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Abstract: Background: Treatment of acute compartment syndrome (ACS)-induced skeletal muscle injury remains a challenge. Previous studies have shown that octanoic acid is a promising treatment for ACS owing to its potential ability to regulate metabolic/epigenetic pathways in ischemic injury. The present study was designed to investigate the efficacy and underlying mechanism of octanoic acid in ACS-induced skeletal muscle injury. Methods: In this study, we established a saline infusion ACS rat model. Subsequently, we assessed the protective effects of sodium octanoate (NaO, sodium salt of octanoic acid) on ACS-induced skeletal muscle injury. Afterward, the level of acetyl-coenzyme A and histone acetylation in the skeletal muscle tissue were quantified. Moreover, we investigated the activation of the AMP-activated protein kinas pathway and the occurrence of mitophagy in the skeletal muscle tissue. Lastly, we scrutinized the expression of proteins associated with mitochondrial dynamics in the skeletal muscle tissue. Results: The administration of NaO attenuated muscle inflammation, alleviating oxidative stress and muscle edema. Moreover, NaO treatment enhanced muscle blood perfusion, leading to the inhibition of apoptosis-related skeletal muscle cell death after ACS. In addition, NaO demonstrated the ability to halt skeletal muscle fibrosis and enhance the functional recovery of muscle post-ACS. Further analysis indicates that NaO treatment increases the acetyl-CoA level in muscle and the process of histone acetylation by acetyl-CoA. Lastly, we found NaO treatment exerts a stimulatory impact on the activation of the AMPK pathway, thus promoting mitophagy and improving mitochondrial dynamics. Conclusion: Our findings indicate that octanoic acid may ameliorate skeletal muscle injury induced by ACS. Its protective effects may be attributed to the promotion of acetyl-CoA synthesis and histone acetylation within the muscular tissue, as well as its activation of the AMPK-related mitophagy pathway., (Copyright © 2024 by the Shock Society.)

Zygosaccharomyces rouxii has excellent fermentation performance and good tolerance to osmotic stress. Acetyl-CoA is a crucial intermediate precursor in the central carbon metabolic pathway of yeast. This study investigated the effect of engineering acetyl-CoA metabolism on the membrane functionality and stress tolerance of yeast. Firstly, exogenous supplementation of acetyl-CoA improved the biomass and the ability of unsaturated fatty acid synthesis of Z. rouxii under salt stress. Q-PCR results suggested that the gene ACSS (coding acetyl-CoA synthetase) was significantly up-expressed. Subsequently, the gene ACSS from Z. rouxii was transformed and heterologously expressed in S. cerevisiae. The recombinant cells exhibited better multiple stress (salt, acid, heat, and cold) tolerance, higher fatty acid contents, membrane integrity, and fluidity. Our findings may provide a suitable means to enhance the stress tolerance and fermentation efficiency of yeast under harsh fermentation environments., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest regarding the publication of this article., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The emergence of anti-EGFR therapy has revolutionized the treatment of colorectal cancer (CRC). However, not all patients respond consistently well. Therefore, it is imperative to conduct further research to identify the molecular mechanisms underlying the development of cetuximab resistance in CRC. In this study, we find that the expressions of many metabolism-related genes are downregulated in cetuximab-resistant CRC cells compared to their sensitive counterparts. Specifically, acetyl-CoA acyltransferase 2 (ACAA2), a key enzyme in fatty acid metabolism, is downregulated during the development of cetuximab resistance. Silencing of ACAA2 promotes proliferation and increases cetuximab tolerance in CRC cells, while overexpression of ACAA2 exerts the opposite effect. RTK-Kras signaling might contribute to the downregulation of ACAA2 expression in CRC, and ACAA2 predicts CRC prognosis in patients with Kras mutations. Collectively, our data suggest that modulating ACAA2 expression contributes to secondary cetuximab resistance in Kras wild-type CRC patients. ACAA2 expression is related to Kras mutation and demonstrates a prognostic role in CRC patients with Kras mutation. Thus, ACAA2 is a potential target in CRC with Kras mutation.

Background: Tumor cells display augmented capability to maintain endoplasmic reticulum (ER) homeostasis and hijack ER stress pathway for malignant phenotypes under microenvironmental stimuli. Metabolic reprogramming is a well-known hallmark for tumor cells to provide specific adaptive traits to the microenvironmental alterations. However, it's unknown how tumor cells orchestrate metabolic reprogramming and tumor progression in response to ER stress. Herein, we aimed to explore the pivotal roles of SEC63-mediated metabolic remodeling in hepatocellular carcinoma (HCC) cell metastasis after ER stress., Methods: The expression levels of SEC63 in HCC tissues and adjacent non-cancerous tissues were determined by immunohistochemistry and western blot. The regulatory roles of SEC63 in HCC metastasis were investigated both in vitro and in vivo by RNA-sequencing, metabolites detection, immunofluorescence, and transwell migration/invasion analyses. GST pull-down, immunoprecipitation/mass spectrometry and in vivo ubiquitination/phosphorylation assay were conducted to elucidate the underlying molecular mechanisms., Results: We identified SEC63 as a new regulator of HCC cell metabolism. Upon ER stress, the phosphorylation of SEC63 at T537 by IRE1α pathway contributed to SEC63 activation. Then, the stability of ACLY was upregulated by SEC63 to increase the supply of acetyl-CoA and lipid biosynthesis, which are beneficial for improving ER capacity. Meanwhile, SEC63 also entered into nucleus for increasing nuclear acetyl-CoA production to upregulate unfolded protein response targets to improve ER homeostasis. Importantly, SEC63 coordinated with ACLY to epigenetically modulate expression of Snail1 in the nucleus. Consequently, SEC63 promoted HCC cell metastasis and these effects were reversed by ACLY inhibition. Clinically, SEC63 expression was significantly upregulated in HCC tissue specimens and was positively correlated with ACLY expression. Importantly, high expression of SEC63 predicted unfavorable prognosis of HCC patients., Conclusions: Our findings revealed that SEC63-mediated metabolic reprogramming plays important roles in keeping ER homeostasis upon stimuli in HCC cells. Meanwhile, SEC63 coordinates with ACLY to upregulate the expression of Snail1, which further promotes HCC metastasis. Metastasis is crucial for helping cancer cells seek new settlements upon microenvironmental stimuli. Taken together, our findings highlight a cancer selective adaption to ER stress as well as reveal the potential roles of the IRE1α-SEC63-ACLY axis in HCC treatment., (© 2023. The Author(s).)

Obesity caused by endocrine disruptors (EDCs) has become a hot topic threatening human health. Recently, Nanoselenium Siraitia grosvenorii (NSG) has been shown to have potential health-modulating uses. Based on the results of 16S rRNA sequencing and metabolomics analysis, NSG has the unique function of improving gut microbiota and inhibiting obesity. Specifically, NSG can enhance gut microbiota diversity and change their composition. A significant positive correlation exists between the liver change in lysine and the high-importance dominant species ([Ruminococcus]_gnavus, Alistipes_finegoldii, etc.). NSG metabolites analysis showed that the lysine level increased by 44.45% and showed a significantly negatively correlated with (TG, TC, Leptin, etc.). Significantly, NSG reduces the degradation of lysine metabolism in the liver and inhibits fatty acid β-oxidation. In addition, NSG decreased Acetyl-CoA levels by 24% and regulated the downregulation of TCA genes (CS, Ogdh, Fh1, and Mdh2) and the upregulation of ketone body production genes (BDH1). NSG may have a positive effect on obesity by reducing the participation of Acetyl-CoA in the TCA cycle pathway and enhancing the ketogenic conversion of Acetyl-CoA. In conclusion, the results of this study may provide a new dietary intervention strategy for preventing endocrine disruptor-induced obesity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

There are no medical drugs that provide an acceptable weight loss with minimal adverse effects. This study evaluated the Moringa peregrina (MP) seed extract's anti-obesity effect. Twenty-four (6/each group) male Sprague Dawley rats were divided into group Ι (control), group ΙΙ (high-fat diet [HFD]), group ΙΙΙ (HFD+ MP [250 mg/kg b.wt]), and group ΙV (HFD+ MP [500 mg/kg b.wt]). MP administration significantly ameliorated body weight gains and HFD induced elevation in cholesterol, triglycerides, LDL, and reduced HDL. Moreover, MP seed oil showed high free radical-scavenging activity, delayed β-carotene bleaching and inhibited lipoprotein and pancreatic lipase enzymes. High-performance liquid chromatography (HPLC) revealed three major active components: crypto-chlorogenic acid, isoquercetin, and astragalin. Both quantitative Real-time PCR (RT-PCR) and western blotting revealed that MP seeds oil significantly decreased the expression of lipogenesis-associated genes such as peroxisome proliferator-activated receptors gamma (PPARγ) and fatty acid synthase (FAS) and significantly elevated the expression of lipolysis-associated genes (acetyl-CoA carboxylase1, ACCl). The oil also enhanced phosphorylation of AMP-activated protein kinase alpha (AMPK-α) and suppressed CCAAT/enhancer-binding protein β (C/EBPβ). In conclusion, administration of M. peregrina seeds oil has anti-obesity potential in HFD-induced obesity in rats. PRACTICAL APPLICATIONS: M. peregrina seeds oil had a potential anti-obesity activity that may be attributed to different mechanisms. These included decreasing body weight, and body mass index and improving lipid levels by decreasing total cholesterol, triglycerides and LDL-C, and increasing HDL-C. Also, M. peregrina seeds oil regulated adipogenesis-associated genes, such as downregulating the expression of (PPARγ, C/EBPα, and FAS) and improving and upregulating the expression and phosphorylation of AMPKα and ACCl. Despite that M. peregrina extract has reported clear anti-obesity potential through animal and laboratory studies, the available evidence-based on human clinical trials are very limited. Therefore, further studies are needed that could focus on clinical trials investigating anti-obesity potential different mechanisms of M. peregrina extract in humans., (© 2022 Wiley Periodicals LLC.)

Background: Autophagy is elevated in metastatic tumors and is often associated with active epithelial-to-mesenchymal transition (EMT). However, the extent to which EMT is dependent on autophagy is largely unknown. This study aimed to identify the mechanisms by which autophagy facilitates EMT., Methods: We employed a liquid chromatography-based metabolomic approach with kirsten rat sarcoma viral oncogene (KRAS) and liver kinase B1 (LKB1) gene co-mutated (KL) cells that represent an autophagy/EMT-coactivated invasive lung cancer subtype for the identification of metabolites linked to autophagy-driven EMT activation. Molecular mechanisms of autophagy-driven EMT activation were further investigated by quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting analysis, immunoprecipitation, immunofluorescence staining, and metabolite assays. The effects of chemical and genetic perturbations on autophagic flux were assessed by two orthogonal approaches: microtubule-associated protein 1A/1B-light chain 3 (LC3) turnover analysis by Western blotting and monomeric red fluorescent protein-green fluorescent protein (mRFP-GFP)-LC3 tandem fluorescent protein quenching assay. Transcription factor EB (TFEB) activity was measured by coordinated lysosomal expression and regulation (CLEAR) motif-driven luciferase reporter assay. Experimental metastasis (tail vein injection) mouse models were used to evaluate the impact of calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) or ATP citrate lyase (ACLY) inhibitors on lung metastasis using IVIS luciferase imaging system., Results: We found that autophagy in KL cancer cells increased acetyl-coenzyme A (acetyl-CoA), which facilitated the acetylation and stabilization of the EMT-inducing transcription factor Snail. The autophagy/acetyl-CoA/acetyl-Snail axis was further validated in tumor tissues and in autophagy-activated pancreatic cancer cells. TFEB acetylation in KL cancer cells sustained pro-metastatic autophagy in a mammalian target of rapamycin complex 1 (mTORC1)-independent manner. Pharmacological inhibition of this axis via CAMKK2 inhibitors or ACLY inhibitors consistently reduced the metastatic capacity of KL cancer cells in vivo., Conclusions: This study demonstrates that autophagy-derived acetyl-CoA promotes Snail acetylation and thereby facilitates invasion and metastasis of KRAS-LKB1 co-mutated lung cancer cells and that inhibition of the autophagy/acetyl-CoA/acetyl-Snail axis using CAMKK2 or ACLY inhibitors could be a potential therapeutic strategy to suppress metastasis of KL lung cancer., (© 2022 The Authors. Cancer Communications published by John Wiley & Sons Australia, Ltd. on behalf of Sun Yat-sen University Cancer Center.)

ATG5-induced autophagy is triggered in the early stages after SAH, which plays a vital role in subarachnoid hemorrhage (SAH). Acyl-CoA synthetase short-chain family 2 (ACSS2) is not just involved in energy metabolism but also binds to TEFB to form a complex translocated to related autophagy genes to regulate the expression of autophagy-related genes. However, the contribution of ACSS2 to the activation of autophagy in early brain injury (EBI) after SAH has barely been discussed. The purpose of this study was to investigate the alterations of ACSS2 and its neuroprotective effects following SAH. We first evaluated the expression of ACSS2 at different time points (6, 12, 24, and 72 h after SAH) in vivo and primary cortical neurons stimulated by oxyhemoglobin (OxyHb). Subsequently, adeno-associated virus and lentivirus were used to regulate ACSS2 expression to investigate the effect of ACSS2 after SAH. The results showed that the ACSS2 level decreased significantly in the early stages of SAH and was minimized at 24 h post-SAH. After artificial intervention to overexpress ACSS2, ATG5-induced autophagy was further enhanced in EBI after SAH, and neuronal apoptosis was alleviated to protect brain injury. In addition, brain edema and neurological function scores were improved. These results suggest that ACSS2 plays an important role in the neuroprotection against EBI after SAH by increasing ATG5-induce autophagy and inhibiting apoptosis., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Benign hepatosteatosis, affected by lipid uptake, de novo lipogenesis and fatty acid (FA) oxidation, progresses to non-alcoholic steatohepatitis (NASH) on stress and inflammation. A key macronutrient proposed to increase hepatosteatosis and NASH risk is fructose. Excessive intake of fructose causes intestinal-barrier deterioration and endotoxaemia. However, how fructose triggers these alterations and their roles in hepatosteatosis and NASH pathogenesis remain unknown. Here we show, using mice, that microbiota-derived Toll-like receptor (TLR) agonists promote hepatosteatosis without affecting fructose-1-phosphate (F1P) and cytosolic acetyl-CoA. Activation of mucosal-regenerative gp130 signalling, administration of the YAP-induced matricellular protein CCN1 or expression of the antimicrobial peptide Reg3b (beta) peptide counteract fructose-induced barrier deterioration, which depends on endoplasmic-reticulum stress and subsequent endotoxaemia. Endotoxin engages TLR4 to trigger TNF production by liver macrophages, thereby inducing lipogenic enzymes that convert F1P and acetyl-CoA to FA in both mouse and human hepatocytes.

Glutamate dehydrogenase (GDH) is essential for the brain function and highly regulated, according to its role in metabolism of the major excitatory neurotransmitter glutamate. Here we show a diurnal pattern of the GDH acetylation in rat brain, associated with specific regulation of GDH function. Mornings the acetylation levels of K84 (near the ADP site), K187 (near the active site), and K503 (GTP-binding) are highly correlated. Evenings the acetylation levels of K187 and K503 decrease, and the correlations disappear. These daily variations in the acetylation adjust the GDH responses to the enzyme regulators. The adjustment is changed when the acetylation of K187 and K503 shows no diurnal variations, as in the rats after a high dose of thiamine. The regulation of GDH function by acetylation is confirmed in a model system, where incubation of the rat brain GDH with acetyl-CoA changes the enzyme responses to GTP and ADP, decreasing the activity at subsaturating concentrations of substrates. Thus, the GDH acetylation may support cerebral homeostasis, stabilizing the enzyme function during diurnal oscillations of the brain metabolome. Daytime and thiamine interact upon the (de)acetylation of GDH in vitro. Evenings the acetylation of GDH from control animals increases both IC 50 GTP and EC 50 ADP . Mornings the acetylation of GDH from thiamine-treated animals increases the enzyme IC 50 GTP . Molecular mechanisms of the GDH regulation by acetylation of specific residues are proposed. For the first time, diurnal and thiamine-dependent changes in the allosteric regulation of the brain GDH due to the enzyme acetylation are shown., (© 2019 International Society for Neurochemistry.)

Differentiation of myeloid progenitor cells into macrophages is accompanied by increased PU.1 concentration and increasing cell cycle length, culminating in cell cycle arrest. Induction of PU.1 expression in a cultured myeloid cell line expressing low PU.1 concentration results in decreased levels of mRNA encoding ATP-Citrate Lyase (ACL) and cell cycle arrest. ACL is an essential enzyme for generating acetyl-CoA, a key metabolite for the first step in fatty acid synthesis and for histone acetylation. We hypothesized that ACL may play a role in cell cycle regulation in the myeloid lineage. In this study, we found that acetyl-CoA or acetate supplementation was sufficient to rescue cell cycle progression in cultured BN cells treated with an ACL inhibitor or induced for PU.1 expression. Acetyl-CoA supplementation was also sufficient to rescue cell cycle progression in BN cells treated with a fatty acid synthase (FASN) inhibitor. We demonstrated that acetyl-CoA was utilized in both fatty acid synthesis and histone acetylation pathways to promote proliferation. Finally, we found that Acly mRNA transcript levels decrease during normal macrophage differentiation from bone marrow precursors. Our results suggest that regulation of ACL activity is a potentially important point of control for cell cycle regulation in the myeloid lineage., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Phosphopantetheine adenylyltransferase (PPAT) is a rate-limiting enzyme essential for biosynthesis of coenzyme A (CoA), which in turn is responsible to regulate the secretion of exotoxins via type III secretion system in Pseudomonas aeruginosa, causing severe health concerns ranging from nosocomial infections to respiratory failure. Acetyl coenzyme A (AcCoA) is a newly reported inhibitor of PPAT, believed to regulate the cellular levels of CoA and thereby the pathogenesis. Very little is known so far regarding the mechanistic details of AcCoA binding inside PPAT-binding cleft. Herein, we have used extensive umbrella sampling simulations to decipher mechanistic insight into the inhibitor accommodation inside the binding cavity. We found that R90 and D94 residues act like a gate near the binding cavity to accommodate and stabilize the incoming ligand. Mutational models concerning these residues also show considerable difference in AcCoA-binding thermodynamics. To substantiate our findings, we have solved the first crystal structure of apo-PPAT from P. aeruginosa, which also found to agree with the simulation results. Collectively, these results describe the mechanistic details of accommodation of inhibitor molecule inside PPAT-binding cavity and also offer valuable insight into regulating cellular levels of CoA/AcCoA and thus controlling the pathogenicity.

Background: Epithelial to mesenchymal transition (EMT) is a complex and dynamic molecular event in lung cancer metastasis that has not yet been thoroughly investigated. EMT transcriptional factors, such as Snail, play a central role in regulation of the EMT process. In this study, we sought to identify an association between p300 and Snail in lung cancer, as well as the engagement of p300 in Snail acetylation., Methods: We transfected p300 small interfering RNA into lung cancer cells to detect Snail and E-cadherin expression levels by real time-PCR. Immunoprecipitation assay was conducted to determine Snail acetylation in vivo. Bacteria-expressed Snail was purified to analyze Snail acetylation in vitro. We further mutated lysine 187 for identifying acetylated residue in Snail., Results: Snail transcription in lung cancer cells was repressed by p300 knockdown. E-cadherin expression was increased by transfection of p300 small interfering RNA in a dose-dependent manner. Immunoprecipitation and Western blot assay with anti-acetylated lysine antibody were used to confirm that Snail was acetylated by p300. A sequence coding snail gene was cloned into glutathione S-transferase-tagged vector and the fusion protein was purified using glutathione. We observed Snail acetylation in vitro by incubation of recombinant Snail and p300 histone acetyltransferase domain with acetyl coenzyme A. The reduced Snail acetylation level was related to lysine mutation at position 187 of Snail., Conclusion: There was a correlation between Snail and p300 expressions in lung cancer. Moreover, p300 acetylates Snail both in vivo and in vitro, and K187 may be involved in this modification., (© 2017 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.)

Enzyme activity is commonly controlled by allostery, where ligand binding at one site alters the activities of distant sites. Classical explanations for multisubunit proteins involve conformational transitions that are fundamentally deterministic. For example, in the Monod-Wyman-Changeaux (MWC) paradigm, conformational transitions occur simultaneously in all subunits. In the Koshland-Nemethy-Filmer (KNF) paradigm, conformational transitions only occur in ligand-bound subunits. In contrast, recent models predict conformational changes that are governed by probabilities rather than absolute rules. To better understand allostery at the molecular level, we applied a recently developed spectroscopic and calorimetric method to the interactions of a dimeric enzyme with two different ligands. We found that conformational transitions appear MWC-like for a ligand that binds at the dimer interface and KNF-like for a distal ligand. These results provide strong experimental support for probabilistic allosteric theory predictions that an enzyme can exhibit a mixture of MWC and KNF character, with the balance partly governed by subunit interface energies.

In budding yeast cells, nutrient repletion induces rapid exit from quiescence and entry into a round of growth and division. The G1 cyclin CLN3 is one of the earliest genes activated in response to nutrient repletion. Subsequent to its activation, hundreds of cell-cycle genes can then be expressed, including the cyclins CLN1/2 and CLB5/6. Although much is known regarding how CLN3 functions to activate downstream targets, the mechanism through which nutrients activate CLN3 transcription in the first place remains poorly understood. Here we show that a central metabolite of glucose catabolism, acetyl-CoA, induces CLN3 transcription by promoting the acetylation of histones present in its regulatory region. Increased rates of acetyl-CoA synthesis enable the Gcn5p-containing Spt-Ada-Gcn5-acetyltransferase transcriptional coactivator complex to catalyze histone acetylation at the CLN3 locus alongside ribosomal and other growth genes to promote entry into the cell division cycle.

Background and Purpose: Acyl derivatives of CoA have been shown to act as antagonists at human platelet and recombinant P2Y1 receptors, but little is known about their effects in the cardiovascular system. This study evaluated the effect of these endogenous nucleotide derivatives at P2Y1 receptors natively expressed in rat and porcine blood vessels., Experimental Approach: Isometric tension recordings were used to evaluate the effects of CoA, acetyl CoA, palmitoyl CoA (PaCoA) and 3'-dephospho-palmitoyl-CoA on concentration relaxation-response curves to ADP and uridine triphosphate (UTP). A FlexStation monitored ADP- and UTP-evoked calcium responses in HEK293 cells., Key Results: Acetyl CoA and PaCoA, but not CoA, inhibited endothelium-dependent relaxations to ADP with apparent selectivity for P2Y1 receptors (over P2Y(2/4) receptors) in rat thoracic aorta; PaCoA was more potent than acetyl CoA (331-fold vs. fivefold shift of ADP response curve evoked by 10 μM PaCoA and acetyl CoA, respectively); the apparent pA2 value for PaCoA was 6.44. 3'-dephospho-palmitoyl-CoA (10 μM) was significantly less potent than PaCoA (20-fold shift). In porcine mesenteric arteries, PaCoA and the P2Y1 receptor antagonist MRS2500 blocked ADP-mediated endothelium-dependent relaxations; in contrast, they were ineffective against ADP-mediated endothelium-independent relaxation in porcine coronary arteries (which does not involve P2Y1 receptors). Calcium responses evoked by ADP activation of endogenous P2Y1 receptors in HEK293 cells were inhibited in the presence of PaCoA, which failed to alter responses to UTP (acting at endogenous P2Y(2/4) receptors)., Conclusions and Implications: Acyl derivatives of CoA can act as endogenous selective antagonists of P2Y1 receptors in blood vessels, and this inhibitory effect critically depends on the palmitate and 3'-ribose phosphate substituents on CoA., (© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

ToxT is the central regulatory protein involved in activation of the main virulence genes in Vibrio cholerae. We have identified transposon insertions in central metabolism genes, whose disruption increases toxT transcription. These disrupted genes encode the primary respiration-linked sodium pump (NADH:ubiquinone oxidoreductase or NQR) and certain tricarboxylic acid (TCA) cycle enzymes. Observations made following stimulation of respiration in the nqr mutant or chemical inhibition of NQR activity in the TCA cycle mutants led to the hypothesis that NQR affects toxT transcription via the TCA cycle. That toxT transcription increased when the growth medium was supplemented with citrate, but decreased with oxaloacetate, focused our attention on the TCA cycle substrate acetyl-CoA and its non-TCA cycle metabolism. Indeed, both the nqr and the TCA cycle mutants increased acetate excretion. A similar correlation between acetate excretion and toxT transcription was observed in a tolC mutant and upon amino acid (NRES) supplementation. As acetate and its tendency to decrease pH exerted no strong effect on toxT transcription, and because disruption of the major acetate excretion pathway increased toxT transcription, we propose that toxT transcription is regulated by either acetyl-CoA or some close derivative.

Physiological or α-processing of amyloid-β precursor protein (APP) prevents the formation of Aβ, which is deposited in the aging brain and may contribute to Alzheimer's disease. As such, drugs promoting this pathway could be useful for prevention of the disease. Along this line, we searched through a number of substances and unexpectedly found that a group of high-energy compounds (HECs), namely ATP, phosphocreatine, and acetyl coenzyme A, potently increased APP α-processing in cultured SH-SY5Y cells, whereas their cognate counterparts, i.e., ADP, creatine, or coenzyme A did not show the same effects. Other HECs such as GTP, CTP, phosphoenol pyruvate, and S-adenosylmethionine also promoted APP α-processing with varying potencies and the effects were abolished by energy inhibitors rotenone or NaN(3). The overall efficacy of the HECs in the process ranged from three- to four-fold, which was significantly greater than that exhibited by other physiological stimulators such as glutamate and nicotine. This suggested that the HECs were perhaps the most efficient physiological stimulators for APP α-processing. Moreover, the HECs largely offset the inefficient APP α-processing in aged human fibroblasts or in cells impaired by rotenone or H(2) O(2). Most importantly, some HECs markedly boosted the survival rate of SH-SY5Y cells in the death process induced by energy suppression or oxidative stress. These findings suggest a new, energy-dependent regulatory mechanism for the putative α-secretase and thus will help substantially in its identification. At the same time, the study raises the possibility that the HECs may be useful to energize and strengthen the aging brain cells to slow down the progression of Alzheimer's disease., (Published 2012. This article is a US Government work and is in the public domain in the USA.)

Mitochondria are important in the function and control of Crassulacean acid metabolism (CAM) during organic acid accumulation at night and acid decarboxylation in the day. In plants of the malic enzyme-(ME) type and the phosphoenolpyruvate carboxykinase- (PEPCK) type, mitochondria may exert their role in the control of the diurnal rhythm of malic and citric acids to a differential degree. In plants of both CAM types, the oxidative capacity of mitochondria, as well as the activity of CAM-linked mitochondrial enzymes, and of the alternative and the rotenone-resistant pathways of substrate oxidation were compared. Furthermore, a C₃ succulent was included, as well as both C₃ and CAM forms of Mesembryanthemum crystallinum during a salt-induced C₃-to-CAM shift. Mitochondria of PEPCK-type CAM plants exhibited a lower activity of malate oxidation, ratio of malate to succinate oxidation, and activity of mitochondrial NAD-ME. With the exception of Kalanchoë daigremontiana, leaf mitochondria of all other CAM species were highly sensitive to cyanide (80-100%), irrespective of the oxidant used. This indicates that the alternative oxidase is not of general importance in CAM. By contrast, rotenone-insensitive substrate oxidation was very high (50-90%) in all CAM species. This is the first comparison of the rotenone-insensitive pathway of respiration in plants with different CAM-types. The results of this study confirm that mitochondria are involved in the control of CAM to different degrees in the two CAM types, and they highlight the multiple roles of mitochondria in CAM.

While crystallographic structures of the R. etli pyruvate carboxylase (PC) holoenzyme revealed the location and probable positioning of the essential activator, Mg(2+), and nonessential activator, acetyl-CoA, an understanding of how they affect catalysis remains unclear. The current steady-state kinetic investigation indicates that both acetyl-CoA and Mg(2+) assist in coupling the MgATP-dependent carboxylation of biotin in the biotin carboxylase (BC) domain with pyruvate carboxylation in the carboxyl transferase (CT) domain. Initial velocity plots of free Mg(2+) vs pyruvate were nonlinear at low concentrations of Mg(2+) and a nearly complete loss of coupling between the BC and CT domain reactions was observed in the absence of acetyl-CoA. Increasing concentrations of free Mg(2+) also resulted in a decrease in the K(a) for acetyl-CoA. Acetyl phosphate was determined to be a suitable phosphoryl donor for the catalytic phosphorylation of MgADP, while phosphonoacetate inhibited both the phosphorylation of MgADP by carbamoyl phosphate (K(i) = 0.026 mM) and pyruvate carboxylation (K(i) = 2.5 mM). In conjunction with crystal structures of T882A R. etli PC mutant cocrystallized with phosphonoacetate and MgADP, computational docking studies suggest that phosphonoacetate could coordinate to one of two Mg(2+) metal centers in the BC domain active site. Based on the pH profiles, inhibition studies, and initial velocity patterns, possible mechanisms for the activation, regulation, and coordination of catalysis between the two spatially distinct active sites in pyruvate carboxylase from R. etli by acetyl-CoA and Mg(2+) are described.

Human arylamine N-acetyltransferase 1 (NAT1) is a xenobiotic-metabolizing enzyme that biotransforms aromatic amine chemicals. We show here that biologically-relevant concentrations of inorganic (Hg2+) and organic (CH3Hg+) mercury inhibit the biotransformation functions of NAT1. Both compounds react irreversibly with the active-site cysteine of NAT1 (half-maximal inhibitory concentration (IC50)=250 nM and kinact=1.4x10(4) M(-1) s(-1) for Hg2+ and IC50=1.4 microM and kinact=2x10(2) M(-1) s(-1) for CH3Hg+). Exposure of lung epithelial cells led to the inhibition of cellular NAT1 (IC50=3 and 20 microM for Hg2+ and CH3Hg+, respectively). Our data suggest that exposure to mercury may affect the biotransformation of aromatic amines by NAT1., (Copyright (c) 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Peroxisome proliferators (PPs), non-genotoxic rodent carcinogens, cause the induction of the peroxisomal fatty acid beta-oxidation system, including bifunctional enzyme (BE) and 3-ketoacyl-CoA thiolase (TH), in the liver. GST M1 gene is polymorphic in Sprague-Dawley rats, NC- and KS-type. The KS-type rats showed enhanced susceptibility to ethyl-alpha-chlorophenoxyisobutyrate (clofibrate, CF), one of the PPs. The degree of BE induction was higher in the KS-type and preneoplastic foci developed after 6-8 weeks of treatment, whereas no foci developed in the NC-type. In the preset study, factors involved in different BE inducibility were investigated. There were no differences in hepatic peroxisome proliferator-activated receptor (PPAR) alpha levels between them. Among various coactivators for PPARalpha, only steroid receptor coactivator (SRC)-3 level was higher in the KS-type. To investigate the association between PPARalpha and SRC-3 or other proteins, nuclear extracts from CF-treated livers were applied to a PPARalpha column. In the KS-type, 110, 72, and 42 kDa proteins were bound and these were identified as SRC-3, BE, and TH, respectively. EMSA supported the binding of these proteins to PPARalpha associated to the BE enhancer in CF-treated KS-type, but not in the NC-type. Histone H3 acetylation was increased 11-fold in the KS-type by CF treatment but not in the NC-type. As BE and TH are responsible for acetyl-CoA production and SRC-3 possesses a histone acetyltransferase activity, these results suggest that enhanced BE induction in the KS-type livers is due to acetylation-mediated transcriptional activation and epigenetic mechanisms might be involved in CF-induced rat hepatocarcinogenesis.

The human T cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T cell leukemia/lymphoma. The multifunctional virally encoded oncoprotein Tax is responsible for malignant transformation and potent activation of HTLV-1 transcription. Tax, in complex with phosphorylated cAMP response element binding protein (pCREB), strongly recruits the cellular coactivators CREB binding protein (CBP)/p300 to the viral promoter concomitant with transcriptional activation. Although the mechanism of activator/coactivator-mediated transcriptional activation is poorly understood, the recruitment of CBP/p300 by regulatory factors appears to function, in part, by promoting changes in chromatin architecture that are permissive to transcriptional activation. Here, we show that CBP/p300 recruitment promotes histone acetylation and eviction of the histone octamer from the chromatin-assembled HTLV-1 promoter template in vitro. Nucleosome disassembly is strictly acetyl-CoA dependent and is not linked to ATP utilization. We find that the histone chaperone, nucleosome assembly protein 1 (NAP1), cooperates with CBP/p300 in eviction of the acetylated histones from the chromatin template. These findings reveal a unique mechanism in which the DNA-bound Tax/pCREB complex recruits CBP/p300, and together with NAP1, the coactivators cooperate to dramatically reduce nucleosome occupancy at the viral promoter in an acetylation-dependent and transcription-independent fashion.

Unlike other eukaryotes studied to date, yeast has two genes for pyruvate carboxylase coding for very similar, but not identical, isozymes (Pyc1 and Pyc2), both of which are located in the cytoplasm. We have found that there are marked differences in the kinetic properties of the isozymes potentially leading to differential regulation of Pyc1 and Pyc2 activity by both activators and substrates. For example, Pyc2 is only activated 3.7-fold by acetyl CoA, and 9.6-fold by NH(4)(+), whilst the figures for Pyc1 are 16 and 14.6-fold, respectively. Pyc1 and Pyc2 display different allosteric properties with respect to acetyl CoA activation and aspartate inhibition, with Pyc1 showing a higher degree of cooperativity than Pyc2, even in the absence of aspartate. We have investigated the locus of action in the amino acid sequence of the isozymes of this activator by measuring its regulation of various chimeric constructs of the two isozymes. In this way, we conclude that the main locus of action of acetyl CoA lies in the N-terminal half of the enzyme, within the biotin-carboxylation domain, between amino acids 99 and 478 of Pyc1.

Mitochondrial dysfunction during acute metabolic crises is considered an important pathomechanism in inherited disorders of propionate metabolism, i.e. propionic and methylmalonic acidurias. Biochemically, these disorders are characterized by accumulation of propionyl-CoA and metabolites of alternative propionate oxidation. In the present study, we demonstrate uncompetitive inhibition of PDHc (pyruvate dehydrogenase complex) by propionyl-CoA in purified porcine enzyme and in submitochondrial particles from bovine heart being in the same range as the inhibition induced by acetyl-CoA, the physiological product and known inhibitor of PDHc. Evaluation of similar monocarboxylic CoA esters showed a chain-length specificity for PDHc inhibition. In contrast with CoA esters, non-esterified fatty acids did not inhibit PDHc activity. In addition to PDHc inhibition, analysis of respiratory chain and tricarboxylic acid cycle enzymes also revealed an inhibition by propionyl-CoA on respiratory chain complex III and alpha-ketoglutarate dehydrogenase complex. To test whether impairment of mitochondrial energy metabolism is involved in the pathogenesis of propionic aciduria, we performed a thorough bioenergetic analysis in muscle biopsy specimens of two patients. In line with the in vitro results, oxidative phosphorylation was severely compromised in both patients. Furthermore, expression of respiratory chain complexes I-IV and the amount of mitochondrial DNA were strongly decreased, and ultrastructural mitochondrial abnormalities were found, highlighting severe mitochondrial dysfunction. In conclusion, our results favour the hypothesis that toxic metabolites, in particular propionyl-CoA, are involved in the pathogenesis of inherited disorders of propionate metabolism, sharing mechanistic similarities with propionate toxicity in micro-organisms.

Here we describe an in vitro chromatin transcription system in which chromatin assembly and transcription are carried out with purified and defined factors. With basal (also known as general) transcription factors and sequence-specific DNA-binding activators, we observed chromatin-specific, activation domain-dependent transcription. We then examined the biochemical function of purified p300 in the absence of the endogenous factor and other related activities and found, unexpectedly, that p300 has a chromatin-specific, transcriptional repression activity that can be relieved by the addition of acetyl-CoA. This p300-mediated repression is reversible, requires the p300 bromodomain but not the acetyltransferase region, and does not involve the formation of a stable, nuclease-resistant nucleoprotein complex. Hence, the mechanism of transcriptional repression by p300 is distinct from that of histone H1, PARP-1 or Sir2. These findings reveal a novel chromatin-specific repressive function of p300.

C75, a synthetic inhibitor of FAS (fatty acid synthase), has both anti-tumour and anti-obesity properties. In this study we provide a detailed kinetic characterization of the mechanism of in vitro inhibition of rat liver FAS. At room temperature, C75 is a competitive irreversible inhibitor of the overall reaction with regard to all three substrates, i.e. acetyl-CoA, malonyl-CoA and NADPH, exhibiting pseudo-first-order kinetics of the complexing type, i.e. a weak non-covalent enzyme-inhibitor complex is formed before irreversible enzyme modification. C75 is a relatively inefficient inactivator of FAS, with a maximal rate of inactivation of 1 min(-1) and an extrapolated K(I) (dissociation constant for the initial complex) of approx. 16 mM. The apparent second-order rate constants calculated from these values are 0.06 mM(-1).min(-1) at room temperature and 0.21 mM(-1).min(-1) at 37 degrees C. We also provide experimental evidence that C75 inactivates the beta-ketoacyl synthase (3-oxoacyl synthase) partial activity of FAS. Unexpectedly, C75 also inactivates the enoyl reductase and thioesterase partial activities of FAS with about the same rates as for inactivation of the beta-ketoacyl synthase. In contrast with the overall reaction, the beta-ketoacyl synthase activity and the enoyl reductase activity, substrates do not protect the thioesterase activity of rat liver FAS from inactivation by C75. These results differentiate inactivation by C75 from that by cerulenin, which only inactivates the beta-ketoacyl synthase activity of FAS, by forming an adduct with an active-site cysteine. Interference by dithiothreitol and protection by the substrates, acetyl-CoA, malonyl-CoA and NADPH, further distinguish the mechanism of C75-mediated inactivation from that of cerulenin. The most likely explanation for the multiple effects observed with C75 on rat liver FAS and its partial reactions is that there are multiple sites of interaction between C75 and FAS.

Pantothenate kinase (PanK) catalyzes the first step in the biosynthesis of the essential and ubiquitous cofactor coenzyme A (CoA) in all organisms. Two well characterized isoforms of the enzyme are known: a prokaryotic PanK that predominates in eubacteria and a eukaryotic isoform that has primarily been characterized from mammalian and plant sources. Curiously, the genomes of certain pathogenic bacteria, including Helicobacter pylori and Pseudomonas aeruginosa, do not contain a PanK similar to either isoform, although these organisms possess all the other biosynthetic machinery required for CoA production. In this study we cloned, overexpressed and characterized an enzyme from Bacillus subtilis and its homologue from H. pylori and show that they catalyze the ATP-dependent phosphorylation of pantothenate. These enzymes do not share sequence homology with any known PanK, and unlike the bacterial and eukaryotic PanK isoforms their activity is not regulated by either CoA or acetyl-CoA. They also do not accept the pantothenic acid antimetabolite N-pentylpantothenamide as a substrate or are inhibited by it. Taken together, these results point to the identification of a third distinct isoform of PanK that accounts for the only known activity of the enzyme in pathogens such as H. pylori and P. aeruginosa.

Arylamine N-acetyltransferases (NATs) catalyze an acetyl group transfer from acetyl coenzyme A (AcCoA) to arylamines, hydrazines, and their N-hydroxylated arylamine metabolites. The recently determined three-dimensional structures of prokaryotic NATs have revealed a cysteine protease-like Cys-His-Asp catalytic triad, which resides in a deep and hydrophobic pocket. This catalytic triad is strictly conserved across all known NATs, including hamster NAT2 (Cys-68, His-107, and Asp-122). Treatment of NAT2 with either iodoacetamide (IAM) or bromoacetamide (BAM) at neutral pH rapidly inactivated the enzyme with second-order rate constants of 802.7 +/- 4.0 and 426.9 +/- 21.0 M(-1) s(-1), respectively. MALDI-TOF and ESI mass spectral analysis established that Cys-68 is the only site of alkylation by IAM. Unlike the case for cysteine proteases, no significant inactivation was observed with either iodoacetic acid (IAA) or bromoacetic acid (BAA). Pre-steady state and steady state kinetic analysis with p-nitrophenyl acetate (PNPA) and NAT2 revealed a single-exponential curve for the acetylation step with a second-order rate constant of (1.4 +/- 0.05) x 10(5) M(-1) s(-1), followed by a slow linear rate of (7.85 +/- 0.65) x 10(-3) s(-1) for the deacetylation step. Studies of the pH dependence of the rate of inactivation with IAM and the rate of acetylation with PNPA revealed similar pK(a)(1) values of 5.23 +/- 0.09 and 5.16 +/- 0.04, respectively, and pK(a)(2) values of 6.95 +/- 0.27 and 6.79 +/- 0.25, respectively. Both rates reached their maximum values at pH 6.4 and decreased by only 30% at pH 9.0. Kinetic studies in the presence of D(2)O revealed a large inverse solvent isotope effect on both inactivation and acetylation of NAT2 [k(H)(inact)/k(D)(inact) = 0.65 +/- 0.02 and (k(2)/K(m)(acetyl))(H)/(k(2)/K(m)(acetyl))(D) = 0.60 +/- 0.03], which were found to be identical to the fractionation factors (Phi) derived from proton inventory studies of the rate of acetylation at pL 6.4 and 8.0. Substitution of the catalytic triad Asp-122 with either alanine or asparagine resulted in the complete loss of protein structural integrity and catalytic activity. From these results, it can be concluded that the catalytic mechanism of NAT2 depends on the formation of a thiolate-imidazolium ion pair (Cys-S(-)-His-ImH(+)). However, in contrast to the case with cysteine proteases, a pH-dependent protein conformational change is likely responsible for the second pK(a), and not deprotonation of the thiolate-imidazolium ion. In addition, substitutions of the triad aspartate are not tolerated. The enzyme appears, therefore, to be engineered to rapidly form a stable acetylated species poised to react with an arylamine substrate.

Lipoic acid is an essential coenzyme in the oxidation of pyruvate and alpha-ketoglutarate. It is easily converted to its reduced form, dihydrolipoic acid (DHLA), in vivo thereby forming a redox pair. DHLA is important in the maintenance and integrity of specific neuronal and subcellular membranes. In the present study we investigated the effect of DHLA on the response of isolated rat bladder strips to repetitive field stimulation (FS), a method used to exhaust synaptic stores of acetylcholine resulting in nerve and synaptic damage. Isolated strips of rat urinary bladders were separated into 4 groups. Group 1 strips were incubated with choline + acetyl-CoA; Group 2 strips with choline, acetyl-CoA + DHLA; and Group 3 with DHLA. Group 4 strips were controls. All strips in Groups 1-3 were subjected to 2 h of repetitive FS followed by 2 h of recovery. DHLA had no effect on the progressive decrease in contractile response observed during repetitive stimulation. However, strips incubated in the presence of DHLA showed a significantly greater degree of recovery than strips incubated in the absence of DHLA. We believe that the protection of the contractile response is related to DHLA's ability to protect nerve and/or muscle membranes from oxidative damage.

Pyruvate conversion to acetyl-CoA by the pyruvate dehydrogenase (PDH) multienzyme complex is known as a key node in affecting the metabolic fluxes of animal cell culture. However, its possible role in causing possible nonlinear dynamic behavior such as oscillations and multiplicity of animal cells has received little attention. In this work, the kinetic and dynamic behavior of PDH of eucaryotic cells has been analyzed by using both in vitro and simplified in vivo models. With the in vitro model the overall reaction rate (nu(1)) of PDH is shown to be a nonlinear function of pyruvate concentration, leading to oscillations under certain conditions. All enzyme components affect nu(1) and the nonlinearity of PDH significantly, the protein X and the core enzyme dihydrolipoamide acyltransferase (E2) being mostly predominant. By considering the synthesis rates of pyruvate and PDH components the in vitro model is expanded to emulate in vivo conditions. Analysis using the in vivo model reveals another interesting kinetic feature of the PDH system, namely, multiple steady states. Depending on the pyruvate and enzyme levels or the operation mode, either a steady state with high pyruvate decarboxylation rate or a steady state with significantly lower decarboxylation rate can be achieved under otherwise identical conditions. In general, the more efficient steady state is associated with a lower pyruvate concentration. A possible time delay in the substrate supply and enzyme synthesis can also affect the steady state to be achieved and leads to oscillations under certain conditions. Overall, the predictions of multiplicity for the PDH system agree qualitatively well with recent experimental observations in animal cell cultures. The model analysis gives some hints for improving pyruvate metabolism in animal cell culture.

In the course of a study conducted to isolate genes upregulated by plant cell wall sugars, we identified an arabinose-inducible locus from a transcriptional fusion library of Rhizobium leguminosarum VF39, carrying random insertions of the lacZ transposon Tn5B22. Sequence analysis of the locus disrupted by the transposon revealed a high similarity to uncharacterized malate synthase G genes from Sinorhizobium meliloti, Agrobacterium tumefaciens, and Mesorhizobium loti. This enzyme catalyzes the condensation of glyoxylate and acetyl-CoA to yield malate and CoA and is thought to be a component of the glyoxylate cycle, which allows microorganisms to grow on two carbon compounds. Enzyme assays showed that a functional malate synthase is encoded in the glcB gene of R. leguminosarum and that its expression is induced by arabinose, glycolate, and glyoxylate. An Escherichia coli aceB glcB mutant, complemented with the R. leguminosarum PCR-amplified gene, recovered malate synthase activity. A very similar genome organization of the loci containing malate synthase and flanking genes was observed in R. leguminosarum, S. meliloti, and A. tumefaciens. Pea plants inoculated with the glcB mutant or the wild-type strain showed no significant differences in nitrogen fixation. This is the first report regarding the characterization of a mutant in one of the glyoxylate cycle enzymes in the rhizobia.

The PHO5 gene promoter is an important model for the study of gene regulation in the context of chromatin. Upon PHO5 activation the chromatin structure is reconfigured, but the mechanism of this transition remains unclear. Using templates reconstituted into chromatin with purified recombinant yeast core histones, we have investigated the mechanism of chromatin structure reconfiguration on the PHO5 promoter, a prerequisite for transcriptional activation. Footprinting analyses show that intrinsic properties of the promoter DNA are sufficient for translational nucleosome positioning, which approximates that seen in vivo. We have found that both Pho4p and Pho2p can bind their cognate sites on chromatin-assembled templates without the aid of histone-modifying or nucleosome-remodeling factors. However, nucleosome remodeling by these transcriptional activators requires an ATP-dependent activity in a yeast nuclear extract fraction. Finally, transcriptional activation on chromatin templates requires acetyl-CoA in addition to these other activities and cofactors. The addition of acetyl-CoA results in significant core histone acetylation. These findings indicate that transcriptional activation requires Pho4p, Pho2p, nucleosome remodeling, and nucleosome acetylation. Furthermore, we find that DNA binding, nucleosome remodeling, and transcriptional activation are separable steps, facilitating biochemical analysis of the PHO5 regulatory mechanism.

Pantothenate kinase (PanK) is a rate-determining enzyme in coenzyme A (CoA) biosynthesis. The mouse murine pantothenate kinase (Pank1) gene consists of seven introns and eight exons and is located on chromosome 19 (19C2-3). Two biochemically distinct PanK1 protein isoforms, PanK1 alpha and PanK1 beta, are encoded by the Pank1 gene. Both proteins have the same 363 amino acid catalytic domain encoded by exons 2 through 7. The PanK1 beta transcript begins with exon 1 beta and translates into a ten-residue amino terminus plus the catalytic domain. The PanK1 alpha transcript initiates at an alternate upstream site at exon 1 alpha which is spliced with exon 2, excluding exon 1 beta. Exon 1 alpha encodes a 184-residue regulatory domain at the amino terminus of the PanK1 alpha protein that confers feedback inhibition by free CoA and long-chain acyl-CoA, and increases the regulation of PanK enzyme activity by acetyl-CoA and malonyl-CoA. Differential expression of the PanK1 alpha and PanK1 beta transcripts would alter the amount of CoA produced in cells as a function of the ratio of free CoA to acetyl-CoA, a reflection of the metabolic status of the tissue.

The specific activity of chicken liver pyruvate carboxylase has been shown to decrease with decreasing enzyme concentration, even at 100 microM, which is close to the estimated physiological concentration. The kinetics of the loss of enzyme specific activity following dilution were biphasic. Incubation of dilution-inactivated enzyme with ATP, acetyl CoA, Mg2+ + ATP or, to a lesser degree, with Mg2+ alone resulted in a high degree of reactivation, while no reactivation occurred in the presence of pyruvate. The association state of the enzyme before, during, and after dilution inactivation has been assessed by gel filtration chromatography. These studies indicate that on dilution, there is dissociation of the catalytically active tetrameric enzyme species into inactive dimers. Reactivation of the enzyme resulted in reassociation of enzymic dimers into tetramers. The enzyme was shown to form high molecular weight aggregates at high enzyme concentrations., ((c) 2002 Elsevier Science (USA).)

Nerve growth factor (NGF) is a potent growth factor for cholinergic neurons. The aim of the present study was to investigate if NGF affects cholinergic neurons of the basal nucleus of Meynert (nBM) in organotypic brain slices. In single nBM slices cholinergic neurons rapidly degenerated when incubated without NGF. The number of remaining neurons was rescued by NGF application at any time point. When nBM slices were co-cultured with a cortex slice the number of cholinergic neurons was significantly increased pointing to a trophic influence of the cortex. Incubation with acetylcholine precursors did not affect the survival of cholinergic neurons. There was no significant difference when postnatal day 3 or day 10 nBM slices were cultured. In conclusion, NGF is the most potent growth factor for cholinergic neurons and is a promising candidate for treating Alzheimers disease, however, the delivery of NGF to the brain must the solved.

To study the regulation of lipid transport from the chloroplast envelope to the thylakoid, intact chloroplasts, isolated from fully expanded or still-expanding pea (Pisum sativum) leaves, were incubated with radiolabeled lipid precursors and thylakoid membranes subsequently were isolated. Incubation with UDP[(3)H]Gal labeled monogalactosyldiacylglycerol in both envelope membranes and digalactosyldiacylglycerol in the outer chloroplast envelope. Galactolipid synthesis increased with incubation temperature. Transport to the thylakoid was slow below 12 degrees C, and exhibited a temperature dependency closely resembling that for the previously reported appearance and disappearance of vesicles in the stroma (D.J. Morré, G. Selldén, C. Sundqvist, A.S. Sandelius [1991] Plant Physiol 97: 1558-1564). In mature chloroplasts, monogalactosyldiacylglycerol transport to the thylakoid was up to three times higher than digalactosyldiacylglycerol transport, whereas the difference was markedly lower in developing chloroplasts. Incubation of chloroplasts with [(14)C]acyl-coenzyme A labeled phosphatidylcholine (PC) and free fatty acids in the inner envelope membrane and phosphatidylglycerol at the chloroplast surface. PC and phosphatidylglycerol were preferentially transported to the thylakoid. Analysis of lipid composition revealed that the thylakoid contained approximately 20% of the chloroplast PC. Our results demonstrate that lipids synthesized at the chloroplast surface as well as in the inner envelope membrane are transported to the thylakoid and that lipid sorting is involved in the process. Furthermore, the results also indicate that more than one pathway exists for galactolipid transfer from the chloroplast envelope to the thylakoid.

Assays of beta-ketoacyl-acyl carrier protein synthases III (KASIII; FabH), a key enzyme initiating bacterial type II fatty acid biosynthesis, usually involve incubation of radiolabeled acetyl-coenzyme A and malonyl-acyl carrier protein (MACP). The radiolabeled acetoacetyl-ACP product is precipitated and separated from the substrate before quantitation. We have developed a scintillation proximity assay (SPA) where use of biotinylated MACP (BMACP) allows the generation of a biotinylated acetoacetyl-ACP. This product, when captured by the streptavidin-coated scintillant-impregnated microspheres, generates an SPA signal. A BMACP K(m) of 7.1 microM was determined using this SPA with the Streptomyces glaucescens FabH. A similar MACP K(m) (6 microM) was determined in a precipitation assay, demonstrating that BMACP is an effective substrate for FabH. IC(50) values of 15.2 microM (SPA) and 24.8 microM were obtained with iodoacetamide and the S. glaucescens FabH. Comparable IC(50) values of 160 microM (SPA) and 125 microM were also obtained with the antibiotic thiolactomycin and the Escherichia coli FabH. These observations demonstrate that FabH inhibitors can be readily detected using a SPA with BMACP and that the effectiveness of inhibitors in the SPA is comparable to that obtained using MACP and a standard TCA precipitation assay. A FabH SPA adaptable to high-throughput screening should facilitate the discovery of potential novel antibiotics., (Copyright 2000 Academic Press.)

Pyruvate dehydrogenase kinase (PDK) isoforms 2 and 3 were produced via co-expression with the chaperonins GroEL and GroES and purified with high specific activities in affinity tag-free forms. By using human components, we have evaluated how binding to the lipoyl domains of the dihydrolipoyl acetyltransferase (E2) produces the predominant changes in the rates of phosphorylation of the pyruvate dehydrogenase (E1) component by PDK2 and PDK3. E2 assembles as a 60-mer via its C-terminal domain and has mobile connections to an E1-binding domain and then two lipoyl domains, L2 and L1 at the N terminus. PDK3 was activated 17-fold by E2; the majority of this activation was facilitated by the free L2 domain (half-maximal activation at 3.3 microm L2). The direct activation of PDK3 by the L2 domain resulted in a 12.8-fold increase in k(cat) along with about a 2-fold decrease in the K(m) of PDK3 for E1. PDK3 was poorly inhibited by pyruvate or dichloroacetate (DCA). PDK3 activity was stimulated upon reductive acetylation of L1 and L2 when full activation of PDK3 by E2 was avoided (e.g. using free lipoyl domains or ADP-inhibited E2-activated PDK3). In marked contrast, PDK2 was not responsive to free lipoyl domains, but the E2-60-mer enhanced PDK2 activity by 10-fold. E2 activation of PDK2 resulted in a greatly enhanced sensitivity to inhibition by pyruvate or DCA; pyruvate was effective at significantly lower levels than DCA. E2-activated PDK2 activity was stimulated >/=3-fold by reductive acetylation of E2; stimulated PDK2 retained high sensitivity to inhibition by ADP and DCA. Thus, PDK3 is directly activated by the L2 domain, and fully activated PDK3 is relatively insensitive to feed-forward (pyruvate) and feed-back (acetylating) effectors. PDK2 was activated only by assembled E2, and this activated state beget high responsiveness to those effectors.

The present study examined the sensitivity of carnitine palmitoyltransferase I (CPT I) activity to its inhibitor malonyl-CoA (M-CoA), and simulated metabolic conditions of rest and exercise, in aerobically trained and untrained humans. Maximal CPT I activity was measured in mitochondria isolated from resting human skeletal muscle. Mean CPT I activity was 492.8 +/- 72.8 and 260.8 +/- 33.6 micromol. min(-1). kg wet muscle(-1) in trained and untrained subjects, respectively (pH 7.0, 37 degrees C). The sensitivity to M-CoA was greater in trained muscle; the IC(50) for M-CoA was 0.17 +/- 0.04 and 0.49 +/- 0.17 microM in trained and untrained muscle, respectively. The presence of acetyl-CoA, free coenzyme A (CoASH), and acetylcarnitine, in concentrations simulating rest and exercise conditions did not release the M-CoA-induced inhibition of CPT I activity. However, CPT I activity was reduced at pH 6.8 vs. pH 7.0 in both trained and untrained muscle in the presence of physiological concentrations of M-CoA. The results of this study indicate that aerobic training is associated with an increase in the sensitivity of CPT I to M-CoA. Accumulations of acetyl-CoA, CoASH, and acetylcarnitine do not counteract the M-CoA-induced inhibition of CPT I activity. However, small decreases in pH produce large reductions in the activity of CPT I and may contribute to the decrease in fat metabolism that occurs during moderate and intense aerobic exercise intensities.

Serotonin N-acetyltransferase (AANAT) is the first enzyme in the conversion of serotonin to melatonin. Changes in AANAT activity determine the daily rhythm in melatonin secretion. Two AANAT genes have been identified in the pike, pAANAT-1 and pAANAT-2, expressed in the retina and in the pineal, respectively. The genes preferentially expressed in these tissues encode proteins with distinctly different kinetic characteristics. Like the pike, trout retina primarily expresses the AANAT-1 gene and trout pineal primarily expresses the AANAT-2 gene. Here we show that the kinetic characteristics of AANAT in these tissues differ as in pike. These differences include optimal temperature for activity (pineal: 12 degrees C; retina: 25 degrees C) and relative affinity for indoleethylamines compared to phenylethylamines. In addition, retinal AANAT exhibited substrate inhibition, which was not seen with pineal AANAT. The kinetic differences between AANAT-1 and AANAT-2 appear to be defining characteristics of these gene subfamilies, and are not species specific.

The Gram-negative bacterium Rhizobium meliloti contains two distinct malic enzymes. We report the purification of the two isozymes to homogeneity, and their in vitro characterization. Both enzymes exhibit unusually high subunit molecular weights of about 82 kDa. The NAD(P)(+) specific malic enzyme [EC 1.1.1.39] exhibits positive co-operativity with respect to malate, but Michaelis-Menten type behavior with respect to the co-factors NAD(+) or NADP(+). The enzyme is subject to substrate inhibition, and shows allosteric regulation by acetyl-CoA, an effect that has so far only been described for some NADP(+) dependent malic enzymes. Its activity is positively regulated by succinate and fumarate. In contrast to the NAD(P)(+) specific malic enzyme, the NADP(+) dependent malic enzyme [EC 1.1.1.40] shows Michaelis-Menten type behavior with respect to malate and NADP(+). Apart from product inhibition, the enzyme is not subjected to any regulatory mechanism. Neither reductive carboxylation of pyruvate, nor decarboxylation of oxaloacetate, could be detected for either malic enzyme. Our characterization of the two R. meliloti malic enzymes therefore suggests a number of features uncharacteristic for malic enzymes described so far.

Isolated human mitochondrial trifunctional protein was incubated with 2-hexadecenoyl-CoA, CoA and NAD+ and the resultant CoA esters measured. Steady state with respect to the concentrations of the intermediates 3-hydroxyhexadecanoyl-CoA and 3-ketohexadecanoyl-CoA and the rate of formation of the product tetradecanoyl-CoA was reached within 4 min. Flux was greatly enhanced by the addition of Tween 20 (0.2% v/v) which stimulated 3-ketoacyl-CoA thiolase activity by over 7-fold. When 3-ketoacyl-CoA thiolase was not stimulated, 3-hydroxyhexadecanoyl-CoA was the prominent CoA ester accumulated, presumably due to inhibition of 3-hydroxyacyl-CoA dehydrogenase activity by accumulated 3-ketoacyl-CoA, analogous to the inhibition of short-chain 3-hydroxyacyl-CoA dehydrogenase by 3-ketoacyl-CoA. When [NAD+]/[NADH] was varied at a fixed total [NAD++NADH], the overall flux was only inhibited by [NAD+]/[NADH] less than 1. In contrast, when [acetyl-CoA]/[CoA] was varied at a fixed total [CoA], much greater sensitivity was observed.

A novel hexyl-substituted methylenecyclopropyl acetyl-CoA was tested as an enzyme-specific acyl-CoA dehydrogenase inhibitor. Its CoA ester generated in situ from the carboxylic acid and CoASH, displayed marked differences in inhibition specificity as compared to methylenecyclopropyl acetyl-CoA, consistent with the substrate specificities of the target enzymes. Thus methylenecyclopropyl acetyl-CoA inactivated short-chain-specific acyl-CoA dehydrogenase rapidly, medium-chain-specific acyl-CoA dehydrogenase much more slowly and had no effect on long-chain- or very long-chain-specific acyl-CoA dehydrogenases. The hexyl-substituent on the methylenecyclopropyl ring gave an inhibitor which rapidly inactivated MCAD and LCAD whilst VLCAD was inhibited more slowly and SCAD was essentially unaffected. In some cases (e.g. SCAD and MCPA-CoA) inhibition was accompanied by flavin bleaching. In other cases (e.g. LCAD and C6MCPA) less pronounced bleaching suggests a different chemistry of inhibition.

Pigeon liver fatty acid synthetase (FAS) was rapidly inactivated by pyridoxal 5'-phosphate (PLP). Assays of the partial activities of the PLP-treated synthetase showed that only the enoyl-CoA reductase was decreased significantly. The inactivation of both the overall activity and enoyl-CoA reductase activity of FAS by PLP could be reversed by dialysis or dilution but not by reduction with sodium borohydride. Malonyl-CoA and acetyl-CoA did not protect the enzyme, whereas NADPH provided 68% protection against PLP-inactivation indicating that PLP modified lysine residues present at or near the co-enzyme binding site. PLP-treated enzyme after reduction with sodium borohydride exhibited fluorescence with a maximum at 397 nm (irradiation at 325 nm). Stoichiometric analysis showed that modification of four lysine residues per enzyme molecule resulted in complete inactivation of the overall and enoyl-CoA reductase activities of FAS. NADPH prevented the inactivation by protecting two of these lysine residues from modification, suggesting the presence of two essential lysine residues per enzyme molecule. These results are consistent with the hypothesis that each subunit of the enzyme contains an enoyl-CoA reductase domain in which a lysine residue, at or near the active site, interacts with NADPH.

Tissue distribution and kinetic parameters for the four isoenzymes of pyruvate dehydrogenase kinase (PDK1, PDK2, PDK3 and PDK4) identified thus far in mammals were analysed. It appeared that expression of these isoenzymes occurs in a tissue-specific manner. The mRNA for isoenzyme PDK1 was found almost exclusively in rat heart. The mRNA for PDK3 was most abundantly expressed in rat testis. The message for PDK2 was present in all tissues tested but the level was low in spleen and lung. The mRNA for PDK4 was predominantly expressed in skeletal muscle and heart. The specific activities of the isoenzymes varied 25-fold, from 50nmol/min per mg for PDK2 to 1250nmol/min per mg for PDK3. Apparent Ki values of the isoenzymes for the synthetic analogue of pyruvate, dichloroacetate, varied 40-fold, from 0.2 mM for PDK2 to 8 mM for PDK3. The isoenzymes were also different with respect to their ability to respond to NADH and NADH plus acetyl-CoA. NADH alone stimulated the activities of PDK1 and PDK2 by 20 and 30% respectively. NADH plus acetyl-CoA activated these isoenzymes nearly 200 and 300%. Under comparable conditions, isoenzyme PDK3 was almost completely unresponsive to NADH, and NADH plus acetyl-CoA caused inhibition rather than activation. Isoenzyme PDK4 was activated almost 2-fold by NADH, but NADH plus acetyl-CoA did not activate above the level seen with NADH alone. These results provide the first evidence that the unique tissue distribution and kinetic characteristics of the isoenzymes of PDK are among the major factors responsible for tissue-specific regulation of the pyruvate dehydrogenase complex activity.

The human immature K562 erythroid cell line was studied for its capacity to produce and to metabolize the phospholipid molecule platelet-activating factor (PAF). K562 cells produced PAF under calcium ionophore stimulation. Lyso PAF and acetyl-CoA (the acetate donor molecule for the acetylation of lyso PAF into PAF) had no effect on the amounts of PAF produced by ionophore-stimulated cells. The metabolism of PAF and lyso PAF by K562 cells was compared to that of freshly-isolated human bone marrow erythroblasts and blood erythrocytes. K562 cells rapidly metabolized [3H]PAF and [3H]lyso PAF with 1-alkyl analogue of phosphatidylcholine as the major metabolic product. In contrast, blood erythrocytes did not. PAF acetylhydrolase activity levels in K562 cells and bone marrow erythroblasts were similar and higher than in blood erythrocytes. PAF (1-100 nM) stimulated [3H]thymidine incorporation in K562 cells grown in low serum concentration, a non-metabolizable PAF agonist being more potent than PAF to stimulate thymidine incorporation. PAF receptor mRNA was detected in K562 cells by polymerase chain reaction on reverse transcripts. The present study demonstrates that K562 cells produce and metabolize PAF and underlines the putative role of erythroid precursors in the modulation of bone marrow PAF concentrations. The effect of PAF on the growth of K562 cells might be mediated through PAF receptors suggesting a potential role of PAF on the proliferation and functions of human erythroid marrow precursors.

Glucose, the most potent insulin secretagogue, stimulates insulin secretion by aerobic glycolysis, but other secretagogues stimulate insulin release exclusively by mitochondrial metabolism. It is well known that in the intact pancreatic beta-cell, either kind of secretagogue can induce oscillations in metabolism (e.g., glycolysis, ATP/ADP, NAD(P)/NAD(P)H ratios) that occur with a periodicity similar to oscillations in membrane electrical potential and insulin secretion. In this study, pancreatic islet cytosol or mitochondrial fractions were incubated in the presence of physiological concentrations of substrates. Repeated additions of physiological effectors caused oscillations in the activities of the three enzymes studied. Succinate dehydrogenase activity in islet mitochondrial extracts was made to oscillate by adding oxaloacetate (5 micromol/l) to inhibit the enzyme. The enzyme was reactivated by adding acetyl-CoA (3 micromol/l), which combines with oxaloacetate in the citrate synthase reaction and lowers the concentration of oxaloacetate, thus beginning another oscillation. Pyruvate kinase activity was made to oscillate by adding fructose bisphosphate (10 micromol/l). Fructose bisphosphate was degraded to triose phosphates fairly rapidly, and, as it was degraded, there was a parallel decrease in pyruvate kinase activity. The enzyme was reactivated and made to oscillate with subsequent additions of fructose bisphosphate. The mitochondrial glycerol phosphate dehydrogenase was made to oscillate by adding EGTA to chelate calcium, which activates the enzyme. When the concentration of free calcium was raised to >0.1 micromol/l by adding more calcium, the activity of the enzyme increased. Repeated additions of chelator and calcium caused the enzyme activity to oscillate. The results with these three enzymes and physiological concentrations of naturally occurring effectors raise the possibility that the activities of not only these enzymes but of numerous enzymes oscillate in vivo in response to levels of allosteric effectors and substrates. If this is the case, pacemaker activity may result from complex effects distributed across multiple regulatory sites in both the cytosol and mitochondria, rather than from a single enzyme acting as a primary pacemaker.

Phosphoenolpyruvate carboxylase (PEPC) was purified from an extremely thermophilic bacterium, Rhodothermus obamensis, growing optimally at 80 degrees C, which had recently been isolated from a shallow marine hydrothermal vent in Japan. The native enzyme was a homotetramer of 400 kDa in molecular mass, as estimated by gel filtration chromatography, and the subunit exhibited an apparent molecular mass of 100 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum temperature for enzyme activity was 75 degrees C. The enzyme exhibited an absolute requirement for divalent cations and a pH optimum of 8.0. The enzyme was extremely thermostable and there was no loss of enzyme activity on incubation for 2 h at 85 degrees C. The enzyme exhibited a positive allosteric property with acetyl-CoA and fructose 1,6-bisphosphate, and a negative one with L-aspartate and L-malate. These effectors affected not only the thermophilicity but also the thermostability of the enzyme, and the substrate, co-factors, and salts increased the thermostability as well. The extrinsic thermostabilization might be a possible mechanism for adaptation of the enzyme to high temperature.

The wild-type NAD(+)-dependent malic enzyme (dme) gene of Rhizobium (now Sinorhizobium) meliloti was cloned and localized to a 3.1 kb region isolated on the cosmid pTH69. This cosmid complemented the symbiotic nitrogen fixation (Fix-) phenotype of R. meliloti dme mutants. The dme gene was mapped by conjugation to between the cys-11 and leu-53 markers on the R. meliloti chromosome. beta-Galactosidase activities measured in bacterial strains carrying either dme-lacZ or tme-lacZ gene fusions (the tme gene encodes NADP(+)-dependent malic enzyme) indicated that the dme gene was expressed constitutively in free-living cells and in N2-fixing bacteroids whereas expression of the tme gene was repressed in bacteroids. The R. meliloti dme gene product (DME) was overexpressed in and partially purified from Escherichia coli. The properties of this enzyme, together with those of the NADP(+)-dependent malic enzyme (TME) partially purified from R. meliloti dme mutants, were determined. Acetyl-CoA inhibited DME but not TME activity. This result supports the hypothesis that DME, together with pyruvate dehydrogenase, forms a pathway in which malate is converted to acetyl-CoA.