathogenic variants in TCA cycle genes in dogs.

Mitochondrial complex I promotes kidney cancer metastasis.

Most kidney cancers are metabolically dysfunctional1-4, but how this dysfunction affects cancer progression in humans is unknown. We infused 13C-labelled nutrients in over 80 patients with kidney cancer during surgical tumour resection. Labelling from [U-13C]glucose varies across subtypes, indicating that the kidney environment alone cannot account for all tumour metabolic reprogramming. Compared with the adjacent kidney, clear cell renal cell carcinomas (ccRCCs) display suppressed labelling of tricarboxylic acid (TCA) cycle intermediates in vivo and in ex vivo organotypic cultures, indicating that suppressed labelling is tissue intrinsic. [1,2-13C]acetate and [U-13C]glutamine infusions in patients, coupled with measurements of respiration in isolated human kidney and tumour mitochondria, reveal lower electron transport chain activity in ccRCCs that contributes to decreased oxidative and enhanced reductive TCA cycle labelling. However, ccRCC metastases unexpectedly have enhanced TCA cycle labelling compared with that of primary ccRCCs, indicating a divergent metabolic program during metastasis in patients. In mice, stimulating respiration or NADH recycling in kidney cancer cells is sufficient to promote metastasis, whereas inhibiting electron transport chain complex I decreases metastasis. These findings in humans and mice indicate that metabolic properties and liabilities evolve during kidney cancer progression, and that mitochondrial function is limiting for metastasis but not growth at the original site.

Differential utilization of vitamin B12-dependent and independent pathways for propionate metabolism across human cells.

Propionic acid links the oxidation of branched-chain amino acids and odd-chain fatty acids to the TCA cycle. Gut microbes ferment complex fiber remnants, generating high concentrations of short chain fatty acids, acetate, propionate and butyrate, which are shared with the host as fuel sources. Analysis of vitamin B12-dependent propionate utilization in skin biopsy samples has been used to characterize and diagnose underlying inborn errors of cobalamin (or B12) metabolism. In these cells, the B12-dependent enzyme, methylmalonyl-CoA mutase (MMUT), plays a central role in funneling propionate to the TCA cycle intermediate, succinate. Our understanding of the fate of propionate in other cell types, specifically, the involvement of the ß-oxidation-like and methylcitrate pathways, is limited. In this study, we have used [14C]-propionate tracing in combination with genetic ablation or inhibition of MMUT, to reveal the differential utilization of the B12-dependent and independent pathways for propionate metabolism in fibroblast versus colon cell lines. We demonstrate that itaconate can be used as a tool to investigate MMUT-dependent propionate metabolism in cultured cell lines. While MMUT gates the entry of propionate carbons into the TCA cycle in fibroblasts, colon-derived cell lines exhibit a quantitatively significant or exclusive reliance on the ß-oxidation-like pathway. Lipidomics and metabolomics analyses reveal that propionate elicits pleiotropic changes, including an increase in odd-chain glycerophospholipids, and perturbations in the purine nucleotide cycle and arginine/nitric oxide metabolism. The metabolic rationale and the regulatory mechanisms underlying the differential reliance on propionate utilization pathways at a cellular, and possibly tissue level, warrant further elucidation.

A transcriptional regulatory mechanism of genes in the tricarboxylic acid cycle in the heart.

The tricarboxylic acid (TCA) cycle plays a crucial role in mitochondrial ATP production in the healthy heart. However, in heart failure, the TCA cycle becomes dysregulated. Understanding the mechanism by which TCA cycle genes are transcribed in the healthy heart is an important prerequisite to understanding how these genes become dysregulated in the failing heart. PPARγ coactivator 1α (PGC-1α) is a transcriptional coactivator that broadly induces genes involved in mitochondrial ATP production. PGC-1α potentiates its effects through the coactivation of coupled transcription factors, such as estrogen-related receptor (ERR), nuclear respiratory factor 1 (Nrf1), GA-binding protein-a (Gabpa), and Yin Yang 1 (YY1). We hypothesized that PGC-1α plays an essential role in the transcription of TCA cycle genes. Thus, utilizing localization peaks of PGC-1α to TCA cycle gene promoters would allow the identification of coupled transcription factors. PGC-1α potentiated the transcription of 13 out of 14 TCA cycle genes, partly through ERR, Nrf1, Gabpa, and YY1. ChIP-sequencing showed PGC-1α localization peaks in TCA cycle gene promoters. Transcription factors with binding elements that were found proximal to PGC-1α peak localization were generally essential for the transcription of the gene. These transcription factor binding elements were well conserved between mice and humans. Among the four transcription factors, ERR and Gabpa played a major role in potentiating transcription when compared to Nrf1 and YY1. These transcription factor-dependent PGC-1α recruitment was verified with Idh3a, Idh3g, and Sdha promoters with DNA binding assay. Taken together, this study clarifies the mechanism by which TCA cycle genes are transcribed, which could be useful in understanding how those genes are dysregulated in pathological conditions.

Hexokinase 1 forms rings that regulate mitochondrial fission during energy stress.

Metabolic enzymes can adapt during energy stress, but the consequences of these adaptations remain understudied. Here, we discovered that hexokinase 1 (HK1), a key glycolytic enzyme, forms rings around mitochondria during energy stress. These HK1-rings constrict mitochondria at contact sites with the endoplasmic reticulum (ER) and mitochondrial dynamics protein (MiD51). HK1-rings prevent mitochondrial fission by displacing the dynamin-related protein 1 (Drp1) from mitochondrial fission factor (Mff) and mitochondrial fission 1 protein (Fis1). The disassembly of HK1-rings during energy restoration correlated with mitochondrial fission. Mechanistically, we identified that the lack of ATP and glucose-6-phosphate (G6P) promotes the formation of HK1-rings. Mutations that affect the formation of HK1-rings showed that HK1-rings rewire cellular metabolism toward increased TCA cycle activity. Our findings highlight that HK1 is an energy stress sensor that regulates the shape, connectivity, and metabolic activity of mitochondria. Thus, the formation of HK1-rings may affect mitochondrial function in energy-stress-related pathologies.

Unravelling the metabolic rewiring in the context of doxorubicin-induced cardiotoxicity: Fuel preference changes from fatty acids to glucose oxidation.

Doxorubicin (DOX) is a highly effective chemotherapeutic agent whose clinical use is hindered by the onset of cardiotoxic effects, resulting in reduced ejection fraction within the first year from treatment initiation. Recently it has been demonstrated that DOX accumulates within mitochondria, leading to disruption of metabolic processes and energetic imbalance. We previously described that phosphoinositide 3-kinase γ (PI3Kγ) contributes to DOX-induced cardiotoxicity, causing autophagy inhibition and accumulation of damaged mitochondria. Here we intend to describe the maladaptive metabolic rewiring occurring in DOX-treated hearts and the contribution of PI3Kγ signalling to this process. Metabolomic analysis of DOX-treated WT hearts revealed an accumulation of TCA cycle metabolites due to a cycle slowdown, with reduced levels of pyruvate, unchanged abundance of lactate and increased Acetyl-CoA production. Moreover, the activity of glycolytic enzymes was upregulated, and fatty acid oxidation downregulated, after DOX, indicative of increased glucose oxidation. In agreement, oxygen consumption was increased in after pyruvate supplementation, with the formation of cytotoxic ROS rather than energy production. These metabolic changes were fully prevented in KD hearts. Interestingly, they failed to increase glucose oxidation in response to DOX even with autophagy inhibition, indicating that PI3Kγ likely controls the fuel preference after DOX through an autophagy-independent mechanism. In vitro experiments showed that inhibition of PI3Kγ inhibits pyruvate dehydrogenase (PDH), the key enzyme of Randle cycle regulating the switch from fatty acids to glucose usage, while decreasing DOX-induced mobilization of GLUT-4-carrying vesicles to the plasma membrane and limiting the ensuing glucose uptake. These results demonstrate that PI3Kγ promotes a maladaptive metabolic rewiring in DOX-treated hearts, through a two-pronged mechanism controlling PDH activation and GLUT-4-mediated glucose uptake.

[Identification of the binding proteins of organic acid metabolites by matrix thermal shift assay].

Organic acid metabolites exhibit acidic properties. These metabolites serve as intermediates in major carbon metabolic pathways and are involved in several biochemical pathways, including the tricarboxylic acid (TCA) cycle and glycolysis. They also regulate cellular activity and play crucial roles in epigenetics, tumorigenesis, and cellular signal transduction. Knowledge of the binding proteins of organic acid metabolites is crucial for understanding their biological functions. However, identifying the binding proteins of these metabolites has long been a challenging task owing to the transient and weak nature of their interactions. Moreover, traditional methods are unsuitable for the structural modification of the ligands of organic acid metabolites because these metabolites have simple and similar structures. Even minor structural modifications can significantly affect protein interactions. Thermal proteome profiling (TPP) provides a promising avenue for identifying binding proteins without the need for structural modifications. This approach has been successfully applied to the identification of the binding proteins of several metabolites. In this study, we investigated the binding proteins of two TCA cycle intermediates, i.e., succinate and fumarate, and lactate, an end-product of glycolysis, using the matrix thermal shift assay (mTSA) technique. This technique involves combining single-temperature (52 ℃) TPP and dose-response curve analysis to identify ligand-binding proteins with high levels of confidence and determine the binding affinity between ligands and proteins. To this end, HeLa cells were lysed, followed by protein desalting to remove endogenous metabolites from the cell lysates. The desalted cell lysates were treated with fumarate or succinate at final concentrations of 0.004, 0.04, 0.4, and 2 mmol/L in the experimental groups or 2 mmol/L sodium chloride in the control group. Considering that the cellular concentration of lactate can be as high as 2-30 mmol/L, we then applied lactate at final concentrations of 0.2, 1, 5, 10, and 25 mmol/L in the experimental groups or 25 mmol/L sodium chloride in the control group. Using high-sensitivity mass spectrometry coupled with data-independent acquisition (DIA) quantification, we quantified 5870, 5744, and 5816 proteins in succinate, fumarate, and lactate mTSA experiments, respectively. By setting stringent cut-off values (i.e., significance of changes in protein thermal stability (p-value)<0.001 and quality of the dose-response curve fitting (square of Pearson's correlation coefficient, R2)>0.95), multiple binding proteins for these organic acid metabolites from background proteins were confidently determined. Several known binding proteins were identified, notably fumarate hydratase (FH) as a binding protein for fumarate, and α-ketoglutarate-dependent dioxygenase (FTO) as a binding protein for both fumarate and succinate. Additionally, the affinity data for the interactions between these metabolites and their binding proteins were obtained, which closely matched those reported in the literature. Interestingly, ornithine aminotransferase (OAT), which is involved in amino acid biosynthesis, and 3-mercaptopyruvate sulfurtransferase (MPST), which acts as an antioxidant in cells, were identified as lactate-binding proteins. Subsequently, an orthogonal assay technique developed in our laboratory, the solvent-induced precipitation (SIP) technique, was used to validate the mTSA results. SIP identified OAT as the top target candidate, validating the mTSA-based finding that OAT is a novel lactate-binding protein. Although MPST was not identified as a lactate-binding protein by SIP, statistical analysis of MPST in the mTSA experiments with 10 or 25 mmol/L lactate revealed that MPST is a lactate-binding protein with a high level of confidence. Peptide-level empirical Bayes t-tests combined with Fisher's exact test also supported the conclusion that MPST is a lactate-binding protein. Lactate is structurally similar to pyruvate, the known binding protein of MPST. Therefore, assuming that lactate could potentially occupy the binding site of pyruvate on MPST. Overall, the novel binding proteins identified for lactate suggest their potential involvement in amino acid synthesis and redox balance regulation.

Mutations Affecting Cellular Levels of Cobalamin (Vitamin B12) Confer Tolerance to Bactericidal Antibiotics in Burkholderia cenocepacia.

The Burkholderia cepacia complex (Bcc) consists of opportunistic pathogens known to cause pneumonia in immunocompromised individuals, especially those with cystic fibrosis. Treating Bcc pneumonia is challenging due to the pathogens' high multidrug resistance. Therefore, inhalation therapy with tobramycin powder, which can achieve high antibiotic concentrations in the lungs, is a promising treatment option. In this study, we investigated potential mechanisms that could compromise the effectiveness of tobramycin therapy. By selecting for B. cenocepacia survivors against tobramycin, we identified three spontaneous mutations that disrupt a gene encoding a key enzyme in the biosynthesis of cobalamin (Vitamin B12). This disruption may affect the production of succinyl-CoA by methylmalonyl-CoA mutase, which requires adenosylcobalamin as a cofactor. The depletion of cellular succinyl-CoA may impact the tricarboxylic acid (TCA) cycle, which becomes metabolically overloaded upon exposure to tobramycin. Consequently, the mutants exhibited significantly reduced reactive oxygen species (ROS) production. Both the wild-type and mutants showed tolerance to tobramycin and various other bactericidal antibiotics under microaerobic conditions. This suggests that compromised ROS-mediated killing, due to the impacted TCA cycle, underlies the mutants' tolerance to bactericidal antibiotics. The importance of ROS-mediated killing and the potential emergence of mutants that evade it through the depletion of cobalamin (Vitamin B12) provide valuable insights for developing strategies to enhance antibiotic treatments of Bcc pneumonia.

Citrate serves as a signal molecule to modulate carbon metabolism and iron homeostasis in Staphylococcus aureus.

Pathogenic bacteria's metabolic adaptation for survival and proliferation within hosts is a crucial aspect of bacterial pathogenesis. Here, we demonstrate that citrate, the first intermediate of the tricarboxylic acid (TCA) cycle, plays a key role as a regulator of gene expression in Staphylococcus aureus. We show that citrate activates the transcriptional regulator CcpE and thus modulates the expression of numerous genes involved in key cellular pathways such as central carbon metabolism, iron uptake and the synthesis and export of virulence factors. Citrate can also suppress the transcriptional regulatory activity of ferric uptake regulator. Moreover, we determined that accumulated intracellular citrate, partly through the activation of CcpE, decreases the pathogenic potential of S. aureus in animal infection models. Therefore, citrate plays a pivotal role in coordinating carbon metabolism, iron homeostasis, and bacterial pathogenicity at the transcriptional level in S. aureus, going beyond its established role as a TCA cycle intermediate.

Molecular characterization and clinical relevance of metabolic signature subtypes in gastric cancer.

Metabolic reprogramming dictates tumor molecular attributes and therapeutic potentials. However, the comprehensive metabolic characteristics in gastric cancer (GC) remain obscure. Here, metabolic signature-based clustering analysis identifies three subtypes with distinct molecular and clinical features: MSC1 showed better prognosis and upregulation of the tricarboxylic acid (TCA) cycle and lipid metabolism, combined with frequent TP53 and RHOA mutation; MSC2 had moderate prognosis and elevated nucleotide and amino acid metabolism, enriched by intestinal histology and mismatch repair deficient (dMMR); and MSC3 exhibited poor prognosis and enhanced glycan and energy metabolism, accompanied by diffuse histology and frequent CDH1 mutation. The Shandong Provincial Hospital (SDPH) in-house dataset with matched transcriptomic, metabolomic, and spatial-metabolomic analysis also validated these findings. Further, we constructed the metabolic subtype-related prognosis gene (MSPG) scoring model to quantify the activity of individual tumors and found a positive correlation with cuproptosis signaling. In conclusion, comprehensive recognition of the metabolite signature can enhance the understanding of diversity and heterogeneity in GC.