Small Lipid-binding Proteins Research Articles

The Multifunctional Family of Mammalian Fatty Acid-Binding Proteins.

Fatty acid-binding proteins (FABPs) are small lipid-binding proteins abundantly expressed in tissues that are highly active in fatty acid (FA) metabolism. Ten mammalian FABPs have been identified, with tissue-specific expression patterns and highly conserved tertiary structures. FABPs were initially studied as intracellular FA transport proteins. Further investigation has demonstrated their participation in lipid metabolism, both directly and via regulation of gene expression, and in signaling within their cells of expression. There is also evidence that they may be secreted and have functional impact via the circulation. It has also been shown that the FABP ligand binding repertoire extends beyond long-chain FAs and that their functional properties also involve participation in systemic metabolism. This article reviews the present understanding of FABP functions and their apparent roles in disease, particularly metabolic and inflammation-related disorders and cancers.

Abstract 219: Apolipoprotein C3 Induces Inflammasome Activation In Human And Mouse Monocytes Only In De-lipidated Form

Individuals with diabetes suffer from an elevated risk of coronary artery disease (CAD) events. Increased serum levels of apolipoprotein C3 (APOC3) predict incident CAD in individuals with type 1 diabetes (T1D). APOC3 is a small lipid-binding protein carried by lipoproteins, including VLDL, in circulation. Silencing of APOC3 in a mouse model of T1D-accelerated atherosclerosis has been demonstrated to prevent diabetes-accelerated atherosclerosis, strongly suggesting that APOC3 is a causal mediator. However, the exact mechanism whereby APOC3 promotes atherosclerosis in the setting of T1D is unclear. Mice with T1D demonstrated elevated levels of glucose, triglycerides, APOC3 (non-diabetic [ND] mice=442.6 μg/ml, diabetic [D] mice=755.9 μg/ml, p<0.01, N=21 per group), and IL-18 (ND=124.1 pg/ml, D=256.8 pg/ml, p<0.01, N=18-22 per group) in plasma. The elevated levels of plasma APOC3 and IL-18 in diabetic mice and a recent study demonstrating that de-lipidated APOC3 can stimulate inflammasome activation in human monocytes prompted us to investigate whether lipidated APOC3 has similar inflammasome-activating properties. Our results show that de-lipidated APOC3, but not VLDL (which contains APOC3) significantly stimulates IL-1β release from isolated human monocytes (p<0.01 for vehicle vs APOC3, N=3 per group) and mouse monocytes (p<0.01 for vehicle vs APOC3, N=4-7 per group). Mouse monocytes stimulated with de-lipidated APOC3 also had elevated IL-18 release (vehicle=21.2 pg/mg, APOC3=170.2 pg/mg, p<0.001), Il1b mRNA (APOC3=86.7-fold over control), and Nlrp3 mRNA (APOC3=13.8-fold over control), indicating activation of the NLRP3 inflammasome pathway. However, analysis of serum samples from subjects with T1D showed that virtually all APOC3 is bound to lipoproteins (primarily VLDL and HDL). To further confirm the effect of lipidated APOC3, we bound APOC3 to lipid-based small unilamellar vesicles to mimic the physiological state of APOC3 in plasma. The stimulatory effect of APOC3, measured by IL-1β release from mouse monocytes, was lost. Together, our findings suggest that although de-lipidated APOC3 has the ability to induce inflammasome activation in monocytes, virtually no APOC3 circulates in a lipid-free form in subjects with T1D.

WIPK-NtLTP4 pathway confers resistance to Ralstonia solanacearum in tobacco.

WIPK-NtLTP4 module improves the resistance to R. solanacearum via upregulating the expression of defense-related genes, increasing the antioxidant enzyme activity, and promoting stomatal closure in tobacco. Lipid transfer proteins (LTPs) are a class of small lipid binding proteins that play important roles in biotic and abiotic stresses. The previous study revealed that NtLTP4 positively regulates salt and drought stresses in Nicotiana tabacum. However, the role of NtLTP4 in biotic stress, especially regarding its function in disease resistance remains unclear. Here, the critical role of NtLTP4 in regulating resistance to Ralstonia solanacearum (R. solanacearum), a causal agent of bacterial wilt disease in tobacco, was reported. The NtLTP4-overexpressing lines markedly improved the resistance to R. solanacearum by upregulating the expression of defense-related genes, increasing the antioxidant enzyme activity, and promoting stomatal closure. Moreover, NtLTP4 interacted with wound-induced protein kinase (WIPK; a homolog of MAPK3 in tobacco) and acted in a genetically epistatic manner to WIPK in planta. WIPK could directly phosphorylate NtLTP4 to positively regulate its protein abundance. Taken together, these results broaden the knowledge about the functions of the WIPK-NtLTP4 module in disease resistance and may provide valuable information for improving tobacco plant tolerance to R. solanacearum.

Unconventional Secretion of Nigerolysins A from Aspergillus Species.

Aegerolysins are small lipid-binding proteins particularly abundant in fungi. Aegerolysins from oyster mushrooms interact with an insect-specific membrane lipid and, together with MACPF proteins produced by the same organism, form pesticidal pore-forming complexes. The specific interaction with the same membrane lipid was recently demonstrated for nigerolysin A2 (NigA2), an aegerolysin from Aspergillus niger. In Aspergillus species, the aegerolysins were frequently found as secreted proteins, indicating their function in fungal defense. Using immunocytochemistry and live-cell imaging we investigated the subcellular localization of the nigerolysins A in A. niger, while their secretion was addressed by secretion prediction and Western blotting. We show that both nigerolysins A are leaderless proteins that reach the cell exterior by an unconventional protein secretion. NigA proteins are evenly distributed in the cytoplasm of fungal hyphae. A detailed bioinformatics analysis of Aspergillus aegerolysins suggests that the same function occurs only in a limited number of aegerolysins. From alignment, analysis of chromosomal loci, orthology, synteny, and phylogeny it follows that the same or a similar function described for pairs of pesticidal proteins of Pleurotus sp. can be expected in species of the subgenus Circumdati, section Nigri, series Nigri, and some other species with adjacent pairs of putative pesticidal proteins.

Inhibiting Stearoyl-CoA Desaturase Ameliorates α-Synuclein Cytotoxicity

The lack of disease-modifying treatments for neurodegenerative disease stems in part from our rudimentary understanding of disease mechanisms and the paucity of targets for therapeutic intervention. Here we used an integrated discovery paradigm to identify a new therapeutic target for diseases caused by α-synuclein (α-syn), a small lipid-binding protein that misfolds and aggregates in Parkinson's disease and other disorders. Using unbiased phenotypic screening, we identified a series of compounds that were cytoprotective against α-syn-mediated toxicity by inhibiting the highly conserved enzyme stearoyl-CoA desaturase (SCD). Critically, reducing the levels of unsaturated membrane lipids by inhibiting SCD reduced α-syn toxicity in human induced pluripotent stem cell (iPSC) neuronal models. Taken together, these findings suggest that inhibition of fatty acid desaturation has potential as a therapeutic approach for the treatment of Parkinson's disease and other synucleinopathies.

Ganglioside Metabolism in Health and Disease.

Gangliosides (GGs) are cell type-specific sialic acid-containing glycosphingolipids (GSLs), which are enriched in mammalian brain. Defects in GSL metabolism cause fatal human diseases. GSLs are composed of a hydrophilic oligosaccharide linked in 1-O-position to a hydrophobic ceramide anchor, which itself is composed of a long-chain amino alcohol, the sphingoid base, and an amide-bound acyl chain. Biosynthesis of mammalian GGs and other GSLs starts with the formation of their hydrophobic ceramide anchor in the endoplasmic reticulum, followed by sequential glycosylation reactions along the secretory pathway, mainly at the luminal surface of Golgi and trans-Golgi network (TGN) membranes. Few membrane-anchored and often promiscuous glycosyltransferases allow the formation of cell type-specific glycolipid patterns in a combinatorial process. Inherited defects of these transferases therefore affect not only single structures but defined glycolipid series. GGs and other GSLs are thereafter transported by an exocytotic membrane flow to the plasma membrane where they are expressed in cell type-specific patterns, which can be modified by metabolic reactions at or near the cellular surface. Endocytosed (glyco)sphingolipids are degraded, together with other membrane lipids in a stepwise fashion by endolysosomal enzymes with the help of small lipid-binding proteins, the sphingolipid activator proteins (SAPs), at the surface of intraluminal lysosomal vesicles. Inherited defects in a sphingolipid-degrading enzyme or SAP cause the accumulation of the corresponding lipid substrates. Endolysosomal GSL degradation is strongly modified by the lipid components of the organelle microenvironments.

Effects of macromolecular crowding on a small lipid binding protein probed at the single-amino acid level

Macromolecular crowding is a distinctive feature of the cellular interior, influencing the behaviour of biomacromolecules. Despite significant advancements in the description of the effects of crowding on global protein properties, the influence of cellular components on local protein attributes has received limited attention. Here, we describe a residue-level systematic interrogation of the structural, dynamic, and binding properties of the liver fatty acid binding protein (LFABP) in crowded solutions.Two-dimensional NMR spectral fingerprints and relaxation data were collected on LFABP in the presence of polymeric and biomolecular crowders. Non-interacting crowders produced minimal site-specific spectral perturbations on ligand-free and lipid-bound LFABP. Conformational adaptations upon ligand binding reproduced those observed in dilute solution, but a perturbation of the free oleate state resulted in less favorable uptake. When LFABP engaged in direct interactions with background molecules, changes in local chemical environments were detected for residues of the internal binding pocket and of the external surface.Enhanced complexity was introduced by investigating LFABP in cell lysates, and in membrane-bounded compartments. LFABP was able to capture ligands from prokaryotic and eukaryotic cell lysates, and from artificial cells (water-in-oil emulsion droplets).The data suggest that promiscuous interactions are a major factor influencing protein function in the cell.

Isolation and functional analysis of yeast ubiquitin ligase Rsp5 variants that alleviate the toxicity of human α-synuclein.

The essential ubiquitin ligase Rsp5 is a key enzyme involved in the degradation of abnormal or unfavourable proteins in the yeast Saccharomyces cerevisiae. Overexpression of human α-synuclein (α-syn), a small lipid-binding protein implicated in several neurodegenerative diseases, in S. cerevisiae leads to growth inhibition due to many intracellular defects, including accumulation of reactive oxygen species (ROS). Here, to understand the mechanism of Rsp5-mediated detoxification of α-syn, we isolated novel Rsp5 variants (T255A, D295G, P343S and N427D), which conferred α-syn tolerance to yeast cells. Interestingly, these mutants were phenotypically distinguished from our previously identified RSP5(T357A) mutation, which increases ubiquitination of the general amino acid permease Gap1. Among them, the RSP5(P343S) substitution accelerated the degradation of α-syn, suppressed the accumulation of intracellular ROS and enhanced the interaction with α-syn and its ubiquitination. In contrast, the RSP5(T255A) mutation did not contribute to degradation of α-syn, but improved cell growth under acetate stress conditions, possibly leading to alleviation of the α-syn toxicity. Thus, these novel mutations might be useful not only in elucidating the molecular basis by which disused proteins are specifically recognized and effectively removed but also in screening drug candidates for neurodegenerative diseases or in improving ethanol production under acidic fermentation conditions.

CRABP2 Promotes Myoblast Differentiation and Is Modulated by the Transcription Factors MyoD and Sp1 in C2C12 Cells

Cellular retinoic acid binding protein 2 (CRABP2), a member of a family of specific carrier proteins for Vitamin A, belongs to a family of small cytosolic lipid binding proteins. Our previous study suggested that CRABP2 was involved in skeletal muscle development; however, the molecular function and regulatory mechanism of CRABP2 in myogenesis remained unclear. In this study, we found that the expression of the CRABP2 gene was upregulated during C2C12 differentiation. An over-expression assay revealed that CRABP2 promotes myogenic transformation by regulating the cell cycle during C2C12 differentiation. The region from −459 to −4 bp was identified as the core promoter and contains a TATA box, a GC box and binding sites for the transcription factors MyoD and Sp1. Over-expression, site-directed mutagenesis and EMSA assays indicated that the transcription factors MyoD and Sp1 regulate CRABP2 expression and promote myoblast differentiation in C2C12 cells.

Small lipid-binding proteins in regulating endothelial and vascular functions: focusing on adipocyte fatty acid binding protein and lipocalin-2.

Dysregulated production of adipokines from adipose tissue plays a critical role in the development of obesity-associated cardiovascular abnormalities. A group of adipokines, including adipocyte fatty acid binding protein (A-FABP) and lipocalin-2, possess specific lipid-binding activity and are up-regulated in obese human subjects and animal models. They act as lipid chaperones to promote lipotoxicity in endothelial cells and cause endothelial dysfunction under obese conditions. However, different small lipid-binding proteins modulate the development of vascular complications in distinctive manners, which are partly attributed to their specialized structural features and functionalities. By focusing on A-FABP and lipocalin-2, this review summarizes recent advances demonstrating the causative roles of these newly identified adipose tissue-derived lipid chaperones in obesity-related endothelial dysfunction and cardiovascular complications. The specific lipid-signalling mechanisms mediated by these two proteins are highlighted to support their specialized functions. In summary, A-FABP and lipocalin-2 represent potential therapeutic targets to design drugs for preventing vascular diseases associated with obesity. This article is part of a themed section on Fat and Vascular Responsiveness. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-3.

Solution Structure of a Repeated Unit of the ABA-1 Nematode Polyprotein Allergen of Ascaris Reveals a Novel Fold and Two Discrete Lipid-Binding Sites

BackgroundNematode polyprotein allergens (NPAs) are an unusual class of lipid-binding proteins found only in nematodes. They are synthesized as large, tandemly repetitive polyproteins that are post-translationally cleaved into multiple copies of small lipid binding proteins with virtually identical fatty acid and retinol (Vitamin A)-binding characteristics. They are probably central to transport and distribution of small hydrophobic compounds between the tissues of nematodes, and may play key roles in nutrient scavenging, immunomodulation, and IgE antibody-based responses in infection. In some species the repeating units are diverse in amino acid sequence, but, in ascarid and filarial nematodes, many of the units are identical or near-identical. ABA-1A is the most common repeating unit of the NPA of Ascaris suum, and is closely similar to that of Ascaris lumbricoides, the large intestinal roundworm of humans. Immune responses to NPAs have been associated with naturally-acquired resistance to infection in humans, and the immune repertoire to them is under strict genetic control.Methodology/Principal FindingsThe solution structure of ABA-1A was determined by protein nuclear magnetic resonance spectroscopy. The protein adopts a novel seven-helical fold comprising a long central helix that participates in two hollow four-helical bundles on either side. Discrete hydrophobic ligand-binding pockets are found in the N-terminal and C-terminal bundles, and the amino acid sidechains affected by ligand (fatty acid) binding were identified. Recombinant ABA-1A contains tightly-bound ligand(s) of bacterial culture origin in one of its binding sites.Conclusions/SignificanceThis is the first mature, post-translationally processed, unit of a naturally-occurring tandemly-repetitive polyprotein to be structurally characterized from any source, and it belongs to a new structural class. NPAs have no counterparts in vertebrates, so represent potential targets for drug or immunological intervention. The nature of the (as yet) unidentified bacterial ligand(s) may be pertinent to this, as will our characterization of the unusual binding sites.

Compounds from an unbiased chemical screen reverse both ER-to-Golgi trafficking defects and mitochondrial dysfunction in Parkinson’s disease models

alpha-Synuclein (alpha-syn) is a small lipid-binding protein involved in vesicle trafficking whose function is poorly characterized. It is of great interest to human biology and medicine because alpha-syn dysfunction is associated with several neurodegenerative disorders, including Parkinson's disease (PD). We previously created a yeast model of alpha-syn pathobiology, which established vesicle trafficking as a process that is particularly sensitive to alpha-syn expression. We also uncovered a core group of proteins with diverse activities related to alpha-syn toxicity that is conserved from yeast to mammalian neurons. Here, we report that a yeast strain expressing a somewhat higher level of alpha-syn also exhibits strong defects in mitochondrial function. Unlike our previous strain, genetic suppression of endoplasmic reticulum (ER)-to-Golgi trafficking alone does not suppress alpha-syn toxicity in this strain. In an effort to identify individual compounds that could simultaneously rescue these apparently disparate pathological effects of alpha-syn, we screened a library of 115,000 compounds. We identified a class of small molecules that reduced alpha-syn toxicity at micromolar concentrations in this higher toxicity strain. These compounds reduced the formation of alpha-syn foci, re-established ER-to-Golgi trafficking and ameliorated alpha-syn-mediated damage to mitochondria. They also corrected the toxicity of alpha-syn in nematode neurons and in primary rat neuronal midbrain cultures. Remarkably, the compounds also protected neurons against rotenone-induced toxicity, which has been used to model the mitochondrial defects associated with PD in humans. That single compounds are capable of rescuing the diverse toxicities of alpha-syn in yeast and neurons suggests that they are acting on deeply rooted biological processes that connect these toxicities and have been conserved for a billion years of eukaryotic evolution. Thus, it seems possible to develop novel therapeutic strategies to simultaneously target the multiple pathological features of PD.

Cytoplasmic fatty acid binding protein sensing fatty acids for peroxisome proliferator activated receptor activation.

Translation of nutrient stimuli through intracellular signaling is important for adaptation and regulation of metabolic processes, while deregulation by either genetic or environmental factors predisposes towards the development of metabolic disorders. Besides providing energy, fatty acids act as prominent signaling molecules by altering cell membrane structures, affecting the lipid modification status of proteins, and by modulating ligand-activated nuclear receptor activity. Given their highly hydrophobic nature, fatty acids in the aqueous intracellular compartment are bound to small intracellular lipid binding proteins which function as intracellular carriers of these hydrophobic components. This review describes recent advances in identifying intracellular pathways for cytosolic fatty acid signaling through ligand activated receptors by means of small intracellular lipid binding proteins. The mechanism behind intracellular fatty acid transport and subsequent nuclear receptor activation is an emerging concept, and advances in understanding this process provide new potential therapeutic targets towards the treatment of metabolic disorders.

Comprehensive analysis of Australian hard wheat cultivars shows limited puroindoline allele diversity

Grain hardness defines the primary commercially important characteristics of wheat, its major grades, its uses and export markets. The puroindoline genes Pina-D1 and Pinb-D1, located only on chromosome 5D of common wheat and encoding small lipid-binding proteins, the puroindolines, are considered to be key genes that determine grain texture, the ‘soft’ texture being wild-type and hard texture being determined by either Pina-D1 gene deletion or a number of separate point mutations in Pinb-D1a, a small number of these being reported to offer different grades of hardness. We have analysed the Pina-D1 and Pinb-D1 genes of 55 Australian hard wheat cultivars, by amplification of the genes, identification of any null mutants, determination of variations in Cleaved Amplified Polymorphic Sequences (CAPS) due to certain point mutations in Pinb-D1, and/or DNA sequencing. The results show that 17 of the hard wheats were Pina-D1b null mutants, 33 others had the well-reported Pinb-D1b glycine-to-serine mutation and 5 were mixed seeds, but none had any other point mutations in Pinb-D1b. The results show that the Australian gene pool is limited in the range of genetically determined grades of hardness to only the two major types. In contrast, of the 30 overseas wheat land-races investigated, 3 exhibited 3 other point mutations in the Pinb-D1 that are suggested to provide different degrees of hardness. Such new genetic resources need to be utilised for the Australian breeding programs to create a range of grain textures suitable for different properties and uses of end-products.

Saposin C: Neuronal Effect and CNS Delivery by Liposomes

Saposin C is one of four small lipid-binding proteins that derive from a single precursor protein, named prosaposin (PSAP). PSAP has several neuronal effects, including neurite outgrowth stimulation, neuron preservation, and nerve regeneration enhancement. A minimal domain required for PSAP's neurotrophic function is located in the amino-terminal half of saposin C. Genetic defects of the PSAP gene in humans and mice lead to a complex lysosomal storage disease. The skin fibroblasts from PSAP- and saposin C-deficient patients have a massive accumulation of multivesicular bodies (MVBs). Incorporation of exogenous saposin C-containing liposomes into the cultured PSAP-/- cells reduced the accumulated MVBs to normal levels. Internalized saposin C was localized to late endosomes and lysosomes. MVBs are crucial for maintaining the cellular homeostasis required for neuronal development and growth. PSAP-/- mice have a short life span (30 days) and central nervous system (CNS) neuronal degeneration. Similar to PSAP-/- fibroblasts, excessive MVBs accumulated in CNS neurons and brain tissues of PSAP-null mice. Cultured cortical and hippocampal neurons from PSAP-/- mice had poor survival and displayed a neurite degenerative pattern. Delivery of saposin C ex vivo into cultured neurons and in vivo into brain neuronal cells in mice across the blood-brain barrier was accomplished with intravenously administered dioleoylphosphatidylserine (DOPS) liposomes. These studies may yield a new therapeutic approach for neuron protection, preservation, and regeneration.

Altered Epithelial Cell Lineage Allocation and Global Expansion of the Crypt Epithelial Stem Cell Population Are Associated with Ileitis in SAMP1/YitFc Mice

Crohn's disease is characterized by cycles of mucosal injury and ulceration followed by epithelial regeneration and restoration of normal epithelial function. In this study, we examined whether ileitis in SAMP1/YitFc mice, a recombinant-inbred line that spontaneously develops ileitis resembling human Crohn's disease, was associated with alterations in normal patterns of epithelial differentiation or changes in epithelial regeneration after experimental injury. Increased numbers of Paneth, goblet, and intermediate cells were present focally in the ileum of SAMP1/YitFc mice by 4 weeks of age, before any histological evidence of acute or chronic inflammation. This increase in secretory cells became more pronounced at sites of ileitis with increasing age and inflammation. Additionally, there was mispositioning of Paneth and intermediate cells along the crypt-to-villus unit. A concomitant reduction in the number of absorptive enterocytes was observed. In contrast to the ileal-specific changes in lineage allocation, crypt stem cell numbers began to increase in both the ileum and proximal jejunum at the onset of inflammation in SAMP1/YitFc mice. These data suggest that the alterations in epithelial cell differentiation and increases in the size of the crypt stem cell population observed in SAMP1/YitFc mice are regulated by distinct mechanisms. We speculate that these epithelial alterations may play a role in the pathogenesis of ileitis in this murine model of Crohn's disease.

How helminth lipid-binding proteins offload their ligands to membranes: differential mechanisms of fatty acid transfer by the ABA-1 polyprotein allergen and Ov-FAR-1 proteins of nematodes and Sj-FABPc of schistosomes.

Three different classes of small lipid-binding protein (LBP) are found in helminth parasites. Although of similar size, the ABA-1A1 (also designated As-NPA-A1) and Ov-FAR-1 (formerly known as Ov20) proteins of nematodes are mainly alpha-helical and have no known structural counterparts in mammals, whereas Sj-FABPc of schistosomes is predicted to form a beta-barrel structure similar to the mammalian family of intracellular fatty acid binding proteins. The parasites that produce these proteins are unable to synthesize their own complex lipids and, instead, rely entirely upon their hosts for supply. As a first step in elucidating whether these helminth proteins are involved in the acquisition of host lipid, the process by which these LBPs deliver their ligands to acceptor membranes was examined, by comparing the rates and mechanisms of ligand transfer from the proteins to artificial phospholipid vesicles using a fluorescence resonance energy transfer assay. All three proteins bound the fluorescent fatty acid 2-(9-anthroyloxy)palmitic acid (2AP) similarly, but there were clear differences in the rates and mechanisms of fatty acid transfer. Sj-FABPc displayed a collisional mechanism; 2AP transfer rates increased with acceptor membrane concentration, were modulated by acceptor membrane charge, and were not diminished in the presence of increasing salt concentrations. In contrast, transfer of ligand from Ov-FAR-1 and ABA-1A1 involved an aqueous diffusion step; transfer rates from these proteins were not modulated by acceptor membrane concentration or charge but did decrease with the ionic strength of the buffer. Despite these differences, all of the proteins interacted directly with membranes, as determined using a cytochrome c competition assay, although Sj-FABPc interacted to a greater extent than did Ov-FAR-1 or rABA-1A1. Together, these results suggest that Sj-FABPc is most likely to be involved in the intracellular targeted transport and metabolism of fatty acids, whereas Ov-FAR-1 and ABA-1A1 may behave in a manner analogous to that of extracellular LBPs such as serum albumin and plasma retinol binding protein.

Lipid-binding proteins modulate ligand-dependent trans-activation by peroxisome proliferator-activated receptors and localize to the nucleus as well as the cytoplasm

Peroxisome proliferator-activated receptors (PPARs) are activated by a variety of fatty acids, eicosanoids, and hypolipidemic and insulin-sensitizing drugs. Many of these compounds bind avidly to members of a family of small lipid-binding proteins, the fatty acid-binding proteins (FABPs). Fatty acids are activated to CoA esters, which bind with high affinity to the acyl-CoA-binding protein (ACBP). Thus, the availability of known and potential PPAR ligands may be regulated by lipid-binding proteins. In this report we show by transient transfection of CV-1 cells that coexpression of ACBP and adipocyte lipid-binding protein (ALBP) exerts a ligand- and PPAR subtype-specific attenuation of PPAR-mediated trans-activation, suggesting that lipid-binding proteins, when expressed at high levels, may function as negative regulators of PPAR activation by certain ligands. Expression of ACBP, ALBP, and keratinocyte lipid-binding protein (KLBP) is induced during adipocyte differentiation, a process during which PPARγ plays a prominent role. We present evidence that endogenous ACBP, ALBP, and KLBP not only localize to the cytoplasm but also exhibit a prominent nuclear localization in 3T3-L1 adipocytes. In addition, forced expression of ACBP, ALBP, and KLBP in CV-1 cells resulted in a substantial accumulation of all three proteins in the nucleus. These results suggest that lipid-binding proteins, contrary to the general assumption, may exert their action in the nucleus as well as in the cytoplasm.—Helledie, T., M. Antonius, R. V. Sørensen, A. V. Hertzel, D. A. Bernlohr, S. Kølvraa, K. Kristiansen, and S. Mandrup. Lipid-binding proteins modulate ligand-dependent trans-activation by peroxisome proliferator-activated receptors and localize to the nucleus as well as the cytoplasm. J. Lipid Res. 2000. 41: 1740–1751.

Purification and properties of a small lipid-binding protein from the hemolymph of Triatoma infestans

A small lipid-binding protein (sLBP) was purified from the hemolymph of the blood-sucking bug Triatoma infestans. Its isolation involved size exclusion-high performance liquid chromatography (HPLC) followed by anion exchange chromatography-HPLC. The molecular weight of the protein, as determined by gel permeation chromatography, was 20 kDa. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) resolved the protein into a single polypeptide with M r≅16 kDa. The sLBP contains 6% lipids. Diacylglycerols represent the major lipid class, whereas phosphatidyl-choline, phosphatidyl-ethanolamine, free fatty acids and hydrocarbons were found in minor amounts. The amino acid composition indicated a high content of aspartic and glutamic acids and non-polar aliphatic amino acids. The N-terminal sequence did not resemble the sequence of any other previously reported insect hemolymph protein. Far-UV circular dichroism suggested that sLBP adopts a conformation rich in β-sheet structure. The presence of this protein in hemolymph, fat body and unfertilized eggs was explored throughout the last nymphal and adult stages of the insect by Western blot assays. These assays indicated that sLBP is particularly abundant in hemolymph. A high concentration of sLBP was also detected in the fat body of the nymphs.

Role of membrane-associated and cytoplasmic fatty acid-binding proteins in cellular fatty acid metabolism

A number of membrane-associated and cytoplasmic fatty acid-binding proteins (FABPs) are now being implicated in the cellular uptake and intracellular transport of long-chain fatty acids (FA). These proteins each have the capacity of non-covalent binding of FA, are present in tissues actively involved in FA metabolism, and are upregulated in conditions of increased cellular FA metabolism. To date, five distinct membrane FABPs have been described, ranging in mass from 22 to 88 kDa and each showing a characteristic tissue distribution. Evidence for involvement in cellular fatty acid uptake has been provided for several of them, because it was recently found that isolated cell lines transfected with 88-kDa putative fatty acid translocase (FAT; homologous to CD36) or with 63-kDa fatty acid-transport protein show an increased rate of FA uptake. The (at least nine) FABPs of cytoplasmic origin belong to a family of small (14–15 kDa) lipid binding proteins, all having a similar tertiairy structure but differing in binding properties and in tissue occurrence. The biological functions of the various FABPs, possibly exerted in a concerted action among them, comprise solubilization and compartmentalization of FA, facilitation of the cellular uptake and intracellular trafficking of FA, and modulation of mitosis, cell growth, and cell differentiation. In addition, the FABPs have been suggested to participate in and/or modulate FA-mediated signal transduction pathways and FA regulation of gene expression, and to prevent local high FA concentrations thereby contributing to the protection of cells against the toxic effects of FA. In conclusion, long-chain fatty acids are subject to continuous interaction with multiple proteins, which interplay influences their cellular metabolism.