Abstract
Although it has been proposed that the secondary bile acids, deoxycholic acid and lithocholic acid, increase the number of aberrant crypt foci in the colon and may act as colon tumor promoters, there is little evidence detailing their mechanism of action. Histones play an important role in controlling gene expression, and the posttranslational modification of histones plays a role in regulation of intracellular signal transduction. In particular, the amino-terminal tail domain of histone H3 is sensitive to several posttranslational modifications, and acetylation of this domain changes its electrostatic environment and results in the loss of native folding. Therefore, we studied the modification of epsilon-amino groups on human histone H3 by deoxycholyl adenylate, which is an active intermediate in deoxycholyl thioester biosynthesis. After incubation of recombinant human histone H3 with a smaller amount of acyl adenylate, followed by enzymatic digestion, the peptide fragment mixtures were analyzed by matrix-assisted laser desorption ionization mass spectrometry. These data showed the formation of only one adduct fragment, which corresponded to amino acids 3-8 with a deoxycholate adduct, suggesting that the epsilon-amino group of Lys(4) had the highest reactivity. This novel modification, formation of a bile acid adduct on the histone H3 amino-terminal tail domain through an active acyl adenylate, may relate to the carcinogenesis-promoting effects of secondary bile acids.
Highlights
It has been proposed that the secondary bile acids, deoxycholic acid and lithocholic acid, increase the number of aberrant crypt foci in the colon and may act as colon tumor promoters, there is little evidence detailing their mechanism of action
Human histone H3, which is a small protein consisting of 135 amino acid residues, is very basic due to the presence of 13 lysine and 17 arginine residues in the molecule
The amino-terminal tail domain of histone H3 is sensitive to several posttranslational modifications [4], and more than one-third of the amino acid residues in this portion of the protein are lysine or arginine
Summary
Reagents and Apparatus—Recombinant human histone H3 and endoproteinase Arg-C were purchased from Upstate Biotechnology (Lake Placid, NY) and Roche Diagnostics, respectively. 5 M sodium hydroxide solution (1 l) was added, and the reaction mixture was incubated at 37 °C for 30 min to hydrolyze DCA-AMP. After adding 10 l of water-trifluoroacetic acid (9:1, v/v) solution, the mixture was passed through a column of Sephadex G-25 gel (0.3 ml of gel) to remove salts and low molecular weight compounds such as DCA and DCA-AMP. The fraction (0.125– 0.25 ml) containing histone H3 molecules was lyophilized and subjected to digestion with endoproteinase Arg-C (3 g) in a 1:4 mixture of acetonitrile and 50 mM ammonium carbonate solution containing 5 mM dithiothreitol and 8.5 mM CaCl2 (50 l). The reaction mixture was gently mixed with the immunoaffinity gel immobilized anti-deoxycholate monoclonal antibody in 50 mM ammonium carbonate solution at
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
