Probing the Ligand Binding Sites of Fatty Acid and Sterol Carrier Proteins: Effects of Ethanol

Schroeder, Friedhelm; Sc, Myers-Payne; Jt, Billheimer; Wood, W. G.

doi:10.1021/bi00037a033

Cited by 102 publications

(130 citation statements)

References 48 publications

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“…Use of methanol in the assay could expand the repertoire of ligands that may be tested in this assay by increasing the solubility of lipid compounds under test. While a previous in vitro study showed that ethanol inhibits binding of some fatty acid derivatives to SCP2 [38], in this mass spectrometric assay we did not detect and inhibition of stearoyl CoA binding to SCP2 as a result of addition of methanol up to 25% (v/v). Indeed, although not strictly quantitated, the presence of methanol seemed to enhance stearoyl CoA binding, maybe as a result of improved ligand solubility or more efficient electrospray ionisation (data not shown).…”

Section: Discussioncontrasting

confidence: 92%

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

Stanley

Versluis

Schultz

et al. 2007

Archives of Biochemistry and Biophysics

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Sterol carrier protein 2 (SCP2) has been investigated by nearly native electrospray ionisation mass spectrometry in the presence of long chain fatty acyl CoAs (LCFA-CoAs) and carnitine derivatives of equivalent fatty acid chain length (LCFA-carnitines). Four SCP2 constructs were compared to examine the influence of the N-terminal presequence and the C-terminal peroxisomal targeting signal on ligand binding. Removal of N-or C-terminal residues did not influence ligand binding. The observation that LCFA-CoAs are high affinity ligands for SCP2 was confirmed, while LCFA-carnitines were demonstrated for the first time not to interact with SCP2. LCFACoAs formed non-covalent complexes with SCP2 of 2:1 and 1:1 stoichiometry, which could be dissociated by elevating the energy of the ions upon entrance to the mass spectrometer. A fluorescence-competition assay using Nile Red butyric acid confirmed the mass spectrometric observations in solution. The physiological significance of the lack of LCFA-carnitine binding by SCP2 is discussed.

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Section: Discussioncontrasting

confidence: 92%

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

Stanley

Versluis

Schultz

et al. 2007

Archives of Biochemistry and Biophysics

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“…Furthermore, liver expresses sterol carrier protein-2 (SCP-2), a 13-kDa protein found in peroxisomes and cytosol (25,26), as well as the 58-kDa sterol carrier protein-x (SCP-x) (27). Finally, serum albumin (68 kDa) is also an abundant liver product, although it is not thought to accumulate in the cytosol.…”

Section: Creation Of L-fabp Nullmentioning

confidence: 99%

“…To measure the binding capacity of column fraction III, a fluorescent ligand, saturation-binding assay was performed with cis-parinaric acid. This fatty acid shows low quantum yield and fluorescence intensity in aqueous environment (22) but high quantum yields upon binding to proteins such as L-FABP (22) or SCP-2 (25). Aliquots of fraction III (Fig.…”

Section: Relative Contribution Of L-fabp To the Maximal Fatty Acid Bimentioning

confidence: 99%

“…5A). Mathematical analysis of this curve revealed the presence of a single low affinity-binding site with a K d of 2.78 Ϯ 0.56 M (Table II) and thus a considerably lower affinity for fatty acids than SCP-2 (K d near 0.2 M) (25). Thus, although SCP-2 was present in fraction III (see above), the residual fatty acid binding of this fraction seems to be largely caused by less specific proteins.…”

Section: Relative Contribution Of L-fabp To the Maximal Fatty Acid Bimentioning

confidence: 99%

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Decreased Liver Fatty Acid Binding Capacity and Altered Liver Lipid Distribution in Mice Lacking the Liver Fatty Acid-binding Protein Gene

Martin

Danneberg

Kumar

et al. 2003

Journal of Biological Chemistry

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183

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Although liver fatty acid-binding protein (L-FABP) isan important binding site for various hydrophobic ligands in hepatocytes, its in vivo significance is not understood. We have therefore created L-FABP null mice and report here their initial analysis, focusing on the impact of this mutation on hepatic fatty acid binding capacity, lipid composition, and expression of other lipid-binding proteins. Gel-filtered cytosol from L-FABP null liver lacked the main fatty acid binding peak in the fraction that normally comprises both L-FABP and sterol carrier protein-2 (SCP-2). The binding capacity for cis-parinaric acid was decreased >80% in this region. Molar ratios of cholesterol/cholesterol ester, cholesteryl ester/triglyceride, and cholesterol/phospholipid were 2-to 3-fold greater, reflecting up to 3-fold absolute increases in specific lipid classes in the order cholesterol > cholesterol esters > phospholipids. In contrast, the liver pool sizes of nonesterified fatty acids and triglycerides were not altered. However, hepatic deposition of a bolus of intravenously injected family (1-3), is found in the liver, intestine, and kidney, but only in liver is it not co-expressed with other members of its family. L-FABP is known to bind fatty acids and various other hydrophobic molecules, although its actual contribution to the lipid-binding capacity of liver cytosol is not known. Given that L-FABP is expressed at very high levels (2-5% of cytosolic protein) in the differentiated hepatocyte (4, 5) and that these levels correlate well with lipid metabolism (2), it can be speculated that L-FABP contributes considerably to hepatic lipidbinding and lipid metabolism. Work with cell-free systems and transfected cells has further strengthened this view. For example, in cell-free preparations L-FABP was shown to stimulate the esterification of oleic acid while inhibiting that of palmitic acid (6). L cells overexpressing L-FABP show increased rates of fatty acid uptake and esterification (7) as well as increased contents of phospholipid and cholesterol esters (8, 9). HepG2 hepatoma cells expressing an L-FABP antisense RNA showed a dose-dependent reduction of fatty acid uptake (10). Furthermore, overexpression of L-FABP in McA-RH7777 hepatoma cells incubated with palmitic acid decreased the synthesis and secretion of triglycerides while increasing beta oxidation and the secretion of apolipoprotein B100 (11). Thus, the various in vitro systems have allowed researchers to propose specific functions of L-FABP in vivo.However, in vitro studies of FABPs have inherent limitations. The only firmly established function of FABPs is the reversible binding of hydrophobic ligands, and these proteins do not exhibit any enzymatic function or energy requirement. This suggests that these proteins play passive (facilitative) roles that, almost by definition, are strongly dependent on the cellular context. One context of the highly expressed L-FABP is the highly differentiated hepatocyte, a cell type featuring an intense lipid metabolism that is not eas...

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“…For over a decade, it was thought that one of the intracellular lipid binding proteins, SCP-2, did not bind fatty acids in the Lipidx-1000 assay and was not involved in fatty acid metabolism (40). However, more recent data using improved fluorescence and 13 C NMR binding assays showed that the SCP-2 binds LCFA (41)(42)(43) and LCFACoA (44) with high affinity, K d values of 200 -400 and 2-4 nM, respectively. Furthermore, SCP-2 also binds branched-chain LCFA (43,45) and may be specifically involved in the peroxisomal oxidation of branched chain LCFA (45,46).…”

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confidence: 99%

Holo-sterol Carrier Protein-2

Stolowich¹,

Frolov²,

Petrescu³

et al. 1999

Journal of Biological Chemistry

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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...

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Probing the Ligand Binding Sites of Fatty Acid and Sterol Carrier Proteins: Effects of Ethanol

Cited by 102 publications

References 48 publications

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

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

Decreased Liver Fatty Acid Binding Capacity and Altered Liver Lipid Distribution in Mice Lacking the Liver Fatty Acid-binding Protein Gene

Holo-sterol Carrier Protein-2

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