Identification of SIRT3 as an eraser of H4K16la

Abstract

Lysine lactylation (Kla) is a novel histone post-translational modification discovered in late 2019. Later, HDAC1-3, were identified as the robust Kla erasers. While the Sirtuin family proteins showed weak eraser activities toward Kla, as reported. However, the catalytic mechanisms and physiological functions of HDACs and Sirtuins are not identical. In this study, we observed that SIRT3 exhibits a higher eraser activity against the H4K16la site than the other human Sirtuins. Crystal structures revealed the detailed binding mechanisms between lactyl-lysine peptides and SIRT3. Furthermore, a chemical probe, p-H4K16laAlk, was developed to capture potential Kla erasers from cell lysates. SIRT3 was captured by this probe and detected via proteomic analysis. And another chemical probe, p-H4K16la-NBD, was developed to detect the eraser-Kla delactylation processes directly via fluorescence indication. Our findings and chemical probes provide new directions for further investigating Kla and its roles in gene transcription regulation.

Keywords: Enzymology; Molecular interaction; Protein; Structural biology.

Graphical abstract

Figure 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, $\frac{1}{v}=\left(\frac{K_M}{V_{\mathit{max}}}\right)\left(\frac{1}{\left[S\right]}\right)+\frac{1}{V_{\mathit{max}}}$, calculated K_M = 142.66 ± 2.64 μM, V_max = 0.925 ± 0.101 μmol/L/min, k_cat = 0.08 ± 0.008 s⁻¹, k_cat/K_M = 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.

Figure 2

The structural basis of SIRT3 and lactyl-lysine peptides (A) Left: The overall structures of SIRT3(white)-H3K23la(L)(green) complex structure (white) and the ligand models observed in the SIRT3-H3K23la(L) structure. Right: The overall structures of SIRT3(light pink)-H4K16la(L)(cyan) complex structure and the ligand models observed in the SIRT3-H4K16la(L) structure. The 2Fo-Fc maps displayed for the ligands are rendered at 0.9σ contour. (B) The preconfigured hydrophobic cage of SIRT3 accommodates the hydrocarbon portion of the lactyl group. Upper: SIRT3(white)-H3K23la(L)(green). Lower: SIRT3(light pink)-H4K16la(L)(cyan). (C) Ligand-protein interaction details in SIRT3(light pink)-H4K16la(L)(cyan) structure. Hydrogen bond is represented as a yellow dashed line, and the water molecule is represented as spheres (marine). The length (Å) of the hydrogen bond is labeled next to the dashed line. (D) LIGPLOT diagram list interactions between the H4K16(L) ligand and SIRT3. H4K16la(L) (bond color: light pink) and residues of SIRT3 (bond color: gold) are depicted in ball-and-stick mode. Carbon is represented as a black ball; nitrogen is represented as a blue ball; oxygen is represented as a red ball; water molecule is represented as a marine ball; hydrophobic interactions are shown as red arcs; and hydrogen bond is represented as a green dashed line. The length (Å) of the hydrogen bond is labeled next to the dashed line.

Figure 3

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 group_{p-H4K16laAlk,} group_{p-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).