Identification of the CoA-modified forms of mitochondrial acetyl-CoA acetyltransferase and of glutamate dehydrogenase as nearest-neighbour proteins.
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ABSTRACT:
SUBMITTER: Schwerdt G
PROVIDER: S-EPMC1130553 | biostudies-other | 1991 Dec
REPOSITORIES: biostudies-other
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Project description:Rat liver mitochondrial acetyl-CoA acetyltransferase (acetoacetyl-CoA thiolase, EC 2.3.1.9) exists additionally in the CoA-modified forms A1 and A2. After a pulse of radioactivity using [35S]methionine in hepatocytes, the highest radioactivity was obtained in the unmodified enzyme. Over the chase time, the radioactivity in the unmodified enzyme decreased, but simultaneously increased in both CoA-modified forms, thus proving that the fully active unmodified enzyme exists before the partially active modified forms A1 and A2. Also, the specific radioactivity (ratio % radioactivity/% immunoreactive area) of A1 > A2 demonstrates a sequential CoA modification of form A1 to form A2. Acetyl-CoA acetyltransferase was degraded with an apparent half-life of 38.0 h: the modified forms A1 and A2 have half-lives of 24.5 and 7.2 h. The physiological meaning of the CoA modification of acetyl-CoA acetyltransferase is not yet understood.
| S-EPMC1134201 | biostudies-other
Project description:Acetyl-CoA:L-glutamate N-acetyltransferase (amino acid acetyltransferase, EC 2.3.1.1) was isolated from human liver mitochondria by precipitation with (NH4)2SO4 and chromatography on hydroxyapatite, DEAE-cellulose and Sephacryl 300. This gave a 360-fold purification. The molecular weight was estimated to be approx. 190 000. The kinetic properties in the absence of arginine are compatible with a rapid-equilibrium random Bi Bi mechanism. The estimated constants are: for the substrates Km,acetyl-CoA 4.4 mM, Ki,acetyl-CoA 4.7 mM, Km,glutamate 8.1 mM, Ki,glutamate 8.8 mM; for the products, Ki,acetylglutamate 0.28 mM, Ki,CoA 5.6 mM. The rate constant for the forward direction is 1.24s-1. If in vivo the constants are of the same order of magnitude as in vitro, the synthesis of N-acetylglutamate, an obligate activator of the first step of urea synthesis, can be expected to occur in the mitochondrion under conditions where the amino acid acetyltransferase is not saturated by its substrates. The regulation of the first step of urea synthesis could thus depend mainly on the intramitochondrial substrate and perhaps product concentrations of amino acid acetyltransferase.
| S-EPMC1158454 | biostudies-other
Project description:Mitochondrial acetyl-CoA acetyltransferase 1 (ACAT1) regulates pyruvate dehydrogenase complex (PDC) by acetylating pyruvate dehydrogenase (PDH) and PDH phosphatase. How ACAT1 is "hijacked" to contribute to the Warburg effect in human cancer remains unclear. We found that active, tetrameric ACAT1 is commonly upregulated in cells stimulated by EGF and in diverse human cancer cells, where ACAT1 tetramers, but not monomers, are phosphorylated and stabilized by enhanced Y407 phosphorylation. Moreover, we identified arecoline hydrobromide (AH) as a covalent ACAT1 inhibitor that binds to and disrupts only ACAT1 tetramers. The resultant AH-bound ACAT1 monomers cannot reform tetramers. Inhibition of tetrameric ACAT1 by abolishing Y407 phosphorylation or AH treatment results in decreased ACAT1 activity, leading to increased PDC flux and oxidative phosphorylation with attenuated cancer cell proliferation and tumor growth. These findings provide a mechanistic understanding of how oncogenic events signal through distinct acetyltransferases to regulate cancer metabolism and suggest ACAT1 as an anti-cancer target.
| S-EPMC5135630 | biostudies-literature
Project description:BackgroundEpigenetic regulation relies on the activity of enzymes that use sentinel metabolites as cofactors to modify DNA or histone proteins. Thus, fluctuations in cellular metabolite levels have been reported to affect chromatin modifications. However, whether epigenetic modifiers also affect the levels of these metabolites and thereby impinge on downstream metabolic pathways remains largely unknown. Here, we tested this notion by investigating the function of N-alpha-acetyltransferase 40 (NAA40), the enzyme responsible for N-terminal acetylation of histones H2A and H4, which has been previously implicated with metabolic-associated conditions such as age-dependent hepatic steatosis and calorie-restriction-mediated longevity.ResultsUsing metabolomic and lipidomic approaches, we found that depletion of NAA40 in murine hepatocytes leads to significant increase in intracellular acetyl-CoA levels, which associates with enhanced lipid synthesis demonstrated by upregulation in de novo lipogenesis genes as well as increased levels of diglycerides and triglycerides. Consistently, the increase in these lipid species coincide with the accumulation of cytoplasmic lipid droplets and impaired insulin signalling indicated by decreased glucose uptake. However, the effect of NAA40 on lipid droplet formation is independent of insulin. In addition, the induction in lipid synthesis is replicated in vivo in the Drosophila melanogaster larval fat body. Finally, supporting our results, we find a strong association of NAA40 expression with insulin sensitivity in obese patients.ConclusionsOverall, our findings demonstrate that NAA40 affects the levels of cellular acetyl-CoA, thereby impacting lipid synthesis and insulin signalling. This study reveals a novel path through which histone-modifying enzymes influence cellular metabolism with potential implications in metabolic disorders.
| S-EPMC8781613 | biostudies-literature
Project description:Acetylation of lysine residues is an important posttranslational modification found in all domains of life. ?-Tubulin is specifically acetylated on lysine 40, a modification that serves to stabilize microtubules of axons and cilia. Whereas histone acetyltransferases have been extensively studied, there is no structural and mechanistic information available on ?-tubulin acetyltransferases. Here, we present the structure of the human ?-tubulin acetyltransferase catalytic domain bound to its cosubstrate acetyl-CoA at 1.05 Å resolution. Compared with other lysine acetyltransferases of known structure, ?-tubulin acetyltransferase displays a relatively well-conserved cosubstrate binding pocket but is unique in its active site and putative ?-tubulin binding site. Using acetylation assays with structure-guided mutants, we map residues important for acetyl-CoA binding, substrate binding, and catalysis. This analysis reveals a basic patch implicated in substrate binding and a conserved glutamine residue required for catalysis, demonstrating that the family of ?-tubulin acetyltransferases uses a reaction mechanism different from other lysine acetyltransferases characterized to date.
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Project description:We used stopped-flow to monitor hypochromicity for 43 oligonucleotide duplexes to study nucleic acid kinetics and extract transition-state parameters for association and dissociation. Reactions were performed in 1.0 M NaCl (for literature comparisons) and 2.2 mM MgCl2 (PCR conditions). Dissociation kinetics depended on sequence, increased exponentially with temperature, and transition-state parameters inversely correlated to thermodynamic parameters (r = -0.99). Association had no consistent enthalpic component, varied little with temperature or sequence, and poorly correlated to thermodynamic parameters (r = 0.28). Average association rates decreased 78% in MgCl2 compared to NaCl while dissociation was relatively insensitive to ionic conditions. A nearest-neighbour kinetic model for dissociation predicted rate constants within 3-fold of literature values (n = 11). However, a nearest-neighbour model for association appeared overparameterized and inadequate for predictions. Kinetic predictions were used to simulate published high-speed (<1 min) melting analysis and extreme (<2 min) PCR experiments. Melting simulations predicted apparent melting temperatures increase on average 2.4°C when temperature ramp rates increased from 0.1 to 32°C/s, compared to 2.8°C reported in the literature. PCR simulations revealed that denaturation kinetics are dependent on the thermocycling profile. Simulations overestimated annealing efficiencies at shorter annealing times and suggested that polymerase interactions contribute to primer-template complex stability at extension temperatures.
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Project description:Researchers hoping to elucidate the behaviour of species that aren't readily observed are able to do so using biotelemetry methods. Accelerometers in particular are proving particularly effective and have been used on terrestrial, aquatic and volant species with success. In the past, behavioural modes were detected in accelerometer data through manual inspection, but with developments in technology, modern accelerometers now record at frequencies that make this impractical. In light of this, some researchers have suggested the use of various machine learning approaches as a means to classify accelerometer data automatically. We feel uptake of this approach by the scientific community is inhibited for two reasons; 1) Most machine learning algorithms require selection of summary statistics which obscure the decision mechanisms by which classifications are arrived, and 2) they are difficult to implement without appreciable computational skill. We present a method which allows researchers to classify accelerometer data into behavioural classes automatically using a primitive machine learning algorithm, k-nearest neighbour (KNN). Raw acceleration data may be used in KNN without selection of summary statistics, and it is easily implemented using the freeware program R. The method is evaluated by detecting 5 behavioural modes in 8 species, with examples of quadrupedal, bipedal and volant species. Accuracy and Precision were found to be comparable with other, more complex methods. In order to assist in the application of this method, the script required to run KNN analysis in R is provided. We envisage that the KNN method may be coupled with methods for investigating animal position, such as GPS telemetry or dead-reckoning, in order to implement an integrated approach to movement ecology research.
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Project description:Calorie restriction (CR), an age delaying diet, affects fat oxidation through poorly understood mechanisms. We investigated the effect of CR on fat metabolism gene expression and intermediate metabolites of fatty acid oxidation in the liver. We found that CR changed the liver acylcarnitine profile: acetylcarnitine, short-chain acylcarnitines, and long-chain 3-hydroxy-acylcarnitines increased, and several long-chain acylcarnitines decreased. Acetyl-CoA and short-chain acyl-CoAs were also increased in CR. CR did not affect the expression of CPT1 and upregulated the expression of long-chain and very-long-chain Acyl-CoA dehydrogenases (LCAD and VLCAD, respectively). The expression of downstream enzymes such as mitochondrial trifunctional protein and enzymes in medium- and short-chain acyl-CoAs oxidation was not affected in CR. CR shifted the balance of fatty acid oxidation enzymes and fatty acid metabolites in the liver. Acetyl-CoA generated through beta-oxidation can be used for ketogenesis or energy production. In agreement, blood ketone bodies increased under CR in a time of the day-dependent manner. Carnitine acetyltransferase (CrAT) is a bidirectional enzyme that interconverts short-chain acyl-CoAs and their corresponding acylcarnitines. CrAT expression was induced in CR liver supporting the increased acetylcarnitine and short-chain acylcarnitine production. Acetylcarnitine can freely travel between cellular sub-compartments. Supporting this CR increased protein acetylation in the mitochondria, cytoplasm, and nucleus. We hypothesize that changes in acyl-CoA and acylcarnitine levels help to control energy metabolism and contribute to metabolic flexibility under CR.
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Project description:Since its proposal by Anderson, resonating valence bonds (RVB) formed by a superposition of fluctuating singlet pairs have been a paradigmatic concept in understanding quantum spin liquids. Here, we show that excitations related to singlet breaking on nearest-neighbour bonds describe the high-energy part of the excitation spectrum in YbMgGaO4, the effective spin-1/2 frustrated antiferromagnet on the triangular lattice, as originally considered by Anderson. By a thorough single-crystal inelastic neutron scattering study, we demonstrate that nearest-neighbour RVB excitations account for the bulk of the spectral weight above 0.5?meV. This renders YbMgGaO4 the first experimental system where putative RVB correlations restricted to nearest neighbours are observed, and poses a fundamental question of how complex interactions on the triangular lattice conspire to form this unique many-body state.
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Project description:Insect stage trypanosomes use an "acetate shuttle" to transfer mitochondrial acetyl-CoA to the cytosol for the essential fatty acid biosynthesis. The mitochondrial acetate sources are acetate:succinate CoA-transferase (ASCT) and an unknown enzymatic activity. We have identified a gene encoding acetyl-CoA thioesterase (ACH) activity, which is shown to be the second acetate source. First, RNAi-mediated repression of ASCT in the ACH null background abolishes acetate production from glucose, as opposed to both single ASCT and ACH mutants. Second, incorporation of radiolabeled glucose into fatty acids is also abolished in this ACH/ASCT double mutant. ASCT is involved in ATP production, whereas ACH is not, because the ASCT null mutant is ∼1000 times more sensitive to oligomycin, a specific inhibitor of the mitochondrial F(0)/F(1)-ATP synthase, than wild-type cells or the ACH null mutant. This was confirmed by RNAi repression of the F(0)/F(1)-ATP synthase F(1)β subunit, which is lethal when performed in the ASCT null background but not in the wild-type cells or the ACH null background. We concluded that acetate is produced from both ASCT and ACH; however, only ASCT is responsible, together with the F(0)/F(1)-ATP synthase, for ATP production in the mitochondrion.
| S-EPMC3366814 | biostudies-literature