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Bioorthogonal Chemical Probes of Lysine Acetyltransferases
Author(s) -
Zheng Y. George
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.00242
Subject(s) - bioorthogonal chemistry , lysine , biochemistry , acetyltransferases , cofactor , acetyltransferase , acetylation , chemistry , enzyme , proteome , biotin , context (archaeology) , streptavidin , click chemistry , combinatorial chemistry , biology , amino acid , gene , paleontology
Acetylation of lysine residues is one of the most important posttranslational modifications that diversify protein functions. Thousands of acetylated lysine residues have been identified, suggesting that the effects of lysine acetyltransferases (KATs) go far beyond the chromatin kingdom. However, there is a missing link connecting the compositions of the cellular acetylome networks to the enzymatic activities of different KAT members. The outstanding challenge is how to dissect the subacetylomes of individual KATs and address their functions on the proteomic scale. We have explored a b ioorthogonal p rofiling of p rotein a cetylation strategy to label and interrogate substrates of KAT enzymes. In this strategy, a suite of Ac‐CoA analogs containing either alkynyl or azido functional group (e.g. 3AZ‐CoA, 4AZ‐CoA, 4PY‐CoA, 5HY‐CoA, 6HY‐CoA) were synthesized as potential cofactor surrogate for selective labeling of KAT substrates. Meanwhile, the active site of the KATs was engineered in order to expand the cofactor binding capability of the enzymes to accommodate the bulkier synthetic cofactors. The acylated substrates can be selectively linked through the copper‐catalyzed azide‐alkyne cycloaddition (CuAAC) reaction with fluorescent reporter or biotin affinity tag for optical imaging or protein enrichment on streptavidin‐coated resin. We have successfully used this bioorthogonal technology to profile substrates of p300 and GCN5 in the context of complex cellular proteomes. Together with LC‐MS/MS technology, more than four hundred proteins were identified as GCN5 or p300 substrate candidates. These proteins are extensively involved in various biological events including gene expression, cell cycle, and cellular metabolism. Overall, we have invented a bioorthogonal, chemoproteomic technology to investigate KAT biology. This chemical biology strategy provides a powerful enabling technology for activity‐based lysine acylation profiling on proteomic scale. Indeed, our KAT activity profiling demonstrates extensive engagement of KATs in cellular pathways and provides new molecular insights into understanding their functions in epigenetics and other biological processes.bioorthogonal profiling