The alanyl-tRNA synthetase AARS1 moonlights as a lactyltransferase to promote YAP signaling in gastric cancer

Lactyl-coenzyme A, the molecule that everyone assumed was donating the lactyl group to proteins, sits inside cells at concentrations roughly one thousand times lower than acetyl-CoA, its closest chemical cousin. Let that ratio sink in for a moment. If the lactyl donor is that scarce, the proposed enzyme cannot plausibly be driving a modification that appears on nearly three thousand distinct sites across more than a thousand proteins. Something else must be doing it. A team led by Ju and colleagues in Shanghai found the answer hiding inside a protein that everyone thought had a completely different job. The modification in question is lactylation, a posttranslational modification, meaning a chemical tag added to a protein after it has been built, that alters what that protein does. Lactylation lands on lysine residues of both histone proteins, which package DNA, and non-histone proteins throughout the cell. That breadth matters. When a modification is this widespread, it is doing real regulatory work. Recent studies have linked it to epigenetic control of gene expression and to human disease. So the field needed to know who was putting those tags on. The leading candidate was p300, an acetyltransferase, an enzyme that normally adds acetyl groups, acting via lactyl-CoA as the lactyl donor. But that model encountered a concentration problem. Lactyl-CoA in tumor cells is extremely low, and the enzymes that would generate it from lactate in mammalian cells remain unknown. Ju and colleagues framed that discrepancy as the open question their paper set out to resolve. Their answer was AARS1, alanyl-tRNA synthetase 1. Its canonical job is to charge transfer RNA with the amino acid L-alanine during protein synthesis, a fundamental housekeeping task. But Ju and colleagues discovered that AARS1 moonlights as a bona fide lactyltransferase, directly using free lactate and adenosine triphosphate, or ATP, not lactyl-CoA, to place lactyl groups onto lysine residues of proteins, including histones H3 and H4. The biochemistry is specific and striking. Molecular docking predicted that lactate could occupy AARS1's catalytic pocket, and isothermal titration calorimetry confirmed direct binding. Lactate bound AARS1 with a dissociation constant of 2.06 micromolar, compared to 0.45 micromolar for L-alanine, its canonical substrate. In purified in vitro systems, recombinant AARS1 lactylated histones H3 and H4 in a reaction strictly dependent on both lactate and ATP. The mechanism is a two-step chemistry analogous to tRNA charging. Lactate and ATP react to form a lactyl-adenylate intermediate, releasing pyrophosphate as the reaction proceeds. The amount of pyrophosphate produced in those assays increased with higher lactate concentrations, tracking the enzymatic activity directly. Mutating five residues lining the catalytic pocket, the so-called five M mutant, completely abolished lactylation activity. Critically, when the team supplied the reaction with lactyl-CoA instead, even at concentrations one hundred times higher than physiological levels, AARS1 failed to catalyze lactylation. Any lactylation seen under those conditions was spontaneous and non-enzymatic. This is a mechanistically distinct reaction from anything p300 was proposed to do. Now, knowing that AARS1 is a lactyltransferase raises the next question: what does it lactylate inside a living cell, and what happens when it does? The answer brought the team to one of cancer biology's central signaling systems, the Hippo pathway. In brief, the Hippo pathway is a kinase cascade that normally restrains cell growth by keeping the transcriptional coactivator YAP out of the nucleus. When the pathway is inactivated, YAP enters the nucleus, partners with TEAD transcription factors, and drives proliferation. Ju and colleagues found that AARS1 connects rising intracellular lactate directly to this switch. When intracellular lactate increases, AARS1 moves from the cytoplasm into the nucleus. This translocation depends on a conserved C-terminal nuclear localization signal. The team showed that AARS1 interacts with the importin subunit KPNA4, and that lactate treatment strengthened that interaction, consistent with importin-mediated nuclear import triggered by lactate itself. Once inside the nucleus, AARS1 lactylates YAP at lysine 90 and TEAD1 at lysine 108. These sites were identified through lactylation proteomics on HGC27 gastric cancer cells, a screen that found one thousand one hundred eighty-two lactylated proteins and two thousand seven hundred eighty-nine unique lactylated lysine sites in lactate-treated cells. Mutation of either site to arginine abolished the lactylation signal, and purified AARS1 converted synthetic peptides carrying those lysines to their lactylated forms in vitro. The catalytic-dead five M mutant failed to do so. On the removal side, Ju and colleagues identified SIRT1 as the enzyme that strips lactyl groups from YAP and TEAD1. Nicotinamide, a sirtuin inhibitor, increased YAP lactylation. Overexpressing SIRT1, but not other sirtuins, substantially reduced it. A catalytically dead SIRT1 mutant had no effect. So the system has both a writer and an eraser, with the balance between them controlling YAP-TEAD1 activity. What does lactylation at lysine 90 and lysine 108 actually do? It promotes nuclear retention of YAP-TEAD1 and activates their downstream transcriptional program, including target genes CTGF and CYR61. Mechanistically, this works in part by competing with ubiquitination. The same lysine residues can be either lactylated or ubiquitinated, but not both simultaneously. Most lactylated YAP and TEAD1 is found in the nucleus, while most ubiquitinated and phosphorylated YAP is cytoplasmic. Lactate treatment decreased YAP-TEAD ubiquitination in a dose-dependent manner and inhibited YAP's interaction with the nuclear exportin XPO1. Nuclear lactylation locks the complex in, preventing both export and the cytoplasmic ubiquitination that would mark it for degradation. Here is where the story loops back on itself in a way that matters enormously for cancer. AARS1 is not just upstream of YAP-TEAD; it is a transcriptional target of the very complex it activates. ChIP-Seq after lactate treatment identified eight hundred thirty-two YAP peaks and nine hundred twenty-three TEAD1 peaks, with four hundred twelve overlapping peaks enriched at the AARS1 promoter. Gel shift, ChIP-qPCR, and promoter-reporter assays confirmed that YAP-TEAD1 directly drives AARS1 transcription. The loop runs like this: more intracellular lactate drives more nuclear AARS1 activity, which lactylates and activates YAP-TEAD1, which transcribes more AARS1, which lactylates more YAP-TEAD1. A self-reinforcing circuit that converts a metabolic state into a durable growth signal. A patient-associated mutation makes this causal, not just correlational. R77Q, the most common AARS1 missense variant in gastric cancer databases, located directly in the catalytic pocket, increased AARS1's lactyltransferase efficiency in vitro, produced greater YAP lactylation in cells, upregulated CTGF and CYR61, and drove greater cell growth and colony formation than wild-type AARS1. This is a mutation that turns up the dial on the moonlighting function specifically. In a human gastric cancer tissue array of ninety patients, AARS1 protein was elevated in fifty-nine tumors. Higher AARS1 expression correlated with larger tumors, more lymph node involvement, and advanced stage. To disentangle AARS1's moonlighting role from its housekeeping function, the team reconstituted AARS1-knockout cells with different mutant versions. Wild-type AARS1 rescued YAP-TEAD lactylation, cell proliferation, and tumor growth in both xenograft and orthotopic mouse models. The nuclear localization signal deleted mutant and the catalytic five M mutant failed to rescue any of those outcomes. Importantly, deleting the nuclear localization signal did not impair protein synthesis, confirmed by a puromycin incorporation assay, meaning the loss of tumor-promoting activity was specifically due to the loss of nuclear lactyltransferase function, not to any disruption of transfer RNA charging. That distinction matters. If AARS1's moonlighting activity is separable from its essential housekeeping role, you can potentially inhibit one without crippling the other. Ju and colleagues close by reframing something that cancer metabolism researchers have debated for decades: the Warburg effect, the observation that cancer cells ferment glucose to lactate even when oxygen is plentiful. Lactate has long been thought of as metabolic exhaust, a necessary byproduct of that abnormal metabolism. This paper argues it is a signal. AARS1 reads the lactate concentration, translocates to the nucleus, and converts that metabolic input via ATP-dependent lysine lactylation into a direct instruction to proliferate. The exhaust, it turns out, is the message. This lecture was created by ennepō. Go to https://ennepo.ai to Discover, Create and Follow the latest research in your field. Read when you can. Listen when you want to.