The helical domain of intestinal fatty acid binding protein is critical for collisional transfer of fatty acids to phospholipid membranes

Fatty acid binding proteins (FABPs) exhibit a beta-barrel topology, comprising 10 antiparallel beta-sheets capped by two short alpha-helical segments. Previous studies suggested that fatty acid transfer from several FABPs occurs during interaction between the protein and the acceptor membrane, and that the helical domain of the FABPs plays an important role in this process. In this study, we employed a helix-less variant of intestinal FABP (IFABP-HL) and examined the rate and mechanism of transfer of fluorescent anthroyloxy fatty acids (AOFA) from this protein to model membranes in comparison to the wild type (wIFABP). In marked contrast to wIFABP, IFABP-HL does not show significant modification of the AOFA transfer rate as a function of either the concentration or the composition of the acceptor membranes. These results suggest that the transfer of fatty acids from IFABP-HL occurs by an aqueous diffusion-mediated process, i.e., in the absence of the helical domain, effective collisional transfer of fatty acids to membranes does not occur. Binding of wIFABP and IFABP-HL to membranes was directly analyzed by using a cytochrome c competition assay, and it was shown that IFABP-HL was 80% less efficient in preventing cytochrome c from binding to membranes than the native IFABP. Collectively, these results indicate that the alpha-helical region of IFABP is involved in membrane interactions and thus plays a critical role in the collisional mechanism of fatty acid transfer from IFABP to phospholipid membranes.     

Functional characterization of the Tn5 transposase by limited proteolysis

The 476 amino acid Tn5 transposase catalyzes DNA cutting and joining reactions that cleave the Tn5 transposon from donor DNA and integrate it into a target site. Protein-DNA and protein-protein interactions are important for this tranposition process. A truncated transposase variant, the inhibitor, decreases transposition rates via the formation of nonproductive complexes with transposase. Here, the inhibitor and the transposase are shown to have similar secondary and tertiary folding. Using limited proteolysis, the transposase has been examined structurally and functionally. A DNA binding region was localized to the N-terminal 113 amino acids. Generally, the N terminus of transposase is sensitive to proteolysis but can be protected by DNA. Two regions are predicted to contain determinants for protein-protein interactions, encompassing residues 114-314 and 441-476. The dimerization regions appear to be distinct and may have separate functions, one involved in synaptic complex formation and one involved in nonproductive multimerization. Furthermore, predicted catalytic regions are shown to lie between major areas of proteolysis.     

A neuronal two P domain K+ channel stimulated by arachidonic acid and polyunsaturated fatty acids

TWIK-1, TREK-1 and TASK K+ channels comprise a class of pore-forming subunits with four membrane-spanning segments and two P domains. Here we report the cloning of TRAAK, a 398 amino acid protein which is a new member of this mammalian class of K+ channels. Unlike TWIK-1, TREK-1 and TASK which are widely distributed in many different mouse tissues, TRAAK is present exclusively in brain, spinal cord and retina. Expression of TRAAK in Xenopus oocytes and COS cells induces instantaneous and non-inactivating currents that are not gated by voltage. These currents are only partially inhibited by Ba2+ at high concentrations and are insensitive to the other classical K+ channel blockers tetraethylammonium, 4-aminopyridine and Cs+. A particularly salient feature of TRAAK is that they can be stimulated by arachidonic acid (AA) and other unsaturated fatty acids but not by saturated fatty acids. These channels probably correspond to the functional class of fatty acid-stimulated K+ currents that recently were identified in native neuronal cells but have not yet been cloned. These TRAAK channels might be essential in normal physiological processes in which AA is known to play an important role, such as synaptic transmission, and also in pathophysiological processes such as brain ischemia. TRAAK channels are stimulated by the neuroprotective drug riluzole.     

Modulation of the expression of early genes by polyunsaturated fatty acids

Expression of early genes is a characteristic immediate cellular response to mitogenic or inflammatory stimulation. Various second messenger systems have been found to transduce the signal from the plasma membrane to the nucleus. Recent observations indicate that in addition to well characterized second messenger systems, polyunsaturated fatty acids, especially the n-6 fatty acid arachidonic acid and its endogenously produced metabolites affect the expression of early genes in different cell types. At least in fibroblasts, the stimulatory effect of arachidonic acid can be antagonized by n-3 polyunsaturated fatty acids. Further identification of the mechanisms through which polyunsaturated fatty acids modulate early gene expression and regulate subsequent cellular responses, like cell growth, may help to define novel concepts in the management of cardiovascular diseases.     

Activation of the uncoupling protein by fatty acids is modulated by mutations in the C-terminal region of the protein

The transport properties of the uncoupling protein (UCP) from brown adipose tissue have been studied in mutants where Cys304 has been replaced by either Gly, Ala, Ser, Thr, Ile or Trp. This position is only two residues away from the C-terminus of the protein, a region that faces the cytosolic side of the mitochondrial inner membrane. Mutant proteins have been expressed in Saccharomyces cerevisiae and their activity determined in situ by comparing yeast growth rates in the presence and absence of 2-bromopalmitate. Their bioenergetic properties have been studied in isolated mitochondria by determining the effects of fatty acids and nucleotides on the proton permeability and NADH oxidation rate. It is revealed that substitution of Cys304 by non-charged residues alters the response of UCP to fatty acids. The most effective substitution is Cys for Gly since it greatly enhances the sensitivity to palmitate, decreasing threefold the concentration required for half-maximal stimulation of respiration. The opposite extreme is the substitution by Ala which increases twofold the half-maximal concentration. We conclude that the C-terminal region participates in the fatty acid regulation of UCP activity. The observed correlation between yeast growth rates in the presence of bromopalmitate and the calculated activation constants for respiration in isolated mitochondria validates growth analysis as a method to screen the in situ activity of UCP mutants.     

Regulation of tumour cell fatty acid oxidation by n-6 polyunsaturated fatty acids

The aim of this study was to evaluate acute effects of ethyl tert-butyl ether (ETBE) in man after short-term exposure. ETBE may in the future replace methyl tert-butyl ether, a widely used oxygenate in unleaded gasoline. Eight healthy male volunteers were exposed to ETBE vapor for 2 h at four levels (0, 5, 25, and 50 ppm) during light physical exercise. The subjects rated irritative symptoms, discomfort, and central nervous system effects in a questionnaire. Ocular (eye redness, tear film break-up time, conjunctival epithelial damage, and blinking frequency), nasal (acoustic rhinometry and analysis of inflammatory markers and cells in nasal lavage fluid), and pulmonary (peak expiratory flow, forced expiratory volume in 1 s, forced vital capacity, vital capacity, and transfer factor) measurements were performed. Significantly increased ratings of solvent smell (p = 0.001, repeated-measures ANOVA) were seen during exposures and correlated to exposure levels. Furthermore, significantly elevated ratings of discomfort in throat and airways were seen during and after 50 ppm compared to the control exposure (p = 0.02). Increased nasal swelling (p = 0.001) and blinking frequency (p = 0.01) were noted at all exposure levels, but their magnitudes were not related to exposure levels. A slightly impaired pulmonary function was seen at 25 and 50 ppm, since forced vital capacity (p = 0.02) and vital capacity (p = 0.04) differed significantly from the clean air exposure. Although the impairments seemed to fall within normal inter- and intraindividual variation and have no clinical relevance as such, it cannot be excluded that other individuals may react more severely than eight healthy male volunteers in this study.     

Modulation of exercise-induced immunosuppression by dietary polyunsaturated fatty acids in mice

The possible interaction between intense exercise, known to suppress the immune response, and nutritive factors, such as polyunsaturated fatty acids (PUFA), was examined in inbred female C57Bl/6 mice. The animals received for 8 wk either a natural ingredient diet or a diet supplemented with 10 g/100 g linseed oil containing over 50% of 18:3 (n-3) alpha-linoleic acid. Other groups received PUFA containing only traces of 18:3 (n-3) fatty acid; beef tallow, containing mostly 18:1 (n-9) saturated fat, safflower oil, an 18:2 (n-6) PUFA, and fish oil, containing longer chain (n-3) PUFA. Each dietary group was divided into two subgroups: sedentary diet controls and exercised animals. Exercise consisted of continuous swimming at high intensity until exhaustion. It was shown in three separate experiments that (1) the primary humoral response to sheep red blood cells, determined by the plaque-forming cell (PFC) assay, was affected by PUFA diet in sedentary animals in the order beef tallow > control diet > safflower oil > fish oil > linseed oil, and (2) the PFC response was suppressed by the exhaustive exercise, as compared to sedentary controls, except for animals fed 18:3 (n-3) linseed oil, where the normal response was noted. Phagocytosis of fluorescent microspheres by peritoneal macrophages, determined by flow cytometry, was significantly lower in exercised animals receiving the linseed oil diet, whereas other diets either increased or did not significantly change the macrophage phagocytic activity, compared to the sedentary diet controls. Spleen lymphocyte subsets were unchanged in exercised animals except for a marked shift from the lymphoid peak toward the erythroid peak. Generally, our data showed a marked immunomodulatory effect of 18-3 (n-3) alpha-linoleic acid on the exhaustive exercise-related immunosuppression, as compared to the effects of other selected PUFA.     

Identification of the binding surface on beta-lactamase for GroEL by limited proteolysis and MALDI-mass spectrometry

Escherichia coli beta-lactamase, alone or as a complex with GroEL at 48 degreesC, was partially digested with trypsin, endoproteinase Glu-C, or thermolysin. Peptides were analyzed by matrix-assisted laser desorption and ionization mass spectrometry and aligned with the known sequence. From the protease cleavage sites which become protected upon binding and those which become newly accessible, a model of the complex is proposed in which the carboxy-terminal helix has melted, two loops form the binding interface and the large beta-sheet become partially uncovered by the slight dislocation of other structural elements. This explains how hydrophobic surface on the substrate protein can become accessible while scarcely disrupting the hydrogen bond network of the native structure. An analysis of the GroEL-bound peptides bound after digestion of the beta-lactamase showed no obvious sequence motifs, indicating that binding is provided by hydrophobic patches in the three-dimensional structure.     

Prevention of ischemia-induced cardiac sudden death by n-3 polyunsaturated fatty acids in dogs

The objective of this study was to obtain functional information associated with the prevention by n-3 polyunsaturated fatty acids (PUFA) of ischemia-induced fatal cardiac ventricular arrhythmias in the intact, conscious, exercising dog. Thirteen dogs susceptible to ischemia-induced ventricular fibrillation were prepared surgically by ligation of their anterior descending left coronary artery and placement of an inflatable cuff around their left circumflex artery. After 4 wk of recovery, exercise-plus-ischemia tests were performed without and then with an intravenous infusion of an emulsion of free n-3 PUFA just prior to occluding the left circumflex artery while the animals were running on a treadmill. One week later the exercise-plus-ischemia test was repeated but with a control infusion replacing the emulsion of n-3 PUFA. The infusion of the free n-3 PUFA in quantities of 1.0 to 10 g prevented ventricular fibrillation in 10 of the 13 dogs tested (P < 0.005), apparently without esterification of the PUFA into membrane phospholipids. The antiarrhythmic effect of the n-3 PUFA was associated with slowing of the heart rate, shortening of the QT-interval (electrical action potential duration), reduction of left ventricular systolic pressure, and prolongation of the electrocardiographic atrial-ventricular conduction time (P-R interval). These effects are comparable with those we have reported in studies with cultured neonatal rat cardiac myocytes.     

Probing the partly folded states of proteins by limited proteolysis

The folding of a polypeptide chain of a relatively large globular protein into its unique three-dimensional and functionally active structure occurs via folding intermediates. These partly folded states of proteins are difficult to characterize, because they are usually short lived or exist as a distribution of possible conformers. A variety of experimental techniques and approaches have been utilized in recent years in numerous laboratories for characterizing folding intermediates that occur at equilibrium, including spectroscopic techniques, solution X-ray scattering, calorimetry and gel filtration chromatography, as well as genetic methods and theoretical calculations. In this review, we focus on the use of proteolytic enzymes as probes of the structure and dynamics of folding intermediates and we show that this simple biochemical technique can provide useful information, complementing that obtained by other commonly used techniques and approaches. The key result of the proteolysis experiments is that partly folded states (molten globules) of proteins can be sufficiently rigid to prevent extensive proteolysis and appear to maintain significant native-like structure.     

Enhanced sensitivity of ubiquinone-deficient mutants of Saccharomyces cerevisiae to products of autoxidized polyunsaturated fatty acids

Coenzyme Q (ubiquinone or Q) plays a well known electron transport function in the respiratory chain, and recent evidence suggests that the reduced form of ubiquinone (QH2) may play a second role as a potent lipid-soluble antioxidant. To probe the function of QH2 as an antioxidant in vivo, we have made use of a Q-deficient strain of Saccharomyces cerevisiae harboring a deletion in the COQ3 gene [Clarke, C. F., Williams, W. & Teruya, J. H. (1991) J. Biol. Chem. 266, 16636-16644]. Q-deficient yeast and the wild-type parental strain were subjected to treatment with polyunsaturated fatty acids, which are prone to autoxidation and breakdown into toxic products. In this study we find that Q-deficient yeast are hypersensitive to the autoxidation products of linolenic acid and other polyunsaturated fatty acids. In contrast, the monounsaturated oleic acid, which is resistant to autoxidative breakdown, has no effect. The hypersensitivity of the coq3delta strains can be prevented by the presence of the COQ3 gene on a single copy plasmid, indicating that the sensitive phenotype results solely from the inability to produce Q. As a result of polyunsaturated fatty acid treatment, there is a marked elevation of lipid hydroperoxides in the coq3 mutant as compared with either wild-type or respiratory-deficient control strains. The hypersensitivity of the Q-deficient mutant can be rescued by the addition of butylated hydroxytoluene, alpha-tocopherol, or trolox, an aqueous soluble vitamin E analog. The results indicate that autoxidation products of polyunsaturated fatty acids mediate the cell killing and that QH2 plays an important role in vivo in protecting eukaryotic cells from these products.     

In vitro activity of polyunsaturated fatty acids on Pseudomonas aeruginosa: relationship to lipid peroxidation

The treatment of patients with deep vein thrombosis and pulmonary embolism with contraindications for a thrombolytic therapy is a therapeutic challenge. We report on a 12 year old patient who was treated for large cell lymphoma according to NHL-BFM 95: Block AA protocol. During his therapy, he developed a thrombosis of his right femoral vein and pulmonary embolism affecting the left segments 4, 5, 8, and 9. Because of cerebral metastasis a fibrinolytic therapy was contraindicated. Therefore, we performed a mechanical thrombectomy using the Amplatz thrombectomy device. The postinterventional scintigraphy showed a markedly improved pulmonary perfusion; dopplersonography 4 months postinterventionally showed a patent right femoral vein.     

Influence of fatty acids on the binding of calcium to human serum albumin

Fatty acids have been shown to influence the binding of calcium to human serum albumin. The calcium binding to albumin was enhanced when long-chain fatty acids were added to albumin prepared by two different methods and decreased when fatty acids were removed from albumin. Palmitic, stearic and oleic acids all exhibited this phenomenon. The effects of long-chain fatty acids on the binding of calcium to albumin in vitro appears to be of sufficient magnitude to have in vivo implications in calcium homeostasis and in determining the ratio of free to total calcium. Preliminary in vivo experiments have confirmed the calcium binding of fatty acids in serum and suggest a physiological role for this phenomena.     

Activation by autophosphorylation or cGMP binding produces a similar apparent conformational change in cGMP-dependent protein kinase

Binding of cyclic nucleotide to or autophosphorylation of cGMP-dependent protein kinase (PKG) activates this kinase, but the molecular mechanism of activation for either process is unknown. Activation of PKG by cGMP binding produces a conformational change in the enzyme (Chu, D.-M., Corbin, J. D., Grimes, K. A., and Francis, S. H. (1997) J. Biol. Chem. 272, 31922-31928; Zhao, J., Trewhella, J., Corbin, J., Francis, S., Mitchell, R., Brushia, R., and Walsh, D. (1997) J. Biol. Chem. 272, 39129-31936). In the present studies, activation of type Ibeta PKG by either autophosphorylation or cGMP-binding alone causes (i) an electronegative charge shift on ion exchange chromatography, (ii) a similar increase ( approximately 3.5 A) in the Stokes radius as determined by gel filtration chromatography, and (iii) a similar decrease in the mobility of the enzyme on native gel electrophoresis. Consistent with these results, cGMP binding increases the rate of phosphoprotein phosphatase-1 catalyzed dephosphorylation of PKG which is autophosphorylated only at Ser-63 (not activated); however, dephosphorylation of PKG that is highly autophosphorylated (activated) is not stimulated by cGMP. The combined results suggest that activation of PKG by either autophosphorylation or cGMP binding alone produces a similar apparent elongation of the enzyme, implying that either process activates the enzyme by a similar molecular mechanism.     

Inclusion of chufa in the human diet as a source of polyunsaturated fatty acids

In 2 experiments lasting 30 days each with participation of 6 volunteers the possibility of daily consumption in the diet of chufa in an amount allowing for minimal requirement of the organism in polyunsaturated fatty acids was studied. The experimental food ration accorded with individual requirements in its basic components. None of the participating volunteers demonstrated any untoward deviations of objective and subjective nature in their health status. For a month chufa was introduced daily at the rate of 1.7 g per kg of body weight.     

Feeding trans fatty acids to rats has no effect on the intestinal uptake of glucose, fatty acids or cholesterol

Trans fatty acids are produced in the manufacture of margarine, and these hydrogenated fatty acids may have a deleterious effect on the reduction in fasting levels of serum cholesterol anticipated from the feeding of cis polyunsaturated fatty acids. We undertook this study in rats to test the effect of feeding trans fatty acids on the intestinal uptake of glucose, fatty acids and cholesterol. Adult female Wistar rats were fed for 2 weeks semisynthetic, isocaloric diets containing no oleic acid (18:1), cis 18:1 or trans 18:1. There was no difference between the three dietary groups in the animals' food consumption or body weight gain. Rats fed trans 18:1 had an approximately 20% decline in the total weight of the ileum as compared with controls fed no 18:1, and therefore there was also a decline in the percentage of the ileal tissue comprised of mucosa. When comparing rats fed trans 18:1 with those fed cis 18:1 or no 18:1, there was no difference in the uptake of varying concentrations of D-glucose when expressed as nmol.100 mg tissue-1.min-1 or nmol.100 mg mucosal-1.min-1 for jejunum or for ileum. Also, there was no difference in the value of the maximal transport rate (Vmax), Michaelis constant (Km), or the contribution of passive uptake of glucose assessed with L-glucose. There was no diet-associated change in the jejunal or ileal uptake of a medium-chain length fatty acid (lauric acid), a long-chain length saturated fatty acid (palmitic acid), a monounsaturated fatty acid (oleic acid), two polyunsaturated fatty acids (linoleic and linolenic acids), or cholesterol. Thus, we conclude that 2 weeks' feeding of trans fatty acid to rats has no influence on the jejunal or ileal uptake of glucose, fatty acids or cholesterol.     

Probing the ligand binding domain of the GluR2 receptor by proteolysis and deletion mutagenesis defines domain boundaries and yields a crystallizable construct

Ionotropic glutamate receptors constitute an important family of ligand-gated ion channels for which there is little biochemical or structural data. Here we probe the domain structure and boundaries of the ligand binding domain of the AMPA-sensitive GluR2 receptor by limited proteolysis and deletion mutagenesis. To identify the proteolytic fragments, Maldi mass spectrometry and N-terminal amino acid sequencing were employed. Trypsin digestion of HS1S2 (Chen GQ, Gouaux E. 1997. Proc Natl Acad Sci USA 94:13431-13436) in the presence and absence of glutamate showed that the ligand stabilized the S1 and S2 fragments against complete digestion. Using limited proteolysis and multiple sequence alignments of glutamate receptors as guides, nine constructs were made, folded, and screened for ligand binding activity. From this screen, the S1S21 construct proved to be trypsin- and chymotrypsin-resistant, stable to storage at 4 degrees C, and amenable to three-dimensional crystal formation. The HS1S21 variant was readily prepared on a large scale, the His tag was easily removed by trypsin, and crystals were produced that diffracted to beyond 1.5 A resolution. These experiments, for the first time, pave the way to economical overproduction of the ligand binding domains of glutamate receptors and more accurately map the boundaries of the ligand binding domain.