Activity-based proteomics analysis revealed that DNJ directly targeted ATP5F1 to alleviate high glucose induced cardiomyocyte injury

BACKGROUND: Diabetic cardiomyopathy (DCM) is a significant complication of diabetes mellitus, characterized by impaired cardiac function and mitochondrial injury. The natural alkaloid 1-deoxynojirimycin (DNJ) has exhibited cardioprotective potential; however, its specific molecular targets in cardiomyocytes are yet to be identified. PURPOSE: This work sought to discover the direct targets of DNJ in cardiomyocytes and elucidate the molecular mechanism behind its cardioprotective activity. METHODS: We first established a cardiomyocyte injury model induced by high glucose. Next, activity-based protein profiling (ABPP) was employed to screen for proteins that directly bind to DNJ. The interactions between DNJ and candidate targets were verified using surface plasmon resonance (SPR), cellular thermal shift assay (CETSA), and molecular docking. Finally, the functional outcomes were validated through small interfering RNA (siRNA) knockdown and plasmid overexpression. RESULTS: ABPP screening identified mitochondrial F0 complex subunit B1 (ATP5F1) as the principal binding target of DNJ. SPR analysis confirmed a high-affinity interaction between DNJ and ATP5F1 (Kd = 52.6 nM). Furthermore, CETSA demonstrated that this binding occurred in living cells. Treatment with DNJ (120 µg/mL) significantly ameliorated high glucose-induced mitochondrial dysfunction, oxidative stress, and apoptosis. Knockdown of ATP5F1 via siRNA attenuated the protective effects of DNJ. Conversely, ATP5F1 overexpression potentiated these protective effects. CONCLUSIONS: ATP5F1 was identified as a direct functional target of DNJ for the first time. DNJ provides cardioprotection by mitigating mitochondrial malfunction, oxidative stress, and apoptosis via its direct interaction with ATP5F1. These findings collectively clarify a new mode of action for DNJ and offer a scientific basis for its possible therapeutic use in DCM.