Abstract
Sterol carrier protein-2 (SCP-2) plays an important role in cholesterol trafficking and metabolism in mammalian cells. The purpose of this study was to determine whether SCP-2, under oxidative stress conditions, might also traffic hydroperoxides of cholesterol, thereby disseminating their cytotoxic effects. Two inhibitors, SCPI-1 and SCPI-3, known to block cholesterol binding by an insect SCP-2, were used to investigate this. A mouse fibroblast transfectant clone (SC2F) overexpressing SCP-2 was found to be substantially more sensitive to apoptotic killing induced by liposomal 7α-hydroperoxycholesterol (7α-OOH) than a wild-type control. 7α-OOH uptake by SC2F cells and resulting apoptosis were both inhibited by SCPI-1 or SCPI-3 at a subtoxic concentration. Preceding cell death, reactive oxidant accumulation and loss of mitochondrial membrane potential were also strongly inhibited. Similar SCPI protection against 7α-OOH was observed with two other types of SCP-2-expressing mammalian cells. In striking contrast, neither inhibitor had any effect on H(2)O(2)-induced cell killing. To learn whether 7α-OOH cytotoxicity is due to uptake/transport by SCP-2, we used a fluorescence-based competitive binding assay involving recombinant SCP-2, NBD-cholesterol, and SCPI-1/SCPI-3 or 7α-OOH. The results clearly showed that 7α-OOH binds to SCP-2 in SCPI-inhibitable fashion. Our findings suggest that cellular SCP-2 not only binds and translocates cholesterol but also cholesterol hydroperoxides, thus expanding their redox toxicity and signaling ranges under oxidative stress conditions.
Highlights
Sterol carrier protein-2 (SCP-2) plays an important role in cholesterol trafficking and metabolism in mammalian cells
Immunodetection methods have revealed that the bulk of SCP-2 in most mammalian cells is located in peroxisomes, but significant amounts are found in mitochondria, lysosomes, and cytosol, probably reflecting its broad-scale lipid trafficking activity [7]
Oxidative stress-generated hydroperoxides are well known for their nonspecific cytotoxic effects, in low doses these species may play a more subtle and selective role as redox signaling molecules [32,33,34]
Summary
Sterol carrier protein-2 (SCP-2) plays an important role in cholesterol trafficking and metabolism in mammalian cells. Subsequent work showed that ChOOH transfer could be further accelerated by human recombinant SCP-2, and when isolated mitochondria were used as acceptors, this exacerbated peroxideinduced damage/dysfunction as reflected by loss of membrane potential [18] This was the first reported example of enhanced oxidative toxicity due to LOOH shuttling by a lipid-trafficking protein. In a more recent follow-up to these noncellular studies, we showed that an SCP-2-oxerexpressing transfectant clone of rat hepatoma cells was much more sensitive to apoptotic killing by liposomal 7␣-OOH than a vector control, the evidence linking this to faster peroxide internalization and delivery to mitochondria by the overexpressing cells [19] These and related findings were consistent with direct SCP-2 involvement in these effects, substantive supporting evidence was lacking [19]. Using two recently discovered hydrophobic inhibitors of mosquito SCP-2 that are close to Ch in molecular mass and bind competitively with it to the protein [20, 21], we provide such evidence for three different SCP-2-expressing mammalian cell lines exposed to a 7␣-OOH challenge
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
