Chemoproteomic Profiling of Protein Substrates of a Major Lysine Acetyltransferase in the Native Cellular Context

The
family of lysine acetyltransferases (KATs) regulates epigenetics
and signaling pathways in eukaryotic cells. So far, knowledge of different
KAT members contributing to the cellular acetylome is limited, which
limits our understanding of biological functions of KATs in physiology
and disease. Here, we found that a clickable acyl-CoA reporter, 3-azidopropanoyl
CoA (3AZ-CoA), presented remarkable cell permeability and effectively
acylated proteins in cells. We rationally engineered the major KAT
member, histone acetyltransferase 1 (HAT1), to generate its mutant
forms that displayed excellent bio-orthogonal activity for 3AZ-CoA
in substrate labeling. We were able to apply the bio-orthogonal enzyme–cofactor
pair combined with SILAC proteomics to achieve HAT1 substrate targeting,
enrichment, and proteomic profiling in living cells. A total of 123
protein substrates of HAT1 were disclosed, underlining the multifactorial
functions of this important enzyme than hitherto known. This study
demonstrates the first example of utilizing bio-orthogonal reporters
as a chemoproteomic strategy for substrate mapping of individual KAT
isoforms in the native biological contexts.