C/PPG 2015 – RP1 – 30-line Summary A complex membrane protein called Scap is the central controller of lipid synthesis in animal cells. Located in the endoplasmic reticulum (ER), Scap binds and transports SREBPs (Sterol Regulatory-Element Binding Proteins) to the Golgi where their transcriptionally active portions are released from the membrane so that they can enter the nucleus. Nuclear SREBPs activate transcription of all genes necessary for synthesis of low density lipoprotein (LDL) receptors, cholesterol, and fatty acids. When cholesterol accumulates in ER membranes it binds to Scap, preventing its transport. Processing of SREBPs decreases and this decreases cholesterol synthesis and uptake from LDL. Scap is responsible for statin-induced up-regulation of LDL receptors and lowering of plasma LDL. In liver, insulin stimulates processing of one isoform of SREBP (SREBP- 1c) that activates synthesis of fatty acids and produces hypertriglyceridemia and fatty liver in Type 2 diabetes. Our laboratory discovered the SREBPs, Scap, and the SREBP-processing proteases. We have begun a molecular dissection of Scap, which has 8 transmembrane helices and several large structured hydrophilic loops. We showed that two large luminal loops (Loop1 and Loop7) bind to each other, and this binding is necessary for Scap to exit from the ER. Point mutations that disrupt this intramolecular binding prevent ER exit. When ER membrane cholesterol exceeds a sharp threshold, the cholesterol binds to Loop1 of Scap. We have hypothesized that cholesterol binding disrupts the Loop1-Loop7 complex and this dissociation prevents Scap from binding to COPII proteins that carry the Scap/SREBP complex to the Golgi. Aim 1 is designed to test the Loop1-Loop7 dissociation hypothesis. We recently produced a soluble fusion protein consisting of Loop1 and Loop7 joined by a linker that is cleavable by a protease. After cleavage, the two loops remain associated. So far, cholesterol addition in vitro has not dissociated the loops. We believe that dissociation may require cholesterol to be presented in a membrane. We propose experiments with liposomes to test this hypothesis. We also propose experiments to test the hypothesis that dissociation requires a conformational change in the 6 transmembrane helices that separate Loop1 and Loop7 in native Scap. Aim 2 is designed to identify drug-like molecules that bind to the sterol-binding site on Loop1 and inhibit SREBP processing. We propose a novel screen using a soluble cholesterol-binding bacterial hemolysin as a surrogate for Scap. We will also screen directly for inhibitors of [3H]cholesterol binding to Loop1. Our knockout studies in mice indicate that Scap inhibitors would be effective therapy for hypertriglyceridemia and fatty liver. Our laboratory has pioneered the molecular dissection of Scap and its functional domains, and we are prepared to extend this work to achieve a complete molecular understanding of this important protein machi...