In 2019, Zhang and colleagues discovered K_L-la that is driven by L-lactate (Zhang et al., 2019). Using high-performance liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS) analysis, they observed a mass shift of 72.021 Da on lysine residues in histone proteolytic peptides. The mass shift corresponds precisely to the addition of a lactyl group to the ε-amino group of a lysine residue. Subsequent immunoblotting, metabolic labelling and MS/MS analysis demonstrated that lysine lactylation is derived from L-lactate (Zhang et al., 2019) (Figure 1). These findings demonstrate that lactylation specifically occurs on lysine residues of histones.

Following the discovery of K_L-la, Gaffney et al. identified its stereoisomer, K_D-la, which occurs on both histones and non-histone proteins (Gaffney et al., 2020). K_D-la is formed via a nonenzymatic reaction by transferring a D-lactyl group from LGSH to lysine residue (Gaffney et al., 2020; Trujillo et al., 2024). LGSH is produced through the glyoxalase pathway, which involves two enzymes: glyoxalase 1 (GLO1) and glyoxalase 2 (GLO2). GLO1 catalyzes the reaction between the glycolysis byproduct methylglyoxal (MGO) and glutathione (GSH) to form LGSH. GLO2 hydrolyzes LGSH to produce D-lactate and regenerate GSH (Figure 1). Subsequent studies showed that K_D-la increased in histone H4 when incubated with LGSH. This result was reproducible with the glycolytic enzyme phosphoglycerate kinase 1 (PGK1). These research findings indicate that LGSH levels are the primary driver of K_D-la formation in cells. Additionally, MGO can directly react with lysine, generating N-ε-(carboxyethyl)-lysine (K_ce), another isomer of lysine lactylation (Galligan et al., 2018; Khadha et al., 2020; Kulkarni and Brookes, 2020; Zhang et al., 2024) (Figure 1).

A variety of chemical tools have been developed to investigate lysine lactylation at the proteomics level. These include specific antibodies for enriching lactylation peptides and bio-orthogonal chemical probes for metabolic labeling (Figure 2). In 2019, Zhang and colleagues introduced a pan anti- K_L-la antibody-based immunoprecipitation method, combined with MS/MS to identify target proteins and modification sites of K_L-la in cells (Figure 2A) (Zhang et al., 2019). This technique has been used in numerous studies to examine the presence of K_L-la in various eukaryotic organisms, including Kupffer cells (Sung et al., 2023), Botrytis cinerea (Gao et al., 2020), maize root (Shi et al., 2023), and Caenorhabditis elegans (Ding et al., 2024). In 2024, they developed second-generation antibodies that effectively distinguish between K_L-la, K_D-la, and K_ce.

Bio-orthogonal chemical analogs has emerged as powerful tools for metabolic labeling and proteomic analysis of PTMs (Stone et al., 2017; Gao and Hannoush, 2018; Parker and Pratt, 2020). These analogs, such as the alkynyl or azido derivatives of PTM donor precursors, are metabolically incorporated into lactylated proteins. Then labelled proteins are conjugated with fluorophores or affinity tags through click chemistry for subsequent fluorescence detection or proteomic analysis (Parker and Pratt, 2020). In 2022, Sun and colleagues developed YnLac, an alkynyl-functionalized bio-orthogonal analog of L-lactate, for profiling of protein lactylation (Figure 2B) (Sun et al., 2022a). This study not only identified lactylation sites on histones but also revealed novel lactylation sites on non-histone proteins, such as nucleolar protein NCL, DNA chaperone HMGB1 and poly (ADP-ribose) polymerase 1 (PARP1). For example, lactylation of PARP1 may regulate its ADP-ribosylation activity, which is essential for its role in DNA repair, indicating a potential regulatory role for lactylation in DNA repair mechanisms (Sun et al., 2022a).

In addition to experimental approaches, computational prediction tools have become a cost-effective ways to identify potential modification sites. In 2021, Jiang et al. introduced the first predictive model for lysine lactylation sites, called FSL-Kla (Jiang et al., 2021). After this, more models have been developed, such as DeepKla and Auto-Kla (Lv et al., 2022; Lai and Gao, 2023). More recently, Yang et al. developed two enhanced frameworks, ABFF-Kla and EBFF-Kla, which integrate both protein sequences and 3D structural features, providing the accuracy of prediction (Yang et al., 2024). These tools improve our ability to identify lactylation sites and offer deeper insights into the substrate proteins and the biological processes they regulate.

Recent studies have identified KATs as key writers that regulate K_L-la. KATs can be classified into three families: the p300/CBP, the MYST, and the GNAT (Berndsen and Denu, 2008; Friedmann and Marmorstein, 2013). The p300/CBP and MYST families are involved in regulating K_L-la in eukaryotes (Table 1). For example, the absence or inhibition of p300/CBP was shown to reduce the lactylation level of proteins such as HMGB1 and MRE11 (Yang et al., 2022; Chen et al., 2024). Yu et al. demonstrated that P300 depletion could reduce the lactylation level of histone, disrupting the binding of P300 to YTHDF2 promoter, and affecting gene expression (Yu et al., 2021). Members of MYST family, KAT8/MOF and KAT5/TIP60, were shown to regulate K_L-la in non-histone proteins such as Vps34 and eEF1A2 (Jia et al., 2023; Xie et al., 2024). These findings indicate that p300/CBP and MYST families serve as writers for both histone and non-histone lactylation.

The GNAT family of acetyltransferases is present in both eukaryotes and prokaryotes (Table 1) (Hentchel and Escalante-Semerena, 2015; Burckhardt and Escalante-Semerena, 2020). Recent studies found that the members of this family, like GCN5, YiaC, and GNAT13, play a role in lysine lactylation (Dong et al., 2022; Wang et al., 2022; Li et al., 2023). Wang and colleagues showed that silencing GCN5 in cells lead to a significant reduction in histone K_L-la (Wang et al., 2022). In prokaryotes, Dong et al. identified 79 potential K_L-la sites regulated by YiaC (Dong et al., 2022). Li et al. also demonstrated that GNAT13 catalyzes lysine lactylation in S. mutans (Li et al., 2023).

In addition to KATs, the aminoacyl-tRNA synthetase (AARS) family has recently been found to catalyze lysine lactylation (Table 1). Mao et al. showed that mitochondrial AARS2 is a lysine lactyltransferase, adding lactyl groups to PDHA1 and CPT2 (Mao et al., 2024). Similarly, Zong et al. demonstrated that cytoplasmic AARS1 in tumor cells binds lactate and catalyzes lactylation of p53 at K120 and K139 residues. Recent studies have identified that AARS1/2 are conserved intracellular sensors of L-lactate and play an essential role as lactyltransferases to stimulate the lysine lactylome in cells (Li H. et al., 2024; Zong et al., 2024).

Lysine deacetylases (KDACs) are enzymes that remove acyl groups from lysine residues. They include histone deacetylases (HDACs), which are Zn²⁺ dependent, and sirtuins (SIRTs), which require NAD⁺ as a co-substrate (Table 1) (Tan et al., 2016; Moreno-Yruela et al., 2022; Yang Y. et al., 2023). Moreno-Yruela et al. screened all 18 HDACs to evaluate their ability to cleave ε-N-L-lactyllysine and found that HDAC1-3 and SIRT1-3 exhibit delactylase activity in vitro. Among them, HDAC1-3 are the most efficient enzymes for removing lactyl groups from lysine residues (Moreno-Yruela et al., 2022). Meanwhile, Zu and colleagues discovered SIRT2 as a key eraser of histone lysine lactylation, which inhibits the proliferation and migration of glioblastoma cells (Zu et al., 2022). Furthermore, research by Zessin’s group revealed that HDAC3 is the most effective delactylase for histones. Its delactylase activity is over 1,000 times more than SIRT2 or other HDAC isoforms (Zessin et al., 2022).

In addition to these findings, Sun et al. utilized genetic code expansion technology to identify delactylases in both bacteria and mammalian cells. Their study identified SIRT1 as a potential delactylase for non-histone proteins (Sun et al., 2022b). Similarly, Jennings and colleagues demonstrated that SIRT2 removes K_D-la from protein pyruvate kinase M2 (PKM2) (Jennings et al., 2021). Recent studies have highlighted SIRT3 as the most effective eraser for non-histone lysine lactylation targets, such as Fis1 and CCNE2 (An et al., 2023; Jin et al., 2023; Mao et al., 2024). However, there is still some debate regarding which enzyme should be considered the primary non-histone delactylase.

In prokaryotes, the most studied lysine deacetylase is the sirtuin 2–like protein CobB, found in Salmonella and E. coli (Starai et al., 2002; Zhao et al., 2004; Yang Y. et al., 2023). CobB serves as the primary deacylase in E. coli, responsible for removing acetylation and succinylation modifications (Zhao et al., 2004). Dong et al. recently confirmed that CobB also functions as an endogenous lysine delactylase in E. coli. Their quantitative proteomics analysis identified 446 endogenous K_L-la sites in E. coli, regulated by CobB, highlighting its role in lysine lactylation dynamics in prokaryotes (Dong et al., 2022).

To date, the study of lysine lactylation readers is still in its early stages. So far only one study has identified a specific reader: bromodomain-containing protein Brg1 (Table 1). Hu et al. demonstrated that Brg1 interacts with H3K18la, influencing cell reprogramming by modulating chromatin structure and gene expression (Hu et al., 2024). While this discovery provides a foundation, the broader research of K_L-la readers and their regulatory roles remains largely unexplored.

These finding highlights the importance of understanding the proteins that regulate lysine lactylation, including its writers, erasers, and readers. However, our understanding of these mechanisms is still in its infancy. Continued investigation is needed to elucidate the regulatory network of these proteins and their biological significance in lysine lactylation.

Lactate, an important carbon source in prokaryotic metabolism (Jiang et al., 2014), influences various cellular processes (Brown et al., 2014). The discovery of lysine lactylation has broadened the biological significance of this metabolite. For instance, Dong et al. profiled the K_L-la proteome in E. coli, revealing widespread lysine lactylation in bacteria. The study identified YiaC functions as a writer enzyme that catalyzes the addition of K_L-la using lactyl-CoA, while CobB, an NAD + -dependent eraser, removes this PTM. Additionally, YdiF was found to catalyze lactyl-CoA formation in E. coli (Dong et al., 2022). Using quantitative proteomic analyses, they identified 1,047 K_L-la sites across 478 proteins candidates in E. coli. Among these, YiaC regulated 79 KL-la sites, while CobB targeted 446 sites. Gene Ontology analysis indicated that K_L-la modified proteins were enriched in pathways such as glycolysis, the TCA cycle, and fatty acid biosynthesis (Figure 3). Notably, YiaC- mediated lactylation on citrate synthase (GltA) and nicotinic acid phosphoribosyltransferase (PncB) suppressed enzymatic activity. In contrast, CobB-mediated delactylation of pyruvate kinase I (PykF) at K382 enhanced enzymatic activity, promoting glycolysis and growth (Figure 3) (Dong et al., 2022). Further studies by Zong et al. identified the lysine lactylome in E. coli, showing substantial overlap with lactylation patterns observed in mammalian cells, suggesting potential evolutionary conservation (Zong et al., 2024). This analysis revealed 4,544 K_L-la sites across 1,704 proteins in E. coli. Functional analysis indicated that K_L-la -modified proteins were predominantly involved DNA and RNA processes.

Lactate also serves as a substrate for numerous pathogenic microbes and has been shown to contribute to their pathogenicity. S. mutans, a facultative anaerobe, ferments host dietary carbohydrate to produce large amounts of L-lactate, a key virulence factor linked to its cariogenicity (Aires et al., 2008; Li et al., 2023). Li and colleagues proposed that S. mutans-derived lactate influences lysine lactylation and regulates various physiological functions associated with its cariogenic potential. Their study demonstrated that K_L-la at lysine 173 of the RNA polymerase subunit α (RpoA) decreased under high-sugar conditions. This reduction enhanced the synthesis of exogenous polysaccharides (EPSs), critical components of cariogenic biofilm, by inducing the expression of glucosyltransferases (Gtfs) and levansucrase (Ftf). Additionally, the study identified GNAT13 as responsible for increasing K_L-la at lysine 173 of RpoA. This dynamic adjustment enables S. mutans to rapidly form cariogenic biofilms in high-sucrose environments, accelerating enamel erosion (Li et al., 2023). Beyond dental caries, S. mutans has also been implicated in bacterial endocarditis, cerebral hemorrhage, and atherosclerosis. Investigating the role of K_L-la lactylation in S. mutans and its contribution to these pathologies hold significant potential for advancing prevention and treatment strategies.

Further research by Wang et al. revealed that lysine lactylation occurs in secreted proteins of the human pathogen Staphylococcus aureus (S. aureus). This modification, driven by lactate concentration, particularly affects alpha-toxin, a key virulence factor in S. aureus infections. Lactylation at lysine 84 of alpha-toxin was found to be essential for its full activity and virulence in infection models. Notably, extracellular lactate levels typically rise during infections, suggesting that pathogenic bacteria may use protein lactylation to enhance toxin-mediated virulence (Wang et al., 2024). These findings not only highlight a novel mechanism by which S. aureus adapts to the host environment, but also point out the potential of targeting lactylation enzymes in S. aureus and other pathogens as a strategy for anti-virulence therapy.

K_L-la, the predominant form of lysine lactylation in humans and other eukaryotes (Zhang et al., 2024), plays a pivotal role in regulating neuronal development (Figure 4). In 2022, Dai et al. found that histone lactylation govern gene expression and facilitate transcriptome remodeling during neural development. Using an in vitro P19 cell neural differentiation system, they showed that inhibiting HDAC1-3 activity triggered a cascade of histone lysine acylation, pre-activating the neuron-specific transcription program (Figure 4A) (Dai et al., 2022). Similarly, Merkuri et al. explored the role of histone K_L-la in neural crest cells (NCC) differentiation. They observed that histone K_L-la was enriched on active enhancers of developmental genes within NCC gene regulatory networks (GRNs). This modification not only induced gene expression but also enhanced chromatin accessibility at these regions. Reducing the deposition of this modification led to the downregulation of NC genes and the impairment of cell migration (Merkuri et al., 2024). These findings define an epigenetic mechanism that integrates lysine lactylation with the GRNs that orchestrate embryonic development.

Histone lactylation has also emerged as a crucial epigenetic factor in cellular reprogramming, directly linking metabolic shifts to gene expression regulation. Its function in both Glis1 and Dux during early reprogramming is exemplified. Glis1 (abbreviation of Gli-like transcription factor 1) is a newly identified reprogramming factor, also known to induce somatic cell reprogramming (Maekawa et al., 2011). Li and colleagues demonstrated that Glis1 directly binds to and opens chromatin at glycolytic genes during the early phases of reprogramming, whereas it closes chromatin at somatic genes. This dual action facilitates reprogramming by inducing a metabolic shift from oxidative phosphorylation to glycolysis, thereby increasing lactate production. This elevation in intracellular lactate enhances histone lactylation, subsequently activating key pluripotent genes such as Oct4 and Sall4 (Li L. et al., 2020). This cascade ultimately facilitates efficient cell reprogramming.

Similarly, Double homeobox protein (Dux) enhances reprogramming efficiency through promoting histone lactylation during the initial stages of induced pluripotent stem cell (iPSC) formation (Hendrickson et al., 2017). In mouse embryonic fibroblasts (MEFs), Hu et al. revealed that Dux recruits p300 via the C-terminal region, which significantly upregulated the level of H3K18 lactylation. Elevated levels of H3K18 lactylation regulate the transition from oxidative phosphorylation to glycolysis, thereby creating a metabolic environment conductive to reprogramming efficiency (Hu et al., 2024) (Figure 4B). These studies emphasize the key role of histone lysine lactylation as an initiator of reprogramming in early cell reprogramming.

Myocardial infarction (MI) triggers a complex immune response that is crucial for acute injury and post-infarction repair. It is characterized by the recruitment and activation of monocytes and macrophages (Fan M. et al., 2023). Wang and colleagues highlighted the role of histone lactylation in post-MI cardiac repair (Wang et al., 2022). Their study demonstrated a significant early increase in H3K18 lactylation within monocytes and macrophages, enhancing the transcription of reparative genes such as Lrg1, Vegf-a, and IL-10. These genes promoted anti-inflammatory and proangiogenic activities, creating an environment conducive to tissue regeneration and improved cardiac function after MI (Figure 4C) (Wang et al., 2022). This finding suggest that histone lactylation as a potential epigenetic regulator with therapeutic implications for cardiac repair.

Conversely, lactate can increase cardiac fibrosis and exacerbate cardiac dysfunction, through pathways such as Endothelial-to-Mesenchymal Transition (EndoMT) following MI (Fan M. et al., 2023). Fan et al. revealed that lactate induces EndoMT via lactylation of Snail, mediated by CBP/p300 enzyme and monocarboxylate transporter (MCT) -dependent signaling. Inhibiting Snail1 lactylation mitigates lactate-induced EndoMT and TGF-β/Smad2 activation after hypoxia/MI. These findings highlight lactate’s dual role in cardiac repair and dysfunction, emphasizing the need to balance its reparative and pathological impacts for better therapeutic outcomes.

The Warburg effect, one of the hallmarks of tumors, produces large amounts of lactate due to the metabolism of glucose via glycolysis. Lactate from tumors contributes to tumor growth and progression by promoting protein lactylation, which regulation of gene expression via an epigenetic modification in cancer cells (Vander Heiden et al., 2009; Palsson-McDermott and O'Neill, 2013).

Zong et al. analyzed the TCGA breast cancer dataset and found that serum lactate levels were elevated in patients with wild-type p53, suggesting a direct role of tumor-derived lactate in the regulating p53 function (Zong et al., 2024). Further studies using a breast cancer mouse model discovered that tumor-derived lactate can promote p53 lactylation and is a natural inhibitor of p53. This process is conducted by AARS1, which acts as a lactate sensor, binding lactate and catalyzing the formation of lactate-AMP. This lactate-AMP is then transferred to lysine residues in the DNA-binding domain of p53, impairing its ability to bind DNA and attenuate its tumor-suppressive activity (Figure 4D). Interestingly, β-alanine competes with lactate for binding to AARS1, thereby preventing p53 lactylation and offering potential therapeutic opportunities for improving chemotherapy (Zong et al., 2024). However, considering the dual functionality of AARS1 in protein translation and lactylation, targeted inhibition strategies should be employed judiciously.

Lactate is secreted into the tumor microenvironment (TME), where it contributes to immune suppression by altering T-cell function (Certo et al., 2021). In tumor-specific cytolytic T lymphocytes (CTLs), lactate drives histone lactylation, which activates the transcription of circATXN7. This circular RNA interacts with NF-κB p65 subunit, sequestering it in the cytoplasm and impeding its nuclear signaling, making tumor-specific CTLs more susceptible to activation-induced cell death (AICD) and thereby reducing their ability to eliminate tumor cells (Figure 4E). Zhou et al. demonstrated that the upregulation of circATXN7 is associated with poor clinical outcomes and resistance to immunotherapy (Zhou et al., 2024). Targeting circATXN7 in T cells may offer a novel strategy to prevent tumor-mediated immune suppression and improve responses to immunotherapy.

Lactate also alters the immune landscape by affecting tumor-associated macrophages (TAMs). Li X.-M. et al. (2024) found that tumor-derived lactate promotes H3K18 lactylation in TAMs, inhibiting the expression of RARγ, a nuclear receptor that suppresses NF-κB signaling. This mechanism leads to the persistent activation of NF-κB, increasing interleukin-6 (IL-6) levels in the TME and enhancing the tumor-promoting functions of macrophages via STAT3 signaling (Figure 4F) (Li X.-M. et al., 2024). These findings reveal a new mechanism by which lactate-driven macrophage functional remodeling supports tumorigenesis. Furthermore, the authors identified nordihydroguaiaretic acid (NDGA) as a promising compound that directly targets RARγ, disputing inflammation-associated tomor growth in the TME. This discovery underscores the potential of targeting lactate-driven pathways as therapeutic strategies for cancer, specifically by modulating macrophage signaling and immune suppression.