Dynamic investigation of hypoxia-induced L-lactylation

Abstract

The recently identified histone modification lysine lactylation can be stimulated by L-lactate and glycolysis. Although the chemical group added upon lysine lactylation was originally proposed to be the L-enantiomer of lactate (K_L-la), two isomeric modifications, lysine D-lactylation (K_D-la) and N-ε-(carboxyethyl) lysine (K_ce), also exist in cells, with their precursors being metabolites of glycolysis. The dynamic regulation and differences among these three modifications in response to hypoxia remain poorly understood. In this study, we demonstrate that intracellular K_L-la, but not K_D-la or K_ce, is up-regulated in response to hypoxia. Depletion of glyoxalase enzymes, GLO1 and GLO2, had minimal impact on K_D-la, K_ce, or hypoxia-induced K_L-la. Conversely, blocking glycolytic flux to L-lactate under hypoxic conditions by knocking out lactate dehydrogenase A/B completely abolished the induction of K_L-la but increased K_D-la and K_ce. We further observed a correlation between the level of K_L-la and hypoxia-inducible factor 1 alpha (HIF-1α) expression under hypoxic conditions and when small molecules were used to stabilize HIF-1α in the normoxia condition. Our result demonstrated that there is a strong correlation between HIF-1α and K_L-la in lung cancer tissues and that patient samples with higher grade tend to have higher K_L-la levels. Using a proteomics approach, we quantified 66 K_L-la sites that were up-regulated by hypoxia and demonstrated that p300/CBP contributes to hypoxia-induced K_L-la. Collectively, our study demonstrates that K_L-la, rather than K_D-la or K_ce, is the prevailing lysine lactylation in response to hypoxia. Our results therefore demonstrate a link between K_L-la and the hypoxia-induced adaptation of tumor cells.

Keywords: LC–MS/MS; hypoxia; lactylation; posttranslational modification (PTM).

Fig. 1.

Schematic representation of lysine L-lactylation (K_L-la) and its two isomers, lysine D-lactylation (K_D-la) and N-ε-(carboxyethyl) lysine (K_ce). K_ce and K_D-la are derived from two glycolysis by-products, MGO and D-LGSH, respectively. Fructose 1,6-P2: Fructose 1,6-bisphosphate; G3P: Glyceraldehyde 3-phosphate; DHAP: Dihydroxyacetone phosphate; 2PG: 2-Phosphoglycerate; PEP: Phosphoenolpyruvate.

Fig. 2.

Hypoxia induces K_L-la, but not K_D-la or K_ce, in WT cultured cells. (A) Schematic representation of hypoxia workflow using WT MCF-7 cells. WT MCF-7 cells were challenged with hypoxia for indicated times (0 h, 12 h, 24 h, and 48 h), followed by measurement of intracellular concentrations of L-lactate, D-lactate, and MGO. Immunoblots were performed to detect intracellular K_L-la, K_D-la, and K_ce levels. (B–D) Relative intracellular levels of L-lactate, D-lactate, and MGO upon hypoxia treatment. Statistical analysis was performed by student’s t test. *** p < 0.001. (E) Immunoblots of metabolic enzymes upon hypoxia treatment. (F–H) Immunoblots of K_L-la, K_D-la, and K_ce upon hypoxia treatment. The immunoblots were run in parallel to (E) and the same amounts of samples were loaded, so the same control was used.

Fig. 3.

Hypoxia induces K_L-la, but not K_D-la or K_ce, in glyoxalase-deficient cells. (A) HEK293T cells deficient in the glyoxalase enzymes (GLO1−/−, GLO2−/−, or GLO1/2−/−) showed similar adaptability to hypoxia to WT cells. (B) Immunoblots of HIF-1α, LDHA, GLO1, and GLO2 were performed to evaluate their expression upon hypoxia treatment in WT or glyoxalase-deficient HEK293T cells. (C–F) Immunoblots of K_L-la, K_D-la, and K_ce in WT (C), GLO1−/− (D), GLO2−/− (E), and GLO1/2 dKO (F) HEK293T cell lines. Whole-cell lysates were collected at the indicated time points of hypoxia treatment and were used for western blot analysis. The immunoblots were run in parallel to (B) and the same amounts of samples were loaded, so the same control was used.

Fig. 4.

Hypoxia-induced K_L-la upregulation is abolished in LDH-deficient cells. (A) Single deletion of LDHA or LDHB did not compromise hypoxia adaptation compared to WT cells. Double knockout of LDHA and LDHB significantly reduced the cell proliferation rate under hypoxic conditions. Statistical analysis was performed by Student’s t test. (B) Immunoblots of HIF-1α, LDHA, LDHB, GLO1, and GLO2 were performed to evaluate their expression upon hypoxia treatment in either WT or LDH-deficient HepG2 cell lines. (C–E) Immunoblots of K_L-la (C), K_D-la (D), and K_ce (E) in either WT or LDH-deficient HepG2 cells with either 24 h hypoxia or 25 mM L-lactate treatment.

Fig. 5.

K_L-la correlates with hypoxia and cancer malignancy. (A) Schematic showing the HIF-1α degradation pathway under normoxia and HIF-1α-induced transcription in response to hypoxia. Molecules in red (antagonists) and green (agonists) are used to modulate the HIF-1α-mediated pathways. (B) Immunoblots of whole-cell lysates of MCF-7 cells in response to VHL inhibitor, which stabilizes HIF-1α in normoxic conditions. (C) Immunoblots of whole-cell lysates of MCF-7 cells in response to hypoxia coupled with PX478, which inhibits HIF-1α-mediated transcription in response to hypoxia. (D) Representative images from human lung adenocarcinoma tissue microarrays stained for K_L-la, HIF-1α, LDHA, and a negative control by omitting the primary antibody. (E) Pearson’s correlation for all tumor samples between K_L-la and HIF-1α staining. (F) Pearson’s correlation for all tumor samples between K_L-la and LDHA staining. (G) Distribution of staining scores for tumor and normal adjacent tissues (n = 90). (H) Distribution of staining scores for grade 1-2 tumor (n = 64) and grade 3 (n = 26) tumor tissues. Statistical analysis was performed by Student’s t test. NAT: normal adjacent tissue.

Fig. 6.

The landscape of hypoxia-regulated histone K_L-la sites. (A) Schematic workflow for identifying and quantifying histone K_L-la in response to hypoxia. (B) Distribution of hypoxia/normoxia ratios for the quantified histone K_L-la sites. A dashed line is drawn to indicate the ratio of 1 (unchanged). (C) Map of the quantified K_L-la sites and their fold changes after hypoxia treatment. A total of 67 K_L-la sites are marked on the corresponding histone proteins, with colors denoting the ratio ranges (red: H/L < 1; orange: 1 ≤ H/L < 1.25; yellow: 1.25 ≤ H/L < 1.5; green: 1.5 ≤ H/L < 1.75; dark blue: 1.75 ≤ H/L < 2; purple: H/L > 2). Homologous K_L-la sites with different flanking sequences are shown separately. Boxed sequences indicate globular domains.