Proteostasis decline is a hallmark of aging and contributes to stress-induced premature senescence (SIPS) through proteotoxic stress and mitochondrial dysfunction. Although mitochondria-derived reactive oxygen species (ROS) are critically involved in SIPS, the behavior of nuclear-encoded mitochondrial antioxidant proteins under proteostasis impairment remains poorly understood. Here, we investigated the dynamics of superoxide dismutase 2 (SOD2) during SIPS induced by the proteasome inhibitor MG132 or the lysosomal inhibitor bafilomycin A1 (BAFA1) in human fibroblasts. Both MG132 and BAFA1 induced cellular senescence and caused an early reduction of SOD2 protein levels in the mitochondrial fraction without a corresponding decrease in SOD2 mRNA or total cellular SOD2 protein levels. Immunofluorescence analysis revealed that SOD2 was partially dissociated from mitochondria under these conditions, indicating impaired mitochondrial localization rather than decreased protein abundance. Quantitative colocalization analysis confirmed a significant reduction in SOD2-mitochondria overlap, which was partially restored by short-term co-treatment with antimycin A (AA), a mitochondrial electron transport chain inhibitor that attenuates excessive ROS production. Furthermore, mislocalized SOD2 was frequently associated with aggregate-like structures, and biochemical analyses demonstrated an increase in high-molecular-weight SOD2-containing complexes under proteostasis stress. These findings suggest that proteostasis disruption is associated with early changes in the intracellular distribution and aggregate-associated accumulation of SOD2, leading to a transient reduction of mitochondrial SOD2 and potentially contributing to mitochondrial oxidative imbalance and the progression of cellular senescence.Key words: cellular senescence, MnSOD, SOD2, mitochondria, oxidative stress, proteostasis, MG132, proteasome.
