Ginsenoside Rg5 targets the KAT8-CISD2 axis to maintain mitochondrial homeostasis and antagonize senescence

• CISD2 was identified by acetylomics as a novel substrate of the KAT8/MSL complex that antagonizes cellular senescence by maintaining mitochondrial homeostasis. • KAT8-mediated acetylation of CISD2 at K74 antagonizes STUB1-dependent ubiquitination at K105. • In C. elegans , MYS-2 interacts with CISD-1 to cooperatively regulate mitochondrial homeostasis and lifespan. • Ginsenoside Rg5 targets the KAT8-CISD2 axis to antagonize cellular senescence by maintaining mitochondrial homeostasis. Mitochondria are central regulators of cellular energy metabolism and its dysfunction drives cellular senescence. CISD2, a mitochondrial outer membrane protein and longevity gene, declines with age, highlighting its role in cellular senescence; however, how its post-translational modifications (PTMs) regulate cellular senescence remains poorly understood. This study aimed to elucidate the molecular mechanisms by which ginsenoside Rg5, as a regulator of histone acetyltransferase KAT8 activity, modulates CISD2 PTMs and thereby exerts anti-aging effects. Using SILAC-based acetyl-proteomics, CISD2 was identified as a substrate of the KAT8/MSL acetyltransferase complex. The interaction between KAT8 and CISD2 was examined by Co-immunoprecipitation, GST pull-down, cycloheximide chase, and protein stability assays. KAT8-mediated acetylation of CISD2 was evaluated using in vitro lysine acetyltransferase assays, modification-specific antibodies, and site-directed mutagenesis. The functional impact of CISD2 acetylation on mitochondrial homeostasis was assessed by comparing CISD2 wild-type and K74 mutant cell lines using a series of assays, including ROS production, JC-1 staining, ATP measurement, SA-β-Gal staining, and analyses of mitochondrial morphology. The interaction between Rg5 and KAT8 was investigated using cellular thermal shift assays (CETSA), Rg5-PEGA pull-down, competitive binding assays, and UV-absorption spectroscopy. Evolutionary conservation was evaluated in C. elegans through genetic depletion of mys-2 (the KAT8 homolog) and cisd-1 (the CISD2 homolog). Mitochondrial function (JC-1, DCFH-DA, mtDNA content, and ATP levels) and cellular senescence (SA-β-Gal staining, EdU incorporation, and CCK-8 assays) were assessed in senescent cells and in C. elegans following CISD-1 and/or MYS-2 RNAi. We demonstrate for the first time that the KAT8/MSL complex acetylates CISD2 at K74, thereby preventing STUB1-mediated ubiquitination and degradation at K105. Importantly, acetylation of CISD2 at K74 preserves mitochondrial homeostasis and enhances cellular resistance to oxidative stress and aging. In C. elegans , simultaneous knockdown of MYS-2 and CISD-1 exacerbates mitochondrial dysfunction and shortens lifespan. Moreover, ginsenoside Rg5 directly binds to KAT8, promotes CISD2 acetylation at the K74 site, maintaining mitochondrial homeostasis, and alleviates aging-associated phenotypes in both cells and nematodes. This study reveals that KAT8-mediated acetylation of CISD2 at K74 preserves mitochondrial homeostasis by inhibiting ubiquitin-mediated degradation, representing a critical mechanism for counteracting cellular senescence. Ginsenoside Rg5 acts as a KAT8 agonist to target and activate this pathway, thereby providing a novel strategy for anti-aging intervention. Cellular senescence is associated with downregulation of CISD2 and KAT8 accompanied by reduced CISD2 K74 acetylation. STUB1 promotes CISD2 ubiquitination at K105, thereby accelerating CISD2 protein degradation. Loss of CISD2 acetylation leads to results mitochondrial dysfunction, which in turn exacerbates cellular senescence. In C. elegans, the homologous proteins MYS-2 and CISD-1 interact synergistically to regulate mitochondrial homeostasis and lifespan. Ginsenoside Rg5 enhances CISD2 acetylation by interacting with KAT8, thereby increasing CISD2 stability and expression, maintaining mitochondrial homeostasis, and antagonizing cellular senescence. Moreover, Rg5 partially rescues the shortened lifespan of C. elegans caused by loss of CISD-1 and MYS-2.