Fluorescent Probes for Single-Step Detection and Proteomic Profiling of Histone Deacetylases

Abstract

Histone deacetylases (HDACs) play important roles in regulating various physiological and pathological processes. Developing fluorescent probes capable of detecting HDAC activity can help further elucidate the roles of HDACs in biology. In this study, we first developed a set of activity-based fluorescent probes by incorporating the Kac residue and the O-NBD group. Upon enzymatic removal of the acetyl group in the Kac residue, the released free amine reacted intramolecularly with the O-NBD moiety, resulting in turn-on fluorescence. These designed probes are capable of detecting HDAC activity in a continuous fashion, thereby eliminating the extra step of fluorescence development. Remarkably, the amount of turn-on fluorescence can be as high as 50-fold, which is superior to the existing one-step HDAC fluorescent probes. Inhibition experiments further proved that the probes can serve as useful tools for screening HDAC inhibitors. Building on these results, we moved on and designed a dual-purpose fluorescent probe by introducing a diazirine photo-cross-linker into the probe. The resulting probe was not only capable of reporting enzymatic activity but also able to directly identify and capture the protein targets from the complex cellular environment. By combining a fluorometric method and in-gel fluorescence scanning technique, we found that epigenetic readers and erasers can be readily identified and differentiated using a single probe. This is not achievable with traditional photoaffinity probes. In light of the prominent properties and the diverse functions of this newly developed probe, we envision that it can provide a robust tool for functional analysis of HDACs and facilitate future drug discovery in epigenetics.

A sulfonium tethered peptide ligand rapidly and selectively modifies protein cysteine in vicinity

“…To assess the ability of peptide PD3-I to label PDZ DRGS3 in a complex proteome environment, 293 T cell lysates (300 mg) were spiked with PDZ DRGS3 (10 mg), and then treated with 50 mM FAM labeled peptide PD3-I as shown in Fig. 3E, referring to the work of Sun et al 45 The gel data showed a clear single uorescence band with the right molecular weight indicating a clean and selective conjugation of peptide PD3-I to PDZ DRGS3 . The successful conjugation was further conrmed by a pull down assay using Ni-NTA agarose beads as shown in Fig.…”

Lysine Deacetylases Exhibit Distinct Changes in Activity Profiles Due to Fluorophore Conjugation of Substrates

“…Other recently developed substrates allow deacetylation to be monitored in a single-step. 24,50,51 For some of these substrates, the dyes are positioned further away from the acetylated lysine than for the AMC substrates; 52,53 however, it will be important to determine how the configuration of these substrates affects KDAC activity. An assumption that AMC or other fluorophores might mimic large non-polar residues after the acetyllysine is clearly incorrect, as tryptophan in the +1 position causes distinctly different activity effects than AMC.…”

Recent Applications of Diazirines in Chemical Proteomics

“…Recently,X ie and co-workers developed bifunctional probe 37 that was capable of monitoring HDAC activity,a s well as identifying cellular targets through tandemP AL ( Figure 10). [27] In the presence of HDACs, the appendant Nacetyl group is deacetylated to form the free amine, which undergoes an intramolecular substitution reactionw itht he O-ni-trobenzoxadiazole (NBD) to form the N-NBD as ah ighly fluorescent species. [59] They demonstrated the ability of 37 to effectively label SIRT1 and SIRT2 in ac omplex proteomic environment as well as its efficacy to differentiate between epigenetic eraser BRD4-1 and readerS IRT2 in cell culture.…”