Association of alpha-synuclein and mutants with lipid membranes: spin-label ESR and polarized IR

Alpha-synuclein is a presynaptic protein, the A53T and A30P mutants of which are linked independently to early-onset familial Parkinson's disease. The association of wild-type alpha-synuclein with lipid membranes was characterized previously by electron spin resonance (ESR) spectroscopy with spin-labeled lipids [Ramakrishnan, M., Jensen, P. H., and Marsh, D. (2003) Biochemistry 42, 12919-12926]. Here, we study the interaction of the A53T and A30P alpha-synuclein mutants and a truncated form that lacks the acidic C-terminal domain with phosphatidylglycerol bilayer membranes, using anionic phospholipid spin labels. The strength of the interaction with phosphatidylglycerol membranes lies in the order: wild type approximately truncated > A53T > A30P > fibrils approximately 0, and only the truncated form interacts with phosphatidylcholine membranes. The selectivity of the interaction of the mutant alpha-synucleins with different spin-labeled lipid species is reduced considerably, relative to the wild-type protein, whereas that of the truncated protein is increased. Polarized infrared (IR) spectroscopy is used to study the interactions of the wild-type and truncated proteins with aligned lipid membranes and additionally to characterize the fibrillar form. Wild-type alpha-synuclein is natively unfolded in solution and acquires secondary structure upon binding to membranes containing phosphatidylglycerol. Up to 30-40% of the amide I band intensity of the membrane-bound wild-type and truncated proteins is attributable to beta-sheet structure, at the surface densities used for IR spectroscopy. The remainder is alpha-helix and residual unordered structure. Fibrillar alpha-synuclein contains 62% antiparallel beta-sheet and is oriented on the substrate surface but does not interact with deposited lipid membranes. The beta-sheet secondary-structural elements of the wild-type and truncated proteins are partially oriented on the surface of membranes with which they interact.     

Nicotine-induced changes in the metabolism of specific brain proteins

The effect of acute and chronic nicotine on the metabolism of specific brain proteins was examined by measuring incorporation of labeled valine into protein, with densitometric scanning of proteins resolved by gel electrophoresis. Acute and chronic administration of nicotine (0.4 mg/kg per 30 min for 2 hours, s.c., or 0.5 mg/kg per 30 min for 5 days (Alzet mini-pump implanted subcutaneously) reduced incorporation of [¹⁴C]valine administered by approximately 6–7%. The results with chronic nicotine administration indicated a lack of tolerance for this effect of nicotine. Mecamylamine, a nicotinic ganglionic antagonist, does not seem to block the inhibition of protein synthesis. Small increases in protein content were observed in a high- and a low-molecular-weight region of SDS-polyacrylamide gel, used to separate proteins from newborn brain. In adult brain after chronic nicotine administration, selective increases and a decrease were seen in selective bands. Results are consonant with selective effects of nicotine on the synthesis or degradation of specific brain proteins.Special Issue Dedicated to Dr. Abel Lajtha.     

Effects of vitamin E on nicotine-induced lipid peroxidation in rat granulosa cells: Folliculogenesis

In this study, we investigated the mechanism of oxidative damage induced by nicotine and the efficacy of vitamin E, an integral component of cellular membranes, against the damage in follicular/granulosa cells of rat ovaries. The animals were randomly divided into 4 groups; control, nicotine, nicotine + vitaminE, vitamin E (n = 8, per each group). Nicotine and vitamin E were administrated intraperitoneally 1 mg/kg/day and 200 mg/kg/day, respectively, once daily for 2 weeks. Nicotine increased lipid peroxide levels such as lipid peroxide (LPO) and malondialdehyde (MDA) in serum, 4-hydroxynonenal (4-HNE) in granulosa cells and apoptotic granulosa cells in the ovary. Positive correlation occurred between the findings of LPO markers and TUNEL labeling. Level of 17-β estradiol (E₂), number of follicles and granulosa cell proliferation decreased with nicotine treatment and negatively correlated with LPO levels and apoptosis in granulosa cells. Ultrastructural study of nicotine treated rat ovaries showed mitochondrial damage and autophagosomes in the granulosa cells. The administration of nicotine and vitamin E together, revealed an increase in E₂ level, granulosa cell proliferation and the number of healthy follicles associated with decrease in LPO, MDA, 4-HNE levels and TUNEL reactivity in a manner correlated with each other, compared to the nicotine group. Vitamin E showed to alleviate mitochondrial damage and decrease the number of autophagosomes in granulosa cells. These results suggest that lipid peroxidation may be one of the nicotine’ damage mechanisms on folliculogenesis and vitamin E may prevent nicotine-induced follicular damage through reducing lipid peroxidation level in granulosa cells.     

d-amphetamine binding to brain lipid

The interaction ofd-amphetamine with several brain tissue components has been investigated. A brain lipid extract and a number of individual phospholipids were found to bindd-amphetamine when measured by means of a hexane-buffer partition coefficient technique. Phosphatidylserine showed the greatest binding and phosphatidylethanolamine and dipalmitoyllecithin were also effective. Phospholipid binding ofd-amphetamine was shown to be related to fatty acid composition. Cholesterol and ganglioside did not bind. It is suggested that phospholipid binding ofd-amphetamine may play a role in the pharmacological action of this drug.     

Accelerated accumulation of amyloid beta proteins on oxidatively damaged lipid membranes

The fully developed lesion of Alzheimer's Disease is a dense plaque composed of fibrillar amyloid beta-proteins with a characteristic and well-ordered beta-sheet secondary structure. Because the incipient lesion most likely develops when these proteins are first induced to form beta-sheet secondary structure, it is important to understand factors that induce amyloid beta-proteins to adopt this conformation. In this investigation we used a novel form of infrared spectroscopy that can characterize the conformation, orientation, and rate of accumulation of the protein on various lipid membranes to determine whether oxidatively damaged phospholipid membranes induce the formation of beta-sheet secondary structure in a 42-residue amyloid beta-protein. We found that membranes containing oxidatively damaged phospholipids accumulated amyloid beta-protein significantly faster than membranes containing only unoxidized or saturated phospholipids. Accelerated accumulation was also seen when 3 mol % G(M1) ganglioside was incorporated into a saturated phosphatidylcholine membrane. The accumulated protein more completely adopted a beta-sheet conformation on oxidized membranes, and the plane of the beta-sheet was oriented parallel to the plane of the membrane. These results indicate that oxidatively damaged phospholipid membranes promote beta-sheet formation by amyloid beta-proteins, and they suggest a possible role for lipid peroxidation in the pathogenesis of Alzheimer's Disease.     

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.     

LIPID PEROXIDE FORMATION IN RAT BRAIN

Abstract— Lipid peroxide formation as measured by the thiobarbituric acid reaction was demonstrated in subcellular fractions of rat brain. The ascorbic acid induced nonenzymic lipid peroxidation was distributed in all the subcellular fractions with a maximum in microsomes. The NADPH dependent enzymic lipid peroxidation occurred mainly in microsomes and to a smaller extent in synaptosomes; NADH could replace NADPH for the enzymic lipid peroxidation under the assay conditions employed. Fe²⁺ but not Fe³⁺ stimulated the NADPH or NADH dependent lipid peroxide formation. The optimum conditions with respect to pH, ascorbic acid or NADPH concentration, time of incubation and protein concentration were studied. Heating the microsomes at 100^oCdid not influence the ascorbate-induced lipid peroxidation but completely abolished the NADPH linked peroxidation. Several heavy metal ions, surface active agents and EDTA were inhibitory to lipid peroxidation. The effect of thiol agents indicated that -SH groups were involved in the enzymic lipid peroxidation. Studies on subcellular fractions of developing rat brain showed an increasing trend in lipid peroxidation with the advancing age of the animal. No significant difference in lipid peroxidation was observed between brains from normal rats and those from rats affected by experimental allergic encephalomyelitis.     

Structure of sterol carrier protein 2 at 1.8 A resolution reveals a hydrophobic tunnel suitable for lipid binding

Sterol carrier protein 2, also known as nonspecific lipid transfer protein is a ubiquitous, small, basic protein of 13 kDa found in animals. Its primary structure is highly conserved between different species, and it has been implicated in the intracellular transport of lipids and in a wide range of other in vitro functions related to sterol and fatty acid metabolism. Sterol carrier protein 2 deficiency in mice leads to elevated concentrations of phytanic acid in the serum and causes hepatocarcinogenesis. However, its actual physiological role is still unknown. Although sterol carrier protein 2 has been studied extensively in the past 20 years, very little is known concerning its three-dimensional structure. The crystal structure of rabbit sterol carrier protein 2, determined at 1.8 A resolution with the MIRAS method, shows a unique alpha/beta-fold. The core of this protein forms a five-stranded antiparallel beta-sheet flanked by five helices. A C-terminal segment (residues 114-123), together with part of the beta-sheet and four alpha-helices, form a hydrophobic tunnel providing the environment for apolar ligands such as fatty acids and fatty acyl-coenzyme As. Structurally well-characterized nonspecific lipid transfer proteins from plants have hydrophobic tunnel-like cavities, which were identified as the binding site for fatty acids and related apolar ligands. Despite the fact that plant nonspecific lipid transfer proteins are smaller proteins than sterol carrier protein 2, show no sequence homology to sterol carrier protein 2, and are structurally unrelated, the cavities of these two classes of proteins are very similar with respect to size, shape, and hydrophobicity, suggesting a common functional role.     

The effectiveness of a lipid peroxide in oxidizing protein and non-protein thiols

1. Thiol oxidation by a lipid peroxide or hydrogen peroxide was as efficient in denatured non-haem proteins as in small thiols. Both peroxides were relatively ineffective in oxidizing haemoprotein thiols, especially at low pH. Increased amounts of haematin decreased greatly the efficiency of GSH oxidation by peroxides especially at low pH. 2. Other than the haematin ring, the thiol group was found to be probably the group in proteins most sensitive to modification by peroxides. 3. At low concentrations, the fatty acid moiety of a lipid peroxide appeared to impede thiol oxidation in proteins, probably by hydrophobic bonding to the protein, rather than to stimulate thiol oxidation by denaturing the protein and thereby increasing the exposure and reactivity of the thiol group. 4. The relative rates of thiol oxidation by peroxides in the different thiols were: haemoprotein thiols>small thiols>other protein thiols. In all cases, thiol oxidation was much more rapid by the lipid peroxide than by hydrogen peroxide.     

The conformation of T4 bacteriophage dihydrofolate reductase from circular dichroism

The secondary and tertiary structure of T4 bacteriophage dihydrofolate reductase is investigated by vacuum ultraviolet circular dichroism (CD) spectroscopy and probability analysis of the primary amino acid sequence. The far ultraviolet CD spectrum of the enzyme in the range of 260-178 nm is analyzed by the generalized inverse and variable selection methods developed by our laboratory. Variable selection yields an average content of 26% alpha-helix, 21% antiparallel beta-sheet, 10% parallel beta-sheet, 20% beta-turns, and 32% "other" structures within the T4 protein. The characteristic peaks of the CD spectrum indicate that the enzyme has a lot of antiparallel beta-sheet, which is typical of the alpha + beta tertiary class of globular proteins. The secondary structure of the protein is also analyzed by using four statistical methods on the amino acid sequence. Although the secondary structures predicted by each individual statistical method vary to a considerable extent, the fractions of each structure jointly predicted by a majority of the methods are in excellent agreement with our CD analysis. The alternating arrangement for some segments of alpha-helix and beta-sheet predicted from primary structure to be within the enzyme is characteristic of proteins containing parallel beta-sheet. This supports our conclusion that the protein contains both parallel and antiparallel beta-sheet structures, but finding both types of beta-sheet also means that the protein may have the variation on alpha/beta tertiary structure recently found in EcoRI endonuclease and thymidylate synthase. These observations, in conjunction with other physical properties of the T4 reductase, suggest that the enzyme perhaps shares an evolution in common with the dihydrofolate reductases derived from type I R-plasmids rather than with the host-cell protein.     

Amyloid-beta peptide disruption of lipid membranes and the effect of metal ions

Beta-amyloid peptide (Abeta), which is cleaved from the larger trans-membrane amyloid precursor protein, is found deposited in the brain of patients suffering from Alzheimer's disease and is linked with neurotoxicity. We report the results of studies of Abeta1-42 and the effect of metal ions (Cu2+ and Zn2+) on model membranes using 31P and 2H solid-state NMR, fluorescence and Langmuir Blodgett monolayer methods. Both the peptide and metal ions interact with the phospholipid headgroups and the effects on the lipid bilayer and the peptide structure were different for membrane incorporated or associated peptides. Copper ions alone destabilise the lipid bilayer and induced formation of smaller vesicles but when Abeta1-42 was associated with the bilayer membrane copper did not have this effect. Circular dichroism spectroscopy indicated that Abeta1-42 adopted more beta-sheet structure when incorporated in a lipid bilayer in comparison to the associated peptide, which was largely unstructured. Incorporated peptides appear to disrupt the membrane more severely than associated peptides, which may have implications for the role of Abeta in disease states.     

Heparin-binding growth-associated molecule contains two heparin-binding beta -sheet domains that are homologous to the thrombospondin type I repeat

Heparin-binding growth-associated molecule (HB-GAM) is an extracellular matrix-associated protein implicated in the development and plasticity of neuronal connections of brain. Binding to cell surface heparan sulfate is indispensable for the biological activity of HB-GAM. In the present paper we have studied the structure of recombinant HB-GAM using heteronuclear NMR. These studies show that HB-GAM contains two beta-sheet domains connected by a flexible linker. Both of these domains contain three antiparallel beta-strands. In addition to this domain structure, HB-GAM contains the N- and C-terminal lysine-rich sequences that lack a detectable structure and appear to form random coils. Studies using CD and NMR spectroscopy suggest that HB-GAM undergoes a conformational change upon binding to heparin, and that the binding occurs primarily to the beta-sheet domains of the protein. Search of sequence data bases shows that the beta-sheet domains of HB-GAM are homologous to the thrombospondin type I repeat (TSR). Sequence comparisions show that the beta-sheet structures found previously in midkine, a protein homologous with HB-GAM, also correspond to the TSR motif. We suggest that the TSR sequence motif found in various extracellular proteins defines a beta-sheet structure similar to that found in HB-GAM and midkine. In addition to the apparent structural similarity, a similarity in biological functions is suggested by the occurrence of the TSR sequence motif in a wide variety of proteins that mediate cell-to-extracellular matrix and cell-to-cell interactions, in which the TSR domain mediates specific cell surface binding.     

Secondary structural features of the bacteriophage Mu-encoded A and B transposition proteins.

The role of the bacteriophage Mu-encoded A and B proteins is to direct the transposition of Mu DNA. These are the first active DNA transposition proteins to have been purified and their mechanism of action at the biochemical level is under intensive study. Structural studies on these proteins, however, have lagged behind their biochemical characterization. We report here near- and far-u.v. c.d. spectra for these proteins and their secondary structural features derived from these data. The Mu A protein appears to be composed of primarily beta-sheet (40%) with 24% alpha-helix, 9% beta-turn and 27% random coil. In contrast, the Mu B protein contains 55% alpha-helix with only 13% beta-sheet and 3+ beta-turn and 29% random coil. The near-u.v. c.d. spectrum of the A protein was not unusual; however, the profile of the B protein suggested either buried or restricted chromophores within the protein or short-range interactions between aromatic residues.     

The Functional Significance of the Pentose Phosphate Pathway in Synaptosomes: Protection Against Peroxidative Damage by Catecholamines and Oxidants

Abstract: Catecholamines added in vitro in rat brain synaptosomes activate the decarboxylation of glucose radioactively labelled on carbon 1, suggesting an effective activation of the pentose phosphate pathway. Stimulation also occurred with phenazine methosulphate, reduced glutathione and hydrogen peroxide. The activation of the pentose phosphate pathway by 5-hydroxytryptamine, noradrenaline and dopamine is ascribed to the activation of monoamine oxidase, producing both the respective biogenic aldehyde and hydrogen peroxide. Evidence is presented that the further metabolism of the aldehyde by aldehyde reductase and the removal of hydrogen peroxide by glutathione peroxidase both release the limitation of N ADP⁺ availability for the pentose phosphate pathway by leading to the oxidation of NADPH. The relevance of the maintenance of reduced NADP⁺ on brain is discussed in relation to the metabolism of glutathione and to lipid peroxidation.     

Effects of proteins on phospholipid vesicle aggregation and lipid vesicle-monolayer interactions

The effects of proteins on divalent cation-induced phospholipid vesicle aggregation and phospholipid vesicle-monolayer membrane interactions (fusion) were examined. Glycophorin (from human erythrocytes) suppressed the membrane interactions more than N-2 protein (from human brain myelin) when these proteins were incorporated into acidic phospholipid vesicle membranes. The threshold concentrations of divalent cations which induced vesicle aggregation were increased by protein incorporation, and the rate of vesicle aggregation was reduced. A similar inhibitory effect by the proteins, incorporated into lipid vesicle membranes, was observed for Ca²⁺-induced lipid vesicle-monolayer interactions. However, when these proteins were incorporated only in the acidic phospholipid monolayers, the interaction (fusion) of the lipid vesicle-monolayer membranes, induced by divalent cations, was not appreciably altered by the presence of the proteins.In contrast to these two proteins, the presence of synexin in the solution did enhance the Ca²⁺-induced aggregation of phosphatidylserine vesicles, but did not seem to affect the degree of Ca²⁺-induced fusion between phosphatidylserine/phosphatidylcholine (1:1) and phosphatidylserine vesicles and monolayer membranes.     

Secondary structure and topology of human interleukin 4 in solution

Human interleukin 4 (IL-4) has been studied by 2D and 3D NMR techniques using uniformly 15N-labeled recombinant protein. Assignment of resonances for all but 3 of the 130 residues of the recombinant protein has been achieved, enabling the secondary structure of the protein to be defined. This consists of four major alpha-helical regions and one short section of double-stranded antiparallel beta-sheet. Analysis of distance and angle restraints derived from NMR experiments has enabled the overall molecular topology to be determined. This is related to that found for other four-helix proteins but has several distinctive features including cross-linking of helices by means of three disulfide bonds and a short section of beta-sheet. The structural analysis gives support to the hypothesis that many helical cytokines have a common fold and provides a basis for understanding the biological function of IL-4.     

Adsorptive and catalytic properties of cholinesterase on phospholipid monolyers in different oxidation states]

Adsorption isotherm and enzymatic activity of protein interacting with the surface of solid carrier formed by oriented fat lipid chains of differently oxidized phospholipids have been studied. It has been found that the appearance of peroxide groups in fat acid chains results in a twofold decrease of protein limiting adsorption on the lipid monolayer. The data on enzymatic activity of proteins at variously filled suface with protein molecules indicate that the peroxide groups produce an activating effect under the conditions when protein interactions can be neglected. Superoxidation of phospholipid fat acids is suggested to be one of the mechanisms involved in the control of the processes proceeding on the membranes.     

Lipid-protein interactions in membranes

The interactions of lipids with integral and peripheral proteins can be studied in reconstituted and natural membranes using spin label electron spin resonance (ESR) spectroscopy. The ESR spectra reveal a reduction in mobility of the spin-labelled lipid species, and in certain cases evidence is obtained for a partial penetration of the peripheral proteins into the membrane. The latter may be relevant to the import mechanism of apocytochrome c into mitochondria. Integral proteins induce a more direct motional restriction of the spin-labelled lipid chains, allowing the stoichiometry and specificity of the interaction, and the lipid exchange rate at the protein interface to be determined from the ESR spectra. In this way, a population of very slowly exchanging cardiolipin associated with the mitochondrial ADP-ATP carrier has been identified. The residues involved in the specificity for charged lipids of the myelin proteolipid protein have been localized to the deletion in the DM-20 mutant, and the difference in lipid-protein interactions with the beta-sheet and alpha-helical conformations of the M-13 coat protein, has been characterized.     

Proteomic profiling of proteins associated with methamphetamine-induced neurotoxicity in different regions of rat brain

It is well documented that methamphetamine (MA) can cause obvious damage to the brain, but the exact mechanism is still unknown. In the present study, proteomic methods of two-dimensional gel electrophoresis in combination with mass spectrometry analysis were used to identify global protein profiles associated with MA-induced neurotoxicity. For the first time, 30 protein spots have been found differentially expressed in different regions of rat brain, including 14 in striatum, 12 in hippocampus and 4 in frontal cortex. The proteins identified by tandem mass spectrometry were Cu, Zn superoxide dismutase, dimethylarginine dimethylaminohydrolase 1, alpha synuclein, ubiquitin-conjugating enzyme E2N, stathmin 1, calcineurin B, cystatin B, subunit of mitochondrial H-ATP synthase, ATP synthase D chain, mitochondrial, NADH dehydrogenase(ubiquinone) Fe-S protein 8, glia maturation factor, beta, Ash-m, neurocalcin delta, myotrophin, profiling IIa, D-dopachrome tautomerase, and brain lipid binding protein. The known functions of these proteins were related to the pathogenesis of MA-induced neurotoxicity, including oxidative stress, degeneration/apoptosis, mitochontrial/energy metabolism and others. Of these proteins, alpha-synuclein was up-regulated, and ATP synthase D chain, mitochondrial was down-regulated in all brain regions. Two proteins, Cu, Zn superoxide dismutase, subunit of mitochondrial H-ATPsynthase were down-regulated and Ubiquitin-conjugating enzyme E2N, NADH dehydrogenase (ubiquinone) Fe-S protein 8 were up-regulated simultaneously in striatum and hippocaltum. The expression of dimethylarginine dimethylaminohydrolase 1 (DDAH 1) increased both in striatum and frontal cortex. The parallel expression patterns of these proteins suggest that the pathogenesis of MA neurotoxicity in different brain regions may share some same pathways.     

Inhibitory effects of echinoisoflavanone and sophoraisoflavanone D in Sophora chrysophylla SEEM on lipid peroxidation of mice brain homogenate by interaction of ferrous ion and hydrogen peroxide, in vitro

We present the results of an in vitro investigation of the inhibitory effects of echinoisoflavanone and sophoraisoflavanone D isolated from Sophora chrysophylla SEEM on lipid peroxidation of mice brain homogenate by interaction of ferrous ion and hydrogen peroxide, in vitro. They inhibited lipid peroxidation. The order of inhibitory effects of these isoflavanones and mannitol as a hydroxy radical scavenger was echinoisoflavanone > mannitol > sophoraisoflavanone D. The results suggest that some isoflavanones may be of use in cases where oxidative stress is present.