Exoplasmic cysteine Cys384 of the HDL receptor SR-BI is critical for its sensitivity to a small-molecule inhibitor and normal lipid transport activity

Abstract

The HDL receptor, scavenger receptor, class B, type I (SR-BI), is a homooligomeric cell surface glycoprotein that controls HDL structure and metabolism by mediating the cellular selective uptake of lipids, mainly cholesteryl esters, from HDL. The mechanism underlying SR-BI-mediated lipid transfer, which differs from classic receptor-mediated endocytosis, involves a two-step process (binding followed by lipid transport) that is poorly understood. Our previous structure/activity analysis of the small-molecule inhibitor blocker of lipid transport 1 (BLT-1), which potently (IC 50 ∼ 50 nM) blocks SR-BI-mediated lipid transport, established that the sulfur in BLT-1's thiosemicarbazone moiety was essential for activity. Here we show that BLT-1 is an irreversible inhibitor of SR-BI, raising the possibility that cysteine(s) in SR-BI interact with BLT-1. Mass spectrometric analysis of purified SR-BI showed two of its six exoplasmic cysteines have free thiol groups (Cys251 and Cys384). Converting Cys384 (but not Cys251) to serine resulted in complete BLT-1 insensitivity, establishing that the unique molecular target of BLT-1 inhibition of cellular SR-BI dependent lipid transport is SR-BI itself. The C384S substitution reduced the receptor's intrinsic lipid uptake activity by approximately 60% without dramatically altering its surface expression, homooligomerization, or HDL binding. Thus, a small-molecule screening approach identified a key residue in SR-BI involved in lipid transport, providing a powerful springboard into the analyses of the structure and mechanism of SR-BI, and highlighting the power of this approach for such analyses.

Mechanisms of selective delivery of xanthophylls to retinal pigment epithelial cells by human lipoproteins

“…It has been effectively used to inhibit carotenoid uptake by SR-B1 (36,37,49) in intestinal cells and it does not inhibit other protein transporters (50). Interestingly, it has been found that BLT-1 inhibits cholesterol uptake by SR-B1 in a Cys384-dependent manner (51). BLT-1 attaches to Cys384, which is located in the lumen of the SR-B1 tunnel and blocks cholesterol transport.…”

Biomimetic, synthetic HDL nanostructures for lymphoma

“…Blocker of lipid transport-1 (BLT-1) is a small molecule that binds cysteine-384 in the extracellular loop domain of SR-B1 and inhibits cholesterol flux through SR-B1 without altering the binding of HDL particles to the receptor (19). Thus, treatment of SUDHL-4 cells with BLT-1 allowed a measurement of engagement and cholesterol flux through SR-B1 (Materials and Methods).…”

Intestinal Scavenger Receptors Are Involved in Vitamin K1 Absorption

“…Uptake Experiment-The specific chemical inhibitor of SR-BI, BLT1, significantly decreased apical Caco-2 cell phylloquinone uptake by up to 38.5% when added at 50 nM (ϳIC 50 (28)). When added in excess (10 M (13, 15, 21, 25, 29)), it led to an inhibition of 84.5% of phylloquinone uptake (Fig.…”