Encoding immune responses: the moonlighting function of alanyl-tRNA synthetase

The emerging role of L-lactate and L-lactylation has been functionally reported in various biological processes. In a recent Nature paper, Li et al. identified alanyl-tRNA synthetases 1/2 as the sensors of L-lactate and lactyltransferases of L-lactylation, conveying repression of innate immune surveillance.

Aside from a metabolic byproduct, L-lactate is increasingly recognized as an important regulator in intracellular signal transduction and cell function remodeling.¹ Unlike cancer cells which utilize aerobic glycolysis and release L-lactate into the extracellular environment, immune cells usually import L-lactate to regulate their function.^2,3 L-lactate can modify proteins via a process known as lactylation (short for L-lactylation). Lactylation was first identified on histone lysine residues as a reversible post-translational modification (PTM) of proteins on the side chain of lysine residues through an acylation reaction in tissue-repairing macrophages in 2019.⁴ In the following years, the roles of lactylation on nonhistone proteins were also functionally characterized.^5,6 Although the erasers of lactylation or delactylases have then been identified as class I histone deacetylases (HDAC1–3)⁷ and Sirtuin 2,⁸ the enzymes that mediate lactylation are still not fully understood, nor the specific mechanisms underlying this process.

The cGAS/STING pathway is essential for host defense and immune homeostasis. Besides sensing microbial DNA, the DNA sensor cGAS can also be activated by endogenous DNA, including cell stress-induced extranuclear chromatin and DNA released from mitochondria, thus promoting autoimmunity, sterile inflammation, and aging. The recognition and defense functions of the cGAS/STING pathway against pathogens are evolutionarily highly conserved, which can be traced back to bacterial defenses against phages.⁹ cGAS/STING signaling regulation is precisely controlled at multiple levels and many PTMs have been identified on protein cGAS,¹⁰ such as SUMOylation, ubiquitination, and phosphorylation. However, it is still elusive whether metabolite-induced modification exists in cGAS.

In a recent study published in Nature, Li et al. ¹¹ found that L-lactate levels were inversely correlated with cGAS-mediated immune responses in patients with human cytomegalovirus infection. Using pharmacological inhibitors and myeloid-specific knockout mice, they found that intracellular L-lactate imported by monocarboxylate transporter 1 (MCT1), rather than that produced by lactate dehydrogenase A, could inhibit cGAMP synthesis and innate immune surveillance, indicating that the inactivation of cGAS is controlled by MCT1-mediated lactate transportation into the cytoplasm. In vitro experiments further confirmed that L-lactate could induce cGAS lactylation in non-denaturing conditions of cell lysates, suggesting that L-lactate inhibits cGAS activity by mediating its lactylation through an enzyme-dependent manner.

Through genome-wide CRISPR screening, alanyl-tRNA synthetases 1 and 2 (AARS1 and AARS2) were identified as key regulatory enzymes in the process of cGAS lactylation. The classical function of AARS1/2 is to catalyze the attachment of L-alanine to tRNA (Ala). Since L-lactate has a similar molecular structure to L-alanine, L-lactate can also specifically bind to AARS1/2, albeit with a lower affinity than L-alanine. Furthermore, global lysine lactylome analysis with LC-MS/MS showed that knockdown of AARS1 or AARS2 inhibits L-lactate-induced lactylation, while overexpression of AARS1 or AARS2 has the opposite effect. These results suggest that AARS1/2 are intracellular L-lactate sensors and lactyltransferases (Fig. 1).

Fig. 1: Regulation of cGAS activity and innate immunity by AARS2-mediated lactylation.

In hyperlactatemia (H-Lac), lactic acidosis (LA), and anxiety patients, excess L-lactates are transported into the cytoplasm by MCT1. Then AARS2 senses L-lactate and mediates the lactylation of cGAS proteins. The lactylation reverses cGAS function from sensing DNA to repelling DNA, resulting in the attenuation of cGAMP production and downstream innate immune responses.

Based on molecular docking and site-directed mutagenesis, AARS1/2 were found to use ATP as the energy source to catalyze protein lactylation through a two-step reaction. First, L-lactate reacts with ATP to generate a lactate-adenylate intermediate (lactate-AMP); then, the lactyl moiety is transferred to specific lysine residues’ epsilon amine group of the target protein. The mechanism of AARS1/2-catalyzed protein lactylation is evolutionarily conserved from bacteria to mammals. Further experiments confirmed that AARS2, rather than AARS1, is the key enzyme that mediates the lactylation of cGAS and attenuates its downstream immune activation. To investigate the mechanism of cGAS inhibition by lactylation, a genetic code expansion orthogonal system was established to incorporate lactylation at a specific site of cGAS. In vitro and in vivo experiments showed that lactylated cGAS has a weaker affinity for DNA and could not effectively form droplets with higher fluidity. Finally, through analysis of mouse models and clinical samples, the authors confirmed that L-lactate-mediated cGAS lactylation results in abnormal immune surveillance in anxiety patients, immunosuppression in vivo, and susceptibility to viral infection.

This work revealed that environmental L-lactate could be taken up by innate immune cells through MCT1 and then intracellular L-lactate could be sensed and catalyzed by AARS1/2 to mediate global lysine lactylation. Moreover, AARS2-mediated lactylation of cGAS plays a key negative regulatory role in cGAS activation and subsequent innate immune responses (Fig. 1). This discovery extends our understanding of the crosstalk between L-lactate metabolism and innate immune regulation, providing a new explanation for the immunosuppressive mechanism of lactate in immune cells. Considering that L-lactate is produced under many different physiological or pathological conditions, such as tumor microenvironment, ischemia, and hyperlactatemia, it warrants future investigations on whether lactate-induced lactylation may also participate in the regulation of cGAS/STING activation and immune response. Moreover, developing therapeutic approaches targeting MCT1 or AARS1/2 may be efficacious to treat or regulate these conditions.

The acetyltransferase activity of AARS was first reported by Shimin Zhao’s group at Cell Research earlier this year.¹² Here, Li et al. comprehensively characterized the process of AARS1/2-mediated lactylation reaction at the molecular level, and performed detailed analyses on how L-lactate is transported into immune cells and how AARS1/2 catalyzes an ATP-dependent conjugation of L-lactate to lysine residue of target proteins. Given this new insight into cGAS regulation and the general effect of lactylation, it will be important to investigate whether other immune-related proteins, such as sensors, adaptors, and transcription factors, can be lactylated by AARS1/2 or through other nonenzymatic manners, under which physiological or pathological setting this occurs, and how this is linked to metabolism and immune states.