Shape and lipid-binding site of the nonspecific lipid-transfer protein (sterol carrier protein 2): A steady-state and time-resolved fluorescence study

Gadella, Theodorus W. J.; Bastiaens, Philippe I. H.; Visser, Antonie J. W. G.; Wirtz, K.W.A.

doi:10.1021/bi00236a031

Cited by 37 publications

(25 citation statements)

References 65 publications

(101 reference statements)

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“…SCP-2 (44). The range in light scatter distribution of 1 mM SCP-2, from 25-58 Å, was also consistent with the fact that SCP-2 is not perfectly spherical but is somewhat ellipsoidal as previously determined by fluorescence techniques (44,61). Thus, the light scatter data demonstrated that both unliganded SCP-2 and liganded SCP-2 were not aggregated or in multimeric form at the millimolar concentrations necessary for 13 C NMR experiments.…”

Section: Discussionsupporting

confidence: 88%

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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Section: Discussionsupporting

confidence: 88%

Holo-sterol Carrier Protein-2

Stolowich¹,

Frolov²,

Petrescu³

et al. 1999

Journal of Biological Chemistry

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“…Finally, mammalian tissues contain a 58-kDa protein, SCP-x, sharing the same C-terminal 123 amino acid sequence (13.2 kDa) as found in SCP-2 (42). The SCP-x is localized exclusively in peroxisomes (13,38,39) and, furthermore, displays fatty acylCoA thiolase activity, an enzyme involved in ␤-oxidation of fatty acids (42).…”

Section: Discussionmentioning

confidence: 99%

Sterol Carrier Protein-2, a New Fatty Acyl Coenzyme A-binding Protein

Frolov

Cho

Billheimer

et al. 1996

Journal of Biological Chemistry

136

174

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The ability of sterol carrier protein-2 (SCP-2) to interact with long chain fatty acyl-CoAs was examined. SCP-2 bound fluorescent fatty acyl-CoAs at a single site with high affinity. K d values for cis-and trans-parinaroylCoA were 4.5 and 2.8 nM, respectively. Saturated 10 -18-carbon and unsaturated 14 -20-carbon fatty acyl-CoAs displaced SCP-2-bound fluorescent ligand. Oleoyl-CoA and oleic acid (but not coenzyme A) significantly altered SCP-2 Trp 50 emission and anisotropy decay, thereby increasing SCP-2 rotational correlation time, SCP-2 hydrodynamic radius, and SCP-2 Trp 50 remaining anisotropy up to 1.7-, 1.2-, and 1.3-fold, respectively. These changes were not accompanied by significant alterations in protein secondary structure as determined by circular dichroism. Finally, SCP-2 differentially altered the fluorescence emission and anisotropy decays of bound cis-and trans-parinaroyl-CoA. Both fluorescent fatty acyl-CoAs were located within a very ordered (limited cone angle of rotation) environment within SCP-2, as shown by a remaining anisotropy of 0.365 and 0.361 and a wobbling cone angle of 12 and 13°, respectively. These anisotropy values were very close to those of such ligands in a propylene glass. However, the rotational relaxation times exhibited by SCP-2-bound cis-and trans-parinaroyl-CoA, 8.4 -8.8 ns, were longer than those for the corresponding free fatty acid, 7.5-6.6 ns. These data show for the first time that SCP-2 is a fatty acylCoA-binding protein.Long chain fatty acyl-CoAs are not only important in the intermediary metabolism of fatty acids and the production of cellular energy; they are also potent regulators of many intracellular functions including neutrophil signaling, Ca 2ϩ signaling, insulin release, protein acylation, protein kinase activation, and gene transcription (reviewed in Ref. 1). The fatty acyl-CoAs are extremely potent, with regulatory activities occurring in the low nanomolar range (2-6).Cellular concentrations of fatty acyl-CoAs range from 2 to 248 nmol/g of protein, depending on tissue, and are as high as 110 -150 M in the cytosol of normal liver (reviewed in Ref. 1). Furthermore, fatty acyl-CoA levels can vary over a 20-fold range, depending on nutrition, drugs, endocrine status, or oxygenation (reviewed in Ref. 1). Cytosolic fatty acyl-CoAs are found bound to cell membranes (7) and to small nonenzymatic cytosolic proteins.It was recognized over 20 years ago that cytosolic fatty acidbinding proteins, representing 2-6% of cytosolic protein, can bind fatty acyl-CoAs (reviewed in Ref. 1). More than 17 members of this family have thus far been identified, with most of them having low micromolar affinities for fatty acyl-CoAs. More recently a totally different fatty acyl-CoA-binding protein (ACBP), 1 unrelated to the fatty acid-binding protein superfamily, was reported (8). ACBP has higher affinity for fatty acylCoAs than the fatty acid-binding proteins (9, 10).In contrast to the ACBP, the cellular sterol carrier protein-2 (SCP-2) has long been associated with the metabolism...

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“…By taking advantage of the fact that 13-kDa SCP-2 contains only a single Trp residue (amino acid position 50) and no tyrosines, the intrinsic fluorescence properties of Trp 50 could be utilized to ascertain the location of the Trp 50 and the overall shape, and hydrodynamic radius of 13 kDa SCP-2 [31;50]. Steady state fluorescence emission spectra as well as Stern-Volmer quenching (acrylamide and iodide) studies revealed that the Trp 50 was located in a hydrophobic locus.…”

Section: Fluorescencementioning

confidence: 99%

“…Steady state fluorescence emission spectra as well as Stern-Volmer quenching (acrylamide and iodide) studies revealed that the Trp 50 was located in a hydrophobic locus. According to time-resolved fluorescence spectroscopy of Trp 50 , 13-kDa SCP-2 (isolated from bovine liver) exhibited a rotational correlation time of 15 ns [50]. In contrast, phase-and modulation-resolved fluorescence spectroscopy of Trp 50 in human recombinant 13-kDa SCP-2 showed a significantly smaller rotational correlation time near 7.8 ns, consistent with SCP-2 being a globular protein with a hydrodynamic radius of 20.5 Å, diameter 41 Å [31].…”

Section: Fluorescencementioning

confidence: 99%

Sterol carrier protein-2: structure reveals function

Stolowich

Petrescu²,

Huang³

et al. 2002

CMLS, Cell. Mol. Life Sci.

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The multiple actions of sterol carrier protein-2 (SCP-2) in intracellular lipid circulation and metabolism originate from its gene and protein structure. The SCP-x/pro-SCP-2 gene is a fusion gene with separate initiation sites coding for 15-kDa pro-SCP-2 (no enzyme activity) and 58-kDa SCP-x (a 3-ketoacyl CoA thiolase). Both proteins share identical cDNA and amino acid sequences for 13-kDa SCP-2 at their C-termini. Cellular 13-kDa SCP-2 derives from complete, posttranslational cleavage of the 15-kDa pro-SCP-2 and from partial posttranslational cleavage of 58-kDa SCP-x. Putative physiological functions of SCP-2 have been proposed on the basis of enhancement of intermembrane lipid transfer (e.g., cholesterol, phospholipid) and activation of enzymes involved in fatty acyl CoA transacylation (cholesterol esters, phosphatidic acid) in vitro, in transfected cells, and in genetically manipulated animals. At least four important SCP-2 structural domains have been identified and related to specific functions. First, the 46-kDa N-terminal presequence present in 58-kDa SCP-x is a 3-ketoacyl-CoA thiolase specific for branched-chain acyl CoAs. Second, the N-terminal 20 amino acid presequence in 15-kDa pro-SCP-2 dramatically modulates the secondary and tertiary structure of SCP-2 as well as potentiating its intracellular targeting coded by the C-terminal peroxisomal targeting sequence. Third, the N-terminal 32 amino acids form an amphipathic a-helical region, one face of which represents a membrane-binding domain. Positively charged amino acid residues in one face of the amphipathic helices allow SCP-2 to bind to membrane surfaces containing anionic phospholipids. Fourth, the hydrophobic faces of the N-terminal amphipathic a helices along with beta strands 4, 5, and helix D form a ligand-binding cavity able to accommodate multiple types of lipids (e. g., fatty acids, fatty acyl CoAs, cholesterol, phospholipids, isoprenoids). Two-dimensional 1H-15N heteronuclear single quantum coherence spectra of both apo-SCP-2 and of the 1:1 oleate-SCP-2 complex, obtained at pH 6.7, demonstrated the homogenous formation of holo-SCP-2. While comparison of the apo- and holoprotein amide fingerprints revealed about 60% of the resonances remaining essentially unchanged, 12 assigned amide residues underwent significant chemical-shift changes upon oleic acid binding. These residues were localized in three regions: the juncture of helices A and B, the mid-section of the beta sheet, and the interface formed by the region of beta strands 4, 5, and helix D. Circular dichroism also showed that these chemical-shift changes, upon oleic acid binding, did not alter the secondary structure of SCP-2. The nuclear magnetic resonance chemical shift difference data, along with mapping of the nearby hydrophobic residues, showed the oleic acid-binding site to be comprised of a pocket created by the face of the beta sheet, helices A and B on one end, and residues associated with beta strands 4, 5, and helix D at the other end of the binding cavity. Furthermore, the ...

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Shape and lipid-binding site of the nonspecific lipid-transfer protein (sterol carrier protein 2): A steady-state and time-resolved fluorescence study

Abstract: Shape and lipid binding site of the nonspecific lipid-transfer protein (sterol carrier protein 2): a steady-state and time-resolved fluorescense study Gadella, T

Cited by 37 publications

References 65 publications

Holo-sterol Carrier Protein-2

Holo-sterol Carrier Protein-2

Sterol Carrier Protein-2, a New Fatty Acyl Coenzyme A-binding Protein

Sterol carrier protein-2: structure reveals function

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