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

Abstract

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...

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

“…1a). These assays demonstrated tight binding of a set of LCFA-CoAs, concurring with published data [14][15][16][17]. However, a set of LCFA-carnitines-a pool of which are present in the peroxisomal matrix [21,22]-were found not to bind to any SCP2 variant.…”

“…Acetyl CoA has previously been observed to lack the ability to displace cis-parinaric acid from SCP2 [14] and has thus provided a convenient negative control ligand in this study. In our assays, no complex was observed with any SCP2 variant after incubation with threefold or even 100-fold molar excess of the acetyl CoA.…”

“…Fig. 2a shows spectra from each variant prepared in the holo-state by saturation with the high-affinity ligand stearoyl CoA [14,16]. Spectra of preSCP2 and preSCP2DAKL are nearly identical to each other, as are mSCP2 and mSCP2DAKL spectra, with the spectral quality deteriorating below about 200 nm.…”

“…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]).…”

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

“…Concomitantly SCP-2 overexpression decreased the slower, vesicular cholesterol transfer -likely by binding and sequestering ligands (i.e. phosphatidylinositides, fatty acyl CoAs) that regulate vesicular trafficking (43,(47)(48)(49)(50)(51)(52)(53).…”