Liver Fatty Acid-Binding Protein Colocalizes with Peroxisome Proliferator Activated Receptor α and Enhances Ligand Distribution to Nuclei of Living Cells

Although it is hypothesized that long-chain fatty acyl CoAs (LCFA-CoAs) and long-chain fatty acids (LCFAs) regulate transcription in the nucleus, little is known regarding factors that determine the distribution of these ligands to nuclei of living cells. Immunofluorescence colocalization showed tha...

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Veröffentlicht in:Biochemistry (Easton) 2004-03, Vol.43 (9), p.2484-2500
Hauptverfasser: Huang, Huan, Starodub, Olga, McIntosh, Avery, Atshaves, Barbara P, Woldegiorgis, Gebre, Kier, Ann B, Schroeder, Friedhelm
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container_end_page 2500
container_issue 9
container_start_page 2484
container_title Biochemistry (Easton)
container_volume 43
creator Huang, Huan
Starodub, Olga
McIntosh, Avery
Atshaves, Barbara P
Woldegiorgis, Gebre
Kier, Ann B
Schroeder, Friedhelm
description Although it is hypothesized that long-chain fatty acyl CoAs (LCFA-CoAs) and long-chain fatty acids (LCFAs) regulate transcription in the nucleus, little is known regarding factors that determine the distribution of these ligands to nuclei of living cells. Immunofluorescence colocalization showed that liver fatty acid-binding protein (L-FABP; binds LCFA-CoA as well as LCFA) significantly colocalized with PPARα in nuclei of transfected L-cell fibroblasts. Colocalization with a DNA binding dye (SYTO59) revealed that, within the nucleus of control L-cells, the nonhydrolyzable fluorescent LCFA-CoA (BODIPY-C16-S-S-CoA) was distributed primarily in a punctate pattern throughout the nucleoplasm, while nonmetabolizable fluorescent LCFAs (BODIPY-C16 and BODIPY-C12) were localized primarily near the nuclear envelope membranes. L-FABP overexpression selectively increased the targeting of BODIPY-C16-S-S-CoA by 1.9- and 2.7-fold into the nuclear membrane and nucleoplasm, respectively. L-FABP also increased the targeting of fluorescent LCFAs (especially long-chain-length BODIPY-C16) by 1.7-fold to the nuclear membrane and 7.4-fold into the nucleoplasm. A cis-parinaric acid displacement assay showed that L-FABP bound BODIPY-C12 and BODIPY-C16 with K is of 10.1 ± 2.5 and 20.7 ± 1.5 nM, respectively, in the same range as naturally occurring LCFAs. Finally, solid-phase extraction and HPLC analysis revealed that, depending on the fatty acid content of the culture medium, L-FABP expression also increased the cellular LCFA-CoA pool size and altered the LCFA-CoA acyl chain composition. Thus, L-FABP may function as a carrier for selectively enhancing the distribution of LCFA-CoA, as well as LCFA, to nuclei for potential interaction with nuclear receptors.
doi_str_mv 10.1021/bi0352318
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Immunofluorescence colocalization showed that liver fatty acid-binding protein (L-FABP; binds LCFA-CoA as well as LCFA) significantly colocalized with PPARα in nuclei of transfected L-cell fibroblasts. Colocalization with a DNA binding dye (SYTO59) revealed that, within the nucleus of control L-cells, the nonhydrolyzable fluorescent LCFA-CoA (BODIPY-C16-S-S-CoA) was distributed primarily in a punctate pattern throughout the nucleoplasm, while nonmetabolizable fluorescent LCFAs (BODIPY-C16 and BODIPY-C12) were localized primarily near the nuclear envelope membranes. L-FABP overexpression selectively increased the targeting of BODIPY-C16-S-S-CoA by 1.9- and 2.7-fold into the nuclear membrane and nucleoplasm, respectively. L-FABP also increased the targeting of fluorescent LCFAs (especially long-chain-length BODIPY-C16) by 1.7-fold to the nuclear membrane and 7.4-fold into the nucleoplasm. A cis-parinaric acid displacement assay showed that L-FABP bound BODIPY-C12 and BODIPY-C16 with K is of 10.1 ± 2.5 and 20.7 ± 1.5 nM, respectively, in the same range as naturally occurring LCFAs. Finally, solid-phase extraction and HPLC analysis revealed that, depending on the fatty acid content of the culture medium, L-FABP expression also increased the cellular LCFA-CoA pool size and altered the LCFA-CoA acyl chain composition. 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Immunofluorescence colocalization showed that liver fatty acid-binding protein (L-FABP; binds LCFA-CoA as well as LCFA) significantly colocalized with PPARα in nuclei of transfected L-cell fibroblasts. Colocalization with a DNA binding dye (SYTO59) revealed that, within the nucleus of control L-cells, the nonhydrolyzable fluorescent LCFA-CoA (BODIPY-C16-S-S-CoA) was distributed primarily in a punctate pattern throughout the nucleoplasm, while nonmetabolizable fluorescent LCFAs (BODIPY-C16 and BODIPY-C12) were localized primarily near the nuclear envelope membranes. L-FABP overexpression selectively increased the targeting of BODIPY-C16-S-S-CoA by 1.9- and 2.7-fold into the nuclear membrane and nucleoplasm, respectively. L-FABP also increased the targeting of fluorescent LCFAs (especially long-chain-length BODIPY-C16) by 1.7-fold to the nuclear membrane and 7.4-fold into the nucleoplasm. A cis-parinaric acid displacement assay showed that L-FABP bound BODIPY-C12 and BODIPY-C16 with K is of 10.1 ± 2.5 and 20.7 ± 1.5 nM, respectively, in the same range as naturally occurring LCFAs. Finally, solid-phase extraction and HPLC analysis revealed that, depending on the fatty acid content of the culture medium, L-FABP expression also increased the cellular LCFA-CoA pool size and altered the LCFA-CoA acyl chain composition. Thus, L-FABP may function as a carrier for selectively enhancing the distribution of LCFA-CoA, as well as LCFA, to nuclei for potential interaction with nuclear receptors.</abstract><cop>United States</cop><pub>American Chemical Society</pub><pmid>14992586</pmid><doi>10.1021/bi0352318</doi><tpages>17</tpages></addata></record>
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subjects Acyl Coenzyme A - analysis
Acyl Coenzyme A - metabolism
Animals
Biomarkers - chemistry
Boron Compounds - metabolism
Carrier Proteins - biosynthesis
Carrier Proteins - metabolism
Cell Nucleus - chemistry
Cell Nucleus - metabolism
Cytoplasm - metabolism
Fatty Acid-Binding Protein 7
Fatty Acid-Binding Proteins
Fatty Acids - analysis
Fatty Acids - metabolism
Fibroblasts - chemistry
Fibroblasts - metabolism
Fluorescent Antibody Technique, Indirect
Fluorescent Dyes - metabolism
Hydrolysis
L Cells (Cell Line)
Ligands
Liver - metabolism
Mice
Nerve Tissue Proteins
Nuclear Envelope - metabolism
Peroxisomes - metabolism
Protein Binding
Receptors, Cytoplasmic and Nuclear - metabolism
Transcription Factors - metabolism
Transfection
title Liver Fatty Acid-Binding Protein Colocalizes with Peroxisome Proliferator Activated Receptor α and Enhances Ligand Distribution to Nuclei of Living Cells
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