RESUMEN
Lipid homeostasis is crucial in human health. Barth syndrome (BTHS), a life-threatening disease typically diagnosed with cardiomyopathy and neutropenia, is caused by mutations in the mitochondrial transacylase tafazzin. By high-resolution 31P nuclear magnetic resonance (NMR) with cryoprobe technology, recently we found a dramatic loss of choline plasmalogen in the tafazzin-knockdown (TAZ-KD) mouse heart, besides observing characteristic cardiolipin (CL) alterations in BTHS. In inner mitochondrial membrane where tafazzin locates, CL and diacyl phosphatidylethanolamine are known to be essential via lipid-protein interactions reflecting their cone shape for integrity of respiratory chain supercomplexes and cristae ultrastructure. Here, we investigate the TAZ-KD brain, liver, kidney, and lymphoblast from patients compared with controls. We identified common yet markedly cell type-dependent losses of ethanolamine plasmalogen as the dominant plasmalogen class therein. Tafazzin function thus critically relates to homeostasis of plasmalogen, which in the ethanolamine class has conceivably analogous and more potent molecular functions in mitochondria than diacyl phosphatidylethanolamine. The present discussion of a loss of plasmalogen-protein interaction applies to other diseases with mitochondrial plasmalogen loss and aberrant forms of this organelle, including Alzheimer's disease.
Asunto(s)
Mitocondrias/metabolismo , Plasmalógenos/metabolismo , Aciltransferasas , Animales , Síndrome de Barth/genética , Síndrome de Barth/fisiopatología , Cardiolipinas/metabolismo , Cardiomiopatías/metabolismo , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Membranas Mitocondriales/metabolismo , Plasmalógenos/fisiología , Factores de Transcripción/metabolismoRESUMEN
Signaling events at membranes are often mediated by membrane lipid composition or membrane physical properties. These membrane properties could act either by favoring the membrane binding of downstream effectors or by modulating their activity. Several proteins can sense/generate membrane physical curvature (i.e. shape). However, the modulation of the activity of enzymes by a membrane's shape has not yet been reported. Here, using a cell-free assay with purified diacylglycerol kinase ϵ (DGKϵ) and liposomes, we studied the activity and acyl-chain specificity of an enzyme of the phosphatidylinositol (PI) cycle, DGKϵ. By systematically varying the model membrane lipid composition and physical properties, we found that DGKϵ has low activity and lacks acyl-chain specificity in locally flat membranes, regardless of the lipid composition. On the other hand, these enzyme properties were greatly enhanced in membrane structures with a negative Gaussian curvature. We also found that this is not a consequence of preferential binding of the enzyme to those structures, but rather is due to a curvature-mediated allosteric regulation of DGKϵ activity and acyl-chain specificity. Moreover, in a fine-tuned interplay between the enzyme and the membrane, DGKϵ favored the formation of structures with greater Gaussian curvature. DGKϵ does not bear a regulatory domain, and these findings reveal the importance of membrane curvature in regulating DGKϵ activity and acyl-chain specificity. Hence, this study highlights that a hierarchic coupling of membrane physical property and lipid composition synergistically regulates membrane signaling events. We propose that this regulatory mechanism of membrane-associated enzyme activity is likely more common than is currently appreciated.
Asunto(s)
Diacilglicerol Quinasa/química , Liposomas/química , Fosfatidilinositoles/química , Animales , Línea Celular , Colesterol/química , Diglicéridos/química , Pruebas de Enzimas , Humanos , Fusión de Membrana , Micelas , Estructura Molecular , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Fosfatidilserinas/química , Spodoptera , Propiedades de SuperficieRESUMEN
Tafazzin is the mitochondrial enzyme that catalyzes transacylation between a phospholipid and a lysophospholipid in remodeling. Mutations in tafazzin cause Barth syndrome, a potentially life-threatening disease with the major symptom being cardiomyopathy. In the tafazzin-deficient heart, cardiolipin (CL) acyl chains become abnormally heterogeneous unlike those in the normal heart with a single dominant linoleoyl species, tetralinoleoyl CL. In addition, the amount of CL decreases and monolysocardiolipin (MLCL) accumulates. Here we determine using high-resolution 31P nuclear magnetic resonance with cryoprobe technology the fundamental phospholipid composition, including the major but oxidation-labile plasmalogens, in the tafazzin-knockdown (TAZ-KD) mouse heart as a model of Barth syndrome. In addition to confirming a lower level of CL (6.4 ± 0.1 â 2.0 ± 0.4 mol % of the total phospholipid) and accumulation of MLCL (not detected â 3.3 ± 0.5 mol %) in the TAZ-KD, we found a substantial reduction in the level of plasmenylcholine (30.8 ± 2.8 â 18.1 ± 3.1 mol %), the most abundant phospholipid in the control wild type. A quantitative Western blot revealed that while the level of peroxisomes, where early steps of plasmalogen synthesis take place, was normal in the TAZ-KD model, expression of Far1 as a rate-determining enzyme in plasmalogen synthesis was dramatically upregulated by 8.3 (±1.6)-fold to accelerate the synthesis in response to the reduced level of plasmalogen. We confirmed lyso-plasmenylcholine or plasmenylcholine is a substrate of purified tafazzin for transacylation with CL or MLCL, respectively. Our results suggest that plasmenylcholine, abundant in linoleoyl species, is important in remodeling CL in the heart. Tafazzin deficiency thus has a major impact on the cardiac plasmenylcholine level and thereby its functions.
Asunto(s)
Síndrome de Barth/metabolismo , Mitocondrias Cardíacas/metabolismo , Proteínas Mitocondriales/metabolismo , Plasmalógenos/biosíntesis , Factores de Transcripción/deficiencia , Acilación , Aciltransferasas , Animales , Síndrome de Barth/genética , Síndrome de Barth/patología , Modelos Animales de Enfermedad , Ratones , Ratones Transgénicos , Mitocondrias Cardíacas/genética , Proteínas Mitocondriales/genética , Plasmalógenos/genética , Factores de Transcripción/metabolismoRESUMEN
Cardiolipin is a specific mitochondrial phospholipid that has a high affinity for proteins and that stabilizes the assembly of supercomplexes involved in oxidative phosphorylation. We found that sequestration of cardiolipin in protein complexes is critical to protect it from degradation. The turnover of cardiolipin is slower by almost an order of magnitude than the turnover of other phospholipids. However, in subjects with Barth syndrome, cardiolipin is rapidly degraded via the intermediate monolyso-cardiolipin. Treatments that induce supercomplex assembly decrease the turnover of cardiolipin and the concentration of monolyso-cardiolipin, whereas dissociation of supercomplexes has the opposite effect. Our data suggest that cardiolipin is uniquely protected from normal lipid turnover by its association with proteins, but this association is compromised in subjects with Barth syndrome, leading cardiolipin to become unstable, which in turn causes the accumulation of monolyso-cardiolipin.
Asunto(s)
Síndrome de Barth/metabolismo , Cardiolipinas/metabolismo , HumanosRESUMEN
In addition to specific intermolecular interactions, biological processes at membranes are also modulated by the physical properties of the membrane. One of these properties is membrane curvature. NMR methods are useful for studying how membrane curvature affects the binding and insertion of proteins into membranes as well as how proteins can affect membrane curvature properties. In many cases these interactions result in a marked change in protein activity. We have reviewed examples from a range of systems having varied mechanisms by which membrane curvature is linked to protein activity. Among the examples discussed are antimicrobial peptides, proteins affecting membrane fusion, rhodopsin, protein kinase C, phospholipase C-delta1, phosphatidylinositol-3 kinase-related kinases and tafazzin.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Proteínas de la Membrana/química , Aciltransferasas , Péptidos Catiónicos Antimicrobianos/química , Fusión de Membrana , Rodopsina/química , Factores de Transcripción/químicaRESUMEN
The diacylglycerol kinase from E. coli transfers some of the γ-phosphate of ATP to water as well as to diacylglycerol. We also demonstrate that glycerol can act as an acceptor for the phosphate of ATP. We have compared this behavior with that of the only mammalian isoform of diacylglycerol kinase that exhibits acyl chain specificity, i.e. DGKÉ. The purpose of the study was to determine if differences in the competition between ATPase activity and lipid phosphorylation could contribute to the observed acyl chain specificity with different diacylglycerols. Neither with the highly specific substrate of DGKÉ, 1-stearoyl-2-arachidonoyl glycerol, nor with a less specific substrate, 1-stearoyl-2-linoleoyl glycerol, is there any evidence for ATP hydrolysis accompanying substrate phosphorylation. Thus, at least for this isoform of diacylglycerol kinase, water does not compete with diacylglycerol as an acceptor of the γ-phosphate of ATP. The results demonstrate that the substrate specificity of mammalian DGKÉ is not a consequence of different degrees of ATP hydrolysis in the presence of different species of diacylglycerol.
Asunto(s)
Adenosina Trifosfato/metabolismo , Diacilglicerol Quinasa/metabolismo , Diglicéridos/metabolismo , Animales , Línea Celular , Escherichia coli/enzimología , Humanos , Hidrólisis , Resonancia Magnética Nuclear Biomolecular , Fosforilación , Isoformas de Proteínas/metabolismo , Especificidad por SustratoRESUMEN
Cardiolipin is a mitochondrial phospholipid with a characteristic acyl chain composition that depends on the function of tafazzin, a phospholipid-lysophospholipid transacylase, although the enzyme itself lacks acyl specificity. We incubated isolated tafazzin with various mixtures of phospholipids and lysophospholipids, characterized the lipid phase by (31)P-NMR and measured newly formed molecular species by MS. Substantial transacylation was observed only in nonbilayer lipid aggregates, and the substrate specificity was highly sensitive to the lipid phase. In particular, tetralinoleoyl-cardiolipin, a prototype molecular species, formed only under conditions that favor the inverted hexagonal phase. In isolated mitochondria, <1% of lipids participated in transacylations, suggesting that the action of tafazzin was limited to privileged lipid domains. We propose that tafazzin reacts with non-bilayer-type lipid domains that occur in curved or hemifused membrane zones and that acyl specificity is driven by the packing properties of these domains.
Asunto(s)
1-Acilglicerofosfocolina O-Aciltransferasa/metabolismo , Proteínas de Drosophila/metabolismo , Metabolismo de los Lípidos , Acilación , Animales , Drosophila , Membrana Dobles de Lípidos , Micelas , Resonancia Magnética Nuclear Biomolecular , Especificidad por SustratoRESUMEN
NMR spin-lattice relaxation efficiency is similar at all carbon and silicon sites in aluminum-doped 4H- and 6H-polytype silicon carbide samples, indicating that the valence band edge (the top of the valence band), where the holes are located in p-doped materials, has similar charge densities at all atomic sites. This is in marked contrast to nitrogen-doped samples of the same polytypes where huge site-specific differences in relaxation efficiency indicate that the conduction band edge (the bottom of the conduction band), where the mobile electrons are located in n-doped materials, has very different charge densities at the different sites. An attempt was made to observe (27)Al NMR signals directly, but they are too broad, due to paramagnetic line broadening, to provide useful information about aluminum doping.
Asunto(s)
Aluminio/química , Compuestos Inorgánicos de Carbono/química , Espectroscopía de Resonancia Magnética/métodos , Semiconductores , Compuestos de Silicona/químicaRESUMEN
The beef heart adenine nucleotide carrier protein (Anc) of the inner mitochondrial membrane can be purified in a form stabilized by binding the inhibitor carboxyatractyloside. The protein is copurified with bound lipid. We show for the first time that phosphatidic acid, although a minor component, is one of the lipids bound to Anc. The short spin-lattice relaxation time found by (31)P magic angle spinning nuclear magnetic resonance (MAS/NMR) for phosphatidic acid indicates that it is tightly bound to the protein. However, this lipid also has a comparatively small chemical shift anisotropy, suggesting that it can undergo rapid reorientation in space. In contrast, most of the lipid bound to Anc shows anisotropic motion typical of a bilayer arrangement. The phosphatidic acid that is detected in the purified preparation of Anc is also shown to be present initially in the unfractionated mitochondria, prior to the isolation of Anc. In Triton-solubilized mitochondria, phosphatidic acid, cardiolipin, phosphatidylethanolamine, and phosphatidylcholine exhibit resonance lines in the static (31)P NMR spectra, but in the purified Anc, only the phosphatidylethanolamine and phosphatidylcholine can be detected by this method, even though the other lipids are still present. This demonstrates that the phosphatidic acid and cardiolipin are interacting with the Anc. The thermal denaturation of the Anc was determined by differential scanning calorimetry. The protein denatures at 74 degrees C both before and after the NMR studies with the same characteristics.
Asunto(s)
Translocasas Mitocondriales de ADP y ATP/química , Ácidos Fosfatidicos/química , Animales , Cardiolipinas/química , Bovinos , Translocasas Mitocondriales de ADP y ATP/aislamiento & purificación , Conformación Molecular , Resonancia Magnética Nuclear Biomolecular , Unión ProteicaRESUMEN
The only human cathelicidin, the 37-residue peptide LL-37, exhibits antimicrobial activity against both gram-positive and gram-negative bacteria. We studied the ability of several fragments of LL-37, exhibiting different antimicrobial activities, to interact with membranes whose compositions mimic the cytoplasmic membranes of gram-positive or of gram-negative bacteria. These fragments are as follows: KR-12, the smallest active segment of LL-37, with the sequence KRIVQRIKDFLR, which exhibits antimicrobial activity only against gram-negative bacteria; a slightly smaller peptide, RI-10, missing the two cationic residues at the N and C termini of KR-12, which has been shown not to have any antimicrobial activity; a longer peptide, GF-17, which shows antimicrobial activity against gram-positive as well as gram-negative bacteria; and GF-17D3, with 3 D-amino-acid residues, which is also selective only for gram-negative bacteria. Those fragments with the capacity to cluster anionic lipids away from zwitterionic lipids in a membrane exhibit selective toxicity toward bacteria containing zwitterionic as well as anionic lipids in their cytoplasmic membranes but not toward bacteria with only anionic lipids. This finding allows for the prediction of the bacterial-species selectivity of certain agents and paves the way for designing new antimicrobials targeted specifically toward gram-negative bacteria.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Rastreo Diferencial de Calorimetría , Humanos , Espectroscopía de Resonancia Magnética , Pruebas de Sensibilidad Microbiana , CatelicidinasRESUMEN
A wide range of chemical structures having antimicrobial activity have been studied in an effort to treat the increasing emergence of bacteria that are resistant to traditional antibiotics. These agents have varying degrees of toxicity against different bacterial species. We demonstrate, using members of a novel class of antimicrobial agents, the oligomers of acyllysine, that one cause for the difference in species selectivity is the ability to induce the clustering of anionic lipids, resulting in their segregation into domains. This phenomenon occurs only in bacterial membranes composed of both anionic and zwitterionic lipids and not with bacteria whose membrane lipids are largely anionic. As a consequence it can be predicted which bacterial species will be most affected by antimicrobial agents that function principally by this mechanism. This finding allows for the design of new antibiotics with selective toxicity against different groups of bacteria.
Asunto(s)
Antibacterianos/química , Proteínas Bacterianas/química , Membrana Celular/química , Lisina/química , Antibacterianos/farmacología , Proteínas Bacterianas/efectos de los fármacos , Calorimetría/métodos , Membrana Celular/efectos de los fármacos , Dicroismo Circular/métodos , Farmacorresistencia Bacteriana/efectos de los fármacos , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Membrana Dobles de Lípidos/química , Lisina/análogos & derivados , Lisina/farmacología , Espectroscopía de Resonancia Magnética/métodos , Espectroscopía de Resonancia Magnética/normas , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Valor Predictivo de las Pruebas , Estándares de Referencia , Relación Estructura-Actividad , Temperatura , Factores de TiempoRESUMEN
Two homologous apoA-I mimetic peptides, 3F-2 and 3F(14), differ in their in vitro antiatherogenic properties (Epand, R. M., Epand, R. F., Sayer, B. G., Datta, G., Chaddha, M., and Anantharamaiah, G. M. (2004) J. Biol. Chem. 279, 51404-51414). In the present work, we demonstrate that the peptide 3F-2, which has more potent anti-inflammatory activity in vitro when administered intraperitoneally to female apoE null mice (20 microg/mouse/day) for 6 weeks, inhibits atherosclerosis (lesion area 15,800 +/- 1000 microm(2), n = 29), whereas 3F(14) does not (lesion area 20,400 +/- 1000 microm(2), n = 26) compared with control saline administered (19,900 +/- 1400 microm(2), n = 22). Plasma distribution of the peptides differs in that 3F-2 preferentially associates with high density lipoprotein, whereas 3F(14) preferentially associates with apoB-containing particles. After intraperitoneal injection of (14)C-labeled peptides, 3F(14) reaches a higher maximal concentration and has a longer half-time of elimination than 3F-2. A study of the effect of these peptides on the motional and organizational properties of phospholipid bilayers, using several NMR methods, demonstrates that the two peptides insert to different extents into membranes. 3F-2 with aromatic residues at the center of the nonpolar face partitions closer to the phospholipid head group compared with 3F(14). In contrast, only 3F(14) affects the terminal methyl group of the acyl chain, decreasing the (2)H order parameter and at the same time also decreasing the molecular motion of this methyl group. This dual effect of 3F(14) can be explained in terms of the cross-sectional shape of the amphipathic helix. These results support the proposal that the molecular basis for the difference in the biological activities of the two peptides lies with their different interactions with membranes.
Asunto(s)
Apolipoproteína A-I/química , Aterosclerosis/metabolismo , Animales , Biomimética , Carbono/química , Femenino , Membrana Dobles de Lípidos/química , Lipoproteínas/química , Espectroscopía de Resonancia Magnética , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Péptidos/química , Fosfolípidos/química , Unión ProteicaRESUMEN
A variety of dipyrromethanes and dipyrromethenes have been prepared, and their 15N NMR chemical shifts have been measured by two-dimensional correlation to 1H NMR signals. The nitrogen atoms in five examples of dipyrromethanes consistently exhibit chemical shifts around -231 ppm, relative to nitromethane. Seven examples of hydrobromide salts of meso-unsubstituted dipyrromethenes consistently display 15N chemical shifts around -210 ppm, while their corresponding zinc(II) complexes exhibit chemical shifts around -170 ppm. The presence of electron-withdrawing substituents on one of the pyrrolic rings of dipyrromethenes affects the chemical shifts of both of the nitrogen nuclei in the molecule. Boron difluoride complexes of meso-unsubstituted dipyrromethenes display 15N chemical shifts around -190 ppm. Two examples of free-base dipyrromethenes bearing substituents at the meso-position exhibit 15N chemical shifts at approximately -156 ppm, and for the zinc complexes of these compounds at -162 ppm. One-bond nitrogen-hydrogen coupling constants, when measurable, were consistently in the range of -96 Hz. Since the measured 15N chemical shifts have such a high regularity correlated to structure, they can be used as diagnostic indications for identifying the structure of dipyrrolic compounds.
RESUMEN
Calcium polyphosphate antibiotic delivery matrices were prepared using a unique processing technique involving the exposure of calcium polyphosphate pastes to high humidity for 0, 5, 24 or 48 h to induce gelling. Subsequently, samples were dried for a minimum of 24 h. The mild conditions associated with matrix fabrication readily allowed for vancomycin incorporation within an environment that did not disrupt antibiotic activity. While reproducible from a processing standpoint, the gelling and drying process did contribute to a decrease in matrix tensile strength and the formation of significant pores near the surface of the matrices. Generally, the core of the gelled matrices appeared to be denser than their non-gelled counterparts. The degree of phosphate chain lysis during the gelling and drying stages was quantified using solution 31P nuclear magnetic resonance (NMR) spectroscopy. Both NMR and Raman spectroscopy indicated that the presence of vancomycin did not appreciably alter the matrix formation process. The ability to incorporate clinically relevant levels of antibiotic within this degradable bone substitute matrix suggests the potential of this approach for creating a localized antibiotic delivery system to treat osteomyelitis infections.