ABSTRACT
BACKGROUND: Lunasin is a chemopreventive peptide produced in a number of plant species. It comprises a helical region with homology to a region of chromatin binding proteins, an Arg-Gly-Asp cell adhesion motif and eight aspartic acid residues. In vitro studies indicate that lunasin suppresses chemical and oncogene driven transformation of mammalian cells. We have explored efficient recombinant production of lunasin by exploiting the Clostridium thermocellum CipB cellulose binding domain (CBD) as a fusion partner protein. RESULTS: We used a pET28 vector to express a CBD-lunasin fusion with a hexahistidine tag and Tobacco Etch Virus protease site, to allow protease-mediated release of native lunasin. Autoinduction in E. coli BL21 (DE3) Star cells achieved expression of 3.35 g/L of CBD-lunasin fusion protein. The final yield of lunasin was 210 mg/L corresponding to 32% of the theoretical yield. Purification by cellulose binding and nickel affinity chromatography were tested with the latter proving more satisfactory.The effects of CBD-lunasin expression on growth and morphology of the E. coli cells were examined by light and electron microscopy revealing an altered morphology in a proportion of cells. Cell division appeared to be inhibited in these cells resulting in elongated, non-septated cells. CONCLUSIONS: The use of CBD as a fusion partner gave high protein yields by autoinduction, with lunasin release by TEV protease cleavage. With some optimisation this approach could provide a potentially valuable route for production of this therapeutic peptide. Over-expression in the host cells manifest as a cell division defect in a population of the cells, presumably mimicking some aspect of the chemopreventive function observed in mammalian cells.
Subject(s)
Bacterial Proteins/biosynthesis , Plant Proteins/biosynthesis , Plant Proteins/genetics , Bacterial Proteins/genetics , Chromatography, Affinity , Clostridium thermocellum/metabolism , Endopeptidases/metabolism , Genetic Vectors/genetics , Genetic Vectors/metabolism , Histidine/biosynthesis , Histidine/genetics , Oligopeptides/biosynthesis , Oligopeptides/genetics , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purificationABSTRACT
Since its discovery three decades ago, sterol carrier protein-2 (SCP-2) has remained a fascinating protein whose physiological function in lipid metabolism remains an enigma. Its multiple proposed functions arise from its complex gene structure, post-translational processing, intracellular localization, and ligand specificity. The SCP-2 gene has two initiation sites coding for proteins that share a common 13 kDa SCP-2 C-terminus: (1) One site codes for 58 kDa SCP-x which is partially post-translationally cleaved to 13 kDa SCP-2 and a 45 kDa protein. (2) A second site codes for 15 kDa pro-SCP-2 which is completely post-translationally cleaved to 13 kDa SCP-2. Very little is yet known regarding how the relative proportions of the two transcripts are regulated. Although all three proteins contain a C-terminal SKL peroxisomal targeting sequence, it is unclear why all three proteins are not exclusively localized in peroxisomes. However, the recent demonstration that the SCP-2 N-terminal presequence in pro-SCP-2 dramatically modulated the intracellular targeting coded by the C-terminal peroxisomal targeting sequence may account for the observation that as much as half of total SCP-2 is localized outside the peroxisome. The tertiary and secondary structure of the 13 kDa SCP-2, but not that of 15 kDa pro-SCP-2 and 58 kDa SCP-x, are now resolved. Increasing evidence suggests that the 58 kDa SCP-x and 45 kDa proteins are peroxisomal 3-ketoacyl-CoA-thiolases involved in the oxidation of branched chain fatty acids. Since 15 kDa pro-SCP-2 is post-translationally completely cleaved to 13 kDa SCP-2, relatively little attention has been focused on this protein. Finally, although the 13 kDa SCP-2 is the most studied of these proteins, because it exhibits diversity of its ligand partners (fatty acids, fatty acyl CoAs, cholesterol, phospholipids), new potential physiological function(s) are still being proposed and questions regarding potential compensation by other proteins with overlapping specificity are only beginning to be resolved.
Subject(s)
Carrier Proteins/genetics , Plant Proteins , Promoter Regions, Genetic/genetics , Acetyl-CoA C-Acetyltransferase/genetics , Acetyl-CoA C-Acetyltransferase/metabolism , Acyl Coenzyme A/metabolism , Animals , Bile/metabolism , Bile Acids and Salts/metabolism , Binding Sites , Biological Transport , Carrier Proteins/metabolism , Carrier Proteins/physiology , Cholesterol/metabolism , Cytosol/metabolism , Fatty Acids/metabolism , Humans , Lipid Metabolism , Mice , Mice, Transgenic , Mitochondria/metabolism , Peroxisomes/metabolism , Protein Binding , Protein Biosynthesis , Protein Processing, Post-Translational , Protein Structure, Secondary , Protein Structure, TertiaryABSTRACT
Cellular cholesterol homeostasis is a balance of influx, catabolism and synthesis, and efflux. Unlike vascular lipoprotein cholesterol transport, intracellular cholesterol trafficking is only beginning to be resolved. Exogenous cholesterol and cholesterol ester enter cells via the low-density lipoprotein (LDL) receptor/lysosomal and less so by nonvesicular, high-density lipoprotein (HDL) receptor/caveolar pathways. However, the mechanism(s) whereby cholesterol enters the lysosomal membrane, translocates, and transfers out of the lysosome to the cell interior are unknown. Likewise, the steps whereby cholesterol enters the cytofacial leaflet of the plasma membrane caveolae, rapidly translocates, leaves the exofacial leaflet, and transfers to extracellular HDL are unclear. Increasing evidence obtained with model and isolated cell membranes, transfected cells, genetic mutants, and gene-ablated mice suggests that proteins such as caveolin, sterol carrier protein-2 (SCP-2), Niemann-Pick C1 protein, steroidogenic acute regulatory protein (StAR), and other intracellular proteins mediate intracellular cholesterol transfer. While these proteins bind cholesterol and/or interact with cholesterol-rich membrane microdomains (e.g., caveolae, rafts, and annuli), their relative contributions to direct molecular versus vesicular cholesterol transfer remain to be resolved. The formation, regulation, and role of membrane microdomains in regulating cholesterol uptake/efflux and trafficking are unclear. Some cholesterol-binding proteins exert opposing effects on cellular cholesterol uptake/efflux, transfer of cholesterol out of the lysosomal membrane, and/or intracellular cholesterol trafficking to select membranous organelles. Resolving these cholesterol pathways and the role of membrane cholesterol microdomains is essential to our understanding not only of processes that affect cholesterol metabolism, but also of the abnormal regulation that may lead to disease (diabetes, obesity, atherosclerosis, neutral lipid storage, Niemann-Pick C, congenital lipoid adrenal hyperplasia, etc.).
Subject(s)
Cell Membrane Structures/metabolism , Cell Membrane/metabolism , Cholesterol/metabolism , Plant Proteins , Proteins/metabolism , Animals , Biological Transport , Carrier Proteins/metabolism , Caveolae , Cell Membrane/ultrastructure , Cell Membrane Structures/chemistry , Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Humans , Intracellular Signaling Peptides and Proteins , Lysosomes/metabolism , Membrane Glycoproteins/metabolism , Mitochondria/metabolism , Niemann-Pick C1 ProteinABSTRACT
Although expression of liver fatty acid binding protein (L-FABP) modulates cell growth, it is not known if L-FABP also alters cell morphology and differentiation. Therefore, pluripotent embryonic stem cells were transfected with cDNA encoding L-FABP and a series of clones expressing increasing levels of L-FABP were isolated. Untransfected ES cells, as well as ES cells transfected only with empty vector, spontaneously differentiated from rounded adipocyte-like to fibroblast-like morphology, concomitant with marked reduction in expression of stage-specific embryonic antigen (SSEA-1). These changes in morphology and expression of SSEA-1 were greatest in ES cell clones expressing L-FABP above a threshold level. Immunofluorescence confocal microscopy revealed that L-FABP was primarily localized in a diffuse-cytosolic pattern along with a lesser degree of punctate L-FABP expression in the nucleus. Nuclear localization of L-FABP was preferentially increased in clones expressing higher levels of L-FABP. In summary, L-FABP expression altered ES cell morphology and expression of SSEA-1. Taken together with the fact that L-FABP was detected in the nucleus, these data suggested that L-FABP may play a more direct, heretofore unknown, role in regulating ES cell differentiation by acting in the nucleus as well as cytoplasm.
Subject(s)
Carrier Proteins/metabolism , Carrier Proteins/physiology , Cell Differentiation/physiology , Cell Division/physiology , Fatty Acids/metabolism , Interleukin-6 , Liver/metabolism , Neoplasm Proteins , Nerve Tissue Proteins , Stem Cells/physiology , Animals , Carrier Proteins/genetics , Cell Nucleus/metabolism , Cells, Cultured , Clone Cells , Embryo, Mammalian/cytology , Fatty Acid-Binding Protein 7 , Fatty Acid-Binding Proteins , Flow Cytometry , Gene Expression Regulation, Developmental , Growth Inhibitors/pharmacology , Leukemia Inhibitory Factor , Lewis X Antigen/physiology , Lymphokines/pharmacology , Mice , Microscopy, Confocal , Stem Cells/cytology , TransfectionABSTRACT
Although sterol carrier protein-2 (SCP-2; also called nonspecific lipid transfer protein) binds fatty acids and fatty acyl-CoAs, its role in fatty acid metabolism is not fully understood. L-cell fibroblasts stably expressing SCP-2 were used to resolve the relationship between SCP-2 intracellular location and fatty acid transacylation in the endoplasmic reticulum. Indirect immunofluorescence double labeling and laser scanning confocal microscopy detected SCP-2 in peroxisomes > endoplasmic reticulum > mitochondria > lysosomes. SCP-2 enhanced incorporation of exogenous [(3)H]oleic acid into phospholipids and triacylglycerols of overexpressing cells 1.6- and 2.5-fold, respectively, stimulated microsomal incorporation of [1-(14)C]oleoyl-CoA into phosphatidic acid in vitro 13-fold, and exhibited higher specificity for unsaturated versus saturated fatty acyl-CoA. SCP-2 enhanced the rate-limiting step in microsomal phosphatidic acid biosynthesis mediated by glycerol-3-phosphate acyltransferase. SCP-2 also enhanced microsomal acyl-chain remodeling of phosphatidylethanolamine up to fivefold and phosphatidylserine twofold, depending on the specific fatty acyl-CoA, but had no effect on other phospholipid classes. In summary, these results were consistent with a role for SCP-2 in phospholipid synthesis in the endoplasmic reticulum.
Subject(s)
Carrier Proteins/metabolism , Endoplasmic Reticulum/metabolism , Fibroblasts/metabolism , Phospholipids/biosynthesis , Plant Proteins , Acyl Coenzyme A/metabolism , Acylation/drug effects , Animals , Carrier Proteins/genetics , Carrier Proteins/pharmacology , Fatty Acids/metabolism , Fibroblasts/cytology , Glycerol-3-Phosphate O-Acyltransferase/metabolism , Immunohistochemistry , Intracellular Fluid/metabolism , L Cells , Mice , Microscopy, Fluorescence , Microsomes/metabolism , Peroxisomes/metabolism , Phosphatidic Acids/biosynthesis , Rabbits , Substrate Specificity , Transfection , Triglycerides/biosynthesisABSTRACT
Although sterol carrier protein-2 (SCP-2) participates in the uptake and intracellular trafficking of cholesterol, its effect on "reverse cholesterol transport" has not been explored. As shown herein, SCP-2 expression inhibited high density lipoprotein (HDL)-mediated efflux of [(3)H]cholesterol and fluorescent 22-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3b-ol (NBD-cholesterol) up to 61 and 157%, respectively. Confocal microscopy of living cells allowed kinetic analysis of two intracellular pools of HDL-mediated NBD-cholesterol efflux: the highly fluorescent lipid droplet pool and the less fluorescent pool outside the lipid droplets, designated the cytoplasmic compartment. Both the whole cell and the cytoplasmic compartment exhibited two similar kinetic pools, the half-times of which were consistent with protein (t(b)(12) near 1 min) and vesicular (t(d)(12) = 10-20 min) mediated sterol transfer. Although SCP-2 expression did not alter cytoplasmic sterol pool sizes, the rapid t(b)(12) decreased 36%, while the slower t(d)(12) increased 113%. Lipid droplets also exhibited two kinetic pools of NBD-cholesterol efflux but with half-times over 200% shorter than those of the cytoplasmic compartment. The lipid droplet slower effluxing pool size and t(d)(12) were increased 48% and 115%, respectively, in SCP-2-expressing cells. Concomitantly, the level of the lipid droplet-specific adipose differentiation-related protein decreased 70%. Overall, HDL-mediated sterol efflux from L-cell fibroblasts reflected that of the cytoplasmic rather than lipid droplet compartment. SCP-2 differentially modulated sterol efflux from the two cytoplasmic pools. However, net efflux was determined primarily by inhibition of the slowly effluxing pool rather than by acceleration of the rapid protein-mediated pool. Finally, SCP-2 expression also inhibited sterol efflux from lipid droplets, an effect related to decreased adipose differentiation-related protein, a lipid droplet surface protein that binds cholesterol with high affinity.
Subject(s)
Carrier Proteins/metabolism , Cholesterol/metabolism , Lipoproteins, HDL/metabolism , Plant Proteins , 4-Chloro-7-nitrobenzofurazan/analogs & derivatives , 4-Chloro-7-nitrobenzofurazan/metabolism , Animals , Blotting, Western , Cell Compartmentation , Cholesterol/analogs & derivatives , L Cells , Mice , Microscopy, Confocal , TransfectionABSTRACT
Although the majority of exogenous cholesterol and cholesterol ester enters the cell by LDL-receptor-mediated endocytosis and the lysosomal pathway, the assumption that cholesterol transfers out of the lysosome by rapid (minutes), spontaneous diffusion has heretofore not been tested. As shown herein, lysosomal membranes were unique among known organellar membranes in terms of cholesterol content, cholesterol dynamics, and response to cholesterol-mobilizing proteins. First, the lysosomal membrane cholesterol:phospholipid molar ratio, 0.38, was intermediate between those of the plasma membrane and other organellar membranes. Second, a fluorescence sterol exchange assay showed that the initial rate of spontaneous sterol transfer out of lysosomes and purified lysosomal membranes was extremely slow, t(1/2) >4 days. This was >100-fold longer than that reported in intact cells (2 min) and 40-60-fold longer than from any other known intracellular membrane. Third, when probed with several cholesterol-binding proteins, the initial rate of sterol transfer was maximally increased nearly 80-fold and the organization of cholesterol in the lysosomal membrane was rapidly altered. Nearly half of the essentially nonexchangeable sterol in the lysosomal membrane was converted to rapidly (t(1/2) = 6 min; fraction = 0.06) and slowly (t(1/2) = 154 min; fraction = 0.36) exchangeable sterol domains/pools. In summary, the data revealed that spontaneous cholesterol transfer out of the lysosome and lysosomal membrane was extremely slow, inconsistent with rapid spontaneous diffusion across the lysosomal membrane. In contrast, the very slow spontaneous transfer of sterol out of the lysosome and lysosomal membrane was consistent with cholesterol leaving the lysosome earlier in the endocytic process and/or with cholesterol transfer out of the lysosome being mediated by additional process(es) extrinsic to the lysosome and lysosomal membrane.
Subject(s)
Cholesterol/chemistry , Ergosterol/analogs & derivatives , Lysosomes/chemistry , Animals , Binding Sites , Biological Transport , Cell Cycle Proteins/biosynthesis , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cells, Cultured , Cholesterol/metabolism , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Ergosterol/chemistry , Ergosterol/metabolism , Fibroblasts/chemistry , Fibroblasts/metabolism , Fluorescence Polarization , Fluorescent Antibody Technique, Indirect , Intracellular Membranes/chemistry , Intracellular Membranes/metabolism , Lipid Metabolism , Lipids/chemistry , Lysosomes/metabolism , Mice , Microscopy, Confocal , Proteins/chemistry , Proteins/metabolism , Reference Standards , Sterols/chemistry , Sterols/metabolism , TransfectionABSTRACT
Although the 20-amino acid presequence present in 15-kDa pro-sterol carrier protein-2 (pro-SCP-2, the precursor of the mature 13-kDa SCP-2) alters the function of SCP-2 in lipid metabolism, the molecular basis for this effect is unresolved. The presequence dramatically altered SCP-2 structure as determined by circular dichroism, mass spectroscopy, and antibody accessibility such that pro-SCP-2 had 3-fold less alpha-helix, 7-fold more beta-structure, 6-fold more reactive C terminus to carboxypeptidase A, 2-fold less binding of anti-SCP-2, and did not enhance sterol transfer from plasma membranes. These differences were not due to protein stability since (i) the same concentration of guanidine hydrochloride was required for 50% unfolding, and (ii) the ligand binding sites displayed the same high affinity (nanomolar K(d) values) in the order: cholesterol straight chain fatty acid > kinked chain fatty acid. Laser scanning confocal microscopy and double immunofluorescence demonstrated that pro-SCP-2 was more efficiently targeted to peroxisomes. Transfection of l-cells or McAR7777 hepatoma cells with cDNA encoding pro-SCP-2 resulted in 45% and 59% of SCP-2, respectively, colocalizing with the peroxisomal marker PMP70. In contrast, l-cells transfected with cDNA encoding SCP-2 exhibited 3-fold lower colocalization of SCP-2 with PMP70. In summary, the data suggest for the first time that the 20-amino acid presequence of pro-SCP-2 alters SCP-2 structure to facilitate peroxisomal targeting mediated by the C-terminal SKL peroxisomal targeting sequence.
Subject(s)
ATP-Binding Cassette Transporters , Carrier Proteins/chemistry , Peroxisomes/metabolism , Plant Proteins , Protein Precursors/chemistry , Animals , Blotting, Western , Carboxypeptidases/metabolism , Carboxypeptidases A , Carrier Proteins/physiology , Cell Line , Cell Membrane/metabolism , Cholesterol/metabolism , Circular Dichroism , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Fatty Acids/metabolism , Fluorescent Antibody Technique , Fluorescent Antibody Technique, Indirect , Guanidine/pharmacology , Humans , Immunoblotting , Kinetics , Ligands , Membrane Proteins/metabolism , Microscopy, Confocal , Protein Folding , Protein Precursors/physiology , Protein Structure, Secondary , Protein Structure, Tertiary , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Structure-Activity Relationship , Transfection , Tumor Cells, CulturedABSTRACT
Although sterol carrier protein 2 (SCP-2) has long been regarded primarily as a sterol transfer protein, its actual physiological function is not known. The recent discovery that SCP-2 binds long chain fatty acyl-CoAs (LCFA-CoAs) with high affinity suggests additional roles for SCP-2 in cellular utilization of LCFA-CoAs for synthesis of glycerides and cholesterol esters. Concomitant to these anabolic pathways, LCFA-CoAs are also degraded by cellular hydrolases. The purpose of the work presented herein was to determine if SCP-2 altered the aqueous pool of LCFA-CoA by (i) extracting LCFA-CoA from microsomal membranes, and (ii) protecting LCFA-CoA from microsomal hydrolase activity. The data demonstrated for the first time that SCP-2 increases the aqueous pool of oleoyl-CoA by increasing the aqueous/membrane distribution oleoyl-CoA by 2.4-fold. In addition, SCP-2 inhibited the hydrolysis of oleoyl-CoA by microsomal acyl-CoA hydrolase 1.6-2.4 fold, depending on the concentration of oleoyl-CoA. By simultaneously extracting LCFA-CoA from membranes and inhibiting LCFA-CoA degradation SCP-2 may potentiate LCFA-CoA transacylation and modulate the role of LCFA-CoAs as intracellular signaling molecules.
Subject(s)
Acyl Coenzyme A/metabolism , Carrier Proteins/metabolism , Microsomes, Liver/metabolism , Plant Proteins , Animals , Dose-Response Relationship, Drug , Endoplasmic Reticulum/metabolism , Escherichia coli/metabolism , Humans , Hydrolysis , Male , Rats , Rats, Sprague-Dawley , Recombinant Proteins/metabolismABSTRACT
Mitochondrial cholesterol oxidation rapidly depletes cholesterol from the relatively cholesterol-poor mitochondrial membranes. However, almost nothing is known regarding potential mechanism(s) whereby the mitochondrial cholesterol pool is restored. Since most exogenous cholesterol enters the cell via the lysosomal pathway, this could be a source of mitochondrial cholesterol. In the present study, an in vitro fluorescent sterol transfer assay was used to examine whether the lysosomal membrane could be a putative cholesterol donor to mitochondria. First, it was shown that spontaneous sterol transfer from lysosomal to mitochondrial membranes was very slow (initial rate, 0.316 +/- 0.032 pmol/min). This was due, in part, to the fact that 90% of the lysosomal membrane sterol was not exchangeable, while the remaining 10% also had a relatively long half-time of exchange t(1/2) = 202 +/- 19 min. Second, the intracellular sterol carrier protein-2 (SCP-2) and its precursor (pro-SCP-2) increased the initial rate of sterol transfer from the lysosomal to mitochondrial membrane by 5.2- and 2.0-fold, respectively, but not in the reverse direction. The enhanced sterol transfer was due to a 3.5-fold increase in exchangeable sterol pool size and to induction of a very rapidly (t(1/2) = 4.1 +/- 0.6 min) exchangeable sterol pool. Confocal fluorescence imaging and indirect immunocytochemistry colocalized significant amounts of SCP-2 with the mitochondrial marker enzyme cytochrome oxidase in transfected L-cells overexpressing SCP-2. In summary, SCP-2 and pro-SCP-2 both stimulated molecular sterol transfer from lysosomal to mitochondrial membranes, suggesting a potential mechanism for replenishing mitochondrial cholesterol pools depleted by cholesterol oxidation.
Subject(s)
Carrier Proteins/metabolism , Cholesterol/metabolism , Mitochondria/metabolism , Plant Proteins , Animals , Biological Transport , Carrier Proteins/genetics , Ergosterol/analogs & derivatives , Ergosterol/metabolism , Fluorescence Polarization , Fluorescent Antibody Technique , Intracellular Membranes/metabolism , Kinetics , L Cells , Lysosomes/metabolism , Mice , Microscopy, Confocal , Recombinant Proteins/metabolism , TransfectionABSTRACT
Brain membrane lipid fatty acid composition and consequently membrane fluidity change with increasing age. Intracellular fatty acid binding proteins (FABPs) such as heart H-FABP and the brain specific B-FABP, detected by immunoblotting of brain tissue, are thought to be involved in fatty acid uptake, metabolism, and differentiation in brain. Yet, almost nothing is known regarding the effect of age on the expression of the cytosolic fatty acid binding proteins (FABPs) or their content in brain subfractions. Electrophoresis and quantitative immunoblotting were used to examine the content of these FABPs in synaptosomes in brains from 4, 15, and 25 month old C57BL/6NNia male mice. Brain H-FABP and B-FABP were differentially expressed in mouse brain subcellular fractions. Brain H-FABP was highly concentrated in synaptosomal cytosol. The level of brain H-FABP in synaptosomes, synaptosomal cytosol, and intrasynaptosomal membranes was decreased 33, 35, and 43%, respectively, in 25 month old mice. B-FABP was detected in lower quantity than H-FABP. More important, B-FABP decreased in synaptosomes, synaptic plasma membranes, and synaptosomal cytosol from brains of 25 month old mice. In contrast to H-FABP, B-FABP was not detectable in the intrasynaptosomal membranes in any of the three age groups of mice. In conclusion, expression of both H-FABP and B-FABP was markedly reduced in aged mouse brain. Age differences in brain H-FABP and B-FABP levels in synaptosomal plasma membranes and synaptosomal cytosol may be important factors modulating neuronal differentiation and function.
Subject(s)
Aging/metabolism , Brain/metabolism , Carrier Proteins/metabolism , Myelin P2 Protein/metabolism , Neoplasm Proteins , Nerve Tissue Proteins , Animals , Blotting, Western , Cell Membrane/metabolism , Electrophoresis, Polyacrylamide Gel , Fatty Acid-Binding Protein 7 , Fatty Acid-Binding Proteins , Male , Mice , Mice, Inbred C57BL , Synaptosomes/metabolismABSTRACT
The recent discovery that sterol carrier protein-2 (SCP-2) binds long chain++ (LCFA-CoA) with high affinity (A. Frolov et al., J. Biol. Chem. 271 (1997) 31878-31884) suggests new possible functions of this protein in LCFA-CoA metabolism. The purpose of the present investigation was to determine whether SCP-2 differentially modulated microsomal LCFA-CoA transacylation to cholesteryl esters, triacylglycerols, and phospholipids in vitro. Microsomal acyl-CoA:cholesterol acyltransferase (ACAT) activity measured with liposomal membrane cholesterol donors depended on substrate LCFA-CoA level, mol% cholesterol in the liposomal membrane, and total amount of liposomal cholesterol. As compared to basal activity without liposomes, microsomal ACAT was inhibited 30-50% in the presence of cholesterol poor (1.4 mol%) liposomes. In contrast, cholesterol rich (>25 mol%) liposomes stimulated ACAT up to 6.4-fold compared to basal activity without liposomes and nearly 10-fold as compared to cholesterol pool (1.4 mol%) liposomes. Increasing oleoyl-CoA reversed the inhibition of microsomal ACAT by cholesterol poor (1.4 mol%) liposomes, but did not further stimulate ACAT in the presence of cholesterol rich (35 mol%) liposomes. In contrast, high (100 microM) oleoyl-CoA inhibited ACAT nearly 3-fold. This inhibition was reversed by LCFA-CoA binding proteins, bovine serum albumin (BSA) and SCP-2. SCP-2 was 10-fold more effective (mole for mole) than BSA in reversing LCFA-CoA inhibited microsomal ACAT. Concomitantly, under conditions in which SCP-2 stimulated ACAT it equally enhanced transacylation of oleoyl-CoA into phospholipids, and 5.2-fold enhanced oleoyl-CoA transacylation to triacylglycerols. In summary, SCP-2 appeared to exert its greatest effects on microsomal transacylation in vitro by reversing LCFA-CoA inhibition of ACAT and by differentially targeting LCFA-CoA to triacylglycerols. These data suggest that the high affinity interaction of SCP-2s with LCFA-CoA may be physiologically important in microsomal transacylation reactions.
Subject(s)
Acyl Coenzyme A/metabolism , Carrier Proteins/metabolism , Microsomes, Liver/enzymology , Plant Proteins , Animals , Carrier Proteins/pharmacology , Cholesterol/analysis , Cholesterol Esters/metabolism , Enzyme Activation , Liposomes/chemistry , Male , Phospholipids/metabolism , Rats , Rats, Sprague-Dawley , Sterol O-Acyltransferase/antagonists & inhibitors , Sterol O-Acyltransferase/metabolism , Triglycerides/metabolismABSTRACT
While aspects of cellular fatty acid uptake have been studied as early as 50 years ago, recent developments in this rapidly evolving field have yielded new functional insights on the individual mechanistic steps in this process. The extremely low aqueous solubility of long chain fatty acids (LCFA) together with the very high affinity of serum albumin and cytoplasmic fatty acid binding proteins for LCFA have challenged the limits of technology in resolving the individual steps of this process. To date no single mechanism alone accounts for regulation of cellular LCFA uptake. Key regulatory points in cellular uptake of LCFA include: the aqueous solubility of the LCFA; the driving force(s) for LCFA entry into the cell membrane; the relative roles of diffusional and protein mediated LCFA translocation across the plasma membrane; cytoplasmic LCFA binding protein-mediated uptake and/or intracellular diffusion; the activity of LCFA-CoA synthetase; and cytoplasmic protein mediated targeting of LCFA or LCFA-CoAs toward specific metabolic pathways. The emerging picture is that the cell has multiple, overlapping mechanisms that assure adequate uptake and directed intracellular movement of LCFA required for maintenance of physiological functions. The upcoming challenge is to take advantage of new advances in this field to elucidate the differential interactions between these pathways in intact cells and in tissues.
Subject(s)
Carrier Proteins/metabolism , Cell Membrane/metabolism , Fatty Acids/metabolism , Myelin P2 Protein/metabolism , Neoplasm Proteins , Biological Transport , Cell Compartmentation , Cytosol/metabolism , Fatty Acid-Binding Proteins , Fatty Acids, Nonesterified/metabolism , Glycerides/metabolism , Serum Albumin/metabolismABSTRACT
Although the sterol carrier protein 2 (SCP-2) gene encodes for two proteins, almost nothing is known of the function and potential processing of the larger transcript corresponding to the 58 kDa sterol carrier protein-2/3-oxoacyl-CoA thiolase (SCP-x), in intact cells. L-cell fibroblasts transfected with cDNA encoding for the 58 kDa SCP-x protein had a 4.5-fold increase in SCP-x mRNA transcript levels. Western blot analysis showed SCP-x protein expression reached 0.011% of total protein, representing a 4.1-fold increase over basal levels. Surprisingly, the 13.2 kDa SCP-2 protein also increased 2-fold in the transfected cells. This was consistent with part of the 58 kDa SCP-x being proteolytically processed to 13.2 kDa SCP-2 as there was no evidence of an mRNA transcript corresponding to a 13.2/15.2 kDa gene product in the transfected L-cell clones. Confocal immunofluorescence microscopy of transfected L-cells showed that SCP-x/SCP-2 co-localized in highest concentration with catalase in peroxisomes, but significant amounts appeared extra-peroxisomal. Overexpression of SCP-x significantly altered cholesterol uptake and metabolism. Uptake of exogenous [3H]cholesterol and total cholesterol mass were increased 1.9- and 1.4-fold, respectively, in SCP-x expressors. Although cholesterol ester mass was unaltered, incorporation of exogenous [3H]cholesterol and [3H]oleic acid into cholesteryl esters increased 2.3- and 2.5-fold, respectively. These results from intact cells suggest the 13.2 kDa SCP-2 can arise from the larger SCP-2 gene product and indicate a role for the 58 kDa SCP-x protein in cholesterol uptake and intracellular cycling.
Subject(s)
Acetyl-CoA C-Acyltransferase/genetics , Carrier Proteins/genetics , Fibroblasts/metabolism , Gene Expression , Plant Proteins , Acetyl-CoA C-Acyltransferase/metabolism , Animals , Blotting, Northern , Blotting, Western , Carrier Proteins/metabolism , Cell Line , Cholesterol/metabolism , Cholesterol Esters/metabolism , Esterification , Fibroblasts/ultrastructure , Fluorescent Antibody Technique , Kinetics , Mice , Microbodies/metabolism , Molecular Weight , Oleic Acid/metabolism , Transfection , TritiumABSTRACT
During the last half-century pathologists have explored the biologic mechanisms associated with inherited human and veterinary diseases by using inbred and inbred mutant (spontaneous) strains of mice. The first successful gene transfer to mice by pronuclear injection of the herpes simplex virus thymidine kinase gene and rabbit and human beta-globulin genes was achieved in the early 1980s. This accomplishment was followed a few years later with the creation of a mouse bearing a disrupted hypoxanthine phosphoribosyl transferase (hrpt) gene (targeted mutation based on ES cell blastocyst injection). Since then, hundreds of genetically engineered models of biomedical importance have been created. The unprecedented scale and scope of development of engineered models present great opportunities as well as experimental challenges to the investigator. The aim of the present review is to provide a framework of information on engineered mouse models from the perspective of experimental and comparative pathology research. Sections include: 1) a brief historical account of the development of mouse models of disease, with increasing progression of genetic refinement as represented by inbred (spontaneous) and congenic (targeted) mutant strains of mice; 2) a synopsis of spontaneous and targeted mutations, with anecdotal examples of expression of individual genes and interactions between multiple mutant genes; 3) selected examples of targeted mutations of interest to developmental and cancer biologists and immunologists; 4) an overview of the technology of development of transgenic mice; and 5) an introduction to on-line database resources of current multi-species genomic information.
Subject(s)
Disease Models, Animal , Genetic Engineering , Mice, Mutant Strains , Animals , Gene Targeting , Gene Transfer Techniques/history , Genetic Engineering/history , History, 20th Century , Humans , Mice , Mice, Transgenic , Mutation , Transfer, PsychologyABSTRACT
Although the existing literature suggests that the sterol carrier protein-2 (SCP-2) gene has only two initiation sites encoding for a 58- and a 15-kDa protein, respectively, this does not explain the profusion of other putative SCP-2-related proteins detectable on Western blotting. Two of these additional anti-SCP-2 immunoreactive proteins, 13.2 and 46 kDa, appear due to proteolytic processing of the two gene transcripts. However, the origin of additional immunoreactive rat liver proteins near 26 and 30 kDa is unclear. The latter proteins were consistently detected on Western blotting by three independent types of polyclonal antisera: anti-13.2-kDa SCP-2, anti-synthetic peptide from the amino-terminus of the 13.2-kDa SCP-2, and Protein A affinity-purified anti-synthetic peptide to the amino-terminus of 13.2-kDa SCP-2. To resolve whether the 26- and 30-kDa proteins are SCP-2 gene products, each protein was isolated from rat liver and purified to homogeneity as indicated by Tricine-SDS polyacrylamide gel electrophoresis, isoelectric focusing, and/or mass spectroscopy. Their masses, determined by MALDI-TOF mass spectroscopy, were 25.7 and 29.8 kDa, respectively. However, the mass spectral data were not consistent with either protein being an SCP-2 gene product. Peptide mass mapping of the 25.7-kDa protein revealed identity to the rat 25,784.79-Da glutathione-S-transferase. Furthermore, neither the mass nor the amino acid composition of the 29.8-kDa protein correlated with any SCP-2 gene product or dimerized SCP-2 gene product. A database search of the amino acid composition identified the protein as rat carbonic anhydrase. In summary, although the 26- and 29.8-kDa proteins may share some common epitopes with the 13.2-kDa SCP-2, they were not SCP-2 gene products.
Subject(s)
Antibodies/immunology , Carbonic Anhydrases/isolation & purification , Carrier Proteins/immunology , Glutathione Transferase/isolation & purification , Liver/metabolism , Plant Proteins , Amino Acid Sequence , Animals , Blotting, Western , Carbonic Anhydrases/immunology , Carbonic Anhydrases/metabolism , Chromatography, Ion Exchange , Cross Reactions , Electrophoresis, Polyacrylamide Gel , Female , Glutathione Transferase/immunology , Glutathione Transferase/metabolism , Isoelectric Focusing , Male , Molecular Sequence Data , Rabbits , Rats , Rats, Sprague-Dawley , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
The effect of cellular differentiation on fatty acid uptake and intracellular diffusion was examined in transfected pluripotent mouse embryonic stem (ES) cells stably expressing intestinal fatty acid binding protein (I-FABP). Control ES cells, whether differentiated or undifferentiated, did not express I-FABP. The initial rate and maximal uptake of the fluorescent fatty acid, 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-octadec anoic acid (NBD-stearic acid), was measured in single cells by kinetic digital fluorescence imaging. I-FABP expression in undifferentiated ES cells increased the initial rate and maximal uptake of NBD-stearic acid 1.7- and 1.6-fold, respectively, as well as increased its effective intracellular diffusion constant (Deff) 1.8-fold as measured by the fluorescence recovery after photobleaching technique. In contrast, ES cell differentiation decreased I-FABP expression up to 3-fold and decreased the NBD-stearic acid initial rate of uptake, maximal uptake, and Deff by 10-, 4.7-, and 2-fold, respectively. There were no significant differences in these parameters between the differentiated control and differentiated I-FABP-expressing ES cell lines. In summary, differentiation and expression of I-FABP oppositely modulated NBD-stearic acid uptake parameters and intracellular diffusion in ES cells.
Subject(s)
Carrier Proteins/biosynthesis , Fatty Acids/metabolism , Myelin P2 Protein/biosynthesis , Neoplasm Proteins , Nerve Tissue Proteins , 4-Chloro-7-nitrobenzofurazan/analogs & derivatives , 4-Chloro-7-nitrobenzofurazan/pharmacokinetics , Animals , Carrier Proteins/genetics , Cell Differentiation , Cells, Cultured , Clone Cells/metabolism , Diffusion , Fatty Acid-Binding Protein 7 , Fatty Acid-Binding Proteins , Indicators and Reagents/pharmacokinetics , Mice , Myelin P2 Protein/genetics , Polymerase Chain Reaction , Restriction Mapping , Stearates/pharmacokinetics , Stem Cells/metabolism , TransfectionABSTRACT
Acyl-CoA binding protein (ACBP) is a ubiquitous cytosolic protein found in high levels in tumorigenic cells. However, the molecular basis for the elevated levels of ACBP in malignant cells, ligand binding characteristics, and function in microsomal phospholipid synthesis have not been resolved. To address whether tumorigenic ACBP differs from the native protein, ACBP was purified from LM cells, a tumorigenic subline of mouse L-929 fibroblasts, and its primary structure was examined by delayed-extraction MALDI-linear TOF mass spectrometry. Proteolytic digestion and peptide sequence analysis confirmed that ACBP from LM cells was identical to native mouse ACBP (based on cDNA-derived amino acid sequence) with no amino acid substitutions, deletions, or posttranslational modifications. A fluorescent binding assay revealed that mouse ACBP bound cis-parinaroyl-CoA with high affinity, Kd 7.6 +/- 2.3 nM, at a single binding site. Furthermore, mouse ACBP enhanced microsomal phosphatidic acid formation from oleoyl-CoA 2.3-fold. Mouse ACBP also inhibited microsomal phospholipid acyl chain remodeling of choline-containing phospholipids, phosphatidylcholine and sphingomyelin, by 50 and 64%, respectively. These effects were specific compared to those of native rat liver or recombinant rat ACBP. Mouse and rat ACBPs differed by three amino acid substitutions at positions 4, 68, and 78. Although these small differences in amino acid sequence did not alter binding affinity for cis-parinaroyl-CoA, rat liver ACBP stimulated utilization of oleoyl-CoA 3.8-fold by microsomal glycerol-3-phosphate acyltransferase, significantly higher than that observed with mouse ACBP, but did not alter microsomal phospholipid acyl chain remodeling from oleoyl-CoA. In addition, these ACBPs protected oleoyl-CoA against hydrolysis. Finally, both mouse and rat ACBP shifted the incorporation of oleoyl-CoA from microsomal phospholipid acyl chain remodeling to phosphatidic acid biosynthesis. These data for the first time show a role for ACBP in stimulating microsomal phosphatidic acid biosynthesis and acyl chain remodeling in vitro. While ACBP from tumorigenic cells did not differ from normal, ACBPs from different murine species displayed subtle differences in their effects on microsomal phospholipid metabolism in vitro.
Subject(s)
Acyl Coenzyme A/metabolism , Neoplasm Proteins/chemistry , Neoplasm Proteins/physiology , Amino Acid Sequence , Animals , Isoelectric Focusing , Mice , Microsomes, Liver/metabolism , Molecular Sequence Data , Molecular Weight , Oleic Acid/metabolism , Phosphatidic Acids/biosynthesis , Phospholipids/metabolism , Protein Binding , Rats , Sequence Analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tumor Cells, CulturedABSTRACT
Fatty acyl-CoA affect many cellular functions as well as serving as cellular building blocks. Several families of cytosolic fatty acyl-CoA binding proteins may modulate the activities of fatty acyl-CoA. Intestinal enterocytes contain at least three unique families of cytosolic proteins that bind fatty acyl-CoA: acyl-CoA binding protein (ACBP), fatty acid binding proteins (including the liver, L-FABP and intestinal, I-FABP), and sterol carrier protein-2 (SCP-2). Immortalized rat colon epithelial cell lines expressed only ACBP and SCP-2 at levels of 0.75 +/- 0.13 and 0.42 +/- 0.02 ng/microgram protein. Ras and src transformation increased colon cell density and differentially altered ACBP and SCP-2 expression without affecting I-FABP or L-FABP levels. ACBP levels were 1.8-fold and 1.5-fold increased in ras- and src-transformed cells, respectively. In contrast, SCP-2 expression was significantly decreased 55 and 67% in ras- and src-transformed cells, respectively. Butyrate treatment of ras- and src-transformed cells decreased cell proliferation up to 60-85% as compared to 25-30% in control cells. Butyrate treatment decreased ACBP expression in all cell lines but had no effect on the levels of SCP-2, I-FABP, or L-FABP. These studies suggest that the differential expression of ACBP and SCP-2 in rat colonic cell lines, as well as their modulation by butyrate, may be altered by cell transformation.
Subject(s)
Butyrates/pharmacology , Carrier Proteins/metabolism , Cell Transformation, Neoplastic/metabolism , Colon/metabolism , Neoplasm Proteins , Nerve Tissue Proteins , Plant Proteins , Actins/drug effects , Actins/metabolism , Animals , Butyric Acid , Carrier Proteins/drug effects , Cell Division/drug effects , Cell Division/genetics , Cell Line, Transformed , Cell Transformation, Neoplastic/genetics , Cells, Cultured , Colon/drug effects , Diazepam Binding Inhibitor , Epithelial Cells , Epithelium/drug effects , Epithelium/metabolism , Fatty Acid-Binding Protein 7 , Fatty Acid-Binding Proteins , Genes, ras , Genes, src , Immunoblotting , Myelin P2 Protein/drug effects , Myelin P2 Protein/metabolism , Protein Synthesis Inhibitors/pharmacology , Rabbits , RatsABSTRACT
The role of apoptosis in the spontaneous regression of Sinclair swine melanoma was investigated in vitro with swine melanoma cell lines. Growth characteristics and sensitivity to cycloheximide-induced apoptosis were determined in melanoma cell lines derived from tumours that were progressing or undergoing regression in vivo. In contrast to cell lines derived from progressing tumours, those derived from regressing tumours showed induction of apoptosis; this phenomenon was dependent on dose but independent of cell growth stage in culture. Chromatin condensation, cell shrinkage, and fragmentation into apoptotic bodies, as well as DNA fragmentation into large kilobase fragments, occurred in cell lines from regressing tumours but not from progressing tumours. These findings suggest that swine melanoma cells are inherently more sensitive to cell death during tumour regression. The apoptosis-sensitive and resistant cell lines will be important for further studies of the roles of cell signalling pathways and gene expression in tumour regression.