Deep-Coverage Ubiquitylomics with Low Starting Materials Reveals Dysregulation of Ubiquitinated FASN as a Key Regulator of MAFLD Pathogenesis

Metabolic-associated fatty liver disease (MAFLD), characterized by excessive hepatic lipid deposition, poses a global health threat, increasing the risk of severe liver complications. Here, we optimized a <b>f</b>ast <b>i</b>n-<b>t</b>ube <b>m</b>icroscale <b>ubi</b>quitylomics strategy (abbreviated as FIT-MUbi, requiring less than 10 mg of liver tissue samples) and identified over 7384 ubiquitinated peptides, constructing a dysregulated ubiquitination network in the MAFLD model. Through bioinformatics analyses, ubiquitinated proteins were significantly enriched in lipid metabolism, particularly in the <i>fatty acid metabolism</i> pathway. Compared with global proteomics analysis alone, this ubiquitylomics approach better captures the typical features of lipid-metabolic dysregulation in the MAFLD model. Protein-protein interaction analysis revealed the fatty acid synthase (FASN) as a key regulator in fatty acid metabolism, showing an elevated protein level and ubiquitination with MS data, which was further confirmed by Western blot analysis. Notably, the PKS/mFAS DH domain of the FASN protein exhibits a substantially higher ubiquitination density (55.6%). By comparison, the overall ubiquitination density of the entire FASN protein is 28%, while that of its ketosynthase family 3 (KS3) domain is even lower, at only 25%. Precisely, we identified K967 as a potential function site regulating FASN stabilization and activity, suggesting that the impaired ubiquitination signal might drive its paradoxical accumulation. Our study explored the application of microscale ubiquitylomics in MAFLD research, unveiling ubiquitination-dependent protein stabilization as a novel regulatory axis. These findings provide mechanistic insights for developing targeted diagnostic and therapeutic strategies to combat MAFLD progression.