acyl-CoA Binding Research Articles

Unconventional Secretion of AcbA in Dictyostelium discoideum through a Vesicular Intermediate

The acyl coenzyme A (CoA) binding protein AcbA is secreted unconventionally and processed into spore differentiation factor 2 (SDF-2), a peptide that coordinates sporulation in Dictyostelium discoideum. We report that AcbA is localized in vesicles that accumulate in the cortex of prespore cells just prior to sporulation. These vesicles are not observed after cells are stimulated to release AcbA but remain visible after stimulation in cells lacking the Golgi reassembly stacking protein (GRASP). Acyl-CoA binding is required for the inclusion of AcbA in these vesicles, and the secretion of AcbA requires N-ethylmaleimide-sensitive factor (NSF). About 1% of the total cellular AcbA can be purified within membrane-bound vesicles. The yield of vesicles decreases dramatically when purified from wild-type cells that were stimulated to release AcbA, whereas the yield from GRASP mutant cells was only modestly altered by stimulation. We suggest that these AcbA-containing vesicles are secretion intermediates and that GRASP functions at a late step leading to the docking/fusion of these vesicles at the cell surface.

Overexpression of Arabidopsis Acyl-CoA Binding Protein ACBP3 Promotes Starvation-Induced and Age-Dependent Leaf Senescence

In Arabidopsis thaliana, a family of six genes (ACBP1 to ACBP6) encodes acyl-CoA binding proteins (ACBPs). Investigations on ACBP3 reported here show its upregulation upon dark treatment and in senescing rosettes. Transgenic Arabidopsis overexpressing ACBP3 (ACBP3-OEs) displayed accelerated leaf senescence, whereas an acbp3 T-DNA insertional mutant and ACBP3 RNA interference transgenic Arabidopsis lines were delayed in dark-induced leaf senescence. Acyl-CoA and lipid profiling revealed that the overexpression of ACBP3 led to an increase in acyl-CoA and phosphatidylethanolamine (PE) levels, whereas ACBP3 downregulation reduced PE content. Moreover, significant losses in phosphatidylcholine (PC) and phosphatidylinositol, and gains in phosphatidic acid (PA), lysophospholipids, and oxylipin-containing galactolipids (arabidopsides) were evident in 3-week-old dark-treated and 6-week-old premature senescing ACBP3-OEs. Such accumulation of PA and arabidopsides (A, B, D, E, and G) resulting from lipid peroxidation in ACBP3-OEs likely promoted leaf senescence. The N-terminal signal sequence/transmembrane domain in ACBP3 was shown to be essential in ACBP3-green fluorescent protein targeting and in promoting senescence. Observations that recombinant ACBP3 binds PC, PE, and unsaturated acyl-CoAs in vitro and that ACBP3 overexpression enhances degradation of the autophagy (ATG)-related protein ATG8 and disrupts autophagosome formation suggest a role for ACBP3 as a phospholipid binding protein involved in the regulation of leaf senescence by modulating membrane phospholipid metabolism and ATG8 stability in Arabidopsis. Accelerated senescence in ACBP3-OEs is dependent on salicylic acid but not jasmonic acid signaling.

Abstract 2727: Predicting lung cancer risk based on a novel proteomic signature of bronchial lesions

Abstract A biomarker signature to identify individuals at-risk for lung cancer would be of great clinical value. We hypothesized that the bronchial epithelium expresses a subset of proteins that once increased confer an elevated risk for lung cancer. We have therefore developed a proteomic approach to assess the risk for lung cancer. From our previous studies, we selected 11 matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) features to distinguish normal bronchial epithelium and low grade preinvasive lesions from high grade preinvasive lesions and invasive lung tumors. We tested this signature in an independent validation set consisting of bronchial specimens from 60 patients. The prediction accuracy was 78% with 73% sensitivity and 81% specificity. There was a 19-fold increased risk of having lung cancer for individuals with a signature intensity above the third quartile (95% CI, 5.65-64.81). We identified 10 of the 11 features corresponding to 7 proteins, thymosin ß4, ubiquitin, acyl-coA binding protein, cystatin A, S100A11, cytochrome c and macrophage migration inhibitory factor. The identity of these proteins was validated by immunohistochemistry, Western blotting and MALDI imaging mass spectrometry (IMS). MALDI IMS localized the candidate biomarker proteins specifically to high grade preinvasive lesions and invasive tumors. This novel proteomic signature may facilitate the selection and monitoring of high risk individuals for lung cancer early detection and chemoprevention strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2727.

A genetic basis for the phenotypic differentiation between siscowet and lean lake trout (Salvelinus namaycush)

In Lake Superior there are three principal forms of lake trout (Salvelinus namaycush): lean, siscowet and humper. Wild lean and siscowet differ in the shape and relative size of the head, size of the fins, location and size of the eyes, caudal peduncle shape and lipid content of the musculature. To investigate the basis for these phenotypic differences, lean and siscowet lake trout, derived from gametes of wild populations in Lake Superior, were reared communally under identical environmental conditions for 2.5 years. Fish were analysed for growth, morphometry and lipid content, and differences in liver transcriptomics were investigated using Roche 454 GS-FLX pyrosequencing. The results demonstrate that key phenotypic differences between wild lean and siscowet lake trout such as condition factor, morphometry and lipid levels, persist in these two forms when reared in the laboratory under identical environmental conditions. This strongly suggests that these differences are genetic and not a result of environmental plasticity. Transcriptomic analysis involving the comparison of hepatic gene frequencies (RNA-seq) and expression (quantitative reverse transcription-polymerase chain reaction (qPCR)) between the two lake trout forms, indicated two primary gene groups that were differentially expressed; those involving lipid synthesis, metabolism and transport (acyl-CoA desaturase, acyl-CoA binding protein, peroxisome proliferator-activated receptor gamma, and apolipoproteins), and those involved with immunity (complement component C3, proteasome, FK506 binding protein 5 and C1q proteins). The results demonstrate that RNA-seq can be used to identify differentially expressed genes; however, some discrepancies between RNA-seq analysis and qPCR indicate that methods for deep sequencing may need to be refined and/or different RNA-seq platforms utilized.

ACBP Knockdown Leads to Down-regulation of Genes Encoding Rate-limiting Enzymes in Cholesterol and Fatty Acid Metabolism

The human Acyl-CoA binding protein (ACBP) is a structural and functional highly conserved protein. As an intracellular pool former and carrier of acyl-CoAs, ACBP influences overall lipid metabolism. Its nuclear abundance and physical interaction with hepatocyte nuclear factor 4Α suggested a gene regulatory function of ACBP. To identify ACBP target genes we performed genome-wide transcript profiling under siRNA-mediated ACBP knockdown in human liver HepG2 cells. Based on a single sided permutation T-test (p<0.05) we identified 256 down-regulated and 198 up-regulated transcripts with a minimal fold change of 1.32 (log 0.5). Gene annotation enrichment analysis revealed ACBP-mediated down-regulation of 18 genes encoding key enzymes in glycerolipid (i.e. mitochondrial glycerol-3-phosphate acyltransferase), cholesterol (i.e. HMG-CoA synthase and HMG-CoA reductase) and fatty acid (i.e. fatty acid synthase) metabolism. Integration of these genes in common pathways suggested decreased lipid biosynthesis. Accordingly, saturated (16:0) and monosaturated (16:1, 18:1) fatty acids were significantly reduced to 75% in ACBP-depleted cells. Taken together, we obtained evidence that ACBP functions in lipid metabolism at the level of gene expression. This effect seems to be translated into certain metabolites. The identified 454 ACBP regulated genes present a first reference for further studies to define the ACBP regulon in mammalian cells.

Crystallographic and mass spectrometric analyses of a tandem GNAT protein from the clavulanic acid biosynthesis pathway

(3R,5R)-Clavulanic acid (CA) is a clinically important inhibitor of Class A beta-lactamases. Sequence comparisons suggest that orf14 of the clavulanic acid biosynthesis gene cluster encodes for an acetyl transferase (CBG). Crystallographic studies reveal CBG to be a member of the emerging structural subfamily of tandem Gcn5-related acetyl transferase (GNAT) proteins. Two crystal forms (C2 and P2(1) space groups) of CBG were obtained; in both forms one molecule of acetyl-CoA (AcCoA) was bound to the N-terminal GNAT domain, with the C-terminal domain being unoccupied by a ligand. Mass spectrometric analyzes on CBG demonstrate that, in addition to one strongly bound AcCoA molecule, a second acyl-CoA molecule can bind to CBG. Succinyl-CoA and myristoyl-CoA displayed the strongest binding to the "second" CoA binding site, which is likely in the C-terminal GNAT domain. Analysis of the CBG structures, together with those of other tandem GNAT proteins, suggest that the AcCoA in the N-terminal GNAT domain plays a structural role whereas the C-terminal domain is more likely to be directly involved in acetyl transfer. The available crystallographic and mass spectrometric evidence suggests that binding of the second acyl-CoA occurs preferentially to monomeric rather than dimeric CBG. The N-terminal AcCoA binding site and the proposed C-terminal acyl-CoA binding site of CBG are compared with acyl-CoA binding sites of other tandem and single domain GNAT proteins.

A new topology of ACBP from Moniliophthora perniciosa

Acyl-CoA binding protein (ACBP) is a housekeeping protein and is an essential protein in human cell lines and in Trypanosoma brucei. The ACBP of Moniliophthora perniciosa is composed of 104 amino acids and is possibly a non-classic isoform exclusively from Basidiomycetes. The M. perniciosa acbp gene was cloned, and the protein was expressed and purified. Acyl-CoA ester binding was analyzed by isoelectric focusing, native gel electrophoresis and isothermal titration calorimetry. Our results suggest an increasing affinity of ACBP for longer acyl-CoA esters, such as myristoyl-CoA to arachidoyl-CoA, and best fit modeling indicates two binding sites. ACBP undergoes a shift from a monomeric to a dimeric state, as shown by dynamic light scattering, fluorescence anisotropy and native gel electrophoresis in the absence and presence of the ligand. The protein's structure was determined at 1.6 Å resolution and revealed a new topology for ACBP, containing five α-helices instead of four. α-helices 1, 2, 3 and 4 adopted a bundled arrangement that is unique from the previously determined four-helix folds of ACBP, while α-helices 1, 2, 4 and 5 formed a classical four-helix bundle. A MES molecule was found in the CoA binding site, suggesting that the CoA site could be a target for small compound screening.

Arg‐85 and Thr‐430 in murine 5‐aminolevulinate synthase coordinate acyl‐CoA‐binding and contribute to substrate specificity

5-Aminolevulinate synthase (ALAS) controls the rate-limiting step of heme biosynthesis in mammals by catalyzing the condensation of succinyl-coenzyme A and glycine to produce 5-aminolevulinate, coenzyme-A (CoA), and carbon dioxide. ALAS is a member of the alpha-oxoamine synthase family of pyridoxal 5'-phosphate (PLP)-dependent enzymes and shares high degree of structural similarity and reaction mechanism with the other members of the family. The X-ray crystal structure of ALAS from Rhodobacter capsulatus reveals that the alkanoate component of succinyl-CoA is coordinated by a conserved arginine and a threonine. The functions of the corresponding acyl-CoA-binding residues in murine erthyroid ALAS (R85 and T430) in relation to acyl-CoA binding and substrate discrimination were examined using site-directed mutagenesis and a series of CoA-derivatives. The catalytic efficiency of the R85L variant with octanoyl-CoA was 66-fold higher than that of the wild-type protein, supporting the proposal of this residue as key in discriminating substrate binding. Substitution of the acyl-CoA-binding residues with hydrophobic amino acids caused a ligand-induced negative dichroic band at 420 nm in the CD spectra, suggesting that these residues affect substrate-mediated changes to the PLP microenvironment. Transient kinetic analyses of the R85K variant-catalyzed reactions confirm that this substitution decreases microscopic rates associated with formation and decay of a key reaction intermediate and show that the nature of the acyl-CoA tail seriously affect product binding. These results show that the bifurcate interaction of the carboxylate moiety of succinyl-CoA with R85 and T430 is an important determinant in ALAS function and may play a role in substrate specificity.

Prediction of the most favorable configuration in the ACBP–membrane interaction based on electrostatic calculations

Acyl-CoA binding proteins (ACBPs) are highly conserved 10 kDa cytosolic proteins that bind medium- and long-chain acyl-CoA esters. They act as intracellular carriers of acyl-CoA and play a role in acyl-CoA metabolism, gene regulation, acyl-CoA-mediated cell signaling, transport-mediated lipid synthesis, membrane trafficking and also, ACBPs were indicated as a possible inhibitor of diazepam binding to the GABA-A receptor. To estimate the importance of the non-specific electrostatic energy in the ACBP–membrane interaction, we computationally modeled the interaction of HgACBP with both anionic and neutral membranes. To compute the Free Electrostatic Energy of Binding ( dE), we used the Finite Difference Poisson Boltzmann Equation (FDPB) method as implemented in APBS. In the most energetically favorable orientation, ACBP brings charged residues Lys18 and Lys50 and hydrophobic residues Met46 and Leu47 into membrane surface proximity. This conformation suggests that these four ACBP amino acids are most likely to play a leading role in the ACBP–membrane interaction and ligand intake. Thus, we propose that long range electrostatic forces are the first step in the interaction mechanism between ACBP and membranes.

An Arabidopsis family of six acyl-CoA-binding proteins has three cytosolic members

In Arabidopsis thaliana, a gene family of six members encodes acyl-CoA-binding proteins (ACBPs). These Arabidopsis ACBPs (designated ACBP1 to ACBP6) range in size from 10.4kDa to 73.1kDa and display varying affinities for acyl-CoA esters, suggesting that they have different roles in plant lipid metabolism. In contrast, only the 10-kDa ACBPs have been well-characterized from other eukaryote species. Our previous studies have revealed that ACBP1 and ACBP2 are membrane-associated proteins, while ACBP3 is extracellularly-targeted. More recently, we have reported that the remaining three members in this protein family (namely ACBP4, ACBP5 and ACBP6) are subcellularly localized to the cytosol in Arabidopsis. The subcellular localizations of ACBP4, ACBP5 and ACBP6 in the cytosol were demonstrated using a number of different approaches incorporating biochemical fractionation, confocal microscopy of transgenic Arabidopsis expressing autofluorescence-tagged fusions and immunoelectron microscopy using ACBP-specific antibodies. Our results indicate that all three ACBPs in the cytosol are potential candidates for acyl-CoA binding and trafficking in plant cells. In this review, the functional redundancy and differences among the three cytosolic ACBPs are discussed by comparison of their light-regulated expression and substrate affinities to acyl-CoA esters, and from biochemical analyses on their knockout mutants and/or overexpression in transgenic Arabidopsis. The transcriptionally light-induced ACBP4 and ACBP5, which encode the two largest forms of Arabidopsis ACBPs, bind oleoyl-CoA esters and likely transfer oleoyl-CoAs from the plastids (the site of de novo fatty acid biosynthesis) to the endoplasmic reticulum for the biosynthesis of non-plastidial membrane lipids in Arabidopsis.

Gene Expression Analysis of a Porcine Native Abdominal Aortic Aneurysm Model

Introduction. We sought to characterize the gene expression patterns occurring during the development of aneurysms in the native porcine aorta. Methods. In Yorkshire swine, the infrarenal aorta was balloon dilated and infused with a solution of type I collagenase/pancreatic porcine elastase (16,000 U/1,000 U). Aneurysmal and control aortic samples were obtained at 1 (n = 3), 2 (n = 6), and 4 (n = 5) weeks following aneurysm induction. RNA was isolated, converted to biotin-modified antisense RNA and hybridized to porcine genome arrays. Aneurysmal and control gene intensities were compared using the 2-sample-for-means z-test. P < .01 was considered statistically significant. Results. Extracellular matrix remodeling genes that were upregulated in aneurysmal compared with control tissue included matrix metalloproteinase-1, -2, -3, and -9; MT-MMP; cathepsin-D, -H, -K, and -S; tissue inhibitor of metalloproteinase-1; and collagen I-a1 chain (P < .01). Elastin exhibited temporally downregulated gene expression (P < .01). Inflammatory genes that were upregulated included intercellular adhesion molecule-2, tumor necrosis factor-a, interleukin (IL)-1b, IL-10, chemokine receptor-4, and tissue plasminogen activator (P < .01). Atherosclerosis and cancer genes that were upregulated included apolipoprotein E, acyl-CoA binding protein, friend leukemia virus integration-1, and E26 transformation-specific sequence (P < .01). Conclusion. The porcine model replicates the gene expression patterns that are observed during the development of aneurysms in human studies as well as in rodent models. The porcine model thereby represents a novel method to study the impact of endovascular, cell-based, and other therapeutic interventions on AAA pathophysiology. (Surgery 2008;144:252-8.)

A Novel High-Throughput Screening Assay for Putative Antidiabetic Agents through PPARα Interactions

As natural peroxisome proliferator-activated receptor-alpha (PPARalpha) ligands, high levels of fatty acids and glucose could lead to hyperactivation of PPARalpha, like that seen in diabetes. Important diabetes research goals are to uncover new metabolic or signaling pathways involved in hyperglycemic cellular injury and to develop therapeutics for preventing or reversing this injury. Consequently, 1040 putative antidiabetic agents were screened for their ability to 1) affect PPARalpha lipid binding, 2) directly bind PPARalpha, and 3) alter PPARalpha transactivation in the presence of high glucose. A high-throughput fluorescent binding assay was developed to examine each compound's ability to restore fatty acyl-CoA binding to PPARalpha in the presence of high glucose concentrations. Approximately 1% of the compounds restored acyl-CoA binding by 60% or more. These compounds directly interacted with PPARalpha with high affinity (nM K(d)s), validating the primary screen. Furthermore, these compounds altered PPARalpha transactivation, and 1 strongly reversed the hyperactivation of PPARalpha found in the presence of clofibrate and high glucose levels.

Structure of a σ 28-Regulated Nonflagellar Virulence Protein from Campylobacter jejuni

Campylobacter jejuni, a Gram-negative motile bacterium, is a leading cause of human gastrointestinal infections. Although the mechanism of C. jejuni-mediated enteritis appears to be multifactorial, flagella play complex roles in the virulence of this human pathogen. Cj0977 is a recently identified virulence factor in C. jejuni and is expressed by a σ 28 promoter that controls late genes in the flagellar regulon. A Cj0977 mutant strain is fully motile but significantly reduced in the invasion of intestinal epithelial cells in vitro. Here, we report the crystal structure of the major structural domain of Cj0977, which reveals a homodimeric “hot-dog” fold architecture. Of note, the characteristic hot-dog fold has been found in various coenzyme A (CoA) compound binding proteins with numerous oligomeric states. Structural comparison with other known hot-dog fold proteins locates a putative binding site for an acyl-CoA compound in the Cj0977 protein. Structure-based site-directed mutagenesis followed by invasion assays indicates that key residues in the putative binding site are indeed essential for the Cj0977 virulence function, suggesting a possible function of Cj0977 as an acyl-CoA binding regulatory protein.

Effect of medium‐ and long‐chain fatty acid diets on PPAR and SREBP‐1 expression and glucose homeostasis in ACBP‐overexpressing transgenic rats

Acyl-CoAs are important intermediates and regulators of lipid metabolism. Binding proteins like acyl-CoA binding protein (ACBP) can influence their regulatory functions. ACBP has also been shown to exert direct effects on gene regulation in vitro. As the physiological relevance of ACBP in the regulation of lipid metabolism under high fat diets is unclear, we investigated the influence of such diets on the metabolic responses in ACBP-overexpressing rats. A transgenic rat line overexpressing the ACBP gene was used to study the effects of 4 weeks of feeding with medium- (MC) or long-chain (LC) fatty acid-containing diets. Glucose tolerance tests were performed. Expression of transcription factors was measured by quantitative RT-PCR and protein levels of AMP-activated protein kinase were determined by western blotting. Transgenic animals fed the MC diet had an improved glucose tolerance and lower serum insulin levels compared with controls. Their liver PPARgamma (by 43%) and SREBP-1 (by 35%) mRNA levels were found to be decreased, while adipose tissue PPARgamma expression was increased by 31%. Tg animals fed the LC diet did not exhibit changes in glucose or insulin levels but exhibited increased mRNA levels of liver PPARs and SREBP-1 (1.5-3.5 times) and decreased protein levels of AMPKalpha (by 48%). Our results demonstrate that ACBP overexpression affects metabolic responses to diets with distinct difference in their fatty acid chain lengths. The molecular regulatory mechanism behind these effects seems to be an ACBP-induced tissue-specific regulation of expression of PPARs and SREBP.

Functional analysis of acyl-CoA binding proteins in Caenorhabditis elegans

Functional analysis of acyl-CoA binding proteins in Caenorhabditis elegans

Gene expression analysis of a porcine native abdominal aortic aneurysm model

We sought to characterize the gene expression patterns occurring during the development of aneurysms in the native porcine aorta. In Yorkshire swine, the infrarenal aorta was balloon dilated and infused with a solution of type I collagenase/pancreatic porcine elastase (16,000 U/1,000 U). Aneurysmal and control aortic samples were obtained at 1 (n = 3), 2 (n = 6), and 4 (n = 5) weeks following aneurysm induction. RNA was isolated, converted to biotin-modified antisense RNA and hybridized to porcine genome arrays. Aneurysmal and control gene intensities were compared using the 2-sample-for-means z-test. P < .01 was considered statistically significant. Extracellular matrix remodeling genes that were upregulated in aneurysmal compared with control tissue included matrix metalloproteinase-1, -2, -3, and -9; MT-MMP; cathepsin-D, -H, -K, and -S; tissue inhibitor of metalloproteinase-1; and collagen I-alpha1 chain (P < .01). Elastin exhibited temporally downregulated gene expression (P < .01). Inflammatory genes that were upregulated included intercellular adhesion molecule-2, tumor necrosis factor-alpha, interleukin (IL)-1 beta, IL-10, chemokine receptor-4, and tissue plasminogen activator (P < .01). Atherosclerosis and cancer genes that were upregulated included apolipoprotein E, acyl-CoA binding protein, friend leukemia virus integration-1, and E26 transformation-specific sequence (P < .01). The porcine model replicates the gene expression patterns that are observed during the development of aneurysms in human studies as well as in rodent models. The porcine model thereby represents a novel method to study the impact of endovascular, cell-based, and other therapeutic interventions on AAA pathophysiology.

Characterization of an acyl-coenzyme A binding protein predominantly expressed in human primitive progenitor cells

Human acyl-coenzyme A binding domain-containing member 6 (ACBD6) is a modular protein that carries an acyl-CoA binding domain at its N terminus and two ankyrin motifs at its C terminus. ACBD6 binds long-chain acyl-CoAs with a strong preference for unsaturated, C18:1-CoA and C20:4-CoA, over saturated, C16:0-CoA, acyl species. Deletion of the C terminus, which is not conserved among the members of this family, did not affect the binding capacity or the substrate specificity of the protein. ACBD6 is not a ubiquitous protein, and its expression is restricted to tissues and progenitor cells with functions in blood and vessel development. ACBD6 was detected in bone marrow, spleen, placenta, cord blood, circulating CD34+ progenitors, and embryonic-like stem cells derived from placenta. In placenta, the protein was only detected in CD34+ progenitor cells present in blood and in CD31+ endothelial cells surrounding the blood vessels. These cells were also positive for the marker CD133, and they probably constitute hemangiogenic stem cells, precursors of both blood and vessels. We propose that human ACBD6 represents a cellular marker for primitive progenitor cells with functions in hematopoiesis and vascular endothelium development.

Identification of a novel phosphorylation site of acyl-CoA binding protein (ACBP) in nodularin-induced apoptotic hepatocytes

The liver specific protein phosphatase inhibiting toxin nodularin (from Nodularia spumigena) rapidly induces hepatocyte apoptosis. Incubation of freshly isolated hepatocytes with this toxin results in hyperphosphorylation of cellular proteins before any morphological signs of apoptosis appear. These phosphorylated proteins may play key roles in the early stage of apoptosis. Here, we identified one of the phosphoproteins to be acyl-CoA binding protein (ACBP), a highly conserved and ubiquitously expressed protein. Phosphorylation-site analysis by matrix-assisted laser desorption ionization time-of-flight MS/MS revealed that the observed phosphorylation is positioned on Ser1 in the N-terminal tryptic peptide Ac-SQADFDKAAE EVKRLK of the rat liver protein. Additionally, we observed a translocation of ACBP towards the cellular membrane in the apoptotic hepatocytes. Moreover, nodularin-induced apoptosis was highly dependent on calpain activation, an event that has previously been shown to be regulated by ACBP. Our findings introduce the possibility that reversible phosphorylation of ACBP regulates its ability to activate calpain in phosphatase inhibitor-induced apoptosis and controls the cellular accessibility of long-chain fatty acid-CoAs for cellular signaling.

Transcriptional Regulation of HMG-CoA Synthase and HMG-CoA Reductase Genes by Human ACBP

The acyl-CoA binding protein (ACBP) is an ubiquitary expressed multi-functional protein which regulates basic cellular functions such as fatty acid and steroid metabolism. Since ACBP is described to interact with the transcription factor hepatocyte nuclear factor 4 alpha (HNF-4α), we investigated the role of human ACBP on transcriptional regulation of the putative HNF-4α target gene HMG-CoA synthase 1 (HMGCS1). As shown by promoter-reporter assays ACBP represses the HNF-4α-induced activity of a 617bp HMGCS1 promoter fragment by approximately 80% in HepG2 cells as well as in non-endodermal HeLa cells devoid of HNF-4α. Interestingly, reporter assays without co-transfection of HNF-4α revealed that ACBP reduces the activity of the HMGCS1 promoter by about 60 to 80% in both cell lines. Activities of 417bp and 317bp HMGCS1 promoter fragments were 2.5 to 4 fold decreased by ACBP. Concordantly, the levels of HMGCS1-mRNA and -protein were diminished to 60% and 70% in ACBP-expressing HeLa cells, respectively. Additionally, ACBP reduces the promoter activity and the mRNA levels of the cholesterogenic HMG-CoA reductase (HMGCR). In conclusion, we provide evidence that ACBP is a transcriptional regulator of the HMGCS1 and HMGCR genes encoding rate-limiting enzymes of cholesterol synthesis pathway.

1P-094 蛍光エネルギー移動キネティクス測定によるACBPフオールディング機構の解明(日本生物物理学会若手奨励賞選考会,若手招待講演,第46回日本生物物理学会年会)

1P-094 蛍光エネルギー移動キネティクス測定によるACBPフオールディング機構の解明(日本生物物理学会若手奨励賞選考会,若手招待講演,第46回日本生物物理学会年会)