Fatty acid-binding protein from human heart localized in native and denaturing two-dimensional gels

Summary A group of low molecular weight fatty acid-binding cytosolic proteins, FABP_c with high abundance in heart, liver, skeletal muscle, intestine and adipose tissue, are anticipated to play a role in long-chain fatty acid metabolism in these tissues. Recently, a FABP_c with M_T 15 kDa has been purified from human heart muscle and found to be present in levels 2–4% of cytosolic proteins of human heart myocytes. In the present study two-dimensional gel electrophoresis under native and denaturing conditions has been used to characterize FABP_c from human heart and this protein is found to be a major protein of human heart myocytes. The pI of FABP_c from human heart was found to be about 5.3 under native conditions and about 6.5 in the presence of 9 M urea.     

Muscle palmitate uptake and binding are saturable and inhibited by antibodies to FABPPM

Studies show that uptake of long-chain fatty acids (LCFA) across the plasma membranes (PM) may occur partly via a carrier-mediated process and that the plasma membrane fatty acid-binding protein (FABP_PM) may be a component of this system. To test the hypothesis that FABP_PM is involved in transsarcolemmal transport of LCFA in muscle, we measured palmitate uptake in giant sarcolemmal vesicles and palmitate binding to PM proteins in rat muscles, (1) in the presence of increasing amounts of unbound palmitate and (2) in the absence or presence of antibody to FABP_PM. Both palmitate uptake and binding were found to be saturable functions of the unbound palmitate concentration with calculated V_max values of 10.5 ± 1.2 pmol/mg protein/15 sec and 45.6 ± 2.9 nmol/mg protein/15 min and K_m values of 12.8 ± 3.8 and 18.4 ± 1.8 nmol/L, respectively. The V_max values for both palmitate uptake and binding were significantly decreased by 75-79% in the presence of a polyclonal antibody to the rat hepatic FABP_PM. Antibody inhibition was found to be dose-dependent and specific to LCFA. Glucose uptake was not affected by the presence of the antibody to FABP_PM. Palmitate uptake and binding were also inhibited in the presence of trypsin and phloretin. These results support the hypothesis that transsarcolemmal LCFA transport occurs in part by a carrier-mediated process and that FABP_PM is a component of this process in muscle.     

Cellular lipid binding proteins as facilitators and regulators of lipid metabolism

Evidence is accumulating that cellular lipid binding proteins are playing central roles in cellular lipid uptake and metabolism. Membrane-associated fatty acid-binding proteins putatively function in protein-mediated transmembrane transport of fatty acids, likely coexisting with passive diffusional uptake. The intracellular trafficking of fatty acids, bile acids, and other lipid ligands, may involve their interaction with specific membrane or protein targets, which are unique properties of some but not of all cytoplasmic lipid binding proteins. Recent studies indicate that these proteins not only facilitate but also regulate cellular lipid utilization. For instance, muscle fatty acid uptake is subject to short-term regulation by translocation of fatty acid translocase (FAT)/CD36 from intracellular storage sites to the plasma membrane, and liver-type cytoplasmic fatty acid-binding protein (L-FABP_c) functions in long-term, ligand-induced regulation of gene expression by directly interacting with nuclear receptors. Therefore, the properties of the lipid-protein complex, rather than those of the lipid ligand itself, determine the fate of the ligand in the cell. Finally, there are an increasing number of reports that deficiencies or altered functioning of both membrane-associated and cytoplasmic lipid binding proteins are associated with disease states, such as obesity, diabetes and atherosclerosis. In conclusion, because of their central role in the regulation of lipid metabolism, cellular lipid binding proteins are promising targets for the treatment of diseases resulting from or characterised by disturbances in lipid metabolism, such as atherosclerosis, hyperlipidemia, and insulin resistance.     

Characterization of a BODIPY-labeled {fl}uorescent fatty acid analogue. Binding to fatty acid-binding proteins, intracellular localization, and metabolism

The BODIPY-labeled fatty acid analogues are a useful addition to the tools employed to study the cellular uptake and metabolism of lipids. In this study, we show that BODIPY FL C₁₆ binds to purified liver and intestinal fatty acid-binding proteins with high affinity at a site similar to that for the physiological fatty acid oleic acid. Further, in human intestinal Caco-2 cells BODIPY FL C₁₆ co-localizes extensively with mitochondria, endoplasmic reticulum/Golgi, and L-FABP. Virtually no esterification of BODIPY FL C₁₆ was observed under the experimental conditions employed. We conclude that BODIPY FL C₁₆ may be a useful tool for studying the distribution and function of FABPs in a cellular environment.     

Fatty acid transport across the cell membrane: Regulation by fatty acid transporters

Transport of long-chain fatty acids across the cell membrane has long been thought to occur by passive diffusion. However, in recent years there has been a fundamental shift in understanding, and it is now generally recognized that fatty acids cross the cell membrane via a protein-mediated mechanism. Membrane-associated fatty acid-binding proteins (‘fatty acid transporters’) not only facilitate but also regulate cellular fatty acid uptake, for instance through their inducible rapid (and reversible) translocation from intracellular storage pools to the cell membrane. A number of fatty acid transporters have been identified, including CD36, plasma membrane-associated fatty acid-binding protein (FABP_pm), and a family of fatty acid transport proteins (FATP1–6). Fatty acid transporters are also implicated in metabolic disease, such as insulin resistance and type-2 diabetes. In this report we briefly review current understanding of the mechanism of transmembrane fatty acid transport, and the function of fatty acid transporters in healthy cardiac and skeletal muscle, and in insulin resistance/type-2 diabetes. Fatty acid transporters hold promise as a future target to rectify lipid fluxes in the body and regain metabolic homeostasis.     

Towards an Understanding of Mesocestoides vogae Fatty Acid Binding Proteins’ Roles

Two fatty acid binding proteins, MvFABPa and MvFABPb were identified in the parasite Mesocestoides vogae (Platyhelmithes, Cestoda). Fatty acid binding proteins are small intracellular proteins whose members exhibit great diversity. Proteins of this family have been identified in many organisms, of which Platyhelminthes are among the most primitive. These proteins have particular relevance in flatworms since de novo synthesis of fatty acids is absent. Fatty acids should be captured from the media needing an efficient transport system to uptake and distribute these molecules. While HLBPs could be involved in the shuttle of fatty acids to the surrounding host tissues and convey them into the parasite, FABPs could be responsible for the intracellular trafficking. In an effort to understand the role of MvFABPs in fatty acid transport of M. vogae larvae, we analysed the intracellular localization of both MvFABPs and the co-localization with in vivo uptake of fatty acid analogue BODIPY FL C₁₆. Immunohistochemical studies on larvae sections using specific antibodies, showed a diffuse cytoplasmic distribution of each protein with some expression in nuclei and mitochondria. MvFABPs distribution was confirmed by mass spectrometry identification from 2D-electrophoresis of larvae subcellular fractions. This work is the first report showing intracellular distribution of MvFABPs as well as the co-localization of these proteins with the BODIPY FL C₁₆ incorporated from the media. Our results suggest that fatty acid binding proteins could target fatty acids to cellular compartments including nuclei. In this sense, M. vogae FABPs could participate in several cellular processes fulfilling most of the functions attributed to vertebrate’s counterparts.     

Quantitation of plasma membrane fatty acid-binding protein by enzyme dilution and monoclonal antibody based immunoassay

Summary A plasma membrane fatty acid-binding protein (h-FABPPm) has been isolated from rat hepatocytes. Analogous proteins have also been identified in adipocytes, jejunal enterocytes and cardiac myocytes, all cells with high transmembrane fluxes of fatty acids. These 43 kDa, highly basic (pl = 9.1) FABP_pm 's appear unrelated to the smaller, cytosolic FABP's (designated FABP's) identified previously in the same tissues. h-FABP_pm appears closely related to the mitochondrial isoform of glutamic-oxaloacetic transaminase (mGOT), and both the purified protein and liver cell plasma membranes (LPM) possess GOT enzymatic activity. From their relative GOT specific activities it is estimated that h-FABP_pm constitutes approximately 2% of LPM protein, or about 0.7 × 10⁷ sites per cell. A monoclonal antibody-based competitive inhibition enzyme immunoassay (CIEIA) for h-FABP_pm is described; it yields an estimate of 3.4 x 10⁷ h-FABP_pm sites per hepatocyte. Quantitated by either method, h-FABPPm appears to be a highly abundant protein constituent of LPM.     

The effect of alcohol on recombinant proteins derived from mammalian adenylyl cyclase

The cyclic AMP (cAMP) signaling pathway is implicated in the development of alcohol use disorder. Previous studies have demonstrated that ethanol enhances the activity of adenylyl cyclase (AC) in an isoform specific manner; AC7 is most enhanced by ethanol, and regions responsible for enhancement by ethanol are located in the cytoplasmic domains of the AC7 protein. We hypothesize that ethanol modulates AC activity by directly interacting with the protein and that ethanol effects on AC can be studied using recombinant AC in vitro. AC recombinant proteins containing only the C_1a or C₂ domains of AC7 and AC9 individually were expressed in bacteria, and purified. The purified recombinant AC proteins retained enzymatic activity and isoform specific alcohol responsiveness. The combination of the C_1a or C₂ domains of AC7 maintained the same alcohol cutoff point as full-length AC7. We also find that the recombinant AC7 responds to alcohol differently in the presence of different combinations of activators including MnCl₂, forskolin, and Gsα. Through a series of concentration-response experiments and curve fitting, the values for maximum activities, Hill coefficients, and EC₅₀ were determined in the absence and presence of butanol as a surrogate of ethanol. The results suggest that alcohol modulates AC activity by directly interacting with the AC protein and that the alcohol interaction with the AC protein occurs at multiple sites with positive cooperativity. This study indicates that the recombinant AC proteins expressed in bacteria can provide a useful model system to investigate the mechanism of alcohol action on their activity.     

Eubacterium cellulosolvens Alters its Membrane Protein,Lipoprotein, and Fatty Acid Composition in Response to Growth on Cellulose

The cellulolytic bacterium, Eubacterium cellulosolvens, altered its cytoplasmic membrane protein composition in response to growth on specific energy substrates. Electrophoresis profiles obtained from membrane protein fractions of cellulose-grown cells were different from that obtained from cells cultivated with other carbohydrates, such as cellobiose or glucose. In addition, [³H]palmitic acid labelling of cellulose-grown E. cellulosolvens revealed two lipoproteins that were not detected in glucose- or cellobiose-grown cultures. These lipoproteins partitioned with the membrane fraction, indicating their association with the cytoplasmic membrane. Proteinase K treatment of whole cells further suggested that these lipoproteins were exposed to the surface of the cell envelope. These membrane proteins and lipoproteins appear to be under some substrate-specific regulatory control with distinct, but as yet undetermined, roles in cellulose utilization. In addition, cellulose-grown E. cellulosolvens was found to posses a higher ratio of oleic acid (C_18:1) to palmitic acid (C_16:0) than cells cultivated on soluble carbohydrates. This change in the ratio of unsaturated to saturated fatty acids was consistent with a comparative increase of membrane fluidity. Further analysis of this shift in the fatty acid profile revealed a correlation with the appearance of protruberances on the cell surface. Such a shift of fatty acid composition may indicate that the assembly and function of proteins for cellulose utilization necessitates an increase of the membrane fluidity.     

Sterol carrier protein‐2:Not just for cholesterol any more

Although sterol carrier protein-2 (SCP-2) mediates cholesterol esterification in L-cell fibroblasts and stimulates an accumulation of cholesterol in these cells, a potential role for SCP-2 in fatty acid uptake and trafficking has not been appreciated. Certainly, recent experiments have shown that SCP-2 binds fatty acids in vitro with an affinity similar to that observed for fatty acid binding proteins. Because of the ubiquitous tissue distribution of SCP-2, as opposed to the specific distribution of fatty acid binding proteins, as well as the need for fatty acid trafficking in all cells, I have recently proposed that SCP-2 is the universal fatty acid trafficking protein. This supposition is based on a number of observations made with L-cell fibroblasts expressing either the 13.2 kDa SCP-2 or the 15 kDa proSCP-2. In L-cells expressing the 13.2 kDa SCP-2, fluorescent fatty acid uptake was increased by 10–30% depending upon the probe used. In 15 kDa proSCP-2 expressing cells, fluorescent fatty acid uptake was increased 20–40% depending upon the probe used. However, only expression of the 15 kDa pro-SCP-2 increased the cytoplasmic diffusion of the fluorescent fatty acid. Expression of either protein increased the uptake of [³H]-oleic acid 1.9-fold compared to control, with targeting of [³H]-oleic acid for esterification into cholesteryl esters. The 13.2 kDa SCP-2 did target a significant amount of [³H]-oleic acid for esterification into the triacylglycerol pool. Expression of either protein markedly reduced total cellular phospholipid levels, however both proteins increased cholesteryl ester levels. Interestingly, expression of the 15 kDa proSCP-2 decreased ethanolamine plasmalogen levels with a concomitant increase in choline plasmalogen. Expression of both proteins increased PUFA content of the phospholipids, although this effect was greater in 15 kDa proSCP-2 expressing cells. Hence, expression of SCP-2 increased fatty acid uptake and targeted fatty acid to unique lipid pools, suggesting that SCP-2 may effectively serve as universal fatty acid binding and trafficking protein.     

Protein interactions with the platelet integrin αIIb regulatory motif

Integrins are transmembrane proteins regulating cellular shape, mobility and the cell cycle. A highly conserved signature motif in the cytoplasmic tail of the integrin α‐subunit, KXGFFKR, plays a critical role in regulating integrin function. To date, six proteins have been identified that target this motif of the platelet‐specific integrin α_IIbβ₃. We employ peptide‐affinity chromatography followed‐up with LC‐MS/MS analysis as well as protein chips to identify new potential regulators of integrin function in platelets and put them into their biological context using information from protein:protein interaction (PPI) databases. Totally, 44 platelet proteins bind with high affinity to an immobilized LAMWKVGFFKR‐peptide. Of these, seven have been reported in the PPI literature as interactors with integrin α‐subunits. 68 recombinant human proteins expressed on the protein chip specifically bind with high affinity to biotin‐tagged α‐integrin cytoplasmic peptides. Two of these proteins are also identified in the peptide‐affinity experiments, one is also found in the PPI databases and a further one is present in the data to all three approaches. Finally, novel short linear interaction motifs are common to a number of proteins identified.     

Fatty acid-binding proteins in the heart

Long-chain fatty acids are important fuel molecules for the heart, their oxidation in mitochondria providing the bulk of energy required for cardiac functioning. The low solubility of fatty acids in aqueous solutions impairs their cellular transport. However, cardiac tissue contains several proteins capable of binding fatty acids non-covalently. These fatty acid-binding proteins (FABPs) are thought to facilitate both cellular uptake and intracellular transport of fatty acids. The majority of fatty acids taken up by the heart seems to pass the sarcolemma through a carrier-mediated translocation mechanism consisting of one or more membrane-associated FABPs. Intracellular transport of fatty acids towards sites of metabolic conversion is most likely accomplished by cytoplasmic FABPs. In this review, the roles of membrane-associated and cytoplasmic FABPs in cardiac fatty acid metabolism under (patho)physiological circumstances are discussed.     

Molecular composition and origin of substrate-attached material from normal and virus-transformed cells

The proteins and polysaccharides which are left adherent to the tissue culture substrate after EGTA-mediated removal of normal, virus-transformed, and revertant mouse cells (so-called SAM, or substrate-attached material), and which have been implicated in the cell-substrate adhesion process, have been characterized by SDS-PAGE and other types of analyses under various conditions of cell growth and attachment. The following components have been identified in SAM: 3 size classes of hyaluronate proteoglycans; glycoprotein C_o (the LETS glycoprotein); protein C_a (a myosin-like protein); protein C_b (MW 85,000); protein C₁ (MW 56,000, which is apparently not tubulin); protein C₂ (actin); proteins C₃–C₅ (histones) which are artifactually bound to the substrate as a result of EGTA-mediated leaching from the cell; and proteins C_c, C_d, C_e, and C_f. The LETS glycoprotein (C_o) and C_d appear in newly-synthesized SAM (which is probably enriched in “footpad” material – “footpads” being focal areas of subsurface membranous contact with the substrate) in greater relative quantities than in the SAM accumulated over a long period of time (which is probably enriched in “footprint” material – remnants of footpads left behind as cells move across the substrate). C_o and C_d turn over very rapidly following short radiolabeling periods during chase analysis. The SAM's deposited during a wide variety of cellular attachment and growth conditions contained the same components in similar relative proportions. This may indicate well-controlled and coordinate deposition of a cell “surface” complex involving the hyaluronate proteoglycans, the LETS glycoprotein, actin-containing microfilaments with associated proteins, and a limited number of additional proteins in the substrate adhesion site. Evidence indicates that SAM is the remnant of “footpad” vesicles by which the cell adheres to the substrate and that EGTA treatment weakens the subsurface cytoskeleton, allowing these footpad vesicles to be pinched off from the rest of the cell. Three different models of cell-substrate adhesion are presented and discussed.     

Biosynthesis of a 42-kD Polypeptide in the Cytoplasmic Membrane of the Cyanobacterium Anacystis nidulans Strain R2 during Adaptation to Low CO(2) Concentration

When cells of Anacystis nidulans strain R2 grown under high CO₂ conditions (3%) were transferred to low CO₂ conditions (0.05%), their ability to accumulate inorganic carbon (C_i) increased up to 8 times. Cytoplasmic membranes (plasmalemma) isolated at various stages of low CO₂ adaptation were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There was a marked increase of a 42-kilodalton polypeptide in the cytoplasmic membrane during adaptation; a linear relationship existed between the amount of this polypeptide and the C_i-accumulating capability of the cells. No significant changes were observed during this process in the amount of other polypeptides in the cytoplasmic membranes or in the polypeptide profiles of the thylakoid membranes, cell walls, and soluble fractions. Spectinomycin, an inhibitor of protein biosynthesis, inhibited both the increase of the 42-kilodalton polypeptide and the induction of high C_i-accumulating capability. The incorporation of [³⁵S]sulfate into membrane proteins was greatly reduced during low CO₂ adaptation. Radioautograms of the ³⁵S-labeled membrane proteins revealed that synthesis of the 42-kilodalton polypeptide in the cytoplasmic membrane was specifically activated during the adaptation, while that of most other proteins was greatly suppressed. These results suggested that the 42-kilodalton polypeptide in the cytoplasmic membrane is involved in the active C_i transport by A. nidulans strain R2 and its synthesis under low CO₂ conditions leads to high C_i-transporting activity.     

Solution structure of bovine heart fatty acid-binding protein (H-FABPc)

Fatty acid-binding protein (FABP) from bovine heart, a 15 kDa cytoplasmic protein has been investigated by multidimensional homonuclear and heteronuclear NMR-spectroscopy. Perdeuterated palmitic acid has been used as fatty acid ligand. The tertiary structure has been determined from distance geometry calculations with the variable target functions algorithm (DIANA) [1] utilizing 1027 interproton distance constraints, which were obtained from¹H-homo-nuclear NOESY spectra. Overlapping NOE crosspeaks were assigned by heteronuclear multidimensional NMR-experiments with a¹⁵N-labelled sample. The tertiary structure resembles a -barrel (-clam) consisting of ten anti-parallel -strands and a short helix-turn-helix motif. The -strands are arranged in two nearly orthogonal -sheets composed of 5 strands each. The solution structure is compared with the x-ray cyrstal structure of bovine heart [4] and rat intestinal FABPs.Abbreviations DOF-COSY Double Quantum Filtered Correlated Spectroscopy - TOCSY Total Correlated Spectroscopy - NOE Nuclear Overhauser Enhancement - NOESY Nuclear Overhauser Enhancement and Exchange Spectroscopy - HMQC Heteronuclear Multiple Quantum Coherence - FABP Fatty Acid-Binding Protein - FABP_c Cellular Fatty Acid-Binding Protein - H-FABP_c Cellular Heart Fatty Acid-Binding Protein - I-FABP_c Cellular Intestinal Fatty Acid-Binding Protein     

ClpB/Hsp100 proteins and heat stress tolerance in plants

High-temperature stress can disrupt cellular proteostasis, resulting in the accumulation of insoluble protein aggregates. For survival under stressful conditions, it is important for cells to maintain a pool of native soluble proteins by preventing and/or dissociating these aggregates. Chaperones such as GroEL/GroES (Hsp60/Hsp10) and DnaK/DnaJ/GrpE (Hsp70/Hsp40/nucleotide exchange factor) help cells minimize protein aggregation. Protein disaggregation is accomplished by chaperones belonging to the Caseinolytic Protease (Clp) family of proteins. ClpB/Hsp100 proteins are strikingly ubiquitous and are found in bacteria, yeast and multi-cellular plants. The expression of these proteins is regulated by heat stress (HS) and developmental cues. Bacteria and yeast contain one and two forms of ClpB proteins, respectively. Plants possess multiple forms of these proteins that are localized to different cellular compartments (i.e. cytoplasm/nucleus, chloroplast or mitochondria). Overwhelming evidence suggests that ClpB/Hsp100 proteins play decisive roles in cell adaptation to HS. Mutant bacteria and yeast cells lacking active ClpB/Hsp100 proteins are critically sensitive to high-temperature stress. Likewise, Arabidopsis, maize and rice mutants lacking cytoplasmic ClpB proteins are very sensitive to heat. In this study, we present the structural and functional attributes of plant ClpB forms.     

Type-specific immunodetection of human heart fatty acid-binding protein with polyclonal anti-peptide antibodies

Summary In order to develop specific antibodies against human heart cytoplasmic fatty acid-binding protein (HFABP_c), four oligo-peptides of 15–20 amino-acids each and corresponding with different antigenic parts of the human H-FABP_c molecule, were synthesized. Polyclonal antibodies against these synthetic peptides were raised in mice (Balb/C) and rabbits (Flemish giant). When tested in enzyme linked immunosorbent assays (ELISA, antibody-capture assay), antisera against three of the four peptides showed a high immunoreactivity with the synthetic peptide selected for immunization as well as with the native human H-FABP_c. Some cross-reactivity with the other synthetic peptides was observed for the rabbit antisera but not for those from mice. Polyclonal antibodies against synthetic peptides can be applied for the specific detection of the native protein in biological preparations containing proteins that show a high degree of homology with the protein to be assayed.