A Proteomic Analysis of Mitochondrial Complex III Inhibition in SH-SY5Y Human Neuroblastoma Cell Line

Background and Objective: Antimycin A (AntA) is a potent Electron Transport System (ETS) inhibitor exerting its effect through inhibiting the transfer of the electrons by binding to the quinone reduction site of the cytochrome bcl complex (Complex III), which is known to be impaired in Huntington's Disease (HD). The current studies were undertaken to investigate the effect of complex III inhibition in the SH-SY5Y cell line to delineate the molecular and cellular processes, which may play a role in the pathogenesis of HD. Method: We treated SH-SY5Y neuroblastoma cells with AntA in order to establish an in vitro mitochondrial dysfunction model for HD. Differential proteome analysis was performed by the nLC-MS/MS system. Protein expression was assessed by western blot analysis. Results: Thirty five differentially expressed proteins as compared to the vehicle-treated controls were detected. Functional pathway analysis indicated that proteins involved in ubiquitin-proteasomal pathway were up-regulated in AntA-treated SH-SY5Y neuroblastoma cells and the ubiquitinated protein accumulation was confirmed by immunoblotting. We found that Prothymosin alpha (ProT alpha) was down-regulated. Furthermore, we demonstrated that nuclear factor erythroid 2-related factor 2 (Nrf2) protein expression was co-regulated with ProT alpha expression, hence knockdown of ProT alpha in SH-SY5Y cells decreased Nrf2 protein level. Conclusion: Our findings suggest that complex III impairment might downregulate ubiquitin-proteasome function and NRF2/Keapl antioxidant response. In addition, it is likely that downregulation of Nrf2 is due to the decreased expression of ProT alpha in AntA-treated SH-SY5Y cells. Our results could advance the understanding of mechanisms involved in neurodegenerative diseases.