Profiling Cellular Substrates of Lysine Acetyltransferases GCN5 and p300 with Orthogonal Labeling and Click Chemistry

Abstract

p300 and GCN5 are two representative lysine acetyltransferases (KATs) in mammalian cells. It was recently reported that they possess multiple acyltransferase activities including acetylation, propionylation, and butyrylation of the ε-amino group of lysine residues of histones and non-histone protein substrates. Although thousands of acetylated substrates and acetylation sites have been identified by mass spectrometry-based proteomic screening, our knowledge about the causative connections between individual KAT members and their corresponding sub-acylomes remain very limited. Herein, we applied 3-azidopropionyl CoA (3AZ-CoA) as a bioorthogonal surrogate of acetyl-, propionyl- and butyryl-CoA for KAT substrate identification. We successfully attached the azide as a chemical warhead to cellular substrates of wild-type p300 and engineered GCN5. The substrates were subsequently labeled with biotin tag through the copper-catalyzed azide-alkyne cycloaddition (CuAAC). Following protein enrichment on streptavidin-coated resin, we conducted LC-MS/MS studies from which more than four hundred proteins were identified as GCN5 or p300 substrate candidates. These proteins are either p300- or GCN5-unique or shared by the two KATs and are extensively involved in various biological events including gene expression, cell cycle, and cellular metabolism. We also experimentally validated two novel substrates of GCN5, that is, IQGAP1 and SMC1. These results demonstrate extensive engagement of GCN5 and p300 in cellular pathways and provide new insights into understanding their functions in specific biological processes.

Steric-Free Bioorthogonal Labeling of Acetylation Substrates Based on a Fluorine-Thiol Displacement Reaction (FTDR)

“…On the contrary, all acetyltransferases failed to incorporate the alkyne-modified 4-PY-CoA, which is consistent with previous reports. 6,11 Substitution with the electron-withdrawing fluorine also endowed a much higher hydrolysis rate to F-Ac-CoA, which somehow did not display significantly greater non-enzymatic reactivity ( Figure S3). 27 Taken together ( Figure 1C), fluorine modification on the acetyl group afforded a relatively steric-free chemical reporter that can satisfactorily label substrates of acetyltransferases.…”

“…[1][2] Representative bioorthogonal reactions such as azide-alkyne cycloaddition, 3 Staudinger ligation, 4 and tetrazine cycloaddition 2 have resulted in the successful development of chemical reporters, which in conjunction with detection/affinity tags, have advanced our understanding of important biological pathways including PTMs. 5 For instance, chemical reporters on acetylation have revealed many new protein substrates of p300, [6][7] providing a more robust substrate recovery from proteome in comparison to anti-acetyl lysine antibodies. [8][9] Yet, current chemical reporters are bulky in length and size, thereby impeding their general applications.…”

“…While the "bump-hole" protein engineering strategy could work for a given enzyme, careful balances between mutations, structure folding, and function (potency, selectivity, etc) are required. 14 For many acetyltransferases that function as subunits in protein complexes, mutations in the "hole" may alter their substrate specificities, leading to results different from in vivo sub-acylome, 6 which undermines this approach's broad applications.…”

Chemical Biology Approaches for Investigating the Functions of Lysine Acetyltransferases

“…It is likely that the limited acetyl-CoA binding pocket of these KATs hinders the binding of relatively bulky acetyl-CoA analogues.Therefore,weengineered the active sites of GCN5 and MOF to create mutants with enlarged acetyl-CoA binding pockets,a nd then screened for activity toward the acetyl-CoA surrogates.G CN5-T612G/F622A and GCN5-T612G/L531A were specifically active toward 5HY-CoA. [114] Heck coupling was explored as an alternative detection method for the bioorthogonal labeling of acylated histones. 3AZ-CoA is also an Figure 12.…”

“…Reviews efficient cofactor for the labeling of p300 substrates.T he "clickable" feature of the alkyne-or azido-modified acetyl-CoA analogues provides au nique advantage for selective and facile labeling of KATtargets with fluorescent reporters or affinity tags through click chemistry.W ith 3AZ-CoA as ab ioorthogonal probe,w er ecently identified 472 substrates of GCN5 and 475 substrates of p300 in HEK293T cells. [114] Heck coupling was explored as an alternative detection method for the bioorthogonal labeling of acylated histones. [112c] In the study by Ourailidou et al,s odium 4-pente-noate was incubated with RAW2 64.7 cells to produce intracellular 4-pentenoyl-CoA for 4-pentenoyl-labeled histones.The cells were then lysed, and the histones were extracted and coupled with fluorescein or biotin-labeled phenylboronic acid through aEDTA-Pd II -catalyzed oxidative Heck reaction.…”