Identification and Profiling of Histone Acetyltransferase Substrates by Bioorthogonal Labeling

Abstract

Histone acetyltransferases (HATs, also known as lysine acetyltransferases, KATs) catalyze acetylation of their cognate protein substrates using acetyl-CoA (Ac-CoA) as a cofactor and are involved in various physiological and pathological processes. Advances in mass spectrometry-based proteomics have allowed the discovery of thousands of acetylated proteins and the specific acetylated lysine sites. However, due to the rapid dynamics and functional redundancy of HAT activities, and the limitation of using antibodies to capture acetylated lysines, it is challenging to systematically and precisely define both the substrates and sites directly acetylated by a given HAT. Here, we describe a chemoproteomic approach to identify and profile protein substrates of individual HAT enzymes on the proteomic scale. The approach involves protein engineering to enlarge the Ac-CoA binding pocket of the HAT of interest, such that a mutant form is generated that can use functionalized acyl-CoAs as a cofactor surrogate to bioorthogonally label its protein substrates. The acylated protein substrates can then be chemoselectively conjugated either with a fluorescent probe (for imaging detection) or with a biotin handle (for streptavidin pulldown and chemoproteomic identification). This modular chemical biology approach has been successfully implemented to identify protein substrates of p300, GCN5, and HAT1, and it is expected that this method can be applied to profile and identify the sub-acetylomes of many other HAT enzymes.

Targeting the chromatin structural changes of antitumor immunity

“…Writers and erasers, modified by various histones, participate in the regulation and maintenance of the balance [ 114 ]. Histone is acetylated by various histone acetyltransferases [ 115 ]. Hyperacetylated histones are generally associated with an open chromatin structure that can be utilized by transcription factors and mechanisms.…”

Promiscuous Enzymes for Residue‐Specific Peptide and Protein Late‐Stage Functionalization

“…[ 144 ] Engineering of KATs expanded the substrate profiles and enabled diverse acyl coenzyme A with terminal alkyne or azides to be accepted. [ 145 , 146 ] Recently, a platform based on thiol recycling was used to introduce bioorthogonal handles and isotopic labels into KAT. [ 147 ] Lysine deacetylases (KDACs, mark erasers) catalyze the reverse reaction of KATs and remove acetyl groups from Lys and together with KATs, fine-tunes the Lys acylation in cells.…”

New Histone Lysine Acylation Biomarkers and Their Roles in Epigenetic Regulation

“…Owing to simplicity, convenience, and affordable cost, antibody‐based western blotting (WB) analysis has been the gold standard for measuring relative acylation mark changes. However, this method suffers from several shortcomings: low‐throughput detection of acylation marks at a time; low antibody specificity to distinguish acylation marks that have very subtle structure differences; interference by nearby modifications or epitope occlusion; and cross‐reactivity to other modification due to homologous protein sequence (Huang et al., 2015; Song et al., 2022). Liquid chromatography (LC)‐MS‐based approaches are used for detecting acylation marks based on their presumed mass shifts and can address the caveats above by not only detecting multiple acylation marks simultaneously regardless their positions on peptide sequence, but also distinguishing different acylation marks that have similar structures.…”