Dataset Information

The Acetyl Group Buffering Action of Carnitine Acetyltransferase Offsets Macronutrient-Induced Lysine Acetylation of Mitochondrial Proteins.

ABSTRACT: Lysine acetylation (AcK), a posttranslational modification wherein a two-carbon acetyl group binds covalently to a lysine residue, occurs prominently on mitochondrial proteins and has been linked to metabolic dysfunction. An emergent theory suggests mitochondrial AcK occurs via mass action rather than targeted catalysis. To test this hypothesis, we performed mass spectrometry-based acetylproteomic analyses of quadriceps muscles from mice with skeletal muscle-specific deficiency of carnitine acetyltransferase (CrAT), an enzyme that buffers the mitochondrial acetyl-CoA pool by converting short-chain acyl-CoAs to their membrane permeant acylcarnitine counterparts. CrAT deficiency increased tissue acetyl-CoA levels and susceptibility to diet-induced AcK of broad-ranging mitochondrial proteins, coincident with diminished whole body glucose control. Sub-compartment acetylproteome analyses of muscles from obese mice and humans showed remarkable overrepresentation of mitochondrial matrix proteins. These findings reveal roles for CrAT and L-carnitine in modulating the muscle acetylproteome and provide strong experimental evidence favoring the nonenzymatic carbon pressure model of mitochondrial AcK.

SUBMITTER: Davies MN

PROVIDER: S-EPMC4754083 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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The Acetyl Group Buffering Action of Carnitine Acetyltransferase Offsets Macronutrient-Induced Lysine Acetylation of Mitochondrial Proteins.

Davies Michael N MN Kjalarsdottir Lilja L Thompson J Will JW Dubois Laura G LG Stevens Robert D RD Ilkayeva Olga R OR Brosnan M Julia MJ Rolph Timothy P TP Grimsrud Paul A PA Muoio Deborah M DM

Cell reports 20151231 2

Lysine acetylation (AcK), a posttranslational modification wherein a two-carbon acetyl group binds covalently to a lysine residue, occurs prominently on mitochondrial proteins and has been linked to metabolic dysfunction. An emergent theory suggests mitochondrial AcK occurs via mass action rather than targeted catalysis. To test this hypothesis, we performed mass spectrometry-based acetylproteomic analyses of quadriceps muscles from mice with skeletal muscle-specific deficiency of carnitine acet ...[more]

PMID: 26748706

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Project description:Soil is a complex niche, where survival of microorganisms is at risk due to the presence of antimicrobial agents. Many microbes chemically modify cytotoxic compounds to block their deleterious effects. Streptothricin is a broad-spectrum antibiotic produced by streptomycetes that affects Gram-positive and Gram-negative bacteria alike. Here we identify the SatA (for streptothricin acetyltransferase A, formerly YyaR) enzyme of Bacillus subtilis as the mechanism used by this soil bacterium to detoxify streptothricin. B. subtilis strains lacking satA were susceptible to streptothricin. Ectopic expression of satA+ restored streptothricin resistance to B. subtilissatA (BsSatA) strains. Purified BsSatA acetylated streptothricin in vitro at the expense of acetyl-coenzyme A (acetyl-CoA). A single acetyl moiety transferred onto streptothricin by SatA blocked the toxic effects of the antibiotic. SatA bound streptothricin with high affinity (Kd [dissociation constant] = 1 μM), and did not bind acetyl-CoA in the absence of streptothricin. Expression of B. subtilissatA+ in Salmonella enterica conferred streptothricin resistance, indicating that SatA was necessary and sufficient to detoxify streptothricin. Using this heterologous system, we showed that the SatA homologue from Bacillus anthracis also had streptothricin acetyltransferase activity. Our data highlight the physiological relevance of lysine acetylation for the survival of B. subtilis in the soil.IMPORTANCE Experimental support is provided for the functional assignment of gene products of the soil-dwelling bacilli Bacillus subtilis and Bacillus anthracis This study focuses on one enzyme that is necessary and sufficient to block the cytotoxic effects of a common soil antibiotic. The enzyme alluded to is a member of a family of proteins that are broadly distributed in all domains of life but poorly studied in B. subtilis and B. anthracis The initial characterization of the enzyme provides insights into its mechanism of catalysis.

Project description:1. The nature of the acetyl-CoA hydrolase (EC 3.1.2.1) reaction in rat and sheep liver homogenates was investigated. 2. The activity determined in an incubated system was 5.10 and 3.28nmol/min per mg of protein for rat and sheep liver homogenate respectively. This activity was not affected by the addition of l-carnitine, but was decreased by the addition of d-carnitine. 3. No acetyl-CoA hydrolase activity could be detected in rat or sheep liver homogenates first treated with Sephadex G-25. This treatment decreased the carnitine concentrations of the homogenates to about one-twentieth. Subsequent addition of l-carnitine, but not d-carnitine, restored the apparent acetyl-CoA hydrolase activity. 4. Sephadex treatment did not affect acetyl-carnitine hydrolase activity of the homogenates, which was 5.8 and 8.1nmol/min per mg of protein respectively for rat and sheep liver. 5. Direct spectrophotometric assay of acetyl-CoA hydrolase, based on the reaction of CoA released with 5,5'-dithiobis-(2-nitrobenzoic acid), clearly demonstrated that after Sephadex treatment no activity could be measured. 6. Carnitine acetyltransferase (EC 2.3.1.7) activity measured in the same assay system in response to added l-carnitine was very low in normal rat liver homogenates, owing to the apparent high acetyl-CoA hydrolase activity, but was increased markedly after Sephadex treatment. The V(max.) for this enzyme in rat liver homogenates was increased from 3.4 to 14.8nmol/min per mg of protein whereas the K(m) for l-carnitine was decreased from 936 to 32mum after Sephadex treatment. 7. Acetyl-CoA hydrolase activity could be demonstrated in disrupted rat liver mitochondria but not in separated outer or inner mitochondrial membrane fractions. Activity could be demonstrated after recombination of outer and inner mitochondrial membrane fractions. The outer mitochondrial membrane fraction showed acetylcarnitine hydrolase activity and the inner mitochondrial membrane fraction showed carnitine acetyltransferase activity. 8. The results presented here demonstrate that acetyl-CoA hydrolase activity in rat and sheep liver is an artifact and the activity is due to the combined activity of carnitine acetyltransferase and acetylcarnitine hydrolase.

Dataset Information

The Acetyl Group Buffering Action of Carnitine Acetyltransferase Offsets Macronutrient-Induced Lysine Acetylation of Mitochondrial Proteins.

Publications

The Acetyl Group Buffering Action of Carnitine Acetyltransferase Offsets Macronutrient-Induced Lysine Acetylation of Mitochondrial Proteins.

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