title: 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.
authors: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors: Miao
authors: M
authors: Yu
authors: Katherine A
authors: KA
authors: Romer
authors: Thomas J F
authors: TJ
authors: Nieland
authors: Shangzhe
authors: S
authors: Xu
authors: Veronica
authors: V
authors: Saenz-Vash
authors: Marsha
authors: M
authors: Penman
authors: Ayce
authors: A
authors: Yesilaltay
authors: Steven A
authors: SA
authors: Carr
authors: Monty
authors: M
authors: Krieger
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D001693:Biological Transport, Active; D019556:COS Cells; D002522:Chlorocebus aethiops; D003517:Cyclopentanes / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D050356:Lipid Metabolism; D008075:Lipoproteins, HDL / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051122:Scavenger Receptors, Class B / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013882:Thiosemicarbazones / Q000494:pharmacology
chemical_list: C526651:2-hexyl-1-cyclopentanone thiosemicarbazone; D003517:Cyclopentanes; D008075:Lipoproteins, HDL; D050505:Mutant Proteins; D011994:Recombinant Proteins; C454739:Scarb1 protein, mouse; D051122:Scavenger Receptors, Class B; D013882:Thiosemicarbazones; D003545:Cysteine