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, a reactive 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 HDACs are sufficient to catalyze Kla formation and that HDACs are a major driver of lysine lactylation. 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.

Keywords: glycolysis; histone deacetylase (HDAC); lactate; lactic acid; lysine lactylation; macrophage; post-translational modification (PTM); protein acylation.

Figure 1

HDACs 1, 2, and 3 catalyze lysine lactylation in vitro.A, in vitro lysine lactylation assay with recombinant HDAC1 (rHDAC1), rHDAC2, or rHDAC3/NcoR2 and histone H3 (rH3) in the presence of 1 mM ʟ-Lactate with or without 5 μM TSA. Reactions were performed at 37 °C for 30 min. Protein loading was visualized by ponceau S staining, and Kla was detected by Western blot. Representative of two independent experiments. B–C, HDAC-catalyzed Kla formation kinetics were quantified in a lysine protection assay. B, schematic of experimental design. C, calculated reaction rate of lactylated H3K9 peptide. D–F, HDAC2 KO HEK293T cells expressing 3xFLAG-mHDAC2 were used for immunoprecipitation (IP) with α-FLAG antibody. The immunoprecipitants were used for in vitro lysine lactylation and deacetylation assays. Parental HDAC2 KO HEK293T lacking transgene expression were used as control cells for IP. D, schematic of experimental workflow. E, Western blots for indicated targets of in vitro lysine lactylation using immunoprecipitated 3xF-mHDAC2 WT or the indicated mutants. All lanes also contained L-lactate (5 mM) and rH3 (1 μg). Representative of three independent experiments. F, quantification of lysine lactylation and deacetylation activity for each mutant. Data represent mean ± SEM, relative to WT 3xF-mHDAC2, from three independent experiments. Each symbol represents an individual experiment. G, Schematic of the experimental workflow to assess lactate concentration-dependent reversibility of HDAC using a dialysis system. H, Western blot showing Kla levels before and after dialysis with different ʟ-Lactate concentrations. Representative of two independent experiments. I, proposed model of the forward and reverse reactions catalyzed by class I HDACs. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

Figure 2

Catalytic activities of HDAC1, 2, and 3 are required for Kla formation in cells.A–B, HEK293T cells were treated with the indicated HDAC inhibitors at the specified concentrations for 24 h. A, representative western blots showing anti-Kla and anti-Kac signals. B, quantification of Kla levels relative to untreated control, normalized to ponceau S staining. Data represent mean ± SEM from three independent experiments. Each symbol represents an individual experiment. Statistical significance was determined by one-way ANOVA followed by Dunnett’s correction for multiple comparisons. C–D, HEK293T cells were transfected with siRNAs targeting the indicated genes. C, representative western blots for the indicated targets. D, quantification of Kla levels relative to si-control, normalized by ponceau S staining. Data represent mean ± SEM of three independent experiments. Each symbol represents an individual experiment. Statistical significance was determined by one-way ANOVA followed by Tukey’s test for multiple comparisons. E, quantification of intracellular lactate concentrations in HEK293T cells treated with HDAC inhibitors: Butyrate (5 mM), TSA (1 μM), SAHA (5 μM), MS-275 (5 μM). Data represent mean ± SD from three technical triplicates of a single experiment. Each symbol represents a technical replicate. F, quantification of intracellular lactyl-CoA concentrations in HEK293T cells treated with MS-275 (5 μM). Data are represented as mean ± SD from six technical triplicates of a single experiment, each symbol represents a technical replicate. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 3

Kla formation is regulated by glucose metabolism.A, schematic illustrating anticipated Kla levels in wild-type (WT) and established LDHA/LDHB double knock-out (LDHA/B dKO) cells. B-C, LDHA/B dKO HEK293T cells were treated with ʟ-Lactate at the indicated concentrations for 24 h. B, representative western blots for the indicated targets. C, quantification of Kla levels relative to untreated control, normalized to ponceau S staining. Data are represented as mean ± SEM from four independent experiments. Each symbol represents an individual experiment. Statistical significance was determined by one-way ANOVA followed by Dunnett’s correction for multiple comparisons. D–G, bone marrow-derived macrophages (BMDM) were stimulated with LPS (5 ng/ml) and IFNγ (12 ng/ml) for the indicated time points. MS-275 (0.5 μM) was added 8 h after LPS + IFNγ stimulation, resulting in 16 h of inhibition (from T8 to T24). D, representative western blots showing anti-Kla and anti-Kac signals. E, quantification of Kla levels relative to untreated control, normalized to ponceau S staining. F, quantification of Kac levels relative to untreated control, normalized to ponceau S staining. Data represent mean ± SEM from two independent experiments that each contained two biological replicates; each symbol represents a biological replicate. Statistical significance was determined by one-way ANOVA followed by Tukey’s correction for multiple comparisons. G, quantification of intracellular lactate concentrations in BMDM after 24 h of stimulation with LPS (5 ng/ml) and IFNγ (12 ng/ml), with or without MS-275 (0.5 μM). Data represent mean ± SD from a single experiment with three biological replicates. Each symbol represents a biological replicate. Statistical significance was determined by one-way ANOVA followed by Dunnett’s correction for multiple comparisons. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 4

Acyl transferases are not required for Kla formation.A–C, HEK293T cells were transfected with siRNA targeting the indicated genes. A, representative western blots for the indicated targets. B, quantification of Kla levels relative to si-control. Signals were normalized to ponceau S staining. C, quantification of HBO1 mRNA expression relative to si-control, normalized by RPL13A. B and C, data represent mean ± SEM from three independent experiments and each symbol represents an individual experiment. Statistical significance was determined by one-way ANOVA followed by Dunnett’s correction for multiple comparisons. D–F, LDHA/B dKO HEK293T cells were transfected with siRNAs targeting the indicated genes and treated with ʟ-Lactate 50 mM for 8 h. WT HEK293T and untransfected LDHA/B dKO cells were used as controls. D, representative western blots for the indicated targets. E, quantification of Kla levels relative to si-control, normalized to ponceau S staining. F, quantification of HBO1 mRNA expression relative to si-control, normalized by RPL13A. For (E, F) data are represented as mean ± SEM of three independent experiments and each symbol represents an individual experiment. Statistical significance was determined by one-way ANOVA followed by Dunnett’s correction for multiple comparisons. G-H, LDHA/B dKO HEK293T cells were treated with ʟ-Lactate (20 mM) and either A485 (10 μM) or MS-275 (5 μM) for 24 h. DMSO was used as vehicle control for HDAC inhibitors. WT HEK293T cells not treated with ʟ-Lactate were used as additional controls. G, representative western blots for the indicated targets. H, quantification of Kla levels relative to DMSO-treated controls for each condition, normalized to ponceau S staining. Data represent mean ± SEM from four independent experiments, each symbol represents an individual experiment. Statistical significance was determined by one-way ANOVA followed by Dunnett’s correction for multiple comparisons. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.