In the long journey of life sciences research, scientists have continuously unveiled the mysteries of life activities. Recently, the discovery of a novel post-translational modification (PTM)—protein lactylation—has provided a new perspective on the connection between cellular metabolism and gene expression regulation. This article will introduce the discovery of lactylation, its formation mechanisms, biological functions, and research methods while exploring the latest advancements and prospects in this emerging field.

Discovery of Lactylation: From Metabolic Byproduct to Epigenetic Regulator

Lactate, commonly known as a byproduct of glycolysis, has long been regarded merely as a metabolic waste. However, a groundbreaking study in 2019 reshaped our understanding of lactate. Researchers found that lactate can act as a chemical group that covalently binds to proteins, forming a new type of PTM known as lactylation.

This discovery stemmed from scientists' deep exploration of the relationship between cellular metabolism and gene expression regulation. They observed that under certain physiological and pathological conditions, intracellular lactate concentrations significantly increased, accompanied by changes in specific gene expression patterns. This phenomenon could not be fully explained by known regulatory mechanisms, prompting researchers to explore new ways lactate might participate in regulation.

Using advanced mass spectrometry techniques, scientists discovered a modification on various proteins that increases their mass by 72.021 Da, corresponding exactly to a lactyl group. Further studies confirmed that this modification primarily occurs on lysine residues, forming lysine lactylation (Kla). This discovery not only expanded our understanding of PTMs but also provided a novel mechanism for explaining the role of lactate in cellular function regulation.

Figure 1. Regulation of lactate metabolism in normal and cancer cells. (Chen L, et al., 2022)

Formation Mechanisms of Lactylation: Enzymatic and Non-Enzymatic Pathways

With the discovery of lactylation, scientists began to delve into its formation mechanisms. Two major pathways have been identified: enzymatic-dependent and non-enzymatic-dependent pathways.

1. Enzymatic-Dependent Pathway

In this pathway, lactylation is catalyzed by a series of enzymes. Members of the histone acetyltransferase (HAT) family, such as p300/CBP and GCN5, traditionally known for catalyzing protein acetylation, have been found capable of catalyzing lactylation reactions. These enzymes transfer the lactyl group from lactyl-CoA to lysine residues on proteins.

Lactylation is also regulated by de-lactylation enzymes. Histone deacetylases (HDACs), such as HDAC1-4 and SIRT1-3, have been found to remove lactyl groups from proteins. This dynamic addition and removal process allows lactylation to respond flexibly to changes in the cellular environment.

2. Non-Enzymatic-Dependent Pathway

Besides the enzymatic pathway, researchers have discovered a non-enzymatic mechanism for lactylation formation. In this pathway, methylglyoxal (MGO), a byproduct of glycolysis, plays a key role. MGO can combine with glutathione (GSH) to form lactoylglutathione (LGSH), transferring the lactyl group to lysine residues on proteins, forming lactylation. The discovery of this non-enzymatic pathway provides new insights into the elevated levels of lactylation observed under high glycolytic states (e.g., in tumor cells). It reveals a direct link between cellular metabolic status and protein function regulation.

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Biological Functions of Lactylation: From Gene Expression to Cell Fate Determination

As research on lactylation has progressed, scientists have gradually uncovered its critical roles in various physiological and pathological processes. Here, we will explore the functions of lactylation in different biological processes.

Figure 2. Lactate's role in regulating cellular physiological and pathological processes. ( Li X, et al., 2022)

1. Gene Expression Regulation

Lactylation was first discovered on histones, immediately drawing attention to its potential role in epigenetic regulation. Research has shown that histone lactylation can affect chromatin structure and transcription factor binding, thereby regulating gene transcription activity.

For example, lactylation at lysine 18 on histone H3 (H3K18) has been found to promote the expression of certain genes. In macrophages, H3K18 lactylation promotes the expression of anti-inflammatory genes such as arginase 1 (Arg1) and vascular endothelial growth factor α (VEGFα), thus playing a role in the regulation of inflammatory responses.

Additionally, lactylation may indirectly regulate gene expression by affecting the function of other transcriptional regulators. For instance, lactylation can influence the stability or DNA-binding ability of certain transcription factors, altering their regulatory activities.

2. Cell Differentiation and Development

Lactylation plays a crucial role in cell differentiation and development. During early embryonic development, histone lactylation levels increase significantly, reaching a peak at the blastocyst stage. This dynamic change suggests that lactylation may be involved in regulating the gene expression program during early embryogenesis.

In stem cell research, lactylation has been found to regulate the maintenance of pluripotency and differentiation. For example, GLIS1, a member of the GLIS family of zinc finger proteins, promotes histone lactylation, activating the expression of several stem cell-related genes, thereby enhancing reprogramming efficiency. Additionally, lactate has been shown to promote histone lactylation in the promoter regions of muscle differentiation-related genes, thereby facilitating muscle cell differentiation.

3. Tumorigenesis and Development

Tumor cells often exhibit metabolic reprogramming, especially enhanced glycolysis (Warburg effect), leading to significantly elevated intracellular lactate concentrations. Lactylation plays multiple roles in tumorigenesis, development, and metastasis.

First, lactylation can promote the expression of certain oncogenes. For instance, in uveal melanoma, H3K18 lactylation promotes the expression of the YTHDF2 gene, whose protein product facilitates tumorigenesis by degrading tumor suppressor gene mRNA. Second, lactylation contributes to metabolic reprogramming in tumor cells. In non-small cell lung cancer, lactylation upregulates hexokinase 1 (HK-1) expression while downregulating enzymes related to the tricarboxylic acid cycle, maintaining the metabolic characteristics of tumor cells. Lastly, lactylation is involved in shaping the tumor microenvironment and immune evasion. Lactate produced by tumor cells can influence the functions of surrounding immune cells through lactylation, for example, by promoting the lactylation of certain genes in tumor-infiltrating macrophages, thereby enhancing their immunosuppressive functions and helping tumors evade immune surveillance.

4. Immune Regulation

Lactylation plays a significant role in regulating the immune system, affecting the functions of various immune cells, including T cells, B cells, and macrophages. In T cells, lactylation influences their activation and differentiation. For example, lactylation at the K164 site of the IKZF1 protein promotes the differentiation of Th17 cells, which plays a role in certain autoimmune diseases.

In macrophages, lactylation regulates their polarization. High levels of lactate promote the M2 (anti-inflammatory) polarization of macrophages by enhancing histone lactylation at anti-inflammatory gene loci, which is crucial in controlling inflammation and tissue repair. Furthermore, lactylation is involved in regulating the production and release of inflammatory factors. For instance, lactylation of high-mobility group box 1 (HMGB1) facilitates its translocation from the nucleus to the cytoplasm and its release into the extracellular space through exosomes, contributing to the regulation of inflammation.

5. Metabolic Regulation

As a modification derived from a metabolic byproduct, lactylation's role in metabolic regulation is particularly intriguing. Several glycolysis-related enzymes can undergo lactylation, potentially forming a metabolic feedback mechanism. For example, lactylation of aldolase A (ALDOA) can decrease its enzymatic activity, thereby inhibiting glycolytic flux. This mechanism may help cells maintain metabolic balance under high glycolytic states, preventing excessive lactate accumulation. Another example is the lactylation of pyruvate kinase M2 (PKM2). Lactylation of PKM2 enhances the stability of its tetrameric form, increasing its kinase activity and affecting cellular metabolic patterns. These findings highlight lactylation as a crucial link between cellular metabolic status and protein function regulation, offering new insights into how cells adjust their functions based on metabolic states.

Interplay of Lactylation with Other Post-Translational Modifications: A Complex Regulatory Network

As research deepens, scientists have come to realize that lactylation does not exist in isolation but rather interacts with other PTMs to form a complex regulatory network. This interplay further enriches the complexity and flexibility of protein function regulation.

1. Competitive Modifications

The most apparent interaction of lactylation is with acetylation, as both modifications primarily occur on lysine residues and may compete for the same modification sites. For example, on the PARP1 protein, lactylation and acetylation can occur on the same lysine residue. Lactylation may competitively inhibit the acetylation of PARP1, thereby regulating its ADP-ribosyltransferase activity and DNA repair functions. This competitive modification provides a flexible regulatory mechanism, enabling cells to rapidly adjust protein functions based on metabolic states (e.g., lactate concentration).

2. Synergistic Modifications

Beyond competition, lactylation has also been found to synergize with other modifications. For instance, multiple acylation modifications, including lactylation, crotonylation, and 2-hydroxyisobutyrylation, can coexist on certain proteins. This multi-modification phenomenon may jointly regulate protein functions, forming a more complex and diverse regulatory network.

3. Functional Crosstalk

In some cases, lactylation interacts with other PTMs in a manner akin to crosstalk, wherein the presence of one modification influences the occurrence or effects of another. For example, lactylation at a certain lysine site on a protein might inhibit or promote the phosphorylation of a nearby serine or threonine residue, affecting the protein's activity or stability. This crosstalk highlights the intricate and interconnected nature of PTMs in regulating cellular functions.

Research Methods for Lactylation: Challenges and Advances

The rapid development of research on lactylation has been made possible by the advancement of various experimental techniques. However, challenges remain, especially in the precise identification and functional study of lactylation sites. Here, we will discuss the current research methods and their limitations.

1. Mass Spectrometry

Mass spectrometry is the most widely used method for identifying lactylation sites. Researchers have developed specific antibodies for lysine lactylation, enabling the enrichment of lactylated peptides. Combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS), this method can identify lactylation sites with high sensitivity and specificity. However, challenges remain, such as distinguishing lactylation from other acylation modifications with similar mass changes (e.g., acetylation).

2. Antibody-Based Techniques

In addition to mass spectrometry, specific antibodies against lactylation have been developed for various applications, such as western blotting, immunoprecipitation, and chromatin immunoprecipitation (ChIP). These techniques allow the study of lactylation in specific proteins or genomic regions. However, the quality and specificity of antibodies remain a critical factor, and cross-reactivity with other modifications can lead to inaccurate results.

3. Gene Editing

CRISPR/Cas9-mediated gene editing is increasingly being used to study the functional roles of lactylation in specific proteins. By introducing site-specific mutations (e.g., lysine to arginine or glutamine) that prevent lactylation, researchers can assess the impact of lactylation on protein function and cellular processes. However, this approach may have off-target effects, and compensatory mechanisms may obscure the direct effects of lactylation.

4. Computational Prediction

Computational tools for predicting lactylation sites based on protein sequence features and known modification sites have been developed. These tools can guide experimental studies and provide insights into potential lactylation targets. However, the accuracy of these predictions is limited by the availability of known lactylation data and the complexity of PTMs.

5. Biochemical Assays

Biochemical assays, such as in vitro lactylation and de-lactylation reactions, are essential for studying the enzymatic mechanisms of lactylation. These assays can be used to characterize the activities of lactyltransferases and delactylases, as well as to assess the effects of lactylation on enzyme function. However, reproducing the cellular environment in vitro remains challenging, and the relevance of in vitro findings to in vivo conditions must be carefully validated.

Latest Developments in Lactylation Research: A Glimpse into the Future

Since its discovery, lactylation has quickly become a hot topic in life sciences research, leading to a surge of studies exploring its roles in various biological processes and diseases. Here, we highlight some of the most recent developments and emerging trends in lactylation research.

Figure 3. Strategies to enhance immune responses through targeting lactate biosynthesis and acidosis. (Chen L, et al., 2022)

1. Lactylation in Cancer

Recent studies have revealed that lactylation plays a pivotal role in cancer, particularly in the context of tumor metabolism and immune evasion. Researchers are investigating the potential of targeting lactylation pathways as a novel therapeutic approach for cancer treatment. For example, inhibitors of lactyltransferases or delactylases are being developed and tested in preclinical models, to disrupt the pro-tumorigenic effects of lactylation.

2. Lactylation in Immune Regulation

The role of lactylation in immune regulation is an area of intense research. Recent findings suggest that lactylation modulates the function of various immune cells, including T cells, macrophages, and dendritic cells. This has implications for the development of new immunotherapies, particularly in the context of cancer and autoimmune diseases. For example, strategies to modulate lactylation in tumor-associated macrophages are being explored as a means to enhance anti-tumor immunity.

3. Lactylation and Metabolic Diseases

The link between lactylation and metabolic diseases, such as diabetes and obesity, is an emerging area of research. Elevated lactylation levels have been observed in tissues of patients with metabolic disorders, and studies are underway to understand the functional consequences of this modification. Researchers are investigating whether modulating lactylation could be a therapeutic strategy for metabolic diseases, potentially by targeting key enzymes involved in lactylation pathways.

4. Lactylation in Neurological Disorders

The role of lactylation in the nervous system is a relatively unexplored area, but recent studies suggest that it may be involved in neurological disorders such as Alzheimer's disease and Parkinson's disease. Researchers are investigating whether lactylation affects the function of key proteins involved in neurodegeneration and whether modulating lactylation could offer therapeutic benefits.

5. Lactylation in Epigenetics and Development

The discovery of lactylation on histones has opened up new avenues for studying its role in epigenetics and development. Researchers are exploring how lactylation interacts with other histone modifications and how it influences gene expression during development. Recent studies have also begun to investigate the role of lactylation in stem cell biology and tissue regeneration, with potential implications for regenerative medicine.

Conclusion: Lactylation, a Frontier in Life Sciences

The discovery of lactylation has added a new dimension to our understanding of post-translational modifications and their role in cellular regulation. As research progresses, it is becoming increasingly clear that lactylation is a critical link between metabolism and gene expression, with far-reaching implications for various biological processes and diseases. Looking ahead, the study of lactylation is poised to reveal new insights into the mechanisms of disease and offer novel therapeutic strategies. As researchers continue to unravel the complexities of this modification, lactylation is likely to become a central focus in the field of life sciences, offering exciting opportunities for scientific discovery and innovation.

In summary, lactylation is a frontier in life sciences that bridges metabolism and gene regulation. Its discovery has opened up new avenues for research, and ongoing studies are likely to yield significant advancements in our understanding of biology and disease.

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