Redox remodeling of central metabolism as a driving force for cellular protection, proliferation, differentiation, and dysfunction.

The production of reactive oxygen species (ROS) is elevated via metabolic hyperactivation in response to a variety of stimuli such as growth factors and inflammation. Tolerable amounts of ROS moderately inactivate enzymes via oxidative modification, which can be reversed back to the native form in a redox-dependent manner. The excessive production of ROS, however, causes cell dysfunction and death. Redox-reactive enzymes are present in primary metabolic pathways such as glycolysis and the tricarboxylic acid cycle, and these act as floodgates for carbon flux. Oxidation of a specific form of cysteine inhibits glyceraldehyde-3-phosphate dehydrogenase, which is reversible, and causes an accumulation of upstream intermediary compounds that increases the flux of glucose-6-phosphate to the pentose phosphate pathway. These reactions increase the NADPH and ribose-5-phosphate that are available for reductive reactions and nucleotide synthesis, respectively. On the other hand, oxidative inactivation of mitochondrial aconitase increases citrate, which is then recruited to synthesize fatty acids in the cytoplasm. Decreases in the use of carbohydrate for ATP production can be compensated via amino acid catabolism, and this metabolic change makes nitrogen available for nucleic acid synthesis. Coupling of the urea cycle also converts nitrogen to urea and polyamine, the latter of which supports cell growth. This metabolic remodeling stimulates the proliferation of tumor cells and fibrosis in oxidatively damaged tissues. Oxidative modification of these enzymes is generally reversible in the early stages of oxidizing reactions, which suggests that early treatment with appropriate antioxidants promotes the maintenance of natural metabolism.

Salmonella Typhimurium screen identifies shifts in mixed-acid fermentation during gut colonization.

How enteric pathogens adapt their metabolism to a dynamic gut environment is not yet fully understood. To investigate how Salmonella enterica Typhimurium (S.Tm) colonizes the gut, we conducted an in vivo transposon mutagenesis screen in a gnotobiotic mouse model. Our data implicate mixed-acid fermentation in efficient gut-luminal growth and energy conservation throughout infection. During initial growth, the pathogen utilizes acetate fermentation and fumarate respiration. After the onset of gut inflammation, hexoses appear to become limiting, as indicated by carbohydrate analytics and the increased need for gluconeogenesis. In response, S.Tm adapts by ramping up ethanol fermentation for redox balancing and supplying the TCA cycle with α-ketoglutarate for additional energy. Our findings illustrate how S.Tm flexibly adapts mixed fermentation and its use of the TCA cycle to thrive in the changing gut environment. Similar metabolic wiring in other pathogenic Enterobacteriaceae may suggest a broadly conserved mechanism for gut colonization.

Qin-Yu-Qing-Chang decoction reshapes colonic metabolism by activating PPAR-γ signaling to inhibit facultative anaerobes against DSS-induced colitis.

BACKGROUND: Qin-Yu-Qing-Chang decoction (QYQC), an herbal formula from China, is extensively employed to manage ulcerative colitis (UC) and exhibits potential benefits for colonic function. Nevertheless, the fundamental molecular mechanisms of QYQC remain largely uncharted. METHODS: The primary constituents of QYQC were determined utilizing UHPLC-MS/MS analysis and the effectiveness of QYQC was assessed in a mouse model of colitis induced by dextran sulfate sodium. Evaluations of colon inflammatory responses and mucosal barrier function were thoroughly assessed. RNA sequencing, molecular docking, colonic energy metabolism, and 16S rRNA sequencing analysis were applied to uncover the complex mechanisms of QYQC in treating UC. Detect the signal transduction of the peroxisome proliferator-activated receptor-γ (PPAR-γ) both in the nucleus and cytoplasm. Furthermore, a PPAR-γ antagonist was strategically utilized to confirm the functional targets that QYQC exerts. RESULTS: Utilizing UHPLC-MS/MS, the principal constituents of the nine traditional Chinese medicinal herbs comprising QYQC were systematically identified. QYQC treatment substantially ameliorated colitis in mice, as evidenced by the improvement in symptoms and the reduction in colonic pathological injuries. Besides, QYQC treatment mitigated the inflammatory response and improved mucosal barrier function. Furthermore, QYQC enhanced the mitochondria citrate cycle (TCA cycle) by triggering PPAR-γ signaling and increasing the proportion of PPAR-γ entering the nucleus. This prevented the unconstrained expansion of facultative anaerobes, particularly pathogenic Escherichia coli (E. coli, family Enterobacteriaceae) and thus improved colitis. Results of molecular docking indicated that the representative chemical components of QYQC including Baicalin, Paeoniflorin, Mollugin, and Imperatorin bound well with PPAR-γ. The impact of QYQC on colitis was diminished in the presence of a PPAR-γ antagonist. CONCLUSIONS: In summary, QYQC ameliorates UC by activating PPAR-γ signaling and increasing the proportion of PPAR-γ entering the nucleus, which enhances the energy metabolism of intestinal epithelial cells and thereby preventing the uncontrolled proliferation of facultative anaerobes.

Perturbations in mitochondrial metabolism associated with defective cardiolipin biosynthesis: An in-organello real-time NMR study.

Mitochondria are central to cellular metabolism; hence, their dysfunction contributes to a wide array of human diseases. Cardiolipin, the signature phospholipid of the mitochondrion, affects proper cristae morphology, bioenergetic functions, and metabolic reactions carried out in mitochondrial membranes. To match tissue-specific metabolic demands, cardiolipin typically undergoes an acyl tail remodeling process with the final step carried out by the phospholipid-lysophospholipid transacylase tafazzin. Mutations in tafazzin are the primary cause of Barth syndrome. Here, we investigated how defects in cardiolipin biosynthesis and remodeling impacts metabolic flux through the TCA cycle and associated yeast pathways. Nuclear magnetic resonance was used to monitor in real-time the metabolic fate of 13C3-pyruvate in isolated mitochondria from three isogenic yeast strains. We compared mitochondria from a WT strain to mitochondria from a Δtaz1 strain that lacks tafazzin and contains lower amounts of unremodeled cardiolipin and mitochondria from a Δcrd1 strain that lacks cardiolipin synthase and cannot synthesize cardiolipin. We found that the 13C-label from the pyruvate substrate was distributed through twelve metabolites. Several of the metabolites were specific to yeast pathways including branched chain amino acids and fusel alcohol synthesis. While most metabolites showed similar kinetics among the different strains, mevalonate concentrations were significantly increased in Δtaz1 mitochondria. Additionally, the kinetic profiles of α-ketoglutarate, as well as NAD+ and NADH measured in separate experiments, displayed significantly lower concentrations for Δtaz1 and Δcrd1 mitochondria at most time points. Taken together, the results show how cardiolipin remodeling influences pyruvate metabolism, tricarboxylic acid cycle flux, and the levels of mitochondrial nucleotides.

Mapping of oxidative modifications on the alpha-keto glutarate dehydrogenase complex induced by singlet oxygen: Effects on structure and activity.

The large multi-subunit mitochondrial alpha-keto glutarate dehydrogenase (KGDH) complex plays a key, rate-determining, role in the tricarboxylic acid (Krebs) cycle, catalyzing the conversion of alpha-keto glutarate to succinyl-CoA. This complex is both a source and target of oxidants, but the sites of modification and association with structural changes and activity loss are poorly understood. We report here oxidative modifications induced by Rose Bengal (RB) in the presence of O2, a source of singlet oxygen (1O2). A rapid loss of activity was detected, with this being dependent on light exposure, illumination time, and the presence of RB and O2. Activity loss was enhanced by D2O (consistent with 1O2 involvement), but diminished by both pre- and (to a lesser extent) post-illumination addition of lipoic acid and lipoamide. Aggregates containing all three KGDH subunits were detected on photooxidation. LC-MS experiments provided evidence for oxidation at 45 sites, including specific Met, His, Trp, Tyr residues and the lipoyllysine active-site cofactor. Products include mono- and di-oxygenated species, and kynurenine from Trp. Mapping of the modifications to the 3-D structure showed that these are localized to both the inner channel and the external surface, consistent with reactions of free 1O2, however the sites and extent of modification do not correlate with their solvent accessibility. These products are generated concurrently with loss of activity, indicative of strong links between these events. These data provide evidence for the impairment of KGDH activity by 1O2 via the oxidation of specific residues on the protein subunits of the complex.

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.

Rewiring the reductive TCA pathway and glyoxylate shunt of Escherichia coli for succinate production from corn stover hydrolysate using a two-phase fermentation strategy.

Succinate was found extensive applications in the food additives, pharmaceutical, and biopolymers industries. However, the succinate biosynthesis in E. coli required IPTG, lacked NADH, and produced high yields only under anaerobic conditions, unsuitable for cell growth. To overcome these limitations, the glyoxylate shunt and reductive TCA pathway were simultaneously enhanced to produce succinate in both aerobic and anaerobic conditions and achieve a high cell growth meanwhile. On this basis, NADH availability and sugars uptake were increased. Furthermore, an oxygen-dependent promoter was used to dynamically regulate the expression level of key genes of reductive TCA pathway to avoid the usage of IPTG. The final strain E. coli Mgls7-32 could produce succinate from corn stover hydrolysate without an inducer, achieving a titer of 72.8 g/L in 5 L bioreactor (1.2 mol/mol of total sugars). Those findings will aid in the industrial production of succinate.

Development and Evaluation of Bis-benzothiazoles as a New Class of Benzothiazoles Targeting DprE1 as Antitubercular Agents.

Benzothiazole-bearing compounds have emerged as potential noncovalent DprE1 (decaprenylphosphoryl-ß-d-ribose-2'-epimerase) inhibitors active against Mycobacterium tuberculosis. Based on structure-based virtual screening (PDB ID: 4KW5), a focused library of thirty-one skeletally diverse benzothiazole amides was prepared, and the compounds were assessed for their antitubercular activity against M.tb H37Ra. Most potent compounds 3b and 3n were further evaluated against the M.tb H37Rv strain by the microdilution assay method. Among the compounds evaluated, bis-benzothiazole amide 3n emerged as a hit molecule and demonstrated promising antitubercular activity with minimum inhibitory concentration (MIC) values of 0.45 µg/mL and 8.0 µg/mL against H37Ra and H37Rv, respectively. Based on the preliminary hit molecule (3n), a focused library of 12 more bis-benzothiazole amide derivatives was further prepared by varying the substituents on either side to obtain new leads and generate a structure-activity relationship (SAR). Among these compounds, 6a, 6c, and 6d demonstrated remarkable antitubercular activity with MIC values of 0.5 µg/mL against H37Ra and 1.0, 2.0, and 8.0 µg/mL against H37Rv, respectively. The most active compound, 6a, also displayed significant efficacy against four drug-resistant tuberculosis strains. Compound 6a was assessed for in vitro cytotoxicity against the HepG2 cell line, and it displayed insignificant cytotoxicity. Furthermore, time-kill kinetic studies demonstrated time- and dose-dependent bactericidal activity of this compound. The GFP release assay revealed that compound 6a targets the inhibition of a cell wall component. SNPs in dprE-1 gene assessment revealed that compound 6a binds to tyrosine at position 314 of DprE1 and replaces it with histidine, causing resistance similar to that of standard TCA1. In silico docking studies further suggest that the strong noncovalent interactions of these compounds may lead to the development of potent noncovalent DprE1 inhibitors.

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.

Proteomics Analysis of Human Chorionic Villi Reveals Dysregulated Pathways That Contribute to Recurrent Pregnancy Loss.

PURPOSE: Recurrent pregnancy loss (RPL) represents a common disorder with consequences on family and society. As more than half of the RPL cases do not have a clearly identified cause, uncovering the mechanisms behind the idiopathic RPL is urgently needed. EXPERIMENTAL DESIGN: Using label-free data-independent LC-MS/MS acquisition coupled with ion mobility, we compared the proteome of chorionic villi from 13 RPL cases with 10 age and gestational week-matched elective pregnancies. Transcriptional levels of selected candidate biomarkers were determined in chorionic villi of 35 RPL cases and 25 controls using quantitative polymerase chain reaction (qPCR). RESULTS: Statistically significant difference in abundance (Benjamini-Hochberg [B-H] p ≤ 0.05) and fold change ≥1.5 showed 128 proteins. Bioinformatics analysis identified complement and coagulation cascades, platelet activation, tricarboxylic acid cycle (TCA) cycle, and ferroptosis as pathways with the highest significance. Correlation with transcriptome datasets revealed a weak statistically significant positive correlation with 45% of the co-differentially expressed proteins/genes displaying the same regulation trend. The transcription levels of neurofilament light polypeptide (NEFL), dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex_mitochondrial (DLST), nitric oxide synthase 3 (NOS3), and ceruloplasmin (CP) were significantly increased in the RPL, consistent with the proteomics findings. CONCLUSIONS AND CLINICAL RELEVANCE: Our data suggests alteration of several pathways as potential causes of idiopathic RPL from the fetal side and opens the way for investigations concerning clinical management.

Regulation of TCA cycle genes by srbA sRNA: Impacts on Pseudomonas aeruginosa virulence and survival.

Pseudomonas aeruginosa, an opportunistic bacterial pathogen of public health concern, is known for its metabolic versatility, adaptability in harsh environment, and pathogenic aggressiveness. P. aeruginosa relies on various regulatory networks modulated by small non-coding RNAs, which in turn influence different physiological traits such as metabolism, stress response, and pathogenesis. In this study, srbA sRNA has been shown to play a diverse role in regulating cellular metabolism and the production of different virulence factors in P. aeruginosa. srbA was found to control the TCA cycle, a key regulatory pathway for cellular metabolism and energy production, by regulating three main enzymes: citrate synthase (gltA), isocitrate dehydrogenase (icd), and α-ketoglutarate dehydrogenase E1 subunit (sucA) at both the transcriptional and translational levels. By modulating the TCA cycle, srbA could help the bacteria to adapt nutritional stress by lowering energy consumption. Additionally, srbA has been found to differentially regulate production of various virulence factors such as rhamnolipid, elastase, LasA protease, and pyocyanin under both nutrient-rich and nutrient-limiting conditions. It could also influence motilities in P. aeruginosa, linked to biofilm formation and pathogenicity. Thus, srbA might hold a promise in the research area for identifying virulence pathways and developing novel therapeutic targets to combat the global pathogenic threat of P. aeruginosa.

Tricyclic Antidepressant and Antipsychotic Toxicity: Clomipramine and Ziprasidone Overdose.

This case report details an intentional overdose attempt utilizing tricyclic antidepressants (TCAs) and atypical antipsychotics with significant neurologic, pulmonary, and cardiac toxicity. In conjunction with the local poison control center, progression of the clinical toxidrome was anticipated, aggressively managed, and successfully treated. This case highlights the dangers of significant TCA toxicity, peak onset of toxicity within six hours, and the amplification of clinical toxidromes with co-ingestions.

A brief harvesting-freezing delay significantly alters the kidney metabolome and leads to false positive and negative results.

Abnormalities in distinct metabolic pathways have been associated with many forms of kidney disease. Metabolomics analyses can be used to determine organ-specific metabolic fingerprints. However, conventional harvesting methods depend on post-euthanasia tissue harvest, which results in ischemia conditions and metabolome changes that could introduce artifacts into the final studies. We optimized a clamp-freezing technique for kidney harvesting and freezing, significantly reducing ischemia and freezing times and granting a closer snapshot of in vivo metabolism. In this study, we characterized and compared the metabolome of kidneys harvested using our approach vs. traditional techniques to determine which metabolites are preferentially affected by a brief lapse of ischemia and freezing delay and which are more stable. We used Sprague Dawley rats as a model of wild-type (WT) kidneys and PCK, polycystic kidney disease (PKD) rats as a model of CKD kidneys. Finally, we compared the metabolic profile of clamp-frozen and delayed WT and PKD-kidneys to determine which metabolic changes are most likely observed in vivo in PKD and which could be subjected to false positive or negative results. Our data indicate that a short harvesting-freezing delay is sufficient to impart profound metabolic changes in WT and PKD kidneys. Interestingly, while the delay had a similar effect in WT and PKD, there were notable differences, leading to false positive and negative results when comparing these genotypes. The data obtained indicate that the quick clamp-freezing technique for kidney metabolomics provides a more accurate interpretation of the in vivo metabolic changes associated with the disease state.

Cervical mucus can be used for metabolite screening in cervical cancer.

Approximately 660,000 women are diagnosed with cervical cancer annually. Current screening options such as cytology or human papillomavirus testing have limitations, creating a need to identify more effective ancillary biomarkers for triage. Here, we evaluated whether metabolomic analysis of cervical mucus metabolism could be used to identify biomarkers of cervical intraepithelial neoplasia (CIN) and cervical cancer. The case-control group consisted of 181 CIN, 69 squamous cell carcinoma (SCC) patients, and 48 healthy controls in the primary cohort. We undertook metabolomic analyses using ultra-HPLC-tandem mass spectrometry. Univariate and multivariate analyses were carried out to profile metabolite characteristics, and receiver operating characteristic (ROC) analysis identified biomarker candidates. Five metabolites conferred the highest discriminatory power for SCC: oxidized glutathione (GSSG) (area under the ROC curve, 0.924; 95% confidence interval, 0.877-0.971), malic acid (0.914, 0.859-0.968), kynurenine (0.884, 0.823-0.945), GSSG/glutathione (GSH) (0.936, 0.892-0.979), and kynurenine/tryptophan (0.909, 0.856-0.961). Malic acid was the best marker for detection of CIN2 or worse (0.858, 0.793-0.922) and was a clinically useful metabolite. We confirmed the reproducibility of the results by validation cohort. Additionally, metabolomic analyses revealed eight pathways strongly associated with cervical neoplasia. Of these, only the tricarboxylic acid cycle was strongly associated with all CINs and cancer, indicating active energy production. Aberrant arginine metabolism by decreasing arginine and increasing citrulline might reduce tumor immunity. Changes in cysteine-methionine and GSH pathways might drive the initiation and progression of cervical cancer. These results suggest that metabolic analysis can identify ancillary biomarkers and could improve our understanding of the pathophysiological mechanisms underlying cervical neoplasia.

Mitochondrial copper overload promotes renal fibrosis via inhibiting pyruvate dehydrogenase activity.

Copper is a trace element essential for numerous biological activities, whereas the mitochondria serve as both major sites of intracellular copper utilization and copper reservoir. Here, we investigated the impact of mitochondrial copper overload on the tricarboxylic acid cycle, renal senescence and fibrosis. We found that copper ion levels are significantly elevated in the mitochondria in fibrotic kidney tissues, which are accompanied by reduced pyruvate dehydrogenase (PDH) activity, mitochondrial dysfunction, cellular senescence and renal fibrosis. Conversely, lowering mitochondrial copper levels effectively restore PDH enzyme activity, improve mitochondrial function, mitigate cellular senescence and renal fibrosis. Mechanically, we found that mitochondrial copper could bind directly to lipoylated dihydrolipoamide acetyltransferase (DLAT), the E2 component of the PDH complex, thereby changing the interaction between the subunits of lipoylated DLAT, inducing lipoylated DLAT protein dimerization, and ultimately inhibiting PDH enzyme activity. Collectively, our study indicates that mitochondrial copper overload could inhibit PDH activity, subsequently leading to mitochondrial dysfunction, cellular senescence and renal fibrosis. Reducing mitochondrial copper overload might therefore serve as a strategy to rescue renal fibrosis.

The perfluorooctanoic acid accumulation and release from pipelines promoted growth of bacterial communities and opportunistic pathogens with different antibiotic resistance genes in drinking water.

The spread of opportunistic pathogens (OPs) and antibiotic resistance genes (ARGs) through drinking water has already caused serious human health issues. There is also an urgent need to know the effects of perfluorooctanoic acid (PFOA) on OPs with different ARGs in drinking water. Our results suggested that PFOA accumulation and release from the pipelines induced its concentration in pipelines effluents increase from 0.03 ± 0.01 µg/L to 0.70 ± 0.01 µg/L after 6 months accumulation. The PFOA also promoted the growth of Hyphomicrobium, Microbacterium, and Bradyrhizobium. In addition, PFOA accumulation and release from the pipelines enhanced the metabolism and tricarboxylic acid (TCA) cycle processes, resulting in more extracellular polymeric substances (EPS) production. Due to EPS protection, Pseudomonas aeruginosa and Legionella pneumophila increased to (7.20 ± 0.09) × 104 gene copies/mL, and (8.85 ± 0.11) × 102 gene copies/mL, respectively. Moreover, PFOA also enhanced the transfer potential of different ARGs, including emrB, mdtB, mdtC, mexF, and macB. The main bacterial community composition and the main OPs positively correlated with the main ARGs and mobile genetic elements (MGE)-ARGs significantly. Therefore, PFOA promoted the propagation of OPs with different ARGs. These results are meaningful for controlling the microbial risk caused by the OPs with ARGs and MGE-ARGs in drinking water.

Transcriptomic analysis reveals insights into the responses of Synechocystis sp. PCC 6803 to acidification during cultivation with ammonium salts as a nitrogen source.

Utilizing ammonium in wastewater is a prospective way to reduce costs for bioproduction by photosynthetic organisms. A model cyanobacterium Synechocystis sp. PCC 6803 takes advantage of tolerance to ammonium compared to other microalgae. However, in this study, we report that Synechocystis growth was inhibited when cultured in a medium containing ammonium. This may be due to the pH decreasing below 6 caused by consuming ammonium. Transcriptomic analysis by RNA-seq revealed that the expression of the genes for proteases, chaperones, and antioxidant-scavenging enzymes was induced, but photosynthetic components were repressed. Although these regulations are similar to the previous studies on acidic stress in nitrate-containing culture, the expression of genes such as sigD, slr0042, slr0373, slr0374, and slr1501 was different, indicating that these phenomena are not simply identical to the known responses to acidic stress. The expression of the genes for photosynthesis, gluconeogenesis, and nitrogen assimilation was repressed, and glycolysis and the tricarboxylic acid cycle were induced. Despite the up-regulation of the carbon catabolism and down-regulation of nitrogen assimilation, the 2-oxoglutarate content in the ammonium-grown cells was lower than that in the nitrate-grown cells, and the contents of the major amino acids, such as Glu, Ala, Asp, and Gly were decreased, while the minor amino acids were the same or increased, especially Arg, Lys, Val, and Ile. These results demonstrated that the acidic stress induced by the consumption of ammonium ions differs from the sudden pH drop, and the Synechocystis cell manages amino acid levels to endure carbon limitation under the stress.

Is the TCA cycle malate dehydrogenase-citrate synthase metabolon an illusion?

This review discusses the intriguing yet controversial concept of metabolons, focusing on the malate dehydrogenase-citrate synthase (MDH-CISY) metabolon as a model. Metabolons are multienzyme complexes composed of enzymes that catalyze sequential reactions in metabolic pathways. Metabolons have been proposed to enhance metabolic pathway efficiency by facilitating substrate channeling. However, there is skepticism about the presence of metabolons and their functionality in physiological conditions in vivo. We address the skepticism by reviewing compelling evidence supporting the existence of the MDH-CISY metabolon and highlighting its potential functions in cellular metabolism. The electrostatic interaction between MDH and CISY and the intermediate oxaloacetate, channeled within the metabolon, has been demonstrated using various experimental techniques, including protein-protein interaction assays, isotope dilution studies, and enzyme coupling assays. Regardless of the wealth of in vitro evidence, further validation is required to elucidate the functionality of MDH-CISY metabolons in living systems using advanced structural and spatial analysis techniques.

Body-Wide Inactivation of the Myc-Like Mlx Transcription Factor Network Accelerates Aging and Increases the Lifetime Cancer Incidence.

The "Mlx" and "Myc" transcription factor networks cross-communicate and share many common gene targets. Myc's activity depends upon its heterodimerization with Max, whereas the Mlx Network requires that the Max-like factor Mlx associate with the Myc-like factors MondoA or ChREBP. The current work demonstrates that body-wide Mlx inactivation, like that of Myc, accelerates numerous aging-related phenotypes pertaining to body habitus and metabolism. The deregulation of numerous aging-related Myc target gene sets is also accelerated. Among other functions, these gene sets often regulate ribosomal and mitochondrial structure and function, genomic stability, and aging. Whereas "MycKO" mice have an extended lifespan because of a lower cancer incidence, "MlxKO" mice have normal lifespans and a higher cancer incidence. Like Myc, the expression of Mlx, MondoA, and ChREBP and their control over their target genes deteriorate with age in both mice and humans. Collectively, these findings underscore the importance of lifelong and balanced cross-talk between the two networks to maintain proper function and regulation of the many factors that can affect normal aging.

Perturbations in mitochondrial metabolism associated with defective cardiolipin biosynthesis: An in-organello real-time NMR study.

Mitochondria are central to cellular metabolism; hence, their dysfunction contributes to a wide array of human diseases including cancer, cardiopathy, neurodegeneration, and heritable pathologies such as Barth syndrome. Cardiolipin, the signature phospholipid of the mitochondrion promotes proper cristae morphology, bioenergetic functions, and directly affects metabolic reactions carried out in mitochondrial membranes. To match tissue-specific metabolic demands, cardiolipin typically undergoes an acyl tail remodeling process with the final step carried out by the phospholipid-lysophospholipid transacylase tafazzin. Mutations in the tafazzin gene are the primary cause of Barth syndrome. Here, we investigated how defects in cardiolipin biosynthesis and remodeling impact metabolic flux through the tricarboxylic acid cycle and associated pathways in yeast. Nuclear magnetic resonance was used to monitor in real-time the metabolic fate of 13C3-pyruvate in isolated mitochondria from three isogenic yeast strains. We compared mitochondria from a wild-type strain to mitochondria from a Δtaz1 strain that lacks tafazzin and contains lower amounts of unremodeled cardiolipin, and mitochondria from a Δcrd1 strain that lacks cardiolipin synthase and cannot synthesize cardiolipin. We found that the 13C-label from the pyruvate substrate was distributed through about twelve metabolites. Several of the identified metabolites were specific to yeast pathways, including branched chain amino acids and fusel alcohol synthesis. Most metabolites showed similar kinetics amongst the different strains but mevalonate and α-ketoglutarate, as well as the NAD+/NADH couple measured in separate nuclear magnetic resonance experiments, showed pronounced differences. Taken together, the results show that cardiolipin remodeling influences pyruvate metabolism, tricarboxylic acid cycle flux, and the levels of mitochondrial nucleotides.