SG00019728

1. To identify and characterize specific mRNAs that increase in abundance during differentiation of mouse 3T3-L1 preadipocytes, a cDNA library was constructed from poly(A)+RNA isolated from differentiated 3T3-L1 adipocytes. Mixed probe isotope ratio selection and RNA blot analyses have identified several unique cDNA clones that represent mRNA species expressed either exclusively or at dramatically increased levels in differentiated cells. Further characterization of one such clone (pAL422) revealed that the corresponding mRNA, detectable only after differentiation, is approximately the same length (600 +/- 150 bases) as the cDNA insert (672 bases). The complete nucleotide sequence of the cDNA insert in pAL422 revealed a single long open reading frame that encodes a 132 amino acid polypeptide (the 422 protein) of 14.6 kDa. These and other results suggest that this cDNA may represent a nearly full-length copy of the mRNA. Computer-assisted analyses showed that the 422 protein shares 69% and 64% homology with myelin P2 proteins from rabbit and bovine peripheral nerves, respectively, as well as 23% and 30% homology with fatty-acid binding proteins from rat liver and intestine, respectively. Moreover, the mRNA hybrid selected by pAL422 DNA directs the in vitro translation of an approximately equal to 13 kDa polypeptide, and this protein is specifically immunoprecipitated by antiserum against bovine myelin P2. These observations strongly suggest that the 422 protein is a structural, and possibly functional, analog of myelin P2. PMID: [6206497]

2. We have isolated the mouse gene encoding adipocyte P2, aP2, the differentiation-dependent adipocyte protein homologous to myelin P2. The aP2 gene is present in a single copy in the mouse and is present in single or few copies in species from human to Drosophila. The entire gene spans 4 kilobases and consists of four exons encoding 25, 57, 34, and 16 amino acids; the overall exon structure is similar to the gene encoding liver fatty acid binding protein. A plasmid vector was constructed containing the entire aP2 gene with flanking sequences, modified by linker insertion. When this gene is stably introduced into 3T3-F442A cells, it is expressed only upon adipose differentiation, with a time course of induction very similar to that of the endogenous aP2 gene. We have compared the DNA sequence of the 5'-flanking region of the aP2 gene to the promoter regions of two other genes activated during adipocyte differentiation, glycerol-3-phosphate dehydrogenase and adipsin, and find a 13-base region of homology (Formula: see text) present in multiple copies in the 5'-flanking region of each gene. An adjacent 15-base sequence is present only in glycerol-3-phosphate dehydrogenase and aP2 genes. Both of these elements share homology with putative viral enhancer core sequences. These results indicate that the aP2 gene contains sequence information necessary for differentiation-dependent expression in fat cells; common elements shared by adipocyte-specific genes may play a role in this process. PMID: [3520554]

3. When 3T3 cells undergo adipose differentiation, they are reprogrammed by changes in gene expression of sufficient magnitude to greatly alter the protein composition of the cells. Three participating genes encode glycerophosphate dehydrogenase, a lipid-binding protein, and a serine protease. These three genes have now been cloned and sequenced. Their exon/intron structures are described, together with some interesting peculiarities and some regions of common sequence. It remains to be demonstrated whether the information for common participation of the genes in the program of differentiation resides in controlling elements within the regions sequenced. PMID: [3015943]

4. This study describes comprehensive polling of transcription start and termination sites and analysis of previously unidentified full-length complementary DNAs derived from the mouse genome. We identify the 5' and 3' boundaries of 181,047 transcripts with extensive variation in transcripts arising from alternative promoter usage, splicing, and polyadenylation. There are 16,247 new mouse protein-coding transcripts, including 5154 encoding previously unidentified proteins. Genomic mapping of the transcriptome reveals transcriptional forests, with overlapping transcription on both strands, separated by deserts in which few transcripts are observed. The data provide a comprehensive platform for the comparative analysis of mammalian transcriptional regulation in differentiation and development. PMID: [16141072]

5. The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline. PMID: [15489334]

6. An adipose-specific protein has been purified from murine 3T3-L1 adipocytes to greater than 98% homogeneity. A purification procedure was developed utilizing a combination of gel filtration, cation exchange chromatography, and covalent chromatography on activated-thiol Sepharose 4B. The protein exists as a single polypeptide with a molecular weight of about 15,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein contains 2 mol of reduced sulfhydryl groups per mol of protein and an amino terminus blocked to sequencing. Automated Edman degradation of trypsin and CNBr-derived peptides has verified that the purified protein is that predicted by the mRNA (Bernlohr, D. A., Angus, C. W., Lane, M. D., Bolanowski, M. A., and Kelly, T. J. Jr. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5468-5472). Based on sequence analysis, the 15-kDa adipocyte protein is considered to be a member of a family of tissue-specific, cytosolic lipid-binding proteins. Utilizing a liposome assay, the purified protein binds both oleic acid and retinoic acid saturably with approximately 1 mol of ligand bound per mol of protein. Dissociation constants determined from Scatchard analysis were 3 and 50 microM, respectively. This report represents the first demonstration of a member of this family of structurally related proteins that is capable of binding both fatty acid and retinoic acid. Hence, we propose the name adipocyte lipid-binding protein, or ALBP. PMID: [2844775]

7. We have isolated and characterized a fragment of the gene encoding adipose fatty acid-binding protein (gene 422) from a 3T3-L1 adipocyte genomic library. The 5'-flanking sequence of the 422 gene contains potential regulatory regions for adipose-specific expression. At position -120 there is a fat-specific element that occurs in several genes expressed as preadipocytes differentiate, and at position -393 there is a glucocorticoid regulatory element core sequence. Chimeric constructs were prepared by ligating 858 base pairs or 248 base pairs of 5'-flanking sequence and 22 nucleotides of 5'-untranslated sequence of the 422 gene to the bacterial gene encoding chloramphenicol acetyltransferase (CAT); these constructs (delta 858.CAT and delta 248.CAT) were transfected into 3T3-L1 preadipocytes. When differentiation was initiated by the adipogenic agents methylisobutylxanthine (a cAMP phosphodiesterase inhibitor), dexamethasone, and insulin, expression of both constructs increased, reaching maximal levels within 24 hr. Both constructs were maximally induced 48 hr before appreciable accumulation of the endogenous 422 mRNA. Expression of delta 858.CAT, but not of delta 248.CAT, was induced by dexamethasone, which correlates with deletion of the potential glucocorticoid regulatory element. Expression of both constructs was induced by 8-bromoadenosine 3',5'-cyclic monophosphate, thus implicating the first 248 base pairs of 5'-flanking sequence of the 422 gene in the response to cAMP. Indirect effects by the adipogenic factors on CAT protein or mRNA synthesis and turnover were ruled out, since replacing the 5'-flanking region of the 422 gene constructs with viral promoters abolished the effects of dexamethasone and 8-bromoadenosine 3',5'-cyclic monophosphate on CAT expression. We conclude that the first 858 base pairs of 5'-flanking sequence of the 422 gene contains elements that mediate activation by dexamethasone and cAMP. PMID: [2452440]

8. We have investigated the regulation of mRNA synthesis during 3T3-adipocyte differentiation by measuring the transcription of specific genes in isolated preadipocyte and adipocyte nuclei. Transcription was assayed by hybridization of newly synthesized RNA to cDNA clones coding for glycerophosphate dehydrogenase (GPD), the induced protein of 13K which is shown here to be related to myelin protein P-2, the induced protein of 28K, actin, and two RNAs that are not developmentally regulated. Transcription of GPD and 13K was observed in adipocyte but not preadipocyte nuclei. Actin was transcribed in both types of nuclei but at a lower level in adipocytes. For most of the RNAs examined, there was a consistent relationship between amounts of nuclear transcription and the abundance of the corresponding cytoplasmic mRNA in adipocytes. However, 13K and 28K mRNAs are 10-100 times more abundant than would be predicted by their nuclear transcription alone. Preliminary mRNA turnover experiments in which 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole was used to inhibit mRNA synthesis suggest that these mRNAs are much more stable in the adipocyte cytoplasm than the other mRNAs examined. These results indicate that the transcription of specific genes is increased during adipocyte differentiation and suggest that other levels of control, particularly mRNA stability, may contribute to the relative abundance of certain developmentally-regulated mRNAs in adipocytes. PMID: [3968175]

9. Proteins of 14,000 daltons (SLP-14) were isolated and partially characterized from mouse mammary glands of different developmental stages. The purified proteins were partially sequenced at the amino acid level. The SLP-14 belong to the family of fatty acid-binding (FABP) proteins. The major SLP-14 expressed was a reflection of the differentiation stage of the mammary gland. Within the regions sequenced for each protein, virgin mouse mammary gland (primarily adipocytes) expressed a protein 100% homologous to adipocyte lipid binding protein whereas pregnant and lactating mouse mammary glands expressed a protein highly homologous to mouse heart fatty acid binding protein. In the lactating gland, the isolated protein was 97% identical to the heart FABP over a 98 amino acid stretch. We could not detect the 14Kd protein identified as mammary gland growth inhibitor (MDGI) which is also a member of the FABP family and highly homologous to heart FABP. A rabbit antiserum to the rat mammary SLP-14 recognized the SLP-14 proteins in mouse and rat mammary gland, skeletal muscle and heart, whereas it failed to recognize the SLP-14 in liver, intestine and other organs. These data indicate that the SLP-14 detected in rodent mammary gland is of the fatty acid-binding protein family. On the basis of amino acid sequence, the major form in the differentiated mouse mammary gland is apparently FABP and not MDGI. MDGI may be a protein in low abundance and/or localized to a specific group of mammary epithelial cells. PMID: [8447836]

10. Lipophilic compounds such as retinoic acid and long-chain fatty acids regulate gene transcription by activating nuclear receptors such as retinoic acid receptors (RARs) and peroxisome proliferator-activated receptors (PPARs). These compounds also bind in cells to members of the family of intracellular lipid binding proteins, which includes cellular retinoic acid-binding proteins (CRABPs) and fatty acid binding proteins (FABPs). We previously reported that CRABP-II enhances the transcriptional activity of RAR by directly targeting retinoic acid to the receptor. Here, potential functional cooperation between FABPs and PPARs in regulating the transcriptional activities of their common ligands was investigated. We show that adipocyte FABP and keratinocyte FABP (A-FABP and K-FABP, respectively) selectively enhance the activities of PPARgamma and PPARbeta, respectively, and that these FABPs massively relocate to the nucleus in response to selective ligands for the PPAR isotype which they activate. We show further that A-FABP and K-FABP interact directly with PPARgamma and PPARbeta and that they do so in a receptor- and ligand-selective manner. Finally, the data demonstrate that the presence of high levels of K-FABP in keratinocytes is essential for PPARbeta-mediated induction of differentiation of these cells. Taken together, the data establish that A-FABP and K-FABP govern the transcriptional activities of their ligands by targeting them to cognate PPARs in the nucleus, thereby enabling PPARs to exert their biological functions. PMID: [12077340]

11. We and others showed earlier that liver-type, epidermal-type and adipocyte-type (A-) fatty acid-binding proteins (FABPs) mediate peroxisome proliferator activated receptor (PPAR) dependent gene expression by channelling their ligand (fatty acid or drug) to the nuclear receptors via direct protein/protein interaction. To clarify mechanistic details of this signaling path, we address here A-FABP import into the nucleus and its interaction with PPARgamma. Making use of COS cells transfected with wild-type or mutant A-FABPs, we exclude posttranslational modification of A-FABP as import signal and provide evidence for both, ligand-dependent and ligand-independent nuclear translocation. With the aid of in vitro pull down assay we demonstrate that specific interaction of A-FABP with PPARgamma isoforms does not require ligand. Moreover, A-FABP binds not only to the ligand-binding domain including hinge domain (domains DEF), but also to the DNA-binding domain including AB domains (domains ABC) of PPARgamma. PMID: [16574478]

12. FABP4 delivers specific ligands from the cytosol to the nuclear receptor PPARgamma in the nucleus, thereby facilitating the ligation and enhancing the transcriptional activity of the receptor. Here, we delineate the structural features that underlie the nucleocytoplasmic transport of FABP4. The primary sequence of FABP4 does not harbor a readily identifiable nuclear localization signal (NLS). However, such a signal could be found in the three-dimensional structure of the protein and was mapped to three basic residues that form a functional NLS stabilized by the FABP4/PPARgamma ligand troglitazone. We show that FABP4 is also subject to active nuclear export. Similarly to the NLS, the nuclear export signal (NES) is not apparent in the primary sequence, but assembles in the tertiary structure from three nonadjacent leucine residues to form a motif reminiscent of established NES. The data demonstrate that both nuclear export and nuclear import are critical for enabling FABP4 to enhance the transcriptional activity of PPARgamma. Additionally, the observations provide insight into the fundamental question of how proteins are activated by ligands. Such an activation may be understood by the "induced-fit" model, which states that ligand-induced conformational changes precede activation of a protein. Alternatively, the "pre-existing equilibrium" hypothesis postulates that activated conformations exist within the repertoire of apoproteins, and that ligands do not induce these but merely stabilize them. Studies of the subcellular localization of FABP4 support the validity of the "pre-existing equilibrium" model for the ligand-controlled activation of the nuclear import of FABP4. PMID: [17516629]

13. Adipocyte lipid-binding protein (ALBP) is the adipocyte member of an intracellular hydrophobic ligand-binding protein family. ALBP is phosphorylated by the insulin receptor kinase upon insulin stimulation. The crystal structure of recombinant murine ALBP has been determined and refined to 2.5 A. The final R factor for the model is 0.18 with good canonical properties. Crystalline ALBP has a conformation which is essentially identical to that of intestinal fatty acid binding protein and myelin P2 protein. Although the crystal structure is of the apo- form, a cavity resembling that in other family members is present. It contains a number of bound and implied unbound water molecules and shows no large obvious portal to the external milieu. The cavity of ALBP, which by homology is the ligand-binding site, is formed by both polar and hydrophobic residues among which is tyrosine 19. Y19 is phosphorylated by the insulin receptor kinase as described in the accompanying paper [Buelt, M. K., Xu, Z., Banaszak, L. J., & Bernlohr, D. A. (1992) Biochemistry (following paper in this issue)]. By comparing ALBP with the earlier structural results on intestinal fatty acid binding protein, it is now possible to delineate conserved amino acids which help form the binding site in this family. PMID: [1554730]

14. Crystals of the adipocyte lipid-binding protein which diffract to near atomic resolution have been obtained in Na/K phosphate buffer/precipitant system. The structures of the apo-form and the protein with bound oleic acid and stearic acid have been determined and refined to 1.6-A resolution with R-factor around 18%. The conformations of the bound fatty acids are nearly the same. In both cases, the carboxylate group of the ligand interacts directly with Arg126 and Tyr128, indirectly with Arg106 through a water molecule. The hydrocarbon tail sticks out of the protein surface through a hydrophobic patch. Saturated and unsaturated fatty acids bind in essentially the same conformation. The remaining space of the binding pocket is filled with well ordered water molecules interacting with most of the polar side chains. Comparisons between the holo- and apostructures reveal that the hydrophobic patch on the protein surface formed by a helix and several tight turns might serve as a portal for lipid binding. Since the adipocyte lipid-binding protein is phosphorylated at Tyr19 by the insulin receptor kinase, the position of this side chain has been re-evaluated using the coordinates of the holo-forms. It appears that the position of Tyr19 does not change significantly upon the binding of either of the fatty acids. PMID: [8463311]

15. The fluorescent probe anilinonaphthalene-8-sulfonate binds to adipocyte lipid binding protein at a site that competes with normal physiological ligands, such as fatty acids. Binding to the protein is accompanied by a relatively large increase in fluorescent intensity. To correlate the major change in optical properties and to determine the mechanism of competitive inhibition with fatty acids, the crystal structure of the protein with the bound fluorophore has been determined. In addition, the thermodynamic contributions to the binding reaction have been studied by titration calorimetry. Because the binding site is in a relatively internal position, kinetic studies have also been carried out to determine k(on). The results indicate that binding is not accompanied by any major conformational change. However, the negatively charged sulfonate moiety is not positioned the same as the carboxylate of fatty acid ligands as determined in previous studies. Nonetheless, the binding reaction is still driven by enthalpic effects. As judged by the crystallographic structure, a significant amount of the surface of the fluorophore is no longer exposed to water in the bound state. PMID: [10423455]

16. Adipocyte lipid-binding protein is a 14.6-kDa polypeptide that is responsible for the intracellular trafficking of fatty acids. Its structure previously has been solved in the apo and holo forms complexed with stearate and oleate. To examine the binding of lipids other than those with a carboxylate headgroup, we have determined the structure of ALBP in complex with a sulfonic acid, hexadecanesulfonic acid, and compared its structure with the natural fatty acid analog, palmitate. Crystallographic refinement led to similar models, both with R-factors of about 20% and a resolution of 1.6 A. results can be compared with earlier studies on C18 fatty acids, both saturated and unsaturated. The previously refined complexes with stearate and oleate in combination with the complexes of palmitate and hexadecanesulfonic acid demonstrate specific positions for water molecules bound in the internal cavity. Many of the water-binding sites are present in both the apo form and the holo forms of the protein. With ligand present, a network of 10 internalized water molecules appear to form a hydrophobic hydration region. In spite of the sp3 geometry of the sulfonic acid derivative, the headgroup occupies the same site as that of the planar carboxylate in natural fatty acids. These results demonstrate that intracellular lipid-binding proteins are capable of binding a wider variety of lipids than previously considered and reveal the importance of interior ordered water molecules in the binding cavity. PMID: [8161548]

17. The association of the adipocyte lipid-binding protein (ALBP) with arachidonic acid (all cis, 20:4 delta 5,8,11,14) and oleic acid (cis, 18:1 delta 9) has been examined by titration calorimentry. In addition, the crystal structure of ALBP with bound arachidonic acid has also been obtained. Crystallographic analysis of the arachidonic acid.ALBP complex along with the previously reported oleic acid-ALBP structure (Xu, Z., Bernlohr, D. A., and Banaszak, L. J. (1993) J. Biol. Chem. 268, 7874-7884) provides a framework for the molecular examination of protein-lipid association. Isothermal titration calorimetry revealed high affinity association of both unsaturated fatty acids with the protein. The calorimetric data yielded the following thermodynamic parameters for arachidonic acid: Kd = 4.4 microM, n = 0.8, delta G = -7370 cal/mol, delta H = -6770 cal/mol, and T delta S = +600 cal/mol. For oleic acid, the thermodynamic parameters were Kd = 2.4 microM, n = 0.9, delta G = -7770 cal/mol, delta H = -6050 cal/mol, and T delta S = +1720 cal/mol. The identification of thermodynamically dominating enthalpic factors for both fatty acids are consistent with the crystallographic studies demonstrating the interaction of the fatty acid carboxylate with a combination of Arg106, Arg126, and Tyr128. The crystallographic refinement of the protein-arachidonate complex was carried out to 1.6 A with the resultant R factor of 0.19. Within the cavity of the crystalline binding protein, the arachidonate was found in a hairpin conformation. The conformation of the bound ligand is consistent with acceptable torsional angles and the four cis double bonds in arachidonate. These results demonstrate that arachidonate is a ligand for ALBP. They provide thermodynamic and structural data concerning the physical basis for protein-lipid interaction and suggest that intracellular lipid-binding proteins may mediate the biological effects of polyunsaturated fatty acids in vivo. PMID: [7929228]

18. The family of proteins accountable for the intracellular movement of lipids is characterized by a 10-stranded beta-barrel that forms an internalized cavity varying in size and binding preferences. The loop connecting beta-strands E and F (the fifth and sixth strands) is the most striking conformational difference between adipocyte lipid binding protein (ALBP; fatty acids) and cellular retinoic acid binding protein type I (CRABP I). A three-residue mutation was made in wild-type (WT)-ALBP [ALBP with a three-residue mutation (EF-ALBP)] to mimic CRABP I. Crystal structures of ligand-free and EF-ALBP with bound oleic acid were solved to resolutions of 1.5 A and 1.7 A, respectively, and compared with previous studies of WT-ALBP. The changes in three residues of one loop of the protein appear to have altered the positioning of the C18 fatty acid, as observed in the electron density of EF-ALBP. The crystallographic studies made it possible to compare the protein conformation and ligand positioning with those found in the WT protein. Although the cavity binding sites in both the retinoid and fatty acid binding proteins are irregular, the ligand atoms appear to favor a relatively planar region of the cavities. Preliminary chemical characterization of the mutant protein indicated changes in some binding properties and overall protein stability. PMID: [14594993]

19. Fatty acid-binding protein 4 (FABP4) delivers ligands from the cytosol to the nuclear receptor PPARgamma in the nucleus, thereby enhancing the transcriptional activity of the receptor. Notably, FABP4 binds multiple ligands with a similar affinity but its nuclear translocation is activated only by specific compounds. To gain insight into the structural features that underlie the ligand-specificity in activation of the nuclear import of FABP4, we solved the crystal structures of the protein complexed with two compounds that induce its nuclear translocation, and compared these to the apo-protein and to FABP4 structures bound to non-activating ligands. Examination of these structures indicates that activation coincides with closure of a portal loop phenylalanine side-chain, contraction of the binding pocket, a subtle shift in a helical domain containing the nuclear localization signal of the protein, and a resultant change in oligomeric state that exposes the nuclear localization signal to the solution. Comparisons of backbone displacements induced by activating ligands with a measure of mobility derived from translation, libration, screw (TLS) refinement, and with a composite of slowest normal modes of the apo state suggest that the helical motion associated with the activation of the protein is part of the repertoire of the equilibrium motions of the apo-protein, i.e. that ligand binding does not induce the activated configuration but serves to stabilize it. Nuclear import of FABP4 can thus be understood in terms of the pre-existing equilibrium hypothesis of ligand binding. PMID: [17761196]