Want to avoid immune "surveillance"? No way! Zhejiang University's latest research published in Nature

Lactic acid plays an important role in human health. Abnormal accumulation of lactic acid is closely related to cancer and immune system diseases with high morbidity and mortality . How does lactic acid "hide" immune surveillance and continue to accumulate in cells? The molecular mechanism behind the problem is crucial, but it is still not fully studied.

Professor Zhang Long's team from the Institute of Life Sciences at Zhejiang University used molecular biology and biochemistry to discover the lactate-sensing proteins alanyl-tRNA synthase 1 (AARS1) and synthase 2 (AARS2), as well as their homologous protein AlaRS in Escherichia coli. They revealed that AARS1/2 plays a key role in mediating proteome lactic acid after sensing lactate, clarified the molecular mechanism by which lactic acid accumulation leads to serious diseases, and provided a new treatment strategy for the poor prognosis caused by lactic acid accumulation.

On September 25, Beijing time, the team published a research paper titled AARS1 and AARS2 Sense L-Lactate to Regulate cGAS as Globally Lysine Lactyltransferases in the journal Nature.

01 Uncovering the “Invisibility Cloak” that Escapes Immune Surveillance

In the human body, normal glucose metabolism is carried out through the tricarboxylic acid cycle, a process that efficiently and completely oxidizes glucose. However, in clinical practice, it has been observed that abnormal glucose metabolism exists in many disease states, manifested as insufficient metabolism of glucose and excessive accumulation of lactic acid. Taking tumor cells as an example, their intracellular lactate concentration is often tens or even hundreds of times higher than that of normal cells. High lactate levels not only disrupt cellular homeostasis, but may also affect the body's resistance to disease. Therefore, a deep understanding of the mechanism of action of lactate in the development of human diseases is of great significance for the development of new treatment strategies.

Cyclic guanosine monophosphate-adenylate synthetase (cGAS) is an immune "monitor" in cells. It can recognize abnormal DNA in cells, induce downstream interferon signals, and activate the immune system. Zhang Long's team used human cytomegalovirus as a starting point to compare the L-lactic acid content in the serum of infected patients with the level of cGAS catalytic product cyclic guanosine monophosphate-adenylate (cGAMP), and found that the content of the two was negatively correlated. This means that lactic acid inhibits the activity of cGAS.

Zhang Long's team further explored how lactate inhibits cGAS activity. Through in vitro incubation experiments, they found that the inhibition of cGAS activity requires the participation of specific proteins in the lysate, indicating that the presence of auxiliary factors plays a key role in L-lactic acid-mediated cGAS inhibition. The experiment also showed that the decrease in cGAS activity was well correlated with the increase in cGAS lactylation levels, indicating that L-lactic acid is likely to regulate its activity by promoting the lactylation modification of cGAS.

In order to identify the genes responsible for sensing intracellular L-lactic acid and mediating cGAS inactivation, the research team performed a genome-wide CRISPR screening in the HT1080 cell line. The results showed that after knocking down AARS1 and AARS2, the overall lactic acid modification level in the cell decreased significantly, indicating that AARS1 and AARS2 may be lactic acid receptors and play a key role in the lactic acid modification process.

If cGAS is a "monitor", then lactylation modification is like an "invisible cloak" covering the surveillance camera, making cGAS lose the ability to recognize and induce immune responses. Just like malignant tumor cells, they evade immune surveillance and continue to proliferate and metastasize without being recognized by the body.

The team further found through structural simulation that the binding mode of human AARS1/2 and E. coli AlaRS to L-lactic acid is similar to L-alanine. After mutating the conserved residues, the ability of these enzymes to bind to L-lactic acid decreased significantly, thus confirming that alanyl-tRNA synthase can specifically bind to L-lactic acid molecules.

Zhang Long said: "AARS1/2 was originally alanine tRNA synthase. We speculate that it may be because L-lactic acid and L-alanine have similar molecular conformations that AARS1/2 will 'mistakenly' recognize L-lactic acid in a high lactate environment."

02 New tools for studying lactylation modification

In vitro experiments have shown that both AARS1/2 and AlaRS can directly catalyze one molecule of L-lactic acid and one molecule of ATP to produce a lactylation modification at one site. "This is the first time since the acylation modification was reported more than half a century ago that people have discovered a catalytic reaction mode that does not rely on coenzyme A. This mode can covalently modify L-lactic acid and ATP, the metabolites of glucose, onto proteins, directly changing the function of important proteins." Zhang Long said that this mechanism also reveals the main way in which lactic acid, as a metabolite, participates in the post-translational modification of proteins.

In order to clarify the impact of cGAS amino-terminal lactylation on its function, the research team developed an orthogonal system for genetic code expansion to prepare site-specific and fully lactylated proteins. This technology provides a powerful tool for detecting the function of proteins after lactylation modification.

In this study, the system prepared site-specific and fully lactylated cGAS protein by integrating lactylated lysine into the cGAS protein, which allowed intuitive observation of the functional differences between the lactylated cGAS protein (cGASLac) and the non-lactated control protein (cGASNon-Lac).

Compared with cGASNon-Lac, cGASLac has a poorer binding ability to 45-bp ISD. cGASNon-Lac and 45bp DNA quickly form larger droplets with higher fluorescence intensity and better fluidity in vitro, while cGASLac has weak binding ability to DNA and tends to self-aggregate to form small, gel-like droplets with low fluorescence recovery ability. It is worth noting that after incubation with long-chain DNA such as 100-bp DNA or HT-DNA in vitro, cGASNon-Lac can effectively form droplets with strong fluidity, while cGASLac tends to aggregate to form gel-like droplets. Compared with cGASNon-Lac, the catalytic activity of cGASLac was also greatly inhibited.

The team verified the inhibitory effect of lactylation modification on cGAS activity in multiple mouse models, once again confirming that cGAS lactylation is an important factor in inhibiting immune surveillance and leading to severe disease.

"In the future, we hope to find a specific way to inhibit the lactate recognition ability of AARS1/2, thereby alleviating hyperlactatemia," Zhang Long said.