Ginkgetin alleviates sepsis-induced acute lung injury by promoting autophagy via inhibiting ubiquitination of Laptm5 in macrophages

• Ginkgetin treatment protects against SI-ALI by inhibiting inflammatory response in vitro/vivo. • Ginkgetin upregulates the protein level of Laptm5, a critical player in the fusion of autophagosomes and lysosomes. • Ginkgetin inhibits the K48-linked ubiquitination of Laptm5 at K86 and K122. • Ginkgetin promoted Laptm5-mediated autophagy and anti-inflammatory effects by directly binding to Ube3c Excessive inflammatory responses mediated by innate immunity are a significant cause of sepsis-induced acute lung injury (SI-ALI), underscoring the need for further drug development to improve therapeutic outcomes. Ginkgetin (GK), a natural flavone, has demonstrated potential anti-inflammatory and antioxidative effects. However, it remains unclear whether GK can ameliorate SI-ALI and what the underlying mechanisms might be. We aimed to investigate the therapeutic effects and mechanisms of GK on SI-ALI. We established SI-ALI models using lipopolysaccharide or cecal ligation and puncture to assess the impact of GK. An autophagy agonist and inhibitor were applied to detect the effect of GK on autophagy. Proteomics and targeted gene knockdown experiments were employed to validate lysosomal-associated transmembrane protein 5 (Laptm5) as the key protein. Co-immunoprecipitation and site mutation assays were used to identify the site where GK inhibited Laptm5 ubiquitination. Biotin pulldown coupled with mass spectroscopy, molecular docking, and drug affinity responsive target stability (DARTS) were performed to elucidate the direct target and underlying mechanisms. We demonstrated that GK activated the autophagosome-lysosome pathway by increasing the protein level of Laptm5. This process facilitated autophagy-mediated degradation of tank-binding kinase 1 (TBK1) and inhibited the signal transduction of downstream inflammatory pathways. Further, we found that GK inhibited the K48-linked ubiquitination of Laptm5 and revealed the ubiquitination sites of Laptm5 (K86 and K122) for the first time. Biotin pulldown and DARTS identified ubiquitin-protein ligase E3C (Ube3c) as a target of GK in inhibiting Laptm5 ubiquitination, with TYR707 and ASN832 being the key residues. Our findings indicate that GK exerts its anti-inflammatory effect on macrophages by promoting autophagy via suppressing the ubiquitination of Laptm5, thus offering a promising therapeutic approach for SI-ALI. Ginkgetin interrupts the interaction between Ube3c and Laptm5, inhibiting the K48-linked ubiquitination of Laptm5. This modification accelerates the autophagic degradation of TBK1 and inhibits proinflammatory activation of macrophages in SI-ALI.