A trypsin-sensitive protein is required for utilization of exogenous cholesterol for pregnenolone synthesis by placental mitochondria

The utilization of cholesterol for steroid hormone synthesis by human place ntal mitochondria is poorly understood. The human placenta does not express the steroidogenic acute regulator protein, which is critical for cholester ol delivery to the cholesterol side chain cleavage system in adrenal and go nadal mitochondria. We explored the mechanism underlying cholesterol transp ort in human placental mitochondria by measuring its transformation into pr egnenolone. Mitochondria of syncytiotrophoblast from human term placenta we re isolated by centrifugation through a sucrose gradient. The synthesis of pregnenolone in the presence of exogenous cholesterol was increased two-fol d in syncytiotrophoblast mitochondria. Treatment of mitochondria with tryps in prevented the increase in the synthesis of pregnenolone in the presence of exogenous cholesterol. However, when 22-OH cholesterol, a substrate that readily crosses membranes, was added, the trypsin-treated mitochondria syn thesized increased amounts of pregnenolone. The trypsin-treated mitochondri a were intact, since oxygen consumption, succinate dehydrogenase and the ad enine nucleotide translocase activities were not significantly different fr om in untreated mitochondria. However, activity of NADH cytochrome c oxidor eductase, an outer mitochondrial membrane enzyme, was reduced in the trypsi n-treated mitochondria, reflecting the selective degradation of proteins. I n addition, SDS-PAGE analysis revealed the loss of a prominent 34 kDa band which proved to be a novel porin-like protein that binds to cholesterol. Th ese results support our previous assumption that human placental mitochondr ia employ a novel protein(s)-mediated the mechanism to take up cholesterol for steroidogenesis. (C) 2000 Harcourt Publishers Ltd.