Role for ORPs in 25‐hydroxycholesterol binding to SCAP

Abstract

The mechanism by which 25-hydroxycholesterol influences intracellular lipid homeostasis is not well understood. This oxysterol is known to be ten times more potent inhibitor of SCAP-SREBP translocation to the Golgi than cholesterol itself, however, the question if it acts directly on SCAP or if it influences its function indirectly remains open. Here, we report that radioactive photo-activatable 25-hydroxycholesterol binds directly to SCAP in living cells. Further, we sought to find proteins that could be involved in presenting 25-hydroxycholesterol to SCAP. We focused our efforts on the ORPs (oxysterol binding protein related proteins) family, which members are thought to be involved in the cellular lipid transport and metabolism. First, we investigated the lipid binding properties of ligand binding domains of all twelve ORPs in living cells. We found that five out of twelve ligand binding domains of ORPs bind specifically to 25-hydroxycholesterol and further two of them also bind cholesterol. Second, we demonstrate that one of 25-hydroxycholesterol binding ORPs, namely ORP2, changes its localization after addition of this oxysterol to the culture medium from predominantly cytosolic to a predominant lipid droplet association. Moreover, overexpression of ORP2 affects cellular localization of VAPA (VAMP-associated protein A), recruiting it to the lipid droplets. Further, we show that VAPA interacts with SCAP, a key component of sterol sensing machinery. We postulate that VAPA can act as a bridging protein between SCAP and ORP2, where it would mediate 25-hydroxycholesterol binding to SCAP. Funding was provided by Max Planck Society.