Fig 1: SIRT3 shows higher delactylation on H4K16la compared to other human Sirtuins(A) 3 h NAD+ consumption/cycling assay of SIRT1, 2, 3, 5, 6, 7 on H3K9la, H3K14la, H3K56la and H4K16la peptides.(B) Overnight consumption/cycling assay of SIRT1 and SIRT3 on H3K9la and H4K16la, respectively.(C) ITC fitting curves of SIRT1-3 titrated with H3K9la, H3K14la, H3K56la and H4K16la.(D) HPLC-comparison of the H4K16la erasing capacity between Sirtuins, and HDAC3 was set as the positive control (3 h incubation).(E) HPLC-time-dependent cleavage assay of SIRT3 with H4K16la peptide.(F) The fitted curve of delactylation speed (v) and original concentration of H4K16 peptide ([S]). Based on the equation, 1v=(KMVmax)(1[S])+1Vmax, calculated KM = 142.66 ± 2.64 μM, Vmax = 0.925 ± 0.101 μmol/L/min, kcat = 0.08 ± 0.008 s−1, kcat/KM = 5.40x102 s−1M−1.(G) NAD+ consumption/cycling assay of SIRT3 with H4K16la(D/L) peptide.(H) ITC fitting curves of SIRT3 titrated by H4K16la(D/L) peptides.(I) Western blot analysis of cellular H4K16la level change after 48 h post-knockdown of SIRT3 and HDAC3 via siRNA in HEK293T cells.(J) Western blot analysis of cellular H4K16la level change after 48 h post knockdown of SIRT1-3 via siRNA in HEK293T cells.
Fig 2: Development of the chemical probes to investigate lysine lactylation(A) Chemical structure of the p-H4K16laAlk.(B) Proteomic analysis results of p-H4K16laAlk pull down assay (the dotted lines represent p = 0.05 and the enriched ratio = 2).(C) Western blot analysis of detecting the existence of SIRT3 in the pull-down products of groupp-H4K16laAlk, groupp-H4K16laAlk with competitor and DMSO group (negative control).(D) Chemical structure of the fluorogenic probe, p-H4K16laNBD and the schematic diagram of the reaction mechanism.(E) Fluorescence spectrum of p-H4K16laNBD probe (λex = 480 nm). Probe: 10 μM, SIRT3: 0.2 μM, HDAC3: 0.1 μM, and NAD+: 100 μM.(F) The Fluorescence of p-H4K16laNBD (10 μM) with different enzymes (enzyme concentration: 0.1 μM; λex = 480 nm; λem = 545 nm). Enzymatic reaction condition: enzymes (0.1 μM) in 20 mM HEPES buffer (pH = 8.0) containing 100 μM NAD+ at 37°C for 2 h. (No NAD+ was added in HDAC3 group).
Supplier Page from Abcam for Recombinant HDAC3+NCOR2 complex protein (GST tag N-Terminus, His tag C-Terminus)