Paraquat Inhibits the Processing of Human Manganese-Dependent Superoxide Dismutase by SF-9 Insect Cell Mitochondria

Abstract

Precursor human manganese-dependent superoxide dismutase (hMn-SOD) was expressed using the baculovirus system inSpodoptera fungiperda(Sf-9) insect cells. Following infection of Sf-9 cells with hMn-SOD-expressing baculovirus, mature hMn-SOD was expressed at 15–25% of total cellular protein, with the recombinant protein localized in the mitochondrial matrix. Partial amino acid sequencing and SOD activity assays indicated that the hMn-SOD was correctly processed and assembled by insect mitochondria. This expression system was used to study the effects of paraquat and menadione, two intracellular superoxide generators, on processing of precursor hMn-SOD by insect mitochondria. Paraquat was found to potently inhibit mitochondrial processing of hMn-SOD, leading to the accumulation of precursor hMn-SOD and a decrease in measurable Mn-SOD activity. In contrast, menadione treatment was not found to affect the ratio of precursor to mature Mn-SOD. Paraquat did not lead to lower total production of hMn-SOD or cellular toxicity at the concentrations which were found to block processing of precursor hMn-SOD. These results indicate that mitochondrial processing and import of the precursor protein hMn-SOD are early events susceptible to dysfunction induced by the redox-cycling agent paraquat. These results also emphasize that mitochondrial processing of precursor proteins represents a parameter of cellular function which may be compromised, preceding the appearance of more generalized deterioration of cellular function, under certain toxic or pathological conditions.