Modalities of Cholesterol Binding and Modulation of the NPC Proteins and Scap

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Title: Modalities of Cholesterol Binding and Modulation of the NPC Proteins and Scap
Author: Motamed, Massoud
Abstract: Low density lipoproteins (LDL ) and related plasma lipoproteins deliver cholesterol to cells by receptor -mediated endocytosis . The lipoprotein is degraded in late endosomes and lysosomes , allowing cholesterol to be released . Export of cholesterol from late endosomes and lysosomes (hereafter referred to as lysosomes ) requires two lysosomal proteins : Niemann -Pick C2 (NPC2 ) , a soluble protein of 132 amino acids ; and NPC1 , a membrane protein with 13 putative membrane -spanning helices . Recessive loss -of -function mutations in either NPC2 or NPC1 produce NPC disease , which causes death owing to lipid accumulation in lysosomes of liver , brain , and lung . Consistent with their cholesterol export role , NPC2 and NPC1 both bind to cholesterol . The cholesterol binding site on NPC1 is located in the NH2 -terminal domain (NTD ) , which projects into the lysosomal lumen . This domain , designated NPC1 (NTD ) , can be expressed in vitro as a soluble protein of 240 amino acids that maintains cholesterol binding activity . This thesis studies NPC2 in detail as summarized below . Despite a shared role as cholesterol binding proteins , NPC2 and NPC1 (NTD ) bind to cholesterol in opposite orientations . The crystal structures of NPC2 and NPC1 (NTD ) have been solved , and NPC2 binds cholesterol with the iso -octyl chain facing the interior of the protein , whereas , NPC1 (NTD ) binds cholesterol with the 3ß -hydroxyl facing the interior of the protein . Another striking difference is the kinetics of this cholesterol binding . NPC2 binds and releases cholesterol rapidly (half -time < 2 min at 4oC ) , while NPC1 (NTD ) binds cholesterol very slowly (half -time > 2 hr at 4oC ) . However , NPC2 can stimulate the rate of cholesterol binding to NPC1 (NTD ) ( >15 -fold in vitro ) . This stimulation of cholesterol binding to NPC1 (NTD ) by NPC2 is believed to occur through a direct transfer of cholesterol from NPC2 to NPC1 (NTD ) . Amino acid residues important for binding or transfer of cholesterol on NPC2 were identified through alanine scan mutagenesis . Residues that decreased binding thermodynamics and /or kinetics mapped to areas surrounding the binding pockets on the crystal structures ; residues that decreased transfer , but not binding , mapped to discrete surface patches near the exposed residues of the binding pockets . These surface patches may be sites where the two proteins interact to transfer cholesterol . The most deleterious binding mutant was P120S , a residue in the cholesterol binding pocket ; the most deleterious transfer mutant was V81D , a residue on the hydrophobic patch extending outward from the cholesterol binding pocket . The above mutants of NPC2 were unable to rescue LDL -stimulated cholesteryl ester synthesis in NPC2 -deficient cells , in contrast to wild -type NPC2 . Once LDL -derived cholesterol leaves the lysosomes , it is transported to the endoplasmic reticulum (ER ) , where it serves a regulatory role in cholesterol homeostasis . In the ER , these regulatory functions include activation of acetyl -coenzyme A acetyltransferase (ACAT ) , allowing for esterification of cholesterol for storage , and regulation of sterol regulatory element–binding protein (SREBP ) localization , a transcription factor that regulates key enzymes for cholesterol synthesis . SREBP cleavage -activating protein (Scap ) is the switch that controls SREBP , and therefore cholesterol synthesis . Scap senses cholesterol abundance in the ER and acts as an escort protein . In sterol depleted cells , Scap escorts SREBP to the Golgi complex , where two proteases cleave SREBP , thereby releasing its transcriptionally active domain so that it can go to the nucleus and activate transcription of genes involved in cholesterol synthesis and uptake . When cholesterol in abundant , the sterol binds to Scap and triggers a conformational change in the protein that prevents it from escorting SREBPs to the Golgi for proteolytic cleavage . Scap is a 1276 amino acid protein that consists of two domains : an N -terminal domain with 8 transmembrane spanning regions and a C -terminal domain that projects into the cytosol and associates with SREBPs . Previous studies have localized the cholesterol -binding activity of Scap to its membrane domain . Studies described in this thesis identify the cholesterol binding pocket in Scap and identify key residues that play an important role in the protein’s responsiveness to cholesterol binding . The first loop region of Scap (hereafter referred to as Scap (Loop1 ) ) was purified as a recombinant protein and found to have cholesterol binding activity . The specificity of this sterol binding was determined through competition studies and shown to be physiologically relevant . Additionally , this binding affinity and specificity was similar to that of the membrane domain of Scap . Subsequently , alanine scan mutagenesis was performed on Scap (Loop1 ) . Through this approach , several mutations of Scap were identified that constitutively adopt the cholesterol -bound state . This data demonstrates that Scap (Loop1 ) binds to cholesterol and that the binding then helps induce the conformational change required for Scap to anchor SREBP in ER membranes .
URI: http : / /hdl .handle .net /2152 .5 /950
Date: 2011-12-14

Citation

Modalities of Cholesterol Binding and Modulation of the NPC Proteins and Scap. Graduate School of Biomedical Sciences. Available electronically from http : / /hdl .handle .net /2152 .5 /950 .

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