Fig 2: Delactylase activity of class I HDAC1–3.(A) Conversion of Kac, K(l-la), and K(d-la) short AMC-conjugated peptides. Data represent means ± SD (n = 2). (B) Michaelis-Menten plots for HDACs 1 and 3 against substrates 2b and 2c. Data represent means ± SEM (n = 2) (see fig. S4 for HDAC2 data and sample assay progression curves). (C) Catalytic efficiencies of HDAC1–3 against substrates 2b and 2c. Data represent means ± SEM (n = 2) (see fig. S4 for complete KM and kcat datasets). *HDAC3 incubated with the DAD of NCoR2.

Fig 3: Potentially novel protein substrates of human PARP1 identified by ADO-3'-N3-NAD+. (A) A pie chart of protein substrates identified by ADO-3'-N3-NAD+. (B) and (C) ADP-ribosylation of HDAC2 (B) and HMGA2 (C) by human PARP1. HDAC2-His6 or HMGA2-His6 was incubated with NAD+ in the absence or presence of His6-tag free PARP1 without or with veliparib at 30 °C for overnight. Reactions with only PARP1 were incubated under the same conditions. The samples were then incubated with Ni-NTA beads to separate unbound PARP1 and elute HDAC2 or HMGA2 for immunoblot analysis as detected by an anti-pan-ADP-ribose binding reagent. The total reactions were detected by the anti-pan-ADP-ribose binding reagent (top panel) or anti-His6 antibody (bottom panels). Right panels: densitometric analysis of ADP-ribosylated proteins for eluted samples normalized to respective substrate proteins. *, p < 0.05; **, p < 0.01.

Fig 4: In cellulo deacetylase, decrotonylase, and delactylase activities of HDAC1–3.(A) Changes in overall lactylation and acetylation of histones in HeLa cells upon 5-hour treatment with pan-HDAC inhibitors (sodium butyrate, 2 mM; TSA, 1 µM), the HDAC1–3–selective inhibitor apicidin (1 µM), the class IIa–selective inhibitor TMP195 (5 µM), or the pan-sirtuin inhibitor NAM (10 mM) (see fig. S6 for full Western blots and blots with longer exposure time). (B) Western blot analysis of core histones from HeLa cells with or without transfection of HA-tagged HDAC1, FLAG-tagged HDAC2, or HA-tagged HDAC3. (C) Western blot analysis of core histones from HeLa cells with or without small interfering RNA (siRNA)–mediated knockdown of HDACs 1, 2, 3, or their combination (72-hour treatment). Top blots correspond to whole-cell lysates and show the level of ectopically expressed HDAC (B) or HDAC knockdown (C) relative to ß-actin (see fig. S6 for full Western blots). (D to F) Immunostaining images of HeLa cells transfected with HA-tagged HDAC1, FLAG-tagged HDAC2, or HA-tagged HDAC3 and the levels of histone acetylation, crotonylation, and lactylation. DAPI: 4',6-diamidino-2-phenylindole (nuclear stain).