Lactylation modification of HIF-1α enhances its stability by blocking VHL recognition

Abstract Hypoxia-inducible factor 1α (HIF-1α) is a master regulator of cellular adaptation to hypoxia. Although prolyl hydroxylation-mediated degradation via the von Hippel–Lindau (VHL) ubiquitination complex is a well-established regulatory mechanism, the role of lactate-induced posttranslational modifications in HIF-1α stabilization remains incompletely understood. Here, we demonstrate that lactate induces lysine lactylation of HIF-1α at distinct residues across species—specifically, K644 in mice and K12 in humans and pigs—to increase protein stability by impairing VHL recognition. Mass spectrometry and mutagenesis analyses revealed that lactylation at these sites reduces K48-linked ubiquitination and proteasomal degradation, even when HIF-1α is hydroxylated. Structural modeling and functional assays revealed that lactylation sterically hinders VHL binding without affecting hydroxylation. Notably, lactylated HIF-1α exhibited increased transcriptional activity, as evidenced by increased promoter occupancy and upregulation of hypoxia-responsive genes (Vegfa, Glut1). Cross-species comparisons highlighted evolutionary divergence in lactylation sites while preserving the functional conservation of this modification. Our findings reveal that lactylation is a universal regulatory mechanism that overrides classical hydroxylation-dependent degradation, expanding our understanding of metabolic control over hypoxic signaling.