Fig. 2: HBO1 directly catalyzes lactylation in vitro.
From: HBO1 catalyzes lysine lactylation and mediates histone H3K9la to regulate gene transcription
a, b HBO1 instead of HBO1 (E508Q) enhanced the lactylation level of histones in vitro. HBO1 and HBO1 (E508Q) were overexpressed in HEK293T cells and affinity purified using anti-Flag M2 beads. The purified proteins were incubated with la-CoA (a) or ac-CoA (b) and histone extracted from HEK293T as the reaction substrate for in vitro histone acylation assay. c, d HBO1 has catalytic activity for the lactylation of recombinant protein H3/H4 but not HBO1 (E508Q) in vitro. HBO1 or HBO1 (E508Q) were incubated with lactyl-CoA (c) or acetyl-CoA (d) and recombinant protein H3/H4 as the reaction substrates, respectively, for acylation assay in vitro. e, f The lactylation modification level of histones catalyzed by HBO1 (336-611) were enzyme dose-dependent (e) and reaction time-dependent (f ). HBO1 (336-611) was purified from E.coli. Reaction time points: 10, 30 and 60 min. Enzyme does: 0.5, 1.5, and 2.5 μg. g, h Isothermal titration calorimetry (ITC) analysis of the affinity of recombinant HBO1 (336-611) with lactyl-CoA (e) and acetyl-CoA (f ), respectively. All immunoblots and isothermal titration calorimetry (ITC) analysis had three biological repetitions, with similar results. Source data are provided as a Source Data file.
