Peptide-Based Fluorescent Probes for Deacetylase and Decrotonylase Activity: Toward a General Platform for Real-Time Detection of Lysine Deacylation.

Histone deacetylases regulate the acetylation levels of numerous proteins and play key roles in physiological processes and disease states. In addition to acetyl groups, deacetylases can remove other acyl modifications on lysines, the roles and regulation of which are far less understood. A peptide-based fluorescent probe for single-reagent, real-time detection of deacetylase activity that can be readily adapted for probing broader lysine deacylation, including decrotonylation, is reported. Following cleavage of the lysine modification, the probe undergoes rapid intramolecular imine formation that results in marked optical changes, thus enabling convenient detection of deacylase activity with good statistical Z' factors for both absorption and fluorescence modalities. The peptide-based design offers broader isozyme scope than that of small-molecule analogues, and is suitable for probing both metal- and nicotinamide adenine dinucleotide (NAD+ )-dependent deacetylases. With an effective sirtuin activity assay in hand, it is demonstrated that iron chelation by Sirtinol, a commonly employed sirtuin inhibitor, results in an enhancement in the inhibitory activity of the compound that may affect its performance in vivo.

IspG-catalyzed positional isotopic exchange in methylerythritol cyclodiphosphate of the deoxyxylulose phosphate pathway: mechanistic implications.

H(2)(18)O under the bridge: Recently, the deoxyxylulose phosphate (DXP) pathway was discovered to be a second pathway supplying isoprenoid biosynthetic precursors. One of steps is an IspG-catalyzed reductive deoxygenation of methylerythritol cyclodiphosphate (MEcPP) to 4-hydroxyl-3-methyl-2-(E)-1-diphosphate (HMBPP). Using [2-(13) C,(18) O]-MEcPP, we detected the positional isotopic exchange for the bridging oxygen in MEcPP.

Real-time detection of histone deacetylase activity with a small molecule fluorescent and spectrophotometric probe.

Histone deacetylases (HDACs) are central players in transcription regulation and important targets in cancer treatment. Activity assays are critical tools for the study of the function and regulation of these enzymes, as well as for the screening of potential inhibitors. We report a small-molecule probe for single-step, continuous detection of deacetylase activity based on an acetyl-lysine mimic functionalized with an amine-reactive fluorophore, designed to undergo rapid intramolecular imine formation upon deacetylation. The probe exhibits a bathochromic shift in the absorption spectrum and changes in fluorescence emission intensity that enable unprecedented real-time detection of HDAC activity of purified enzymes or in cell lysates, and offers a means to evaluate HDAC inhibitors via simple spectrophotometric or fluorescence readings without the need of additional reagents.