l-lactate modifies proteins through lactylation¹, but how this process occurs is unclear. Here we identify the alanyl-tRNA synthetases AARS1 and AARS2 (AARS1/2) as intracellular l-lactate sensors required for l-lactate to stimulate the lysine lactylome in cells. AARS1/2 and the evolutionarily conserved Escherichia coli orthologue AlaRS bind to l-lactate with micromolar affinity and they directly catalyse l-lactate for ATP-dependent lactylation on the lysine acceptor end. In response to l-lactate, AARS2 associates with cyclic GMP–AMP synthase (cGAS) and mediates its lactylation and inactivation in cells and in mice. By establishing a genetic code expansion orthogonal system for lactyl-lysine incorporation, we demonstrate that the presence of a lactyl moiety at a specific cGAS amino-terminal site abolishes cGAS liquid-like phase separation and DNA sensing in vitro and in vivo. A lactyl mimetic knock-in inhibits cGAS, whereas a lactyl-resistant knock-in protects mice against innate immune evasion induced through high levels of l-lactate. MCT1 blockade inhibits cGAS lactylation in stressed mice and restores innate immune surveillance, which in turn antagonizes viral replication. Thus, AARS1/2 are conserved intracellular l-lactate sensors and have an essential role as lactyltransferases. Moreover, a chemical reaction process of lactylation targets and inactivates cGAS.

L-乳酸通过乳酰化修饰蛋白质¹，但这一过程如何发生尚不清楚。在这里，我们将丙氨酰-tRNA 合成酶 AARS1 和 AARS2 （AARS1/2） 确定为 l-乳酸刺激细胞中赖氨酸乳酸组所需的细胞内 l-乳酸传感器。AARS1/2 和进化上保守的大肠杆菌直系同源物 AlaRS 以微摩尔亲和力与 l-乳酸结合，它们直接催化 l-乳酸在赖氨酸受体端发生 ATP 依赖性乳酸化。在 l-乳酸的响应下，AARS2 与环状 GMP-AMP 合酶 （cGAS） 结合，并介导其在细胞和小鼠中的乳酰化和失活。通过建立用于乳酰 - 赖氨酸掺入的遗传密码扩增正交系统，我们证明了在特定 cGAS 氨基末端位点存在乳基部分消除了体外和体内的 cGAS 液体样相分离和 DNA 传感。乳酰模拟物敲入抑制 cGAS，而乳酰基抗性敲入保护小鼠免受高水平 l-乳酸诱导的先天免疫逃避。MCT1 阻断抑制应激小鼠的 cGAS 乳酸化并恢复先天免疫监视，进而拮抗病毒复制。因此，AARS1/2 是保守的细胞内 l-乳酸传感器，作为乳酰转移酶起着重要作用。此外，乳酰化的化学反应过程靶向并使 cGAS 失活。