Migration increases mitochondrial oxidative capacity without increasing reactive oxygen species emission in a songbird.

Birds remodel their flight muscle metabolism prior to migration to meet the physiological demands of migratory flight, including increases in both oxidative capacity and defence against reactive oxygen species. The degree of plasticity mediated by changes in these mitochondrial properties is poorly understood but may be explained by two non-mutually exclusive hypotheses: variation in mitochondrial quantity or in individual mitochondrial function. We tested these hypotheses using yellow-rumped warblers (Setophaga coronata), a Nearctic songbird which biannually migrates 2000-5000 km. We predicted higher flight muscle mitochondrial abundance and substrate oxidative capacity, and decreased reactive oxygen species emission in migratory warblers captured during autumn migration compared with a short-day photoperiod-induced non-migratory phenotype. We assessed mitochondrial abundance via citrate synthase activity and assessed isolated mitochondrial function using high-resolution fluororespirometry. We found 60% higher tissue citrate synthase activity in the migratory phenotype, indicating higher mitochondrial abundance. We also found 70% higher State 3 respiration (expressed per unit citrate synthase) in mitochondria from migratory warblers when oxidizing palmitoylcarnitine, but similar H2O2 emission rates between phenotypes. By contrast, non-phosphorylating respiration was higher and H2O2 emission rates were lower in the migratory phenotype. However, flux through electron transport system complexes I-IV, II-IV and IV was similar between phenotypes. In support of our hypotheses, these data suggest that flight muscle mitochondrial abundance and function are seasonally remodelled in migratory songbirds to increase tissue oxidative capacity without increasing reactive oxygen species formation.

Emergence of fractal geometries in the evolution of a metabolic enzyme.

Fractals are patterns that are self-similar across multiple length-scales1. Macroscopic fractals are common in nature2-4; however, so far, molecular assembly into fractals is restricted to synthetic systems5-12. Here we report the discovery of a natural protein, citrate synthase from the cyanobacterium Synechococcus elongatus, which self-assembles into Sierpinski triangles. Using cryo-electron microscopy, we reveal how the fractal assembles from a hexameric building block. Although different stimuli modulate the formation of fractal complexes and these complexes can regulate the enzymatic activity of citrate synthase in vitro, the fractal may not serve a physiological function in vivo. We use ancestral sequence reconstruction to retrace how the citrate synthase fractal evolved from non-fractal precursors, and the results suggest it may have emerged as a harmless evolutionary accident. Our findings expand the space of possible protein complexes and demonstrate that intricate and regulatable assemblies can evolve in a single substitution.

Citrate synthase lysine K215 hypoacetylation contributes to microglial citrate accumulation and pro-inflammatory functions after traumatic brain injury.

AIMS: This study aimed to investigate the relationship between microglial metabolism and neuroinflammation by examining the impact of citrate accumulation in microglia and its potential regulation through Cs K215 hypoacetylation. METHODS: Experimental approaches included assessing Cs enzyme activity through Cs K215Q mutation and investigating the inhibitory effects of hesperidin, a natural flavanone glycoside, on citrate synthase. Microglial phagocytosis and expression of pro-inflammatory cytokines were also examined in relation to Cs K215Q mutation and hesperidin treatment. RESULTS: Cs K215Q mutation and hesperidin exhibited significant inhibitory effects on Cs enzyme activity, microglial citrate accumulation, phagocytosis, and pro-inflammatory cytokine expression. Interestingly, Sirt3 knockdown aggravated microglial pro-inflammatory functions during neuroinflammation, despite its proven role in Cs deacetylation. CONCLUSION: Cs K215Q mutation and hesperidin effectively inhibited microglial pro-inflammatory functions without reversing the metabolic reprogramming. These findings suggest that targeting Cs K215 hypoacetylation and utilizing hesperidin may hold promise for modulating neuroinflammation in microglia.

The role of promoter methylation of the genes encoding the enzymes metabolizing di- and tricarboxylic acids in the regulation of plant respiration by light.

We discuss the role of epigenetic changes at the level of promoter methylation of the key enzymes of carbon metabolism in the regulation of respiration by light. While the direct regulation of enzymes via modulation of their activity and post-translational modifications is fast and readily reversible, the role of cytosine methylation is important for providing a prolonged response to environmental changes. In addition, adenine methylation can play a role in the regulation of transcription of genes. The mitochondrial and extramitochondrial forms of several enzymes participating in the tricarboxylic acid cycle and associated reactions are regulated via promoter methylation in opposite ways. The mitochondrial forms of citrate synthase, aconitase, fumarase, NAD-malate dehydrogenase are inhibited while the cytosolic forms of aconitase, fumarase, NAD-malate dehydrogenase, and the peroxisomal form of citrate synthase are activated. It is concluded that promoter methylation represents a universal mechanism of the regulation of activity of respiratory enzymes in plant cells by light. The role of the regulation of the mitochondrial and cytosolic forms of respiratory enzymes in the operation of malate and citrate valves and in controlling the redox state and balancing the energy level of photosynthesizing plant cells is discussed.

Physiological and muscle histochemical assessment of men and women 10 km runners matched for race performance or training volume.

Women have a disadvantage for performance in long-distance running compared with men. To elaborate on inherent characteristics, 12 subelite women were matched with 12 men for training volume (M-Tm) (56.6 ± 18 vs. 55.7 ± 17 km/wk). The women were also matched to other men for a 10 km staged outdoor time trial (M-Pm) (42:36 min:s) to determine which factors could explain equal running performance. Anthropometry and treadmill tests were done. Fiber type (% Type I and Type IIA) and citrate synthase activities were analyzed in muscle biopsy samples. Consistent sex differences for both comparisons included height, weight, % body fat (P < 0.01), and hematocrit (P < 0.05). Women had lower V̇o2max and peak treadmill speed (PTS) compared with both M-Tm and M-Pm (P < 0.01). Training matched pairs had no sex difference in % PTS at race pace but compared with M-Pm women ran at a higher % PTS (P < 0.05) and %HRmax (P < 0.01) at race pace. On average, the women trained 22.9 km/wk more than M-Pm (+67.5%, P < 0.01). This training was not associated with higher V̇o2max or better running economy. Muscle morphology and oxidative capacity did not differ between groups. Percentage body fat remained significantly higher in women. In conclusion, women matched to men for training volume had slower 10 km performance (-10.5% P < 0.05). Higher training volume, more high-intensity sessions/wk, and time spent training in the 95%-100% HRmax zone may explain the higher % PTS and %HRmax at race pace in women compared with performance-matched men.NEW & NOTEWORTHY When subelite women 10 km runners were matched with male counterparts for 10 km race performance, inherent differences in % body fat, V̇o2max, Hct, and peak treadmill speed were counteracted by significantly higher training volume, more time training at higher %HRmax and consequently, higher %HRmax and %PTS at race pace. Citrate synthase activity and muscle fiber types did not differ. When women and men matched for training, 10 km performance of men was 10.5% faster.

Mitochondrial density in skeletal and cardiac muscle.

Kubat et al. provide a review on the role Mitochondrial density in skeletal and cardiac muscle of mitochondrial dysfunction in muscle atrophy. They stress mitochondria's pivotal function, citing a 52 % density in skeletal muscle. However, the reference to Park et al.'s work misinterprets their findings. Park et al. report citrate synthase (CS) activity, indicating mitochondrial density as 222 ± 13 µmol.min-1.mg-1 for cardiac muscle and 115 ± 2 µmol.min-1.mg-1 for skeletal muscle. Thus, the authors should clarify that skeletal muscle density is approximately 52 % of cardiac muscle, not an absolute 52 %. Mitochondrial volume density assessment, predominantly through TEM, establishes cardiomyocytes at 25-30 % and untrained skeletal muscle at 2-6 %, increasing to 11 % in trained athletes. However, this remains modest compared to myofibrils' 75 %-85 % of muscle fiber volume. Although the utility of CS activity is evident, TEM and other novel approaches such as three-dimensional focused ion beam scanning electron microscopy are likely superior for assessing mitochondrial volume density and morphology.

Degradation of citrate synthase lacking the mitochondrial targeting sequence is inhibited in cells defective in Hsp70/Hsp40 chaperones under heat stress conditions.

Most nucleus-encoded mitochondrial precursor proteins are synthesized in the cytosol and imported into mitochondria in a post-translational manner. In recent years, the quality control mechanisms of nonimported mitochondrial proteins have been intensively studied. In a previous study, we established that in budding yeast a mutant form of citrate synthase 1 (N∆Cit1) that lacks the N-terminal mitochondrial targeting sequence, and therefore mislocalizes to the cytosol is targeted for proteasomal degradation by the SCFUcc1 ubiquitin ligase complex. Here, we show that Hsp70 and Hsp40 chaperones (Ssa1 and Ydj1 in yeast, respectively) are required for N∆Cit1 degradation under heat stress conditions. In the absence of Hsp70 function, a portion of N∆Cit1-GFP formed insoluble aggregates and cytosolic foci. However, the extent of ubiquitination of N∆Cit1 was unaffected, implying that Hsp70/Hsp40 chaperones are involved in the postubiquitination step of N∆Cit1 degradation. Intriguingly, degradation of cytosolic/peroxisomal gluconeogenic citrate synthase (Cit2), an endogenous substrate for SCFUcc1-mediated proteasomal degradation, was not highly dependent on Hsp70 even under heat stress conditions. These results suggest that mitochondrial citrate synthase is thermally vulnerable in the cytosol, where Hsp70/Hsp40 chaperones are required to facilitate its degradation.

HBV suppresses macrophage immune responses by impairing the TCA cycle through the induction of CS/PDHC hyperacetylation.

BACKGROUND: It is now understood that HBV can induce innate and adaptive immune response disorders by affecting immunosuppressive macrophages, resulting in chronic HBV infection. However, the underlying mechanism is not fully understood. Dysregulated protein acetylation can reportedly influence the differentiation and functions of innate immune cells by coordinating metabolic signaling. This study aims to assess whether HBV suppresses macrophage-mediated innate immune responses by affecting protein acetylation and to elucidate the underlying mechanisms of HBV immune escape. METHODS: We investigated the effect of HBV on the acetylation levels of human THP-1 macrophages and identified potential targets of acetylation that play a role in glucose metabolism. Metabolic and immune phenotypes of macrophages were analyzed using metabolomic and flow cytometry techniques. Western blot, immunoprecipitation, and immunofluorescence were performed to measure the interactions between deacetylase and acetylated targets. Chronic HBV persistent infected mice were established to evaluate the role of activating the tricarboxylic acid (TCA) cycle in macrophages for HBV clearance. RESULTS: Citrate synthase/pyruvate dehydrogenase complex hyperacetylation in macrophages after HBV stimulation inhibited their enzymatic activities and was associated with impaired TCA cycle and M2-like polarization. HBV downregulated Sirtuin 3 (SIRT3) expression in macrophages by means of the toll-like receptor 2 (TLR2)-NF-κB- peroxisome proliferatoractivated receptor γ coactivator 1α (PGC-1α) axis, resulting in citrate synthase/pyruvate dehydrogenase complex hyperacetylation. In vivo administration of the TCA cycle agonist dichloroacetate inhibited macrophage M2-like polarization and effectively reduced the number of serum HBV DNA copies. CONCLUSIONS: HBV-induced citrate synthase/pyruvate dehydrogenase complex hyperacetylation negatively modulates the innate immune response by impairing the TCA cycle of macrophages. This mechanism represents a potential therapeutic target for controlling HBV infection.

Bariatric surgery alters mitochondrial function in gut mucosa.

BACKGROUND: The obesity pandemic has worsened global disease burden, including type 2 diabetes, cardiovascular disease, and cancer. Metabolic/bariatric surgery (MBS) is the most effective and durable obesity treatment, but the mechanisms underlying its long-term weight loss efficacy remain unclear. MBS drives substrate oxidation that has been linked to improvements in metabolic function and improved glycemic control that are potentially mediated by mitochondria-a primary site of energy production. As such, augmentation of intestinal mitochondrial function may drive processes underlying the systemic metabolic benefits of MBS. Herein, we applied a highly sensitive technique to evaluate intestinal mitochondrial function ex vivo in a mouse model of MBS. METHODS: Mice were randomized to surgery, sham, or non-operative control. A simplified model of MBS, ileal interposition, was performed by interposition of a 2-cm segment of terminal ileum into the proximal bowel 5 mm from the ligament of Treitz. After a four-week recovery period, intestinal mucosa of duodenum, jejunum, ileum, and interposed ileum were assayed for determination of mitochondrial respiratory function. Citrate synthase activity was measured as a marker of mitochondrial content. RESULTS: Ileal interposition was well tolerated and associated with modest body weight loss and transient hypophagia relative to controls. Mitochondrial capacity declined in the native duodenum and jejunum of animals following ileal interposition relative to controls, although respiration remained unchanged in these segments. Similarly, ileal interposition lowered citrate synthase activity in the duodenum and jejunum following relative to controls but ileal function remained constant across all groups. CONCLUSION: Ileal interposition decreases mitochondrial volume in the proximal intestinal mucosa of mice. This change in concentration with preserved respiration suggests a global mucosal response to segment specific nutrition signals in the distal bowel. Future studies are required to understand the causes underlying these mitochondrial changes.

Upstream migrant sea lamprey (Petromyzon marinus) show signs of increasing oxidative stress but maintain aerobic capacity with age.

Following the parasitic juvenile phase of their life cycle, sea lamprey (Petromyzon marinus) mature into a reproductive but rapidly aging and deteriorating adult, and typically die shortly after spawning in May or June. However, pre-spawning upstream migrant sea lamprey can be maintained for several months beyond their natural lifespan when held in cold water (∼4-8 °C) under laboratory conditions. We exploited this feature to investigate the interactions between senescence, oxidative stress, and metabolic function in this phylogenetically ancient fish. We investigated how life history traits and mitochondria condition, as indicated by markers of oxidative stress (catalase activity, lipid peroxidation) and aerobic capacity (citrate synthase activity), changed in adult sea lamprey from June to December after capture during their upstream spawning migration. Body mass but not liver mass declined with age, resulting in an increase in hepatosomatic index. Both effects were most pronounced in males, which also tended to have larger livers than females. Lamprey experienced greater oxidative stress with age, as reflected by increasing activity of the antioxidant enzyme catalase and increasing levels of lipid peroxidation in liver mitochondrial isolates over time. Surprisingly, the activity of citrate synthase also increased with age in both sexes. These observations implicate mitochondrial dysfunction and oxidative stress in the senescence of sea lamprey. Due to their unique evolutionary position and the technical advantage of easily delaying the onset of senescence in lampreys using cold water, these animals could represent an evolutionary unique and tractable model to investigate senescence in vertebrates.

The Novel Role of Mitochondrial Citrate Synthase and Citrate in the Pathophysiology of Alzheimer's Disease.

Citrate synthase is a key mitochondrial enzyme that utilizes acetyl-CoA and oxaloacetate to form citrate in the mitochondrial membrane, which participates in energy production in the TCA cycle and linked to the electron transport chain. Citrate transports through a citrate malate pump and synthesizes acetyl-CoA and acetylcholine (ACh) in neuronal cytoplasm. In a mature brain, acetyl-CoA is mainly utilized for ACh synthesis and is responsible for memory and cognition. Studies have shown low citrate synthase in different regions of brain in Alzheimer's disease (AD) patients, which reduces mitochondrial citrate, cellular bioenergetics, neurocytoplasmic citrate, acetyl-CoA, and ACh synthesis. Reduced citrate mediated low energy favors amyloid-ß (Aß) aggregation. Citrate inhibits Aß25-35 and Aß1-40 aggregation in vitro. Hence, citrate can be a better therapeutic option for AD by improving cellular energy and ACh synthesis, and inhibiting Aß aggregation, which prevents tau hyperphosphorylation and glycogen synthase kinase-3 beta. Therefore, we need clinical studies if citrate reverses Aß deposition by balancing mitochondrial energy pathway and neurocytoplasmic ACh production. Furthermore, in AD's silent phase pathophysiology, when neuronal cells are highly active, they shift ATP utilization from oxidative phosphorylation to glycolysis and prevent excessive generation of hydrogen peroxide and reactive oxygen species (oxidative stress) as neuroprotective action, which upregulates glucose transporter-3 (GLUT3) and pyruvate dehydrogenase kinase-3 (PDK3). PDK3 inhibits pyruvate dehydrogenase, which decreases mitochondrial-acetyl-CoA, citrate, and cellular bioenergetics, and decreases neurocytoplasmic citrate, acetyl-CoA, and ACh formation, thus initiating AD pathophysiology. Therefore, GLUT3 and PDK3 can be biomarkers for silent phase of AD.

The implications of exercise in Drosophila melanogaster: insights into Akt/p38 MAPK/Nrf2 pathway associated with Hsp70 regulation in redox balance maintenance.

This study investigated the potential effects of exercise on the responses of energy metabolism, redox balance maintenance, and apoptosis regulation in Drosophila melanogaster to shed more light on the mechanisms underlying the increased performance that this emerging exercise model provides. Three groups were evaluated for seven days: the control (no exercise or locomotor limitations), movement-limited flies (MLF) (no exercise, with locomotor limitations), and EXE (with exercise, no locomotor limitations). The EXE flies demonstrated greater endurance-like tolerance in the swimming test, associated with increased citrate synthase activity, lactate dehydrogenase activity and lactate levels, and metabolic markers in exercise. Notably, the EXE protocol regulated the Akt/p38 MAPK/Nrf2 pathway, which was associated with decreased Hsp70 activation, culminating in glutathione turnover regulation. Moreover, reducing the locomotion environment in the MLF group decreased endurance-like tolerance and did not alter citrate synthase activity, lactate dehydrogenase activity, or lactate levels. The MLF treatment promoted a pro-oxidant effect, altering the Akt/p38 MAPK/Nrf2 pathway and increasing Hsp70 levels, leading to a poorly-regulated glutathione system. Lastly, we demonstrated that exercise could modulate major metabolic responses in Drosophila melanogaster aerobic and anaerobic metabolism, associated with apoptosis and cellular redox balance maintenance in an emergent exercise model.

Citrate Synthase GltA Modulates the 2,4-Diacetylphloroglucinol Biosynthesis of Pseudomonas fluorescens 2P24 and is Essential for the Biocontrol Capacity.

Carbon metabolism is critical for microbial physiology and remarkably affects the outcome of secondary metabolite production. The production of 2,4-diacetylphloroglucinol (2,4-DAPG), a bacterial secondary metabolite with a broad spectrum of antibiotic activity, is a major mechanism used by the soil bacterium Pseudomonas fluorescens 2P24 to inhibit the growth of plant pathogens and control disease occurrence. Strain 2P24 has evolved a complex signaling cascade to regulate the production of 2,4-DAPG. However, the role of the central carbon metabolism in modulating 2,4-DAPG production has not been fully determined. In this study, we report that the gltA gene, which encodes citrate synthase, affects the expression of the 2,4-DAPG biosynthesis gene and is essential for the biocontrol capacity of strain 2P24. Our data showed that the mutation of gltA remarkably decreased the biosynthesis of 2,4-DAPG. Consistent with this result, the addition of citrate in strain 2P24 resulted in increased 2,4-DAPG production and decreased levels of RsmA and RsmE. In comparison with the wild-type strain, the gltA mutant was severely impaired in terms of biocontrol activity against the bacterial wilt disease of tomato plants caused by Ralstonia solanacearum. Moreover, the gltA mutant exhibited increased antioxidant activity, and the expression of oxidative, stress-associated genes, including ahpB, katB, and oxyR, was significantly upregulated in the gltA mutant compared to the wild-type strain. Overall, our data indicate that the citrate synthase GltA plays an important role in the production of 2,4-DAPG and oxidative stress and is required for biocontrol capacity.

Effects of salt stress on seed germination and respiratory metabolism in different Flueggea suffruticosa genotypes.

The selection and utilization of ornamental plants that are highly tolerant to salt are helpful for landscape construction and the ecological protection of coastal and arid areas. To evaluate salt tolerance, one of the most used methods is the observation of seed germination under salt stress. Therefore, this work aimed to evaluate the influence of different concentrations of NaCl in water absorption, germination, and respiratory metabolism in seeds of different Flueggea suffruticosa genotypes. P2 and P27, salt-sensitive and salt-tolerant line s of F. suffruticosa, were chosen for treatment with 0, 40, 80, 120, 160, 200, and 240 mM NaCl. F. suffruticosa under salt stress exhibited inhibition of seed germination. The seeds of F. suffruticosa have different times for the physiological phases of water absorption with different NaCl concentrations. Salt stress retarded the seed water absorption process, and it depended on seed genotypes for F. suffruticosa. Soluble sugars accumulated in both P2 and P27 under salt stress. Meanwhile, the activities of hexokinase, 6-phosphofructokinase, pyruvate kinase, pyruvate dehydrogenase, citrate synthase, and glucose-6-phosphate dehydrogenase were overall increased in P27 after salt treatment, which caused increases in pyruvic acid and citric acid. The citrate synthase and glucose-6-phosphate dehydrogenase activities decreased in P2. These results suggest that the respiratory metabolism of salt-tolerant F. suffruticosa was enhanced, compared with the salt-sensitive line, to ameliorate the repression of seed germination under salt stress. The different changes in respiratory metabolism could influence the degree of salt tolerance.

Alveolar Mitochondrial Quality Control During Acute Respiratory Distress Syndrome.

Acute respiratory distress syndrome (ARDS) is a leading cause of respiratory failure and death in patients in the intensive care unit. Experimentally, acute lung injury resolution depends on the repair of mitochondrial oxidant damage by the mitochondrial quality control (MQC) pathways, mitochondrial biogenesis, and mitophagy, but nothing is known about this in the human lung. In a case-control autopsy study, we compared the lungs of subjects dying of ARDS (n = 8; cases) and age-/gender-matched subjects dying of nonpulmonary causes (n = 7; controls). Slides were examined by light microscopy and immunofluorescence confocal microscopy, randomly probing for co-localization of citrate synthase with markers of oxidant stress, mitochondrial DNA damage, mitophagy, and mitochondrial biogenesis. ARDS lungs showed diffuse alveolar damage with edema, hyaline membranes, and neutrophils. Compared with controls, a high degree of mitochondrial oxidant damage was seen in type 2 epithelial (AT2) cells and alveolar macrophages by 8-hydroxydeoxyguanosine and malondialdehyde co-staining with citrate synthase. In ARDS, antioxidant protein heme oxygenase-1 and DNA repair enzyme N-glycosylase/DNA lyase (Ogg1) were found in alveolar macrophages but not in AT2 cells. Moreover, MAP1 light chain-3 (LC3) and serine/threonine-protein kinase (Pink1) staining were absent in AT2 cells, suggesting a mitophagy failure. Nuclear respiratory factor-1 staining was missing in the alveolar region, suggesting impaired mitochondrial biogenesis. Widespread hyperproliferation of AT2 cells in ARDS could suggest defective differentiation into type 1 cells. ARDS lungs show profuse mitochondrial oxidant DNA damage but little evidence of MQC activity in AT2 epithelium. Because these pathways are important for acute lung injury resolution, our findings support MQC as a novel pharmacologic target for ARDS resolution.

Hepatic citrate synthase suppression in the freeze-tolerant wood frog (Rana sylvatica).

The wood frog, Rana sylvatica endures whole body freezing for weeks/months while overwintering at subzero temperatures. Survival of long-term freezing requires not only cryoprotectants but also strong metabolic rate depression (MRD) and reorganization of essential processes in order to maintain a balance between ATP-producing and ATP-consuming processes. Citrate synthase (CS) (E.C. 2.3.3.1) is an important irreversible enzyme of the tricarboxylic acid (TCA) cycle and forms a crucial checkpoint for many metabolic processes. Present study investigated the regulation of CS from wood frog liver during freezing. CS was purified to homogeneity by a two-step chromatographic process. Kinetic and regulatory parameters of the enzyme were investigated and, notably, demonstrated a significant decrease in the Vmax of the purified form of CS from frozen frogs as compared to controls when assayed at both 22 °C and 5 °C. This was further supported by a decrease in the maximum activity of CS from liver of frozen frogs. Immunoblotting also showed changes in posttranslational modifications with a significant decrease in threonine phosphorylation (by 49 %) for CS from frozen frogs. Taken together, these results suggest that CS is suppressed and TCA flux is inhibited during freezing, likely to support MRD survival of harsh winters.

Allosteric role of the citrate synthase homology domain of ATP citrate lyase.

ATP citrate lyase (ACLY) is the predominant nucleocytosolic source of acetyl-CoA and is aberrantly regulated in many diseases making it an attractive therapeutic target. Structural studies of ACLY reveal a central homotetrameric core citrate synthase homology (CSH) module flanked by acyl-CoA synthetase homology (ASH) domains, with ATP and citrate binding the ASH domain and CoA binding the ASH-CSH interface to produce acetyl-CoA and oxaloacetate products. The specific catalytic role of the CSH module and an essential D1026A residue contained within it has been a matter of debate. Here, we report biochemical and structural analysis of an ACLY-D1026A mutant demonstrating that this mutant traps a (3S)-citryl-CoA intermediate in the ASH domain in a configuration that is incompatible with the formation of acetyl-CoA, is able to convert acetyl-CoA and OAA to (3S)-citryl-CoA in the ASH domain, and can load CoA and unload acetyl-CoA in the CSH module. Together, this data support an allosteric role for the CSH module in ACLY catalysis.

Aerobic exercise training-induced alteration of gut microbiota composition affects endurance capacity.

This study aimed to clarify whether aerobic exercise training-induced alterations in the gut microbiota affect physiological adaptation with endurance exercise capacity. In study 1, ICR mice were randomly divided into three groups: vehicle intake + sedentary (V+S), vehicle intake + exercise training (V+Ex) and antibiotic intake + exercise training (AB+Ex). In the exercise training groups, treadmill running was performed for 8 weeks. During the exercise training intervention, the antibiotic-intake group freely drank water containing antibiotics. In study 2, ICR mice were randomly divided into three groups: Sham, transplantation of caecum microbiota from sedentary mice (Sed-CMT) and exercise training mice (Ex-CMT). In study 1, the treadmill running time to exhaustion, an index of maximal aerobic capacity, after aerobic exercise training in the V+Ex group was significantly longer than that in the V+S and AB+Ex groups. Gastrocnemius muscle citrate synthase (CS) activity and PGC-1α protein levels in the V+Ex group were significantly higher than in the V+S and AB+Ex groups. The bacterial Erysipelotrichaceae and Alcaligenaceae families were positively correlated with treadmill running time to exhaustion. In study 2, the treadmill running time to exhaustion after transplantation was significantly higher in the Ex-CMT group than in the Sham and Sed-CMT groups. Furthermore, CS activity and PGC-1α protein levels in the gastrocnemius muscle were significantly higher in the Ex-CMT group than in the Sham and Sed-CMT groups. Thus, gut microbiota altered by aerobic exercise training may be involved in the augmentation of endurance capacity and muscle mitochondrial energy metabolism. KEY POINTS: Aerobic exercise training changes gut microbiota composition, and the Erysipelotrichaceae and Alcaligenaceae families were among the altered gut bacteria. The gut microbiota was associated with endurance performance and metabolic regulator levels in skeletal muscle after aerobic exercise training. Continuous antibiotic treatment attenuated the increase in endurance performance, citrate synthase activity and PGC-1α levels in skeletal muscle induced by aerobic exercise training. Gut microbiota transplantation from exercise-trained mice improved endurance performance and metabolic regulator levels in recipient skeletal muscle, despite the absence of aerobic exercise training.

A Pseudomonas taiwanensis malonyl-CoA platform strain for polyketide synthesis.

Malonyl-CoA is a central precursor for biosynthesis of a wide range of complex secondary metabolites. The development of platform strains with increased malonyl-CoA supply can contribute to the efficient production of secondary metabolites, especially if such strains exhibit high tolerance towards these chemicals. In this study, Pseudomonas taiwanensis VLB120 was engineered for increased malonyl-CoA availability to produce bacterial and plant-derived polyketides. A multi-target metabolic engineering strategy focusing on decreasing the malonyl-CoA drain and increasing malonyl-CoA precursor availability, led to an increased production of various malonyl-CoA-derived products, including pinosylvin, resveratrol and flaviolin. The production of flaviolin, a molecule deriving from five malonyl-CoA molecules, was doubled compared to the parental strain by this malonyl-CoA increasing strategy. Additionally, the engineered platform strain enabled production of up to 84 mg L-1 resveratrol from supplemented p-coumarate. One key finding of this study was that acetyl-CoA carboxylase overexpression majorly contributed to an increased malonyl-CoA availability for polyketide production in dependence on the used strain-background and whether downstream fatty acid synthesis was impaired, reflecting its complexity in metabolism. Hence, malonyl-CoA availability is primarily determined by competition of the production pathway with downstream fatty acid synthesis, while supply reactions are of secondary importance for compounds that derive directly from malonyl-CoA in Pseudomonas.

Defective import of mitochondrial metabolic enzyme elicits ectopic metabolic stress.

Deficiencies in mitochondrial protein import are associated with a number of diseases. However, although nonimported mitochondrial proteins are at great risk of aggregation, it remains largely unclear how their accumulation causes cell dysfunction. Here, we show that nonimported citrate synthase is targeted for proteasomal degradation by the ubiquitin ligase SCFUcc1. Unexpectedly, our structural and genetic analyses revealed that nonimported citrate synthase appears to form an enzymatically active conformation in the cytosol. Its excess accumulation caused ectopic citrate synthesis, which, in turn, led to an imbalance in carbon flux of sugar, a reduction of the pool of amino acids and nucleotides, and a growth defect. Under these conditions, translation repression is induced and acts as a protective mechanism that mitigates the growth defect. We propose that the consequence of mitochondrial import failure is not limited to proteotoxic insults, but that the accumulation of a nonimported metabolic enzyme elicits ectopic metabolic stress.