Immunoprecipitation of Acetyl-lysine And Western Blotting of Long-chain acyl-CoA Dehydrogenases and Beta-hydroxyacyl-CoA Dehydrogenase in Palmitic Acid Treated Human Renal Tubular Epithelial Cells.
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ABSTRACT:
SUBMITTER: Lv T
PROVIDER: S-EPMC7842828 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The acyl-CoA dehydrogenases (ACADs) are enzymes that catalyze the alpha,beta-dehydrogenation of acyl-CoA esters in fatty acid and amino acid catabolism. Eleven ACADs are now recognized in the sequenced human genome, and several homologs have been reported from bacteria, fungi, plants, and nematodes. We performed a systematic comparative genomic study, integrating homology searches with methods of phylogenetic reconstruction, to investigate the evolutionary history of this family. Sequence analyses indicate origin of the family in the common ancestor of Archaea, Bacteria, and Eukaryota, illustrating its essential role in the metabolism of early life. At least three ACADs were already present at that time: ancestral glutaryl-CoA dehydrogenase (GCD), isovaleryl-CoA dehydrogenase (IVD), and ACAD10/11. Two gene duplications were unique to the eukaryotic domain: one resulted in the VLCAD and ACAD9 paralogs and another in the ACAD10 and ACAD11 paralogs. The overall patchy distribution of specific ACADs across the tree of life is the result of dynamic evolution that includes numerous rounds of gene duplication and secondary losses, interdomain lateral gene transfer events, alteration of cellular localization, and evolution of novel proteins by domain acquisition. Our finding that eukaryotic ACAD species are more closely related to bacterial ACADs is consistent with endosymbiotic origin of ACADs in eukaryotes and further supported by the localization of all nine previously studied ACADs in mitochondria.
| S-EPMC4136416 | biostudies-literature
Project description:Long-chain fatty acids are an important source of energy in muscle and heart where the acyl-CoA dehydrogenases (ACADs) participate in consecutive cycles of ?-oxidation to generate acetyl-CoA and reducing equivalents for generating energy. However, the role of long-chain fatty acid oxidation in the brain and other tissues that do not rely on fat for energy is poorly understood. Here we characterize two new ACADs, ACAD10 and ACAD11, both with significant expression in human brain. ACAD11 utilizes substrates with primary carbon chain lengths between 20 and 26, with optimal activity towards C22CoA. The combination of ACAD11 with the newly characterized ACAD9 accommodates the full spectrum of long chain fatty acid substrates presented to mitochondrial ?-oxidation in human cerebellum. ACAD10 has significant activity towards the branched-chain substrates R and S, 2 methyl-C15-CoA and is highly expressed in fetal but not adult brain. This pattern of expression is similar to that of LCAD, another ACAD previously shown to be involved in long branched chain fatty acid metabolism. Interestingly, the ACADs in human cerebellum were found to have restricted cellular distribution. ACAD9 was most highly expressed in the granular layer, ACAD11 in the white matter, and MCAD in the molecular layer and axons of specific neurons. This compartmentalization of ACADs in the human central nerve system suggests that ?-oxidation in cerebellum participates in different functions other than generating energy, for example, the synthesis and/or degradation of unique cellular lipids and catabolism of aromatic amino acids, compounds that are vital to neuronal function.
| S-EPMC3073726 | biostudies-literature
Project description:Acyl-CoA dehydrogenases (ACADs), which are key enzymes in fatty acid and amino acid catabolism, form a large, pan-taxonomic protein family with at least 13 distinct subfamilies. Yet most reported ACAD members have no subfamily assigned, and little is known about the taxonomic distribution and evolution of the subfamilies. In completely sequenced genomes from approximately 210 species (eukaryotes, bacteria and archaea), we detect ACAD subfamilies by rigorous ortholog identification combining sequence similarity search with phylogeny. We then construct taxonomic subfamily-distribution profiles and build phylogenetic trees with orthologous proteins. Subfamily profiles provide unparalleled insight into the organisms' energy sources based on genome sequence alone and further predict enzyme substrate specificity, thus generating explicit working hypotheses for targeted biochemical experimentation. Eukaryotic ACAD subfamilies are traditionally considered as mitochondrial proteins, but we found evidence that in fungi one subfamily is located in peroxisomes and participates in a distinct beta-oxidation pathway. Finally, we discern horizontal transfer, duplication, loss and secondary acquisition of ACAD genes during evolution of this family. Through these unorthodox expansion strategies, the ACAD family is proficient in utilizing a large range of fatty acids and amino acids-strategies that could have shaped the evolutionary history of many other ancient protein families.
| S-EPMC2761260 | biostudies-literature
Project description:Protein post-translational modifications serve to regulate a broad range of cellular functions including signal transduction, transcription, and metabolism. Protein lysine residues undergo many post-translational acylations and are regulated by a range of enzymes, such as histone acetyl transferases (HATs) and histone deacetylases (HDACs). KAT2A, well characterized as a lysine acetyltransferase for both histone and nonhistone substrates, has been reported to tolerate additional acyl-CoA substrates, such as succinyl-CoA, and shows nonacetyl transferase activity in specific biological contexts. In this work, we investigate the acyl-CoA substrate preference of KAT2A and attempt to determine whether and to what extent additional acyl-CoA substrates may be utilized by KAT2A in a cellular context. We show that while KAT2A can bind and utilize malonyl-CoA, its activity with succinyl-CoA or glutaryl-CoA is very weak, and acetylation is still the most efficient activity for KAT2A in vitro and in cells.
| S-EPMC9454090 | biostudies-literature
Project description:5 day RNAi treatment to knockdown Enigma, CG9006, a Drosophila mitochondrial protein with homology to acyl-CoA dehydrogenases. Experiment Overall Design: Kc-167 cells, 5 day treatment with Enigma-specific RNAi or beta-lactamase control RNAi. Specifics:1.6X10^6 cells in 0.5mL of serum-free Sang's M3 media incubated for 1 hour with 20ug of dsRNA, subsequently equal volume of M3 media containing 10% serum was added. Cells harvested after 5 days. RNAi efficiency measured by immunoblot for Enigma.
2007-07-31 | E-GEOD-3566 | biostudies-arrayexpress
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.
| S-EPMC7681051 | biostudies-literature
Project description:FadE, an acyl-CoA dehydrogenase, introduces unsaturation to carbon chains in lipid metabolism pathways. Here, we report that FadE5 from Mycobacterium tuberculosis (MtbFadE5) and Mycobacterium smegmatis (MsFadE5) play roles in drug resistance and exhibit broad specificity for linear acyl-CoA substrates but have a preference for those with long carbon chains. Here, the structures of MsFadE5 and MtbFadE5, in the presence and absence of substrates, have been determined. These reveal the molecular basis for the broad substrate specificity of these enzymes. FadE5 interacts with the CoA region of the substrate through a large number of hydrogen bonds and an unusual π-π stacking interaction, allowing these enzymes to accept both short- and long-chain substrates. Residues in the substrate binding cavity reorient their side chains to accommodate substrates of various lengths. Longer carbon-chain substrates make more numerous hydrophobic interactions with the enzyme compared with the shorter-chain substrates, resulting in a preference for this type of substrate.
| S-EPMC7368279 | biostudies-literature
Project description:Acetyl-CoA synthetase (ACS) is one of several enzymes that generate the key metabolic intermediate, acetyl-CoA. In microbes and mammals ACS activity is regulated by the post-translational acetylation of a key lysine residue. ACS in plant cells is part of a two-enzyme system that maintains acetate homeostasis, but its post-translational regulation is unknown. This study demonstrates that the plant ACS activity can be regulated by the acetylation of a specific lysine residue that is positioned in a homologous position as the microbial and mammalian ACS sequences that regulates ACS activity, occurring in the middle of a conserved motif, near the carboxyl-end of the protein. The inhibitory effect of the acetylation of residue Lys-622 of the Arabidopsis ACS was demonstrated by site-directed mutagenesis of this residue, including its genetic substitution with the non-canonical N-ε-acetyl-lysine residue. This latter modification lowered the catalytic efficiency of the enzyme by a factor of more than 500-fold. Michaelis-Menten kinetic analysis of the mutant enzyme indicates that this acetylation affects the first half-reaction of the ACS catalyzed reaction, namely, the formation of the acetyl adenylate enzyme intermediate. The post-translational acetylation of the plant ACS could affect acetate flux in the plastids and overall acetate homeostasis.
| S-EPMC10062202 | biostudies-literature
Project description:We report that human acetyl-CoA synthetase 2 (AceCS2) is a mitochondrial matrix protein. AceCS2 is reversibly acetylated at Lys-642 in the active site of the enzyme. The mitochondrial sirtuin SIRT3 interacts with AceCS2 and deacetylates Lys-642 both in vitro and in vivo. Deacetylation of AceCS2 by SIRT3 activates the acetyl-CoA synthetase activity of AceCS2. This report identifies the first acetylated substrate protein of SIRT3. Our findings show that a mammalian sirtuin directly controls the activity of a metabolic enzyme by means of reversible lysine acetylation. Because the activity of a bacterial ortholog of AceCS2, called ACS, is controlled via deacetylation by a bacterial sirtuin protein, our observation highlights the conservation of a metabolic regulatory pathway from bacteria to humans.
| S-EPMC1502439 | biostudies-literature
Project description:UNLABELLED:Long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency is an inborn error of fatty acid metabolism that affects the degradation of long chain fatty acids and causes insufficient energy production and accumulation of toxic intermediates. The treatment consists of a diet low in fat, with supplementation of medium-chain triglycerides that bypass the metabolic block. In addition, frequent feeds and extra carbohydrates are given during febrile illnesses to reduce lipolysis. Hence, this diet differs from the general dietary recommendations for growing children. Furthermore, the Swedish dietary instructions for fat intake in LCHAD deficiency are given in grams, which differ from most guidelines that recommend fat intake as percentage shares of total caloric intake. AIMS:To assess growth in patients with LCHAD deficiency, in relation to dietary treatment and to evaluate if overweight/obesity is more common than in the normal population. RESULTS:The growth velocity showed acceleration after diagnosis and the start of treatment, followed by a period of stable or decelerated growth. The majority of the patients developed overweight to a greater extent than children without LCHAD deficiency. Several patients also went through a phase of obesity. Data on final height (FH) showed that three out of five patients had grown according to their genetic potential. CONCLUSIONS:Regular and frequent follow-up and careful monitoring of weight are essential to avoid the development of overweight and obesity. The Swedish dietary instructions defining fat intake in total grams per day may be an alternative approach to achieve a moderate total caloric intake.
| S-EPMC3565647 | biostudies-literature