RESUMEN
Cancer pharmacogenomics studies provide valuable insights into disease progression and associations between genomic features and drug response. PharmacoDB integrates multiple cancer pharmacogenomics datasets profiling approved and investigational drugs across cell lines from diverse tissue types. The web-application enables users to efficiently navigate across datasets, view and compare drug dose-response data for a specific drug-cell line pair. In the new version of PharmacoDB (version 2.0, https://pharmacodb.ca/), we present (i) new datasets such as NCI-60, the Profiling Relative Inhibition Simultaneously in Mixtures (PRISM) dataset, as well as updated data from the Genomics of Drug Sensitivity in Cancer (GDSC) and the Genentech Cell Line Screening Initiative (gCSI); (ii) implementation of FAIR data pipelines using ORCESTRA and PharmacoDI; (iii) enhancements to drug-response analysis such as tissue distribution of dose-response metrics and biomarker analysis; and (iv) improved connectivity to drug and cell line databases in the community. The web interface has been rewritten using a modern technology stack to ensure scalability and standardization to accommodate growing pharmacogenomics datasets. PharmacoDB 2.0 is a valuable tool for mining pharmacogenomics datasets, comparing and assessing drug-response phenotypes of cancer models.
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Bases de Datos Genéticas , Farmacogenética/normas , Pruebas de Farmacogenómica/métodos , Programas Informáticos , Genómica/métodos , HumanosRESUMEN
Self-assembling peptides form aggregates with various nanostructures such as spheres, sheets, and fibers and have potential applications in nanomedicine and drug delivery. The alkylation of peptides is a promising strategy for controlling the self-assembly of peptides. In this study, we investigated the thermodynamic properties associated with the aggregation of alkyl-chain-modified self-assembling peptides. The tripeptide sequence, KYF, which has been reported to form fibrous aggregates via self-assembly, was modified with various fatty acids at the N-terminus. The fibrous morphology of the aggregates was observed by transmission electron microscopy and atomic force microscopy. Thioflavin T fluorescence and circular dichroism spectroscopy revealed the formation of ß-sheet structures. The critical micelle concentration and its temperature dependence were determined to obtain the thermodynamic parameters for aggregation. The results showed that the aggregation was an entropy-driven process at low temperatures, whereas it was enthalpy-driven at high temperatures. The negative heat capacity changes for aggregation suggested that hydrophobic interactions were the major driving force for self-assembly. Other entropic and enthalpic interactions were also contributed in part to the self-assembly. We individually identified the contributions of the peptide and alkyl chain moiety to the self-assembly. These contributions can be explained by the theoretical values for the self-assembly of each component. The results of this study provide fundamental insights into the design of self-associating peptides.
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Micelas , Péptidos , Dicroismo Circular , Ácidos Grasos , Interacciones Hidrofóbicas e Hidrofílicas , Péptidos/química , TermodinámicaRESUMEN
Amphipathic α-helical peptides have been reported to form discoidal particles or nanodiscs with phospholipids, in which a lipid bilayer patch is encircled by peptides. Peptide-based nanodiscs have broad applicability because of their ease of preparation, size flexibility, and structural plasticity. We previously revealed that the nanodiscs formed by apolipoprotein-A-I-derived peptide 18A showed temperature-dependent structural destabilization above the gel-to-liquid-crystalline phase transition temperature of the lipid bilayer. It has been suggested that this destabilization is due to the migration of peptides bound to the edge of the discs to the bilayer surface. In this study, we designed a peptide that could stabilize nanodisc structures against the phase transition of lipid bilayers by disulfide cross-linking of peptides. An 18A-dimer cross-linked by a proline residue, 37pA (Ac-18A-P-18A-CONH2), also showed thermal destabilization of nanodiscs like 18A. However, cross-linking the sides of the two α-helices of the cysteine-substituted analogue 37pA-C2 with disulfide bonds led to the formation of nanodiscs that were more stable to temperature changes. This stabilizing effect was mainly due to the formation of a cyclic 37pA-C2 monomer by intramolecular disulfide cross-linking. These results suggest that the lateral association of two α-helices, which is the basis of the double-belt structure, is an important factor for the implementation of stable nanodiscs. The results of this study will help in development of more stable nanoparticles with membrane proteins in the future.
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Membrana Dobles de Lípidos , Fosfolípidos , Secuencia de Aminoácidos , Disulfuros , Péptidos/química , Fosfolípidos/química , Conformación Proteica en Hélice alfaRESUMEN
BACKGROUND: Bleeding complications during venovenous extracorporeal membrane oxygenation (V-V ECMO) can be critical. However, there is limited information on the associated risk factors. This study investigated the risk factors for bleeding complications during V-V ECMO as a bridge to recovery. METHODS: This single-center retrospective study enrolled 59 patients (bleeding and non-bleeding groups) who received V-V ECMO from 2012 to 2020, to evaluate whether peak activated partial thromboplastin time (APTT) value, lowest platelet count, and mobilization to sitting on the edge of the bed during V-V ECMO were risk factors for bleeding complications, defined according to the Extracorporeal Life Support Organization guidelines. Age, sex, body mass index, Sequential Organ Failure Assessment score, and ECMO duration before bleeding complications were covariates in the multivariate logistic regression analysis. RESULTS: Thirty-one (53%) participants experienced 36 bleeding complications; the ECMO cannulation site, gastrointestinal tract, and nasopharyngeal region were the most common bleeding sites. The use of transfusion products and length of ECMO and intensive care unit stay were significantly and medical costs were non-significantly increased in the bleeding group. Peak APTT (odds ratio [OR] 1.03, 95% confidence interval [CI] 1.01-1.05, p < 0.01) was significantly associated whereas the lowest platelet count (OR 0.96, 95% CI 0.82-1.13, p = 0.66) was unassociated with bleeding complications during ECMO. Achieving mobilization (OR 0.14, 95% CI 0.02-1.17, p = 0.07) decreased the trend of risk for bleeding complications. CONCLUSIONS: Peak APTT might be an independent modifiable factor for bleeding complications during V-V ECMO. The protective effect of mobilization during V-V ECMO requires further investigation.
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Oxigenación por Membrana Extracorpórea , Oxigenación por Membrana Extracorpórea/efectos adversos , Hemorragia/etiología , Hemorragia/terapia , Humanos , Estudios Retrospectivos , Factores de RiesgoRESUMEN
Lipid transbilayer movement (flip-flop) is regulated by membrane proteins that are involved in homeostasis and signaling in eukaryotic cells. In the plasma membrane, an asymmetric lipid composition is maintained by energy-dependent unidirectional transport. Energy-independent flip-flop promotion by phospholipid scramblases disrupts the asymmetry in several physiological processes, such as apoptosis and blood coagulation. In the endoplasmic reticulum, rapid flip-flop is essential for bilayer integrity because phospholipids are synthesized only in the cytoplasmic leaflet. Phospholipid scramblases are also involved in lipoprotein biogenesis, autophagosome formation, and viral infection. Although several scramblases have been identified and investigated, the precise flip-flop promotion mechanisms are not fully understood. Model transmembrane peptides are valuable tools for investigating the general effects of lipid-peptide interactions. We focus on the development of model transmembrane peptides with flip-flop promotion abilities and their mechanisms.
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Membrana Dobles de Lípidos , Fosfolípidos , Membrana Celular/metabolismo , Membrana Dobles de Lípidos/química , Proteínas de la Membrana/química , Péptidos/química , Péptidos/farmacología , Fosfolípidos/químicaRESUMEN
The design of nanoassemblies is an important part of the development of new materials for applications in nanomedicine and biosensors. In our previous study, cysteine substitution of the apolipoprotein A-I-derived peptide 18A at residue 11, 18A[A11C], bound to 1-palmitoyl-2-oleoylphosphatidylcholine to form fibrous aggregates at a lipid-to-peptide molar ratio of ≤2 and a fiber diameter of 10-20 nm. However, the mechanisms underlying the lipid-peptide interactions that enable nanofiber formation remain unclear. Here, we evaluated the phospholipid specificity, concentration dependence, and temperature dependence of the formation of 18A[A11C]-lipid nanofibers. Nanofibers were found to form in the presence of specific phospholipids and have a constant lipid/peptide stoichiometry of 1.2 ± 0.2. Moreover, an increase in the length of the acyl chain in phosphatidylcholines was found to increase the structural stability of the nanofibers. These results indicate that specific molecular interactions between peptides and both the headgroups and acyl chains of phospholipids are involved in nanofiber formation. Furthermore, the formation and disassembly of the nanofibers were reversibly controlled by changes in temperature and concentration. The results of the present study provide an insight into the creation of nanoassembling structures.
RESUMEN
High-density lipoprotein (HDL) is a naturally occurring composite of lipids and lipid-binding proteins. The cholate dialysis method, first reported by Jonas in 1969, is the most widely used approach for reconstituting discoidal HDL (dHDL) in test tubes with phospholipids and the most dominant protein, apolipoprotein A-1 (apoA-I). Here, we show that a dHDL-relevant complex can also be prepared by gently mixing 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and apoA-I or its mutants in ethanol/H2O solutions containing urea at a concentration of a few molar and then incubating the mixture at the gel-liquid crystalline phase transition temperature in test tubes. Subsequent purification steps involve quick dialysis following size exclusion chromatography. The yields (73 ± 3% and 70 ± 1% protein and DMPC, respectively) of the resulting HDL-like nanoparticles, designated as uHDL, were comparable to the values of 68 ± 9% and 71 ± 12% obtained in the cholate dialysis method. Using apoA-I and two mutants, the key factor in this method was found to be urea at the folded and unfolded transition midpoint concentration. By using this urea-assisted method in the presence of a hydrophobic drug, all-trans-retinoic acid (ATRA), one-step preparation of ATRA-loaded uHDL was also possible. The loading efficiency was comparable to that in the mixing of ATRA and uHDL or dHDL reconstituted by the cholate dialysis method. Atomic force microscopy analysis revealed that uHDL and ATRA-loaded uHDL were discoidal. Our urea-assisted method is an easy and efficient method for reconstituting dHDL and can be utilized to prepare various drug-dHDL complexes.
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Lipoproteínas HDL/análisis , Urea/química , Apolipoproteína A-I/química , Apolipoproteína A-I/genética , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Tretinoina/químicaRESUMEN
Sec14, the major yeast phosphatidylcholine (PC)/phosphatidylinositol (PI) transfer protein (PITP), coordinates PC and PI metabolism to facilitate an appropriate and essential lipid signaling environment for membrane trafficking from trans-Golgi membranes. The Sec14 PI/PC exchange cycle is essential for its essential biological activity, but fundamental aspects of how this PITP executes its lipid transfer cycle remain unknown. To address some of these outstanding issues, we applied time-resolved small-angle neutron scattering for the determination of protein-mediated intervesicular movement of deuterated and hydrogenated phospholipids in vitro. Quantitative analysis by small-angle neutron scattering revealed that Sec14 PI- and PC-exchange activities were sensitive to both the lipid composition and curvature of membranes. Moreover, we report that these two parameters regulate lipid exchange activity via distinct mechanisms. Increased membrane curvature promoted both membrane binding and lipid exchange properties of Sec14, indicating that this PITP preferentially acts on the membrane site with a convexly curved face. This biophysical property likely constitutes part of a mechanism by which spatial specificity of Sec14 function is determined in cells. Finally, wild-type Sec14, but not a mixture of Sec14 proteins specifically deficient in either PC- or PI-binding activity, was able to effect a net transfer of PI or PC down opposing concentration gradients in vitro.
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Fosfatidilcolinas/metabolismo , Fosfatidilinositoles/metabolismo , Proteínas de Transferencia de Fosfolípidos/química , Proteínas de Saccharomyces cerevisiae/química , Difracción de Neutrones , Fosfatidilcolinas/química , Fosfatidilinositoles/química , Proteínas de Transferencia de Fosfolípidos/metabolismo , Unión Proteica , Proteínas de Saccharomyces cerevisiae/metabolismo , Dispersión del Ángulo Pequeño , Liposomas Unilamelares/química , Liposomas Unilamelares/metabolismoRESUMEN
The bilayer structure of biomembranes consists of thousands of lipids, the composition of which is different for each organelle. Since most lipids are synthesized in the endoplasmic reticulum, subsequent distribution to each organelle determines the composition and function of the biomembranes. Thus, interbilayer transfer and transbilayer movement (flip-flop) of phospholipids play important roles in maintaining homeostasis. A crucial task in biophysics and cell biology is to understand how rapidly lipids migrate between bilayers spontaneously or through proteins and to control these lipid dynamics. Time-resolved small-angle neutron scattering (TR-SANS) is a powerful technique to determine the intervesicular exchange and flip-flop rates of lipids in situ and real time. In this review, I explain how TR-SANS detects the interbilayer and transbilayer transfer of phospholipids and introduce recent progress of my group on the evaluation of spontaneous and protein- (or peptide-)mediated lipid transfer in several phospholipid dispersion systems.
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Membrana Dobles de Lípidos/química , Fosfolípidos/química , Dispersión del Ángulo Pequeño , Nanoestructuras/química , Difracción de NeutronesRESUMEN
Cubosomes are lipidic nanoparticles containing bicontinuous cubic structures. Their unique architecture and potential as drug delivery vehicles have attracted researchers' attention. However, cubosome systems that are more robust in the presence of plasma components are being sought after for applications in intravenous administration. In this study, we prepared cubosomes consisting of 1,2-dioleoyl-sn-glycero-3-hexylphosphocholine (hexyl-DOPC) and compared their interaction with bovine serum albumin (BSA), the most abundant protein in plasma, with that of conventional cubosome systems consisting of several bicontinuous cubic phase-forming lipids, including 1-monoolein (MO), 1-O-(5,9,13,17-tetramethyloctadecanoyl)erythritol (EROCO C22), or 1-O-(5,9,13,17-tetramethyloctadecyl)-ß-D-xylopyranoside (ß-XP). The average number of lipids bound to each BSA molecule was between 1.2-4.0 for MO, EROCO C22, and ß-XP. On the other hand, hexyl-DOPC exhibited negligible binding to BSA. This result suggests that hexyl-DOPC, which was shown to resist removal from particles by BSA, can be used as a new lipid component of cubosomes, and has higher plasma stability than the other cubic phase-forming lipids.
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Nanopartículas/química , Fosfatidilcolinas/química , Albúmina Sérica Bovina/química , Animales , Bovinos , Estructura Molecular , Fosfatidilcolinas/aislamiento & purificaciónRESUMEN
Most biomembranes have an asymmetric structure with regard to phospholipid distribution between the inner and outer leaflets of the lipid bilayers. Control of the asymmetric distribution plays a pivotal role in several cellular functions such as intracellular membrane fusion and cell division. The mechanism by which membrane asymmetry and its alteration function in these transformation processes is not yet clear. To understand the significance of membrane asymmetry on trafficking and metabolism of intracellular vesicular components, a system that experimentally reproduces the asymmetric nature of biomembranes is essential. Here, we succeeded in obtaining asymmetric vesicles by means of transphosphatidylation reactions with phospholipase D (PLD), which acts exclusively on phosphatidylcholine (PC) present in the outer leaflet of vesicles. By treating PC vesicles with PLD in the presence of 1.7M serine and 0.3M ethanolamine, we obtained asymmetric vesicles that are topologically similar to intracellular vesicles containing phosphatidylserine and phosphatidylethanolamine in the cytosolic leaflet. PLD and other unwanted compounds could be removed by trypsin digestion followed by dialysis. Our established technique has a great advantage over conventional methods in that asymmetric vesicles can be provided at high yield and high efficiency, which is requisite for most physicochemical assays.
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Membrana Celular/metabolismo , Vesículas Citoplasmáticas/metabolismo , Membrana Dobles de Lípidos/metabolismo , Fosfolipasa D/metabolismo , Fosfolípidos/metabolismo , Membrana Celular/química , Vesículas Citoplasmáticas/química , Membrana Dobles de Lípidos/química , Fusión de Membrana , Modelos Químicos , Estructura Molecular , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Fosfolípidos/química , Espectrometría de FluorescenciaRESUMEN
In mammals, four ATP-binding cassette (ABC) proteins belonging to subfamily D have been identified. ABCD1â3 are located on peroxisomal membrane and play an important role in the transportation of various fatty acid-CoA derivatives, including very long chain fatty acid-CoA, into peroxisomes. ABCD4 is located on lysosomal membrane and is suggested to be involved in the transport of vitamin B12 from lysosomes to the cytosol. However, the precise transport mechanism by which these ABC transporters facilitate the import or export of substrate has yet to be well elucidated. In this study, the overexpression of human ABCD1â4 in the methylotrophic yeast Pichia pastoris and a purification procedure were developed. The detergent-solubilized proteins were reconstituted into liposomes. ABCD1â4 displayed stable ATPase activity, which was inhibited by AlF3. Furthermore, ABCD1â4 were found to possess an equal levels of acyl-CoA thioesterase activity. Proteoliposomes is expected to be an aid in the further biochemical characterization of ABCD transporters.
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Subfamilia D de Transportadores de Casetes de Unión al ATP/química , Liposomas/química , Proteolípidos/química , Sitios de Unión , Activación Enzimática , Estabilidad de Enzimas , Cinética , Unión ProteicaRESUMEN
BACKGROUND: In trauma resuscitation with resuscitative endovascular balloon occlusion of the aorta (REBOA), urgent and accurate placement of the catheter in the resuscitation area without fluoroscopy can shorten the time from admission to REBOA, allowing rapid, temporary control of bleeding. DISCUSSION: The experience-based protocol in our center for ultrasound-guided REBOA in the resuscitation area without fluoroscopy is as follows: the femoral artery is punctured and a guidewire inserted; sonography is used to verify that the guidewire is in the abdominal aorta; the position of the balloon is confirmed with ultrasound after estimating the distance to the clavicle, and the pressure in the radial artery and sheath is used to monitor correct positioning; connect the pressure transducer to the catheter sheath for continuous monitoring of the blood pressure in the sheath, and inflate the balloon until the blood pressure tracing at the sheath has disappeared; check the pulse in the left radial artery, and withdraw the catheter slightly if the pulse in the radial artery is not palpable or is decreased (if this pulse is not palpable or decreased, the balloon is in the aortic arch). In this retrospective review of our REBOA protocol, between April 2012 and March 2016, 34 patients were enrolled. Two patients had complications, including dissection of the femoral artery in one and difficult percutaneous vascular access in another. Median time needed to complete the procedure was 8 min. Overall, 24 of 34 patients survived more than 24 h (72%), and overall mortality was 47%. Patients who lived more than 24 h, and then died had severe traumatic brain injury or septic shock. CONCLUSIONS: Ultrasound-guided REBOA is presented. Monitoring the blood pressure in the left radial artery allows us to determine adequate positioning of the balloon, and the blood pressure in the catheter sheath located in the femoral artery should also be monitored to prevent aortic injuries caused by the overinflation of the balloon.
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Oclusión con Balón/métodos , Procedimientos Endovasculares/métodos , Resucitación/métodos , Ultrasonografía/métodos , Anciano , Anciano de 80 o más Años , Aorta/diagnóstico por imagen , Aorta/fisiopatología , Enfermedades de la Aorta/terapia , Oclusión con Balón/normas , Procedimientos Endovasculares/normas , Femenino , Fluoroscopía/efectos adversos , Hemorragia/terapia , Humanos , Masculino , Persona de Mediana Edad , Resucitación/normas , Estudios Retrospectivos , Factores de TiempoRESUMEN
The mechanism whereby phospholipids rapidly flip-flop in the endoplasmic reticulum (ER) membrane remains unknown. We previously demonstrated that the presence of a hydrophilic residue in the center of the model transmembrane peptide sequence effectively promoted phospholipid flip-flop and that hydrophilic residues composed 4.5% of the central regions of the membrane-spanning sequences of human ER membrane proteins predicted by SOSUI software. We hypothesized that ER proteins with hydrophilic residues might play a critical role in promoting flip-flop. Here, we evaluated the flip rate of fluorescently labeled lipids in vesicles containing each of the 11 synthetic peptides of membrane-spanning sequences, using a dithionite-quenching assay. Although the flippase activities of nine peptides were unexpectedly low, the peptides based on the EDEM1 and SPAST proteins showed enhanced flippase activity with three different fluorescently labeled lipids. The substitution of hydrophobic Ala with His or Arg in the central region of the EDEM1 or SPAST peptides, respectively, attenuated their ability to flip phospholipids. Interestingly, substituting Ala with Arg or His at a location outside of the central region of EDEM1 or SPAST, respectively, also affected the enhancement of flip-flop. These results indicated that both Arg and His are important for the ability of these two peptides to increase the flip rates. The EDEM1 peptide exhibited high activity at significantly low peptide concentrations, suggesting that the same side positioning of Arg and His in α-helix structure is critical for the flip-flop promotion and that the EDEM1 protein is a candidate flippase in the ER.
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Adenosina Trifosfatasas/química , Retículo Endoplásmico/química , Péptidos y Proteínas de Señalización Intracelular/química , Proteínas de la Membrana/química , Péptidos/química , Fosfolípidos/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Colesterol/metabolismo , Dicroismo Circular , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Péptidos y Proteínas de Señalización Intracelular/genética , Cinética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Mutación , Péptidos/síntesis química , Péptidos/genética , Péptidos/metabolismo , Fosfolípidos/metabolismo , Estructura Secundaria de Proteína , Programas Informáticos , Espastina , Liposomas Unilamelares/química , Liposomas Unilamelares/metabolismoRESUMEN
Chylomicron remnants, which carry dietary fats and cholesterol, play a role in promoting atherosclerosis. Chylomicron remnants are characterized by high cholesterol content at the surface, different from low-density lipoproteins (LDLs) containing high amounts of esterified cholesterol (CE) in the core. We prepared cholesterol-rich emulsions (TO-PC/cholesterol emulsions) as models for chylomicron remnants and compared their effects on J774 macrophages with acetylated-LDL (ac-LDL). Internalization of TO-PC/cholesterol emulsions into macrophages reduced cell viability, whereas ac-LDL did not. Surprisingly, there was no difference in intracellular free cholesterol content between cells incubated with TO-PC/cholesterol emulsions and with ac-LDL. Furthermore, cholesterol in TO-PC/cholesterol emulsions and ac-LDL both were internalized into J774 macrophages; however, incubation with TO-PC/cholesterol emulsions induced leakage of lysosomal protease, cathepsin-L, to cytosol, which was not observed for incubation with ac-LDL. Inhibition of the activity of cathepsin-L recovered the viability of macrophages that ingested TO-PC/cholesterol emulsions. We suggest an alternative fate of cholesterol-rich emulsions taken up by macrophages, which is different from other atherogenic lipoproteins rich in CE; internalization of TO-PC/cholesterol emulsions into macrophages induces rapid free cholesterol accumulation in lysosomes and cell death due to lysosomal destabilization.
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Colesterol/metabolismo , Remanentes de Quilomicrones/metabolismo , Lisosomas/metabolismo , Macrófagos/metabolismo , Animales , Transporte Biológico , Muerte Celular , Línea Celular , Emulsiones , Lipoproteínas LDL/metabolismo , Macrófagos/patología , Ratones , Fosfatidilcolinas/metabolismo , Trioleína/metabolismoRESUMEN
KcsA is a pH-dependent potassium channel that is activated at acidic pH. The channel undergoes global conformational changes upon activation. We hypothesized that the open-close conformational changes of the transmem brane region could promote the flip-flop of phosphoiipids. Based on this hypothesis, we measured the flip-flop ofNBD-labeled phospholipids in KcsA-incorporated proteoliposomes. Both flip and flop rates of ~NBD-PC were significantly enhanced in the presence of KcsA and were several times higher at pH 4.0 than at pH 7.4, suggesting that KcsA promotes the phospholipid flip in a conformation-dependent manner. Phospholipids were nonselectively flipped with respect to the glycerophospholipid structure. In the active state of KcsA channel,tetrabutylammonium locks the channel in the open conformation at acidic pH; however, it did not alter the fliprate of CGNBD-PC. Thus, the open-close transition of the transmembrane region did not affect the flip-flop of phospholipids. In addition, the KcsA mutant that lacked anN-terminal amphipathic helix (MO-helix) was found to show reduced ability to fl ip C6NBD-phospholipids at acidic pH. The closed conformation is stabilized in the absence of MO-heli x, and thus the attenuated flip could be explained by the reduced prevalence of the open conformation.These results suggest that the open conformation of KcsA can disturb the bilayer integrity and facilitate the flip-flop of phospholipids.
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Proteínas Bacterianas/química , Membrana Dobles de Lípidos/química , Fosfolípidos/química , Canales de Potasio/química , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Colesterol/química , Colesterol/metabolismo , Concentración de Iones de Hidrógeno , Membrana Dobles de Lípidos/metabolismo , Modelos Moleculares , Mutación , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfolípidos/metabolismo , Canales de Potasio/genética , Canales de Potasio/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Compuestos de Amonio Cuaternario/químicaRESUMEN
Here, we report an experimental approach for determining the change in the free energy and the enthalpy that accompanies the mixing of the anionic phosphatidylglycerol and the zwitterionic phosphatidylcholine. The enthalpy change originates in the thermal changes of disrupting lipid bilayer vesicles titrated into a surfactant micelle solution and is monitored using isothermal titration calorimetry. The difference in the solubilization enthalpies between pure and mixed lipid vesicles yields the lipid mixing enthalpy. The Gibbs free energy changes are estimated by determining the thermodynamic equilibrium constants of forming a molecular complex between phospholipids and methyl-ß-cyclodextrin. We provide direct experimental evidence that mixing of the anionic lipid and the zwitterionic lipid is explained well by the entropic term of the electrostatic free energy of a charged surface in the Gouy-Chapman model. The present strategy enables us to determine the precise energetics of lipid-lipid interactions in near-native environments such as liposomes without any chemical modification to lipid molecules.
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Membrana Dobles de Lípidos/química , Fosfolípidos/química , Fosfatidilcolinas , Fosfatidilgliceroles , Termodinámica , beta-Ciclodextrinas/químicaRESUMEN
Methyl-ß-cyclodextrin (MßCD) can transfer phospholipids between vesicles, and its transfer ability has been utilized for the preparation of asymmetric vesicle and lipid incorporation into culture cells. Nevertheless, a detailed kinetic analysis of the MßCD-mediated phospholipid transfer has not yet been carried out. We performed real-time monitoring of intervesicular lipid transfer by means of the fluorescence of pyrene-labeled phospholipids. Intermolecular excimer formation of the pyrene-labeled lipids in a membrane strongly depends on the local concentration of the fluorophore and decreases when the pyrene-labeled lipids are transferred from donor (fluorophore-containing) vesicles to acceptor (fluorophore-free) vesicles. We monitored the fluorescence intensity of the pyrene monomer and excimer simultaneously and found that the excimer/monomer ratio decreased in the presence of MßCD, pointing to MßCD-mediated lipid transfer. The transfer rate depended on the MßCD concentration but not on the lipid concentration, suggesting that dissociation from the membrane via extraction by MßCD is the rate-limiting step of the lipid transfer. Calibration of the excimer/monomer ratio to the molar fraction of the pyrene-labeled lipids enabled us to evaluate the dissociation rate constant correctly. From the temperature dependence of the transfer, we obtained the thermodynamic activation parameters, which revealed that the extraction of phosphatidylcholine by MßCD from membranes is less enthalpically unfavorable than that of phosphatidylglycerol and phosphatidylinositol.
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Fosfolípidos/química , Pirenos , beta-Ciclodextrinas/química , Cinética , Espectrometría de FluorescenciaRESUMEN
We describe a self-reproduction mechanism of nanometer-sized particles (i.e., nanodiscs) through chemical ligation of the precursors and self-assembly of the building blocks. The ligation reaction was accelerated on lipid bilayer surfaces, and the products spontaneously assembled into nanodiscs with lipid molecules. With the increase in the number of nanodiscs, a rapid proliferation of the nanodiscs occurred through the spatial rearrangements of the molecules between the pre-existing nanodiscs and the unreacted materials, rather than template- or complex-enhanced ligation of the precursors. The subsequent process of surface-enhanced ligation of integrated precursors matured the nanoparticles into identical copies of the pre-existing assembly. Our study showed that the synergistic self-assembly mechanism probably underlie the self-replication principles for heterogeneous multimolecular systems.