Pro-sterol Carrier Protein-2

Abstract

Although the 20-amino acid presequence present in 15-kDa pro-sterol carrier protein-2 (pro-SCP-2, the precursor of the mature 13-kDa SCP-2) alters the function of SCP-2 in lipid metabolism, the molecular basis for this effect is unresolved. The presequence dramatically altered SCP-2 structure as determined by circular dichroism, mass spectroscopy, and antibody accessibility such that pro-SCP-2 had 3-fold less ␣-helix, 7-fold more ␤-structure, 6-fold more reactive C terminus to carboxypeptidase A, 2-fold less binding of anti-SCP-2, and did not enhance sterol transfer from plasma membranes. These differences were not due to protein stability since (i) the same concentration of guanidine hydrochloride was required for 50% unfolding, and (ii) the ligand binding sites displayed the same high affinity (7) approaches revealed additional potential roles for SCP-2 in cellular fatty acid metabolism. However, resolving the relative roles of SCP-2 in cholesterol versus fatty acid metabolism has been complicated by the fact that neither the function of the 20-amino acid presequence in 15-kDa pro-SCP-2, the primary SCP-2 gene product, nor how it affects the intracellular targeting of this protein are yet known. Further, little is known about the structure and function pro-SCP-2 because it is posttranslationally completely cleaved to the mature SCP-2 in all tissues examined (reviewed in Ref.2). In addition, pro-SCP-2 is completely (8) or nearly completely cleaved to SCP-2 in cells transfected with the cDNA encoding pro-SCP-2 (9, 10). SCP-2 was originally isolated from liver and other tissues as a 13-kDa soluble protein whose amino acid sequence did not identify any consensus sequences targeting SCP-2 to specific intracellular organelle(s), suggesting a primarily cytosolic localization (11, 12). In contrast, cDNA sequencing revealed that the two SCP-2 gene products, 58-kDa SCP-x and 15-kDa pro-SCP-2, as well as the mature 13-kDa SCP-2 contained an C-terminal SKL peroxisomal targeting sequence, thereby suggesting an exclusive peroxisomal localization (reviewed in Refs. 2 and 13). However, immunogold electron microscopy and immunofluorescence imaging showed that, whereas SCP-x appears almost exclusively peroxisomal, the intracellular localization of SCP-2 is more complex with over half of the total SCP-2 being extraperoxisomal where it is localized diffusely in the cytoplasm as well as associated with endoplasmic reticulum and mitochondria (reviewed in Refs. 2, 3, and 13). The fact that the SCP-x and SCP-2 did not copurify with peroxisomes (catalase) in subcellular fractionation further supported differential intracellular distribution of SCP-2 in the cell.These studies suggest that the 20-amino acid presequence of the pro-SCP-2 may function in some manner to modify the intracellular targeting of this gene product and thereby account for dual functions of SCP-2 gene products in both fatty acid and sterol metabolism. Consistent with this possibility, transfection of cells with the cDNA encoding pro-SCP-2 (8 -10, 14, 15) alters ch...

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

“…Previous spectroscopic studies [32,33] have shown that the presequence of SCP2 does not display regular secondary structure-a conclusion confirmed by the CD data presented here. In contrast to one earlier CD study [34], in which the presence of the presequence was found to significantly reduce the ordered secondary structure content of SCP2, our data are consistent with available NMR data [18,31,32] indicating that the overall fold of preSCP2 and mSCP2, aside from the coiled presequence, does not change. We additionally demonstrate that removal of the PTS1 tripeptide does not alter SCP2 secondary structure and thus is not critical for SCP2 folding, as could be expected from high-resolution structural analyses Fig.…”

“…NR-BA is of similar size and shape to cholesterol and binds SCP2 in a pocket continuous with the LCFA-CoA binding site [17,18]. The NR-BA assay was previously used to demonstrate the 2:1 binding of linoleoyl [16,34] between the isoforms. The presequence and PTS1 are thus implicated in regulating SCP2 localization [9,34].…”

“…The NR-BA assay was previously used to demonstrate the 2:1 binding of linoleoyl [16,34] between the isoforms. The presequence and PTS1 are thus implicated in regulating SCP2 localization [9,34]. The physiological relevance of the lack of LCFA-carnitine binding capacity of SCP2 may relate to the dual role of the peroxisome and the mitochondrion in fatty acid b-oxidation.…”

Inhibition of biogenic membrane flippase activity in reconstituted ER proteoliposomes in the presence of low cholesterol levels

“…We hypothesize that cholesterol directly interacts with biogenic membrane proteins and inhibits activity. Studies have showed that 22-NBD-cholesterol was bound with high affinity (nM) by intracellular lipid binding proteins (SCP-2, ADRP) and was bound to these proteins with orientation similar to cholesterol [37][38][39][40][41][42]. Biogenic membrane flippase which is involved in lipid flip-flop might have similar interaction with cholesterol.…”

Ligand Specificity and Conformational Dependence of the Hepatic Nuclear Factor-4α (HNF-4α)

“…Fluorescent Ligands-Direct binding assays not requiring separation of bound from free ligand were performed using cis-parinaroyl-CoA as described (25)(26)(27)(28)(29)(30). To establish specificity for LCFA-CoA, this assay was repeated using fluorescent fatty acids: cis-parinaric acid and NBDstearic acid.…”