Holo-sterol Carrier Protein-2

Abstract

Although sterol carrier protein-2 (SCP-2) stimulates sterol transfer in vitro, almost nothing is known regarding the identity of the putative cholesterol binding site. Although great advances have been made in our understanding of vascular lipid transport via serum lipoproteins, much less is known regarding intracellular trafficking pathways of lipids. While spontaneous desorption and intracellular diffusion of lipids occur, they do not account for (i) the asymmetric distribution of cellular lipids, (ii) the synthesis of cholesterol in and targeted efflux from the relatively cholesterol poor endoplasmic reticulum, (iii) the lysosomal release and intracellular targeting of cholesterol, fatty acids, and glycerides, or (iv) high density lipoprotein-mediated reverse cholesterol transport. Cellular lipids are transferred and targeted within the cell via vesicular pathways, cytosolic proteins, and possibly via as yet undefined interactions between cytosolic proteins and vesicular pathways.In addition to vesicular pathways, intracellular cholesterol is believed to be transferred and targeted within the cell via proteins such as SCP-2 1 (reviewed in Refs. 1-5), caveolin (reviewed in (Refs. 4 and 6 -11), or steroidogenic acute regulatory protein (12). SCP-2 is primarily a soluble protein, caveolin exists in both membrane-bound and soluble homo-and heterocomplex forms, and steroidogenic acute regulatory protein traffics from the endoplasmic reticulum to the inner mitochondrial membrane by mechanism(s) not yet understood.A variety of studies show positive correlation between SCP-2 expression and intracellular cholesterol transfer in vivo: biliary cholesterol secretion (13-15), lung surfactant formation (16), intestinal cholesterol absorption (17, 18), macrophage foam cell formation (19), diabetes (20), and cholesterol oxidation (21-23). Likewise, studies with intact transfected cells overexpressing SCP-2 confirm a role for SCP-2 in intracellular sterol trafficking (24 -30). Despite these observations, very little is known regarding the mechanism whereby SCP-2 transfers cholesterol between membranes. It has been suggested that interaction of SCP-2 with cholesterol (31-34) is essential for SCP-2-mediated intermembrane sterol transfer in vitro (35). Unfortunately, clear demonstration of cholesterol binding or saturation binding of other sterols to SCP-2 has been difficult to achieve.In addition to its involvement in intermembrane cholesterol transfer, recent reports that SCP-2 also interacts with long chain fatty acids (LCFA) and long chain fatty acyl-CoAs (LCFA-CoA) suggest additional role(s) for this protein in intracellular LCFA and/or LCFA-CoA trafficking. The pathway(s) whereby LCFA and their CoA derivatives traffic and are targeted within the cell are as yet undefined (reviewed in Refs. 36 and 37). Increasing evidence shows that the intracellular fatty acid binding proteins function in intracellular trafficking by enhancing LCFA uptake, intracellular diffusion, or metabolic targeting (reviewed in Refs. 36 -39). For over...

Probing lipid- and drug-binding domains with fluorescent dyes

“…Further, our data support the suggestion that mSCP2 has not one but two very long-chain fatty acyl CoA binding sites. 13,20,21 Finally, the direct and high-fidelity binding assay, ITC, strongly supports the data obtained from our competition assay with 1a. The standard deviations from assays with 1a are rather higher than those obtained from ITC but can be considered tolerable for a relatively quick, easy, and more conservative (in terms of reagent consumption) assay.…”

“…4B), as has previously been demonstrated for several long-chain fatty acid CoAs. 20,21 Site 1 was estimated to have a higher occupancy (n ~ 0.73) and stronger binding properties (K d ~ 339 nM) than Site 2 (n ~ 0.59, K d ~ 947 nM), as shown in Table 1.…”

Investigation of the ligand spectrum of human sterol carrier protein 2 using a direct mass spectrometry assay

“…High affinity LCFA-CoA ligands can be found in 1:1 and 2:1 stoichiometry with SCP2. It has previously been reported that SCP2 has a capacity to bind two LCFA-CoA ligands [10,17], but not more than one cholesterol molecule [10]. These earlier reports have been suggestive of a sequential loading of SCP2, first with one LCFA-CoA ligand and then with the second.…”

“…Further to this, different isoforms of the same gene product can be found in a single cell-the SCPx gene can produce an alternate transcript encoding 15 kDa preSCP2 [1,7], identical to the C-terminal domain of SCPx. Both SCPx and preSCP2 carry a type 1 peroxisomal targeting signal (PTS1) tripeptide at the extreme C-terminus [8] and both appear to be substrates for intraperoxisomal proteolytic cleavage to yield 13 kDa mature mSCP2 [1,9,10].…”

“…Amongst the numerous ligands of SCP2 are LCFAs (discussed extensively in [10,11] with structural analysis in [12,13]) and LCFA-CoAs [14][15][16][17][18] as well as a number of intermediates of LCFA b-oxidation-a primary function of the peroxisome [16]. In addition to the high affinity for these ligands, the observed direct association of peroxisomal SCP2 with b-oxidation enzymes acyl CoA oxidase, thiolase A and MFE-2 [19] has led to the suggestion that a function of SCP2 is to stabilise LCFA b-oxidation intermediates and to present them to the appropriate enzymes for further processing (reviewed in [20]).…”