“…These findings suggested a large α-helix content. Analysis of the CD spectrum as indicated in Materials and Methods showed that mrACBP had 56% α-helix, consistent with NMR data of native ACBP ( , ). 5 Effect of liposome surface curvature and lipid composition on mrACBP circular dichroism spectra.…”
Section: Resultssupporting
confidence: 82%
“…Although several intracellular proteins have been discovered that bind LCFA-CoAs (), only acyl-CoA binding protein exclusively binds C 12 −C 22 LCFA-CoAs with high affinity ( K d = 2−10 nM) (). Acyl-CoA binding protein (ACBP) is primarily a 10 kDa cytosolic protein, highly conserved and widely distributed among all eukaryotic organisms and tissues tested ( , ).…”
Although acyl-CoA binding protein (ACBP) stimulates utilization of long-chain fatty acyl-CoA by a variety of membrane-bound enzymes, it is not known whether ACBP directly interacts with membranes. To test this hypothesis, mouse recombinant (mr) ACBP was engineered to contain the native mouse ACBP amino acid sequence expressed as a fusion protein at high levels (>150 mg/L) in Escherichia coli. Purification and cleavage of the fusion tag resulted in mrACBP identical to native ACBP as shown by mass (10000.5 Da) and amino acid sequence (peptide mapping after proteolysis) determined by matrix-assisted laser desorption time of flight (MALDI-TOF) mass spectroscopy. The mrACBP was functionally active as shown by binding of cis-parinaroyl-CoA with high affinity, K(d) = 12 +/- 2 nM, at a single binding site, stimulating oleoyl-CoA utilization by microsomal glycerol-3-phosphate acyltransferase 3.2-fold and protecting oleoyl-CoA from microsomal acyl-CoA hydrolase. Direct interaction of mrACBP with membranes was demonstrated by two independent methods: (i) Circular dichroism showed an 8% increase in alpha-helix content of mrACBP in the presence of anionic phospholipid-rich, but not neutral, small unilamellar vesicles (SUV). (ii) Membrane filtration confirmed that mrACBP bound to anionic phospholipid-rich SUV but only weakly interacted with neutral SUV or large unilamellar vesicles (LUV), regardless of charge. (iii) The mrACBP-oleoyl-CoA complex transferred 2-3-fold more oleoyl-CoA to anionic phospholipid-rich SUV than to anionic phospholipid-rich LUV and neutral SUV or LUV. Conversely, mrACBP extracted less oleoyl-CoA from anionic phospholipid-rich SUV. Taken together, these data indicated for the first time that mrACBP interacted preferentially with anionic phospholipid-rich, highly curved membranes to facilitate transfer of ACBP-bound ligands.
“…These findings suggested a large α-helix content. Analysis of the CD spectrum as indicated in Materials and Methods showed that mrACBP had 56% α-helix, consistent with NMR data of native ACBP ( , ). 5 Effect of liposome surface curvature and lipid composition on mrACBP circular dichroism spectra.…”
Section: Resultssupporting
confidence: 82%
“…Although several intracellular proteins have been discovered that bind LCFA-CoAs (), only acyl-CoA binding protein exclusively binds C 12 −C 22 LCFA-CoAs with high affinity ( K d = 2−10 nM) (). Acyl-CoA binding protein (ACBP) is primarily a 10 kDa cytosolic protein, highly conserved and widely distributed among all eukaryotic organisms and tissues tested ( , ).…”
Although acyl-CoA binding protein (ACBP) stimulates utilization of long-chain fatty acyl-CoA by a variety of membrane-bound enzymes, it is not known whether ACBP directly interacts with membranes. To test this hypothesis, mouse recombinant (mr) ACBP was engineered to contain the native mouse ACBP amino acid sequence expressed as a fusion protein at high levels (>150 mg/L) in Escherichia coli. Purification and cleavage of the fusion tag resulted in mrACBP identical to native ACBP as shown by mass (10000.5 Da) and amino acid sequence (peptide mapping after proteolysis) determined by matrix-assisted laser desorption time of flight (MALDI-TOF) mass spectroscopy. The mrACBP was functionally active as shown by binding of cis-parinaroyl-CoA with high affinity, K(d) = 12 +/- 2 nM, at a single binding site, stimulating oleoyl-CoA utilization by microsomal glycerol-3-phosphate acyltransferase 3.2-fold and protecting oleoyl-CoA from microsomal acyl-CoA hydrolase. Direct interaction of mrACBP with membranes was demonstrated by two independent methods: (i) Circular dichroism showed an 8% increase in alpha-helix content of mrACBP in the presence of anionic phospholipid-rich, but not neutral, small unilamellar vesicles (SUV). (ii) Membrane filtration confirmed that mrACBP bound to anionic phospholipid-rich SUV but only weakly interacted with neutral SUV or large unilamellar vesicles (LUV), regardless of charge. (iii) The mrACBP-oleoyl-CoA complex transferred 2-3-fold more oleoyl-CoA to anionic phospholipid-rich SUV than to anionic phospholipid-rich LUV and neutral SUV or LUV. Conversely, mrACBP extracted less oleoyl-CoA from anionic phospholipid-rich SUV. Taken together, these data indicated for the first time that mrACBP interacted preferentially with anionic phospholipid-rich, highly curved membranes to facilitate transfer of ACBP-bound ligands.
“…Accumulated lipid was enriched not only with cholesterol and phospholipid, but even more so cholesteryl ester than in LKO. While loss of SCP-2/SCP-x L-FABP would be expected to decrease hepatic uptake of cholesterol and decrease endoplasmic reticulum formation of cholesteryl esters (via ACAT-2) and glycerides, this appeared to be counteracted in part by compensatory: i) increased levels of L-FABP and ACBP, which stimulate microsomal ACAT-2 mediated cholesterol esterification [24;26;99;100] and glyceride formation [30;31;37;39;104]; ii) increased levels of hepatic L-FABP to increase APO B loading consistent with increased appearance of APO B in serum. Finally, loss of SCP-2/SCP-x (DKO, TKO) increased hepatic total non-esterified fatty acid levels (Table 5, NEFA).…”
Although roles for both sterol carrier protein-2/sterol carrier protein-x (SCP-2/SCP-x) and liver fatty acid binding protein (L-FABP) have been proposed in hepatic lipid accumulation, individually ablating these genes has been complicated by concomitant alterations in the other gene product(s). For example, ablating SCP2/SCP-x induces upregulation of L-FABP in female mice. Therefore, the impact of ablating SCP-2/SCP-x (DKO) or L-FABP (LKO) individually or both together (TKO) was examined in female mice. Loss of SCP-2/SCP-x (DKO, TKO) more so than loss of L-FABP alone (LKO) increased hepatic total lipid and total cholesterol content, especially cholesteryl ester. Hepatic accumulation of nonesterified long chain fatty acids (LCFA) and phospholipids occurred only in DKO and TKO mice. Loss of SCP-2/SCP-x (DKO, TKO) increased serum total lipid primarily by increasing triglycerides. Altered hepatic level of proteins involved in cholesterol uptake, efflux, and/or secretion was observed, but did not compensate for the loss of L-FABP, SCP-2/SCP-x or both. However, synergistic responses were not seen with the combinatorial knock out animals-suggesting that inhibiting SCP-2/SCP-x is more correlative with hepatic dysfunction than L-FABP. The DKO-and TKO-induced hepatic accumulation of cholesterol and long chain fatty acids shared significant phenotypic similarities with non-alcoholic fatty liver disease (NAFLD).
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