Alterations in phospholipid and fatty acid lipid profiles in primary neocortical cells during oxidant-induced cell injury

Specific phospholipids and fatty acids altered during oxidant-induced neuronal cell injury were determined using electrospray ionization mass spectrometry (ESI-MS) and ion trapping. The oxidants hydrogen peroxide (H(2)O(2), 0-1000 microM) and tert-butylhydroperoxide (TBHP, 0-400 microM) induced time- and concentration-dependent increases in reactive oxygen species in primary cultures of mouse neocortical cells as determined by 2',7'-dichlorofluorescein diacetate staining and thiobarbituric acid formation. ESI-MS analysis of 26 m/z values, representing 42 different phospholipids, demonstrated that H(2)O(2) and TBHP increased the abundance of phospholipids containing polyunsaturated fatty acids, but had minimal affect on those containing mono- or di-unsaturated fatty acids. These increases correlated to time-dependent increase in 16:1-20:4, 16:0-20:4, 18:1-20:4 and 18:0-20:4 phosphatidylcholine. Oxidant exposure also increased mystric (14:0), palmitic (16:0), and stearic (18:0) acid twofold, oleic acid (18:1) two- to threefold, and arachidonic acid (20:4) fourfold, compared to controls. Increases in arachidonic acid levels occurred prior to increases in the phospholipids, but after increases in ROS, and correlated to increases in oxidized arachidonic acid species, specifically [20:4-OOH]-H(2)O-, 20:4-OH-, and Tri-OH-20:4-arachidonic acid. Treatment of cells with methyl arachidonyl flourophosphonate an inhibitor of Group IV and VI PLA(2), decreased oxidant-induced arachidonic acid release, while bromoenol lactone, an inhibitor of Group VI PLA(2), did not. Collectively, these data identify phospholipids and fatty acids altered during oxidant treatment of neurons and suggest differential roles for Group IV and VI PLA(2) in oxidant-induced neural cell injury.     

An Integrated Model of Eicosanoid Metabolism and Signaling Based on Lipidomics Flux Analysis

There is increasing evidence for a major and critical involvement of lipids in signal transduction and cellular trafficking, and this has motivated large-scale studies on lipid pathways. The Lipid Metabolites and Pathways Strategy consortium is actively investigating lipid metabolism in mammalian cells and has made available time-course data on various lipids in response to treatment with KDO₂-lipid A (a lipopolysaccharide analog) of macrophage RAW 264.7 cells. The lipids known as eicosanoids play an important role in inflammation. We have reconstructed an integrated network of eicosanoid metabolism and signaling based on the KEGG pathway database and the literature and have developed a kinetic model. A matrix-based approach was used to estimate the rate constants from experimental data and these were further refined using generalized constrained nonlinear optimization. The resulting model fits the experimental data well for all species, and simulated enzyme activities were similar to their literature values. The quantitative model for eicosanoid metabolism that we have developed can be used to design experimental studies utilizing genetic and pharmacological perturbations to probe fluxes in lipid pathways.     

Glycerophospholipids and glycerophospholipid-derived lipid mediators: a complex meshwork in Alzheimer's disease pathology

An increasing body of evidence suggested that intracellular lipid metabolism is dramatically perturbed in various cardiovascular and neurodegenerative diseases with genetic and lifestyle components (e.g., dietary factors). Therefore, a lipidomic approach was also developed to suggest possible mechanisms underlying Alzheimer’s disease (AD). Neural membranes contain several classes of glycerophospholipids (GPs), that not only constitute their backbone but also provide the membrane with a suitable environment, fluidity, and ion permeability. In this review article, we focused our attention on GP and GP-derived lipid mediators suggested to be involved in AD pathology. Degradation of GPs by phospholipase A₂ can release two important brain polyunsaturated fatty acids (PUFAs), e.g., arachidonic acid and docosahexaenoic acid, linked together by a delicate equilibrium. Non-enzymatic and enzymatic oxidation of these PUFAs produces several lipid mediators, all closely associated with neuronal pathways involved in AD neurobiology, suggesting that an interplay among lipids occurs in brain tissue. In this complex GP meshwork, the search for a specific modulating enzyme able to shift the metabolic pathway towards a neuroprotective role as well as a better knowledge about how lipid dietary modulation may act to slow the neurodegenerative processes, represent an essential step to delay the onset of AD and its progression. Also, in this way it may be possible to suggest new preventive or therapeutic options that can beneficially modify the course of this devastating disease.     

Correlation between the phospholipids domains of the target cell membrane and the extent of Naja kaouthia PLA2-induced membrane damage: Evidence of distinct catalytic and cytotoxic sites in PLA2 molecules

Two phospholipase A₂ (PLA₂) enzymes (NK-PLA₂-A and NK-PLA₂-B) were purified from the venom of the monocled cobra Naja kaouthia. The molecular weights of NK-PLA₂-A and NK-PLA₂-B, as estimated by mass spectrometry, were 13,619 and 13,303 Da respectively. Both phospholipases were highly thermostable, had maximum catalytic activity at basic pH, and showed preferential hydrolysis of phosphatidylcholine. Intravenous injection of either PLA₂ up to a dose of 10 mg/kg body weight was non-toxic to mice and did not show neurotoxic symptoms. The N. kaouthia PLA₂s displayed anticoagulant and cytotoxic activity, but poor hemolytic activity. Both the PLA₂s were more toxic to Sf9 and Tn cells compared to VERO cells. NK-PLA₂ exhibited selective lysis of wild-type baculovirus-infected Sf9 cells compared to normal cells. Amino acid modification studies and heating experiments suggest that separate sites in the NK-PLA₂ molecules are responsible for their catalytic, anticoagulant and cytotoxic activities.     

Plasmalogens the neglected regulatory and scavenging lipid species

Plasmalogens are a class of phospholipids carrying a vinyl ether bond in sn-1 and an ester bond in sn-2 position of the glycerol backbone. Although they are widespread in all tissues and represent up to 18% of the total phospholipid mass in humans, their physiological function is still poorly understood. The aim of this review is to give an overview over the current knowledge in plasmalogen biology and pathology with an emphasis on neglected aspects of their involvement in neurological and metabolic diseases. Furthermore a better understanding of plasmalogen biology in health and disease could also lead to the development of better diagnostic and prognostic biomarkers for vascular and metabolic diseases such as obesity and diabetes mellitus, inflammation, neuro-degeneration and cancer.     

Effect of pentoxifylline on arachidonic acid metabolism,neutral lipid synthesis and accumulation during induction of the lipocyte phenotype by retinol in murine hepatic stellate cell

In liver fibrosis, the quiescent hepatic stellate cells (HSC) are activated to proliferate and express the activated myofibroblast phenotype, losing fat droplets and the stored vitamin A, and depositing more extracellular matrix. Therapeutic strategies for liver fibrosis are focused on HSC. Pentoxifylline (PTF), an analog of the methylxanthine, prevents the biochemical and histological changes associated with animal liver fibrosis. The aim of the present study was to investigate the phenotypic change of myofibroblasts into quiescent lipocytes by PTF and/or retinol, using a permanent cell line GRX that represents murine HSC. We studied the action of both drugs on the synthesis of neutral lipids, activity of phospholipase A₂ (PLA₂), release of arachidonic acid (AA) and prostaglandins synthesis. Accumulation and synthesis of neutral lipids was dependent upon association of retinol with PTF. PTF (0.5 mg/mL) alone did not induce lipid accumulation and synthesis, but in cells induced by physiologic concentration of retinol (1–2.5 M), it increased the quantity of stored lipids. Retinol and PTF (5 M and 0.1 mg/mL, respectively) had a synergistic effect on neutral lipid synthesis and accumulation. In higher PTF concentrations (0.5 and 0.7 mg/ml), the synthesis was stimulated but accumulation decreased. Membrane-associated PLA₂ activity decreased after PTF treatment, which increased the AA release 8 fold, and significantly increased the production of PGE₂, but not of PGF₂. However, when in presence of retinol, we observed a slightly higher increase in PGE₂ and PGF₂ production. In conclusion, PTF treatment generated an excess of free AA. We propose that retinol counteracts the action of PTF on the AA release and PGs production, even though both drugs stimulated the lipocyte induction in the HSC.     

Action of jasmonates in plant stress responses and development — Applied aspects

Jasmonates (JAs) are lipid-derived compounds acting as key signaling compounds in plant stress responses and development. The JA co-receptor complex and several enzymes of JA biosynthesis have been crystallized, and various JA signal transduction pathways including cross-talk to most of the plant hormones have been intensively studied. Defense to herbivores and necrotrophic pathogens are mediated by JA. Other environmental cues mediated by JA are light, seasonal and circadian rhythms, cold stress, desiccation stress, salt stress and UV stress. During development growth inhibition of roots, shoots and leaves occur by JA, whereas seed germination and flower development are partially affected by its precursor 12-oxo-phytodienoic acid (OPDA). Based on these numerous JA mediated signal transduction pathways active in plant stress responses and development, there is an increasing interest in horticultural and biotechnological applications. Intercropping, the mixed growth of two or more crops, mycorrhization of plants, establishment of induced resistance, priming of plants for enhanced insect resistance as well as pre- and post-harvest application of JA are few examples. Additional sources for horticultural improvement, where JAs might be involved, are defense against nematodes, biocontrol by plant growth promoting rhizobacteria, altered composition of rhizosphere bacterial community, sustained balance between growth and defense, and improved plant immunity in intercropping systems. Finally, biotechnological application for JA-induced production of pharmaceuticals and application of JAs as anti-cancer agents were intensively studied.     

Physio-biochemical and proteome analysis of chickpea in early phases of cold stress

Intensive and short-term strategies can aid in more rapid screening with informative and reliable results for long-term investigations under cold stress (CS). The integration of cellular analysis of chickpea during 0, 2, 4, 8, and 12 h CS supplied us with novel possible responsive components and the possible interactions embedded inside, still remaining a Maze. Seedlings showed a biphasic pattern of responses over time. The transitory phase happened after 8 h, when cells are presumably experiencing a new stage of responses and setting the stage for long-term adjustments. Physio-biochemical analysis confirmed the direct effect of fatty acids composition, lipoxygenase activity and antioxidant systems in cell responses under CS. Also, proteome results using MALDI-TOF-TOF and/or LC–MS/MS were able to differentiate changes in early phases of CS. Two-dimensional gel analysis results showed the possible targets of CS as mitochondria, chloroplast, organelle–nucleus communications, storage resources, stress and defense, protein degradation and signal transduction that confirmed the cell intended to re-establish a new homeostasis, in energy and primary metabolites to adapt to long-term CS. Here we propose a time course dynamic assessing multi-dimensional approaches for CS studies as one of the first studies in short-term treatment to progressively fill in the gaps between physio-biochemical and molecular events and touch the cell architecture for a better comprehension of the nature of plant stress response.     

Eicosapentaenoic acid decreases expression of anandamide synthesis enzyme and cannabinoid receptor 2 in osteoblast-like cells

Anandamide (AEA) is an endogenous agonist for the cannabinoid receptor 2 (CB2) which is expressed in osteoblasts. Arachidonic acid (AA) is the precursor for AEA and dietary n-3 polyunsaturated fatty acids (PUFA) are known to reduce the concentrations of AA in tissues and cells. Therefore, we hypothesized that n-3 PUFA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which reduce AA in cells, could lower AEA in osteoblasts by altering enzyme expression of the endocannabinoid (EC) system. MC3T3-E1 osteoblast-like cells were grown for 6, 10, 15, 20, 25 or 30 days in osteogenic medium. Osteoblasts were treated with 10 μM of AA, EPA, DHA, oleic acid (OA) or EPA+DHA (5 μM each) for 72 h prior to their collection for measurement of mRNA and alkaline phosphatase (ALP) activity. Compared to vehicle control, osteoblasts treated with AA had higher levels of AA and n-6 PUFA while those treated with EPA and DHA had lower n-6 but higher n-3 PUFA. Independent of the fatty acid treatments, osteoblasts matured normally as evidenced by ALP activity. N-acyl phosphatidylethanolamine-selective phospholipase D (NAPE-PLD), fatty acid amide hydrolase (FAAH) and CB2 mRNA expression were higher at 20 days compared to 10 days. NAPE-PLD and CB2 mRNA was lower in osteoblasts treated with EPA compared to all other groups. Thus, mRNA expression for NAPE-PLD, FAAH, and CB2 increased during osteoblast maturation and EPA reduced mRNA for NAPE-PLD and CB2 receptor. In conclusion, EPA lowered mRNA levels for proteins of the EC system and mRNA for AEA synthesis/degradation is reported in osteoblasts.     

Dietary n-6 PUFA deprivation for 15 weeks reduces arachidonic acid concentrations while increasing n-3 PUFA concentrations in organs of post-weaning male rats

Few studies have examined effects of feeding animals a diet deficient in n-6 polyunsaturated fatty acids (PUFAs) but with an adequate amount of n-3 PUFAs. To do this, we fed post-weaning male rats a control n-6 and n-3 PUFA adequate diet and an n-6 deficient diet for 15 weeks, and measured stable lipid and fatty acid concentrations in different organs. The deficient diet contained nutritionally essential linoleic acid (LA,18:2n-6) as 2.3% of total fatty acids (10% of the recommended minimum LA requirement for rodents) but no arachidonic acid (AA, 20:4n-6), and an adequate amount (4.8% of total fatty acids) of α-linolenic acid (18:3n-3). The deficient compared with adequate diet did not significantly affect body weight, but decreased testis weight by 10%. AA concentration was decreased significantly in serum (− 86%), brain (− 27%), liver (− 68%), heart (− 39%), testis (− 25%), and epididymal adipose tissue (− 77%). Eicosapentaenoic (20:5n-3) and docosahexaenoic acid (22:6n-3) concentrations were increased in all but adipose tissue, and the total monounsaturated fatty acid concentration was increased in all organs. The concentration of 20:3n-9, a marker of LA deficiency, was increased by the deficient diet, and serum concentrations of triacylglycerol, total cholesterol and total phospholipid were reduced. In summary, 15 weeks of dietary n-6 PUFA deficiency with n-3 PUFA adequacy significantly reduced n-6 PUFA concentrations in different organs of male rats, while increasing n-3 PUFA and monounsaturated fatty acid concentrations. This rat model could be used to study metabolic, functional and behavioral effects of dietary n-6 PUFA deficiency.     

Old and new generation lipid mediators in acute inflammation and resolution

Originally regarded as just membrane constituents and energy storing molecules, lipids are now recognised as potent signalling molecules that regulate a multitude of cellular responses via receptor-mediated pathways, including cell growth and death, and inflammation/infection. Derived from polyunsaturated fatty acids (PUFAs), such as arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), each lipid displays unique properties, thus making their role in inflammation distinct from that of other lipids derived from the same PUFA. The diversity of their actions arises because such metabolites are synthesised via discrete enzymatic pathways and because they elicit their response via different receptors. This review will collate the bioactive lipid research to date and summarise the findings in terms of the major pathways involved in their biosynthesis and their role in inflammation and its resolution. It will include lipids derived from AA (prostanoids, leukotrienes, 5-oxo-6,8,11,14-eicosatetraenoic acid, lipoxins and epoxyeicosatrienoic acids), EPA (E-series resolvins), and DHA (D-series resolvins, protectins and maresins).     

A possible role of lysophospholipids produced by calcium-independent phospholipase A2 in membrane-raft budding and fission

Phospholipase A₂ (PLA₂) not only plays a role in the membrane vesiculation system but also mediates membrane-raft budding and fission in artificial giant liposomes. This study aimed to demonstrate the same effects in living cells. Differentiated Caco-2 cells were cultured on filter membranes. MDCK cells were challenged with Influenza virus. The MDCK cultures were harvested for virus titration with a plaque assay. Alkaline phosphatase (ALP), a membrane-raft associated glycosylphosphatidylinositol (GPI)-anchored protein, was 70% released by adding 0.2 mmol/l lysophosphatidylcholine, which was abolished by treatment with a membrane-raft disrupter, methyl-β-cyclodextrin. Activation of calcium-independent PLA₂ (iPLA₂) by brefeldin A increased the apical release of ALP by approximately 1.5-fold (p < 0.01), which was blocked by PLA₂ inhibitor bromoenol lactone (BEL). BEL also reduced Influenza virus production into the media (< 10%) in the MDCK culture. These results suggest that cells utilize inverted corn-shaped lysophospholipids generated by PLA₂ to modulate plasma membrane structure and assist the budding of raft-associated plasma membrane particles, which virus utilizes for its budding. Brush borders are enriched with membrane-rafts and undergo rapid turnover; thus, PLA₂ may be involved in the regulatory mechanism in membrane dynamism. Further, iPLA₂ may provide a therapeutic target for viral infections.     

Non-specific effects of leukotriene synthesis inhibitors on HeLa cell physiology

We examined the effects of various leukotriene synthesis inhibitors on calcium signalling in HeLa cells, before and after transfection with BLT₁. All of the inhibitors studied were found to reduce increases in intracellular calcium concentration induced by BLT₁, but also by an ionophore or activation of various G-protein coupled receptors, regardless of BLT₁ expression. In order to explore the mechanism of these apparently general effects we examined HeLa cell expression of leukotriene receptors and biosynthetic enzymes and found that the genes for key leukotriene synthesis enzymes and all of the leukotriene receptors were not expressed. Leukotrienes are involved in the pathology of a variety of cancers, and for HeLa cells leukotrienes have been reported to be important for aspects of the carcinogenic phenotype. We find that leukotriene synthesis inhibitors have non-specific effects, so careful controls are necessary to avoid interpreting non-specific effects as evidence for leukotriene involvement.     

Characterization of a lysophospholipid acyltransferase involved in membrane remodeling in Candida albicans

Phospholipid remodeling involves phospholipase activity to remove acyl chains and acyltransferases to replace acyl chains. We here describe the characterization of a lysophospholipid acyltransferase in the opportunistic fungal pathogen, Candida albicans. Expression of this gene, C.a. LPT1, complemented the lysophospholipid acyltransferase defect in Saccharomyces cerevisiae strains lacking the homologous LPT1 gene. In vitro, lysophospholipid acyltransferase activity in these strains showed acyl-CoA substrate specificity, as measured by apparent Vmax/Km ratios, to be linolenoyl-CoA > oleoyl-CoA > linoleoyl-CoA > stearoyl-CoA. To address the physiological importance of C.a. LPT1, homozygous deletion strains were generated. Lysophospholipid acyltransferase activity with amine containing lysophospholipids was dramatically reduced while lysophosphatidylinositol and lysophosphatidic acid esterification was not significantly lowered. However, C.a. LPT1 over-expression yielded an increased amount of lysophosphatidic acyltransferase activity, suggesting a role in de novo phospholipid synthesis. LPT1 deletion strains showed slightly slowed growth in standard liquid media but no phenotype in media containing three antifungals that target sterols. To assess the role of C.a. Lpt1 in phospholipid remodeling, an in vivo, pulse–chase assay utilizing polysorbitan palmitate and mass spectrometry was developed. Cellular phospholipid composition became atypical with the provision of palmitate and gradually returned to the typical distribution when palmitate was removed. Deletion of C.a. LPT1 showed a modest yet significant effect on remodeling under these conditions.     

Cardiac oxidative stress in a mouse model of neutral lipid storage disease

Cardiac oxidative stress has been implicated in the pathogenesis of hypertrophy, cardiomyopathy and heart failure. Systemic deletion of the gene encoding adipose triglyceride lipase (ATGL), the enzyme that catalyzes the rate-limiting step of triglyceride lipolysis, results in a phenotype characterized by severe steatotic cardiac dysfunction. The objective of the present study was to investigate a potential role of oxidative stress in cardiac ATGL deficiency. Hearts of mice with global ATGL knockout were compared to those of mice with cardiomyocyte-restricted overexpression of ATGL and to those of wildtype littermates. Our results demonstrate that oxidative stress, measured as lucigenin chemiluminescence, was increased ~ 6-fold in ATGL-deficient hearts. In parallel, cytosolic NADPH oxidase subunits p67phox and p47phox were upregulated 4–5-fold at the protein level. Moreover, a prominent upregulation of different inflammatory markers (tumor necrosis factor α, monocyte chemotactant protein-1, interleukin 6, and galectin-3) was observed in those hearts. Both the oxidative and inflammatory responses were abolished upon cardiomyocyte-restricted overexpression of ATGL. Investigating the effect of oxidative and inflammatory stress on nitric oxide/cGMP signal transduction we observed a ~ 2.5-fold upregulation of soluble guanylate cyclase activity and a ~ 2-fold increase in cardiac tetrahydrobiopterin levels. Systemic treatment of ATGL-deficient mice with the superoxide dismutase mimetic Mn(III)tetrakis (4-benzoic acid) porphyrin did not ameliorate but rather aggravated cardiac oxidative stress. Our data suggest that oxidative and inflammatory stress seems involved in lipotoxic heart disease. Upregulation of soluble guanylate cyclase and cardiac tetrahydrobiopterin might be regarded as counterregulatory mechanisms in cardiac ATGL deficiency.     

Mitochondrial localization of cyclooxygenase-2 and calcium-independent phospholipase A2 in human cancer cells: implication in apoptosis resistance

Cyclooxygenase-2 (COX-2) is inducible by myriad stimuli. The inducible COX-2 in primary cultured human cells has been reported to localize to nuclear envelope, endoplasmic reticulum, nucleus and caveolae. As COX-2 plays an important role in tumor growth, we were interested in its subcellular location in cancer cells. We examined COX-2 localization in several cancer cell lines by confocal microscopy. A majority of COX-2 was colocalized with heat shock protein 60, a mitochondrial protein, in colon cancer (HT-29, HCT-15 and DLD-1), breast cancer (MCF7), hepatocellular cancer (HepG2) and lung cancer cells (A549) with a similar distribution pattern. By contrast, COX-2 was not localized to mitochondria in human foreskin fibroblasts or endothelial cells. Immunoblot analysis of COX-2 in mitochondrial and cytosolic fractions confirmed localization of COX-2 to mitochondria in HT-29 and DLD-1 cells but not in fibroblasts. Calcium-independent phospholipase A2 was colocalized with heat shock protein 60 to mitochondria not only in cancer cells (HT-29 and DLD-1) but also in fibroblasts. HT-29 which expressed more abundant mitochondrial COX-2 than DLD-1 was highly resistant to arachidonic acid and H2O2-induced apoptosis whereas DLD-1 was less resistant and human fibroblasts were highly susceptible. Treatment of HT-29 cells with sulindac or SC-236, a selective COX-2 inhibitor, resulted in loss of resistance to apoptosis. These results suggest that mitochondrial COX-2 in cancer cells confer resistance to apoptosis by reducing the proapoptotic arachidonic acid.     

Stimulation of arachidonic acid release by vasopressin in A7r5 vascular smooth muscle cells mediated by Ca2+-stimulated phospholipase A2

Arachidonic acid (AA) regulates many aspects of vascular smooth muscle behaviour, but the mechanisms linking receptors to AA release are unclear. In A7r5 vascular smooth muscle cells pre-labelled with (3)H-AA, vasopressin caused a concentration-dependent stimulation of 3H-AA release that required phospholipase C and an increase in cytosolic [Ca2+]. Ca2+ release from intracellular stores and Ca2+ entry via L-type channels or the capacitative Ca2+ entry pathway were each effective to varying degrees. Selective inhibitors of PLA2 inhibited the 3H-AA release evoked by vasopressin, though not the underlying Ca2+ signals, and established that cPLA2 mediates the release of AA. We conclude that in A7r5 cells vasopressin stimulates AA release via a Ca2+-dependent activation of cPLA2.     

Galactolipase, phospholipase and triacylglycerol lipase activities in the midgut of six species of lepidopteran larvae feeding on different lipid diets

Galactolipase, phospholipase and triacylglycerol lipase activities were measured from the midgut of six species of lepidopteran larvae, two folivores, Epiphyas postvittana (Tortricidae) and Helicoverpa armigera (Noctuidae); two granivores, Plodia interpunctella (Pyralidae) and Ephestia kuehniella (Pyrallidae); a presumptive carnivore, Galleria mellonella (Pyralidae); and a keratinophage, Tineola bisselliella (Tineidae). Galactolipase has not been previously reported in insects. Galactolipase and phospholipase activities were high in the folivores and triacylglycerol lipase activity was low, matching the high galactolipid content of leaves. Conversely, galactolipase and phospholipase activities were low, but not absent, and triacylglycerol lipase activity high in the four other non-folivorous species, matching the high acylglycerol content of their diets. These data suggest the utility of reclassification, for evolutionary studies, of phytophagous lepidoptera into two feeding classes; folivore and granivore, the latter having similarity to the fungivore line of feeders in terms of its lipase activities and ability to retrieve essential polyunsaturated long chain fatty acids from their diets. All the digestive lipases have alkaline pH optima for activity, matching the pH of the lepidopteran midgut and their amino acid content show modifications likely to stabilize the proteins in that environment.     

Correct folding of the beta-barrel of the human membrane protein VDAC requires a lipid bilayer

Spontaneous membrane insertion and folding of beta-barrel membrane proteins from an unfolded state into lipid bilayers has been shown previously only for few outer membrane proteins of Gram-negative bacteria. Here we investigated membrane insertion and folding of a human membrane protein, the isoform 1 of the voltage-dependent anion-selective channel (hVDAC1) of mitochondrial outer membranes. Two classes of transmembrane proteins with either alpha-helical or beta-barrel membrane domains are known from the solved high-resolution structures. VDAC forms a transmembrane beta-barrel with an additional N-terminal alpha-helix. We demonstrate that similar to bacterial OmpA, urea-unfolded hVDAC1 spontaneously inserts and folds into lipid bilayers upon denaturant dilution in the absence of folding assistants or energy sources like ATP. Recordings of the voltage-dependence of the single channel conductance confirmed folding of hVDAC1 to its active form. hVDAC1 developed first beta-sheet secondary structure in aqueous solution, while the alpha-helical structure was formed in the presence of lipid or detergent. In stark contrast to bacterial beta-barrel membrane proteins, hVDAC1 formed different structures in detergent micelles and phospholipid bilayers, with higher content of beta-sheet and lower content of alpha-helix when inserted and folded into lipid bilayers. Experiments with mixtures of lipid and detergent indicated that the content of beta-sheet secondary structure in hVDAC1 decreased at increased detergent content. Unlike bacterial beta-barrel membrane proteins, hVDAC1 was not stable even in mild detergents such as LDAO or dodecylmaltoside. Spontaneous folding of outer membrane proteins into lipid bilayers indicates that in cells, the main purpose of membrane-inserted or associated assembly factors may be to select and target beta-barrel membrane proteins towards the outer membrane instead of actively assembling them under consumption of energy as described for the translocons of cytoplasmic membranes.