No abstract

Metabolism-mediated epigenetic changes represent an adapted mechanism for cellular signaling, in which lysine acetylation and methylation have been the historical focus of interest. We recently discovered a β-hydroxybutyrate–mediated epigenetic pathway that couples metabolism to gene expression. However, its regulatory enzymes and substrate proteins remain unknown, hindering its functional study. Here, we report that the acyltransferase p300 can catalyze the enzymatic addition of β-hydroxybutyrate to lysine (Kbhb), while histone deacetylase 1 (HDAC1) and HDAC2 enzymatically remove Kbhb. We demonstrate that p300-dependent histone Kbhb can directly mediate in vitro transcription. Moreover, a comprehensive analysis of Kbhb substrates in mammalian cells has identified 3248 Kbhb sites on 1397 substrate proteins. The dependence of histone Kbhb on p300 argues that enzyme-catalyzed acylation is the major mechanism for nuclear Kbhb. Our study thus reveals key regulatory elements for the Kbhb pathway, laying a foundation for studying its roles in diverse cellular processes.

Lysine L-lactylation [K(L-la)] is a newly discovered histone mark stimulated under conditions of high glycolysis, such as the Warburg effect. K(L-la) is associated with functions that are different from the widely studied histone acetylation. While K(L-la) can be introduced by the acetyltransferase p300, histone delactylases enzymes remained unknown. Here, we report the systematic evaluation of zinc- and nicotinamide adenine dinucleotide–dependent histone deacetylases (HDACs) for their ability to cleave ε- N -L-lactyllysine marks. Our screens identified HDAC1–3 and SIRT1–3 as delactylases in vitro. HDAC1–3 show robust activity toward not only K(L-la) but also K(D-la) and diverse short-chain acyl modifications. We further confirmed the de-L-lactylase activity of HDACs 1 and 3 in cells. Together, these data suggest that histone lactylation is installed and removed by regulatory enzymes as opposed to spontaneous chemical reactivity. Our results therefore represent an important step toward full characterization of this pathway’s regulatory elements.

No abstract

CEPR2 interacts with some PYLs to promote their phosphorylation and degradation, whereas ABA inhibits this process. Thus, CEPR2 balances the growth regulation and stress response in Arabidopsis.

Cysteine mutagenesis and surface labeling has been used to define more precisely the transmembrane spans of subunit a of the Escherichia coli ATP synthase. Regions of subunit a that are exposed to the periplasmic space have been identified by a new procedure, in which cells are incubated with polymyxin B nonapeptide (PMBN), an antibiotic derivative that partially permeabilizes the outer membrane of E. coli, along with a sulfhydryl reagent, 3-(N-maleimidylpropionyl) biocytin (MPB). This procedure permits reaction of sulfhydryl groups in the periplasmic space with MPB, but residues in the cytoplasm are not labeled. Using this procedure, residues 8, 27, 37, 127, 131, 230, 231, and 232 were labeled and so are thought to be exposed in the periplasm. Using inside-out membrane vesicles, residues near the end of transmembrane spans 1, 64, 67, 68, 69, and 70 and residues near the end of transmembrane spans 5, 260, 263, and 265 were labeled. Residues 62 and 257 were not labeled. None of these residues were labeled in PMBNpermeabilized cells. These results provide a more detailed view of the transmembrane spans of subunit a and also provide a simple and reliable technique for detection of periplasmic regions of inner membrane proteins in E. coli.The ATP synthase from Escherichia coli is typical of the ATP synthases found in mitochondria, chloroplasts, and many other bacteria (for reviews, see Refs. 1-3). It contains an F 1 sector, with subunits for nucleotide binding and catalysis, and an F 0 sector, which conducts protons across the membrane. Five different subunits are found in the E. coli F 1 : ␣, ␤, ␥, ␦, and ⑀, in a stoichiometry of 3:3:1:1:1. Three different subunits named a, b, and c form the E. coli F 0 with a stoichiometry of 1:2:12 (4) . The mechanism by which an electrochemical proton gradient across the membrane drives ATP synthesis is thought to involve a rotary mechanism. The crystallization of F 1 from bovine mitochondria (5) led to a high resolution structure of the ␣ 3 ␤ 3 hexamer, plus parts of ␥ in the central core . Subsequently, the hypothesis of rotation of ␥ and ⑀ and relative to ␣ 3 ␤ 3 has been supported by direct visualization of rotation of fluorescently labeled actin filaments covalently attached to ␥ (6) or ⑀ (7). It has been proposed that F 0 subunit c drives the rotation of ␥ and ⑀ as a rotor (8), whereas subunits a and b function as the stator. Recent theoretical work has indicated the feasibility of this proposal (9), but as of yet there is no direct evidence of rotation by F 0 subunits.Information about the tertiary and quaternary structure of F 0 subunits will be necessary for an understanding of how F 0 translocates protons, and how it might drive rotation of ␥ and ⑀ subunits in F 1 . Subunit b seems to be embedded in the membrane via a span of hydrophobic amino acids at its N terminus. A truncated, soluble form of b has been shown to be extended and dimeric (10) . Recent NMR studies of c have confirmed the ␣-helical hairpin structure of the two predicted transmembrane spans, and also ...

The purpose of the present work is to study the pancreatic lipase inhibitory effects of different subfractions (n-hexane, ethyl acetate (EA), n-butanol, and water) from ethanol extracts of nonfermented and fungi-fermented oats and to delineate the interactions of three primary phenolic acids in the EA subfractions. The EA subfraction showed the highest inhibitory effect on pancreatic lipase activity at 1.5 mg/mL compared to the other subfractions, regardless of whether the oats were fermented. Meanwhile, both of the EA subfractions of two fungi-fermented oats demonstrated more effective inhibitory activity than that of nonfermented oats. A positive correlation between the total phenolics content and inhibitory activity was found. The inhibitory ability of the EA subfraction from nonfermented or fermented oats also displayed a dose-dependent effect. The standards of caffeic, ferulic, and p-coumaric acids, mainly included in EA subfractions of fermented oats, also displayed a dose-dependent inhibitory effect. A synergistic effect of each binary combination of p-coumaric, ferulic, and caffeic acids was observed, especially at 150.0 μg/mL. Those results indicate that fungi-fermented oats have a more effective inhibitory ability on pancreatic lipase and polyphenols may be the most effective component and could be potentially used for dietary therapy of obesity.

The molecular basis of the interaction of DT-diaphorase with a cytotoxic nitrobenzamide CB1954 [5-(aziridin-1-yl)-2, 4-dinitrobenzamide] and five inhibitors was investigated with wild-type DT-diaphorase (human and rat) and five mutants [three rat mutants (rY128D, rG150V, rH194D) and two human mutants (hY155F, hH161Q)]. hY155F and hH161Q were generated to evaluate a hypothesis that Tyr155 and His161 participate in the obligatory two-electron transfer reaction of the enzyme. The catalytic properties of hY155F and hH161Q were compared with a naturally occurring mutant, hP187S. Pro187 to Ser mutation disturbs the structure of the central parallel beta-sheet, resulting in a reduction of the binding affinity of the flavin-adenine dinucleotide prosthetic group. With NADH as the electron donor and menadione as the electron acceptor, the k(cat) values for the wild-type human DT-diaphorase, hY155F, hH161Q, and hP187S were measured as 66 +/- 1, 23 +/- 0, 5 +/- 0 and 8 +/- 2 x 10(3) min(-1), respectively. Because hY155F still has significant catalytic activity, the hydroxyl group on Tyr155 may not be as important as proposed. Interestingly, hY155F was found to be 3. 3 times more active than the human wild-type DT-diaphorase in the reduction of CB1954. Computer modeling based on our results suggests that CB1954 is situated in the active site, with the aziridinyl group pointing toward Tyr155 and the amide group placed near a hydrophobic pocket next to Tyr128. Dicoumarol, Cibacron blue, chrysin, 7,8-dihydroxyflavone, and phenindone are competitive inhibitors of the enzyme with respect to nicotinamide coenzymes. The binding orientations of dicoumarol, flavones, and phenindone in the active site of DT-diaphorase were predicted by results from our inhibitor-binding studies and computer modeling based on published X-ray structures. Our studies generated results that explain why dicoumarol is a potent inhibitor and binds differently from flavones and phenindone in the active site of DT-diaphorase.