Multidimensional Proteomics Reveal Metformin's Impact on Interconnected Regulatory Networks of Protein Turnover, Ubiquitination, DNA Damage, and Cell Cycle

Metformin, a first-line therapy for type 2 diabetes, has also been implicated in regulating diverse physiological and pathological processes, including lifespan extension, cancer, and other disease-related conditions. However, its mechanisms of action remain incompletely understood, with many effects still unexplained. In this study, we investigated the impact of metformin on the cellular ubiquitinome and associated protein turnover. Through an integrated analysis combining ubiquitinome profiling with pulsed metabolic labeling, we found that metformin markedly suppresses global protein ubiquitination, including various types of ubiquitin chain linkages, and concurrently inhibits both protein synthesis and degradation. Notably, metformin induces a marked reduction in the ubiquitination of histone H4, a modification closely associated with DNA damage repair. We further establish a mechanistic link whereby metformin regulates DNA damage repair and cell cycle progression through downregulating ubiquitination. Together, our findings demonstrate that metformin modulates ubiquitination and proteostasis, central processes that regulate numerous cellular functions. By identifying histone H4 ubiquitination as a key target, we elucidate a potential mechanism through which metformin influences DNA repair and cell cycle progression. This comprehensive dataset advances understanding of the drug's multifaceted pharmacological activities and provides a valuable resource for future drug development.