Dataset Information

Kinetic Characterization of Human Histone Deacetylase 8 With Medium-Chain Fatty Acyl Lysine.

ABSTRACT: Histone deacetylases (HDACs) catalyze the removal of Ɛ-acetyl-lysine residues of histones via hydrolysis. Removal of acetyl groups results in condensation of chromatin structure and alteration of gene expression by repression. HDACs are considered targets for the treatment of cancer due to their role in regulating transcription. HDAC8 inhibition may be an important anti-proliferative factor for histone deacetylase inhibitors on cancer cells and may give rise to the progression of apoptosis. HDAC8 activity was analyzed with various peptides where the target lysine is modified with medium-chain fatty acyl group. Kinetic data were determined for each p53 peptide substrate. The results suggest that there was HDAC8 deacetylase activity on peptide substrate as well as deacylase activity with acylated peptide substrate variants. HDAC8 inhibition by hexanoic and decanoic acid was also examined. The Ki for hexanoic and decanoic acid were determined to be 2.35 ± 0.341 and 4.48 ± 0.221 mM, respectively.

SUBMITTER: Yoo H

PROVIDER: S-EPMC8669121 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Kinetic Characterization of Human Histone Deacetylase 8 With Medium-Chain Fatty Acyl Lysine.

Yoo Harrison H Polsinelli Gregory A GA

Epigenetics insights 20211210

Histone deacetylases (HDACs) catalyze the removal of Ɛ-acetyl-lysine residues of histones via hydrolysis. Removal of acetyl groups results in condensation of chromatin structure and alteration of gene expression by repression. HDACs are considered targets for the treatment of cancer due to their role in regulating transcription. HDAC8 inhibition may be an important anti-proliferative factor for histone deacetylase inhibitors on cancer cells and may give rise to the progression of apoptosis. HDAC ...[more]

PMID: 34917889

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Project description:Triheptanoin (triheptanoylglycerol) has shown value as anaplerotic therapy for patients with long chain fatty acid oxidation disorders but is contraindicated in medium-chain acyl-CoA dehydrogenase (MCAD) deficiency. In search for anaplerotic therapy for patients with MCAD deficiency, fibroblasts from three patients homozygous for the most common mutation, ACADMG985A/G985A, were treated with fatty acids hypothesized not to require MCAD for their metabolism, including heptanoic (C7; the active component of triheptanoin), 2,6-dimethylheptanoic (dMC7), 6-amino-2,4-dimethylheptanoic (AdMC7), or 4,8-dimethylnonanoic (dMC9) acids. Their effectiveness as anaplerotic fatty acids was assessed in live cells by monitoring changes in cellular oxygen consumption rate (OCR) and mitochondrial protein lysine succinylation, which reflects cellular succinyl-CoA levels, using immunofluorescence (IF) staining. Krebs cycle intermediates were also quantitated in these cells using targeted metabolomics. The four fatty acids induced positive changes in OCR parameters, consistent with their oxidative catalysis and utilization. Increases in cellular IF staining of succinylated lysines were observed, indicating that the fatty acids were effective sources of succinyl-CoA in the absence of media glucose, pyruvate, and lipids. The ability of MCAD deficient cells to metabolize C7 was confirmed by the ability of extracts to enzymatically utilize C7-CoA as substrate but not C8-CoA. To evaluate C7 therapeutic potential in vivo, Acadm-/- mice were treated with triheptanoin for seven days. Dose dependent increase in plasma levels of heptanoyl-, valeryl-, and propionylcarnitine indicated efficient metabolism of the medication. The pattern of the acylcarnitine profile paralleled resolution of liver pathology including reversing hepatic steatosis, increasing hepatic glycogen content, and increasing hepatocyte protein succinylation, all indicating improved energy homeostasis in the treated mice. These results provide the impetus to evaluate triheptanoin and the medium branched chain fatty acids as potential therapeutic agents for patients with MCAD deficiency.

Project description:Enzyme and metabolic engineering offer the potential to develop biocatalysts for converting natural resources into a wide range of chemicals. To broaden the scope of potential products beyond natural metabolites, methods of engineering enzymes to accept alternative substrates and/or perform novel chemistries must be developed. DNA synthesis can create large libraries of enzyme-coding sequences, but most biochemistries lack a simple assay to screen for promising enzyme variants. Our solution to this challenge is structure-guided mutagenesis in which optimization algorithms select the best sequences from libraries based on specified criteria (i.e. binding selectivity). Here, we demonstrate this approach by identifying medium-chain (C6-C12) acyl-ACP thioesterases through structure-guided mutagenesis. Medium-chain fatty acids, products of thioesterase-catalyzed hydrolysis, are limited in natural abundance compared to long-chain fatty acids; the limited supply leads to high costs of C6-C10 oleochemicals such as fatty alcohols, amines, and esters. Here, we applied computational tools to tune substrate binding to the highly-active 'TesA thioesterase in Escherichia coli. We used the IPRO algorithm to design thioesterase variants with enhanced C12- or C8-specificity while maintaining high activity. After four rounds of structure-guided mutagenesis, we identified three thioesterases with enhanced production of dodecanoic acid (C12) and twenty-seven thioesterases with enhanced production of octanoic acid (C8). The top variants reached up to 49% C12 and 50% C8 while exceeding native levels of total free fatty acids. A comparably sized library created by random mutagenesis failed to identify promising mutants. The chain length-preference of 'TesA and the best mutant were confirmed in vitro using acyl-CoA substrates. Molecular dynamics simulations, confirmed by resolved crystal structures, of 'TesA variants suggest that hydrophobic forces govern 'TesA substrate specificity. We expect that the design rules we uncovered and the thioesterase variants identified will be useful to metabolic engineering projects aimed at sustainable production of medium-chain oleochemicals.

Dataset Information

Kinetic Characterization of Human Histone Deacetylase 8 With Medium-Chain Fatty Acyl Lysine.

Publications

Kinetic Characterization of Human Histone Deacetylase 8 With Medium-Chain Fatty Acyl Lysine.

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