Class I histone deacetylases catalyze lysine lactylation

Abstract

Metabolism and post-translational modifications (PTMs) are intrinsically linked and the number of identified metabolites that can covalently modify proteins continues to increase. This metabolism/PTM crosstalk is especially true for lactate, the product of anaerobic metabolism following glycolysis. Lactate forms an amide bond with the ε-amino group of lysine, a modification known as lysine lactylation, or Kla. Multiple independent mechanisms have been proposed in the formation of Kla, including p300/CBP-dependent transfer from lactyl-CoA, via a high-energy intermediate lactoylglutathione species that non-enzymatically lactylates proteins, and several enzymes are reported to have lactyl transferase capability. We recently discovered that class I histone deacetylases (HDACs) 1, 2, and 3 can all reverse their canonical chemical reaction to catalyze lysine β-hydroxybutyrylation. Here we tested the hypothesis that HDACs can also catalyze Kla formation. Using biochemical, pharmacological, and genetic approaches, we found that HDAC-catalyzed lysine lactylation accounts for the majority of Kla formation in cells. Dialysis experiments confirm this is a reversible reaction that depends on lactate concentration. We also directly quantified intracellular lactyl-CoA and found that Kla abundance can be uncoupled from lactyl-CoA levels. Therefore, we propose a model in which the majority of Kla is formed through enzymatic addition of lactate by HDACs 1, 2, and 3.

Fig. 1.. HDACs 1, 2, and 3 catalyze lysine lactylation in vitro

(A) Western blots of an in vitro lysine lactylation assay with recombinant HDAC1 (rHDAC1), rHDAC2, or rHDAC2/NcoR2 and histone H3 (rH3) in the presence of 1 mM L-Lactate with or without TSA (5 μM). The reactions were performed at 37°C for 30 mins. Protein loading was visualized by ponceau S staining, and Kbhb was detected by western blot. Experiments were repeated independently twice with similar results. (B-C) LC-MS/MS analysis of H3 after in vitro lysine lactylation assay. Detected Kla-modified sites on H3 (B). MS/MS-spectra for the indicated peptide with detected y and b ions (C). (D) Schematic of experimental workflow. HDAC2 KO HEK293T cells expressing 3xFLAG-mHDAC2 were used for immunoprecipitation with α-FLAG antibody. The immunoprecipitants were used for in vitro lysine lactylation assays and in vitro deacetylation assays. (E) Representative western blots of in vitro lysine lactylation with IPed 3xF-mHDAC2 WT or indicated mutants. This experiment was independently repeated three times with similar results. (F) Lysine lactylation and deacetylation activity for each mutant. Data are represented as mean +/− SEM activity relative to WT 3xF-mHDAC2, from n=3 independent experiments. (G) Schematic of the experimental workflow to test the Lactate concentration-dependence of HDAC reversibility using a dialysis system. (H) Representative western blot to assess Kla formation before and after dialysis with different L-Lactate concentrations. This experiment was repeated independently twice with similar results. (I) Schematic of the proposed model of the forward and reverse reactions catalyzed by class I HDACs.

Fig. 2.. Catalytic activities of HDAC1, 2, and 3 are required for Kla formation in cells

(A-B) HEK293T cells were treated with each HDAC inhibitor at the indicated concentrations for 24 hours. Representative western blots are shown in (A). Quantification of anti-Kla signals relative to lane 1 (untreated, unTx) were shown in (B). Signals were normalized to ponceau S staining. Data are represented as mean +/− SEM of three independently performed experiments and each symbol represents an individual experiment. Statistical differences were calculated by 1-way ANOVA followed by Dunnett’s correction for multiple comparisons. (C-D) HEK293T cells were transfected with siRNA against the indicated targets. Representative western blots are shown in (C). Quantification of anti-Kla signals relative to lane 2 (si-control) are shown in (D). Signals were normalized by ponceau S staining. Data are represented as mean +/− SEM of three independently performed experiments and each symbol represents an individual experiment. Statistical differences were calculated by 1-way ANOVA followed by Tukey’s test for multiple comparisons. (E) Quantification of intracellular lactate concentrations in HEK293T cells treated with HDAC inhibitors: Butyrate at 5 mM, TSA at 1 μM, SAHA at 5 μM, MS-275 5 μM. Data are represented as mean +/− SD of technical triplicates. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

Fig. 3.. Kla formation is regulated by glucose metabolism

(A) Schematic of anticipated Kla levels in wild-type and established LDHA and LDHB double knock-out (LDHA/B dKO) cells. (B-C) LDHA/B dKO HEK293T cells were treated with L-Lactate at the indicated concentrations for 24 hours. Representative western blots are shown in (B) and quantification of anti-Kla signals relative to lane 1 (untreated, unTx) were shown in (C). Signals were normalized to ponceau S staining. Data are represented as mean +/− SEM of three independently performed experiments and each symbol represents an individual experiment. Statistical differences were calculated by 1-way ANOVA followed by Dunnett’s correction for multiple comparisons. (D-F) WT HEK293T cells were cultured in 0, 1, or 5 g/L glucose containing medium and treated with 1 μM TSA at the indicated time points. Representative western blots are shown in (D). Quantification of anti-Kla or anti-H3K18la signals relative to lane 3 (untreatment, 5 g/L glucose) were shown in (E) or (F), respectively. Signals were normalized to ponceau S staining. Data are represented as mean +/− SEM of three independently performed experiments and each symbol represents an individual experiment. Statistical differences were calculated by 1-way ANOVA followed by Tukey’s correction for multiple comparisons. (G-I) Bone-marrow derived macrophages (BMDM) were stimulated with LPS (5 ng/mL) and IFNγ (12 ng/mL) for the indicated time. MS-275 (0.5 μM) was added at 8 hr after LPS/IFNγ stimulation, resulting in 16 hours of inhibition from T8 to T24. Representative western blots are shown in (G). Quantification of anti-Kla signals relative to lane 1 (unstimulated) and normalized to ponceau S staining are shown in (H). Data are represented as mean +/− SEM of three independently performed experiments and each symbol represents an individual experiment. Statistical differences were calculated by 1-way ANOVA followed by Tukey’s correction for multiple comparisons. Quantification of intracellular lactate concentrations in BMDM after 24 hours of stimulation, with or without MS-275, are shown in (I). *p<0.05, **p<0.01, ****p<0.0001

Fig. 4.. Lactate and HDAC enzymatic activity are the dominant regulators of Kla formation

(A) Proposed model of AARS enzymes-dependent lysine lactylation. (B-E) HEK293T cells were transfected with siRNA to assess the role of indicated target proteins in Kla formation. Representative western blots are shown in (B). Quantification of anti-Kla signals relative to lane 2 (si-control), normalized to ponceau S staining, are shown in (C). Relative mRNA expression for (D) AARS1, and (E) AARS2 were measured by qPCR and normalized to RPL13A. Data are represented as mean +/− SEM of three independently performed experiments and each symbol represents an individual experiment. Statistical differences were calculated by 1-way ANOVA followed by Dunnett’s correction for multiple comparisons. (F) Proposed model of lactyl-CoA enzyme-dependent lysine lactylation. (G-J) HEK293T cells were transfected with siRNA to assess the role of ACSS2 and SUCLG2 in Kla formation. Western blots containing all three independently performed experiments were shown in (G, labeled Exp1, Exp2, and Exp3). Quantification of anti-Kla signals relative to lane 2 (si-control) are shown in (H). Signals were normalized to ponceau S staining. Relative mRNA expression for (I) ACSS2 and (J) SUCLG2 were measured by qPCR and normalized to RPL13A. Data are represented as mean +/− SEM of three independently performed experiments and each symbol represents an individual experiment. Statistical differences were calculated by 1-way ANOVA followed by Dunnett’s correction for multiple comparisons. (K-L) LDHA/B dKO HEK293T cells were treated with 20 mM L-Lactate and A485/MS-275 inhibitors for 24 hours. Representative western blots are shown in (K) and quantification of anti-Kla signals relative to lane 1 (WT, untreated, unTx) are shown in (L). Signals were normalized to ponceau S staining. Data are represented as mean +/− SEM of three independently performed experiments and each symbol represents an individual experiment. Statistical differences were calculated by 1-way ANOVA followed by Dunnett’s correction for multiple comparisons. (M) Quantification of intracellular lactyl-CoA concentrations in HEK293T cells treated with MS-275 at 5 μM. Data are represented as mean +/− SD of 6 technical replicates. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001