RESUMEN
The complement system is a critical part of our innate immune response, and the terminal products of this cascade, anaphylatoxins C3a and C5a, exert their physiological and pathophysiological responses primarily via two GPCRs, C3aR and C5aR1. However, the molecular mechanism of ligand recognition, activation, and signaling bias of these receptors remains mostly elusive. Here, we present nine cryo-EM structures of C3aR and C5aR1 activated by their natural and synthetic agonists, which reveal distinct binding pocket topologies of complement anaphylatoxins and provide key insights into receptor activation and transducer coupling. We also uncover the structural basis of a naturally occurring mechanism to dampen the inflammatory response of C5a via proteolytic cleavage of the terminal arginine and the G-protein signaling bias elicited by a peptide agonist of C3aR identified here. In summary, our study elucidates the innerworkings of the complement anaphylatoxin receptors and should facilitate structure-guided drug discovery to target these receptors in a spectrum of disorders.
Asunto(s)
Anafilatoxinas , Receptores de Complemento , Transducción de Señal , Anafilatoxinas/metabolismo , Complemento C3a/metabolismo , Inmunidad Innata , Receptores de Complemento/metabolismo , Humanos , Animales , RatonesRESUMEN
The repeating structural unit of metazoan chromatin is the chromatosome, a nucleosome bound to a linker histone, H1. There are 11 human H1 isoforms with diverse cellular functions, but how they interact with the nucleosome remains elusive. Here, we determined the cryoelectron microscopy (cryo-EM) structures of chromatosomes containing 197 bp DNA and three different human H1 isoforms, respectively. The globular domains of all three H1 isoforms bound to the nucleosome dyad. However, the flanking/linker DNAs displayed substantial distinct dynamic conformations. Nuclear magnetic resonance (NMR) and H1 tail-swapping cryo-EM experiments revealed that the C-terminal tails of the H1 isoforms mainly controlled the flanking DNA orientations. We also observed partial ordering of the core histone H2A C-terminal and H3 N-terminal tails in the chromatosomes. Our results provide insights into the structures and dynamics of the chromatosomes and have implications for the structure and function of chromatin.
Asunto(s)
ADN/química , Histonas/química , Nucleosomas/química , Microscopía por Crioelectrón , ADN/ultraestructura , Humanos , Nucleosomas/ultraestructura , Isoformas de Proteínas/químicaRESUMEN
Mammalian small extracellular vesicles (sEVs) can deliver diverse molecules to target cells. However, they are difficult to obtain in large quantities and can activate host immune responses. Plant-derived vesicles may help to overcome these challenges. We optimized isolation methods for two types of plant vesicles, nanovesicles from disrupted leaf and sEVs from the extracellular apoplastic space of Arabidopsis thaliana. Both preparations yielded intact vesicles of uniform size, and a mean membrane charge of approximately -25â¯mV. We also demonstrated applicability of these preparative methods using Brassicaceae vegetables. Proteomic analysis of a subset of vesicles with a density of 1.1-1.19â¯gâ¯mL-1 sheds light on the likely cellular origin and complexity of the vesicles. Both leaf nanovesicles and sEVs were taken up by cancer cells, with sEVs showing an approximately three-fold higher efficiency compared to leaf nanovesicles. These results support the potential of plant-derived vesicles as vehicles for therapeutic delivery.
Asunto(s)
Arabidopsis/química , Sistemas de Liberación de Medicamentos , Vesículas Extracelulares/química , Hojas de la Planta/química , Arabidopsis/genética , Vesículas Extracelulares/genética , Humanos , Hojas de la Planta/genética , Proteómica/métodosRESUMEN
Cyanobacteria are photosynthetic organisms responsible for â¼25% of organic carbon fixation on the Earth. These bacteria began to convert solar energy and carbon dioxide into bioenergy and oxygen more than two billion years ago. Cyanophages, which infect these bacteria, have an important role in regulating the marine ecosystem by controlling cyanobacteria community organization and mediating lateral gene transfer. Here we visualize the maturation process of cyanophage Syn5 inside its host cell, Synechococcus, using Zernike phase contrast electron cryo-tomography (cryoET). This imaging modality yields dramatic enhancement of image contrast over conventional cryoET and thus facilitates the direct identification of subcellular components, including thylakoid membranes, carboxysomes and polyribosomes, as well as phages, inside the congested cytosol of the infected cell. By correlating the structural features and relative abundance of viral progeny within cells at different stages of infection, we identify distinct Syn5 assembly intermediates. Our results indicate that the procapsid releases scaffolding proteins and expands its volume at an early stage of genome packaging. Later in the assembly process, we detected full particles with a tail either with or without an additional horn. The morphogenetic pathway we describe here is highly conserved and was probably established long before that of double-stranded DNA viruses infecting more complex organisms.
Asunto(s)
Bacteriófagos/crecimiento & desarrollo , Bacteriófagos/ultraestructura , Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/métodos , Synechococcus/ultraestructura , Synechococcus/virología , Ensamble de Virus , Organismos Acuáticos/citología , Organismos Acuáticos/ultraestructura , Organismos Acuáticos/virología , Modelos Biológicos , Synechococcus/citologíaRESUMEN
Trypanosoma brucei is a parasitic protozoan that causes African sleeping sickness. It contains a flagellum required for locomotion and viability. In addition to a microtubular axoneme, the flagellum contains a crystalline paraflagellar rod (PFR) and connecting proteins. We show here, by cryoelectron tomography, the structure of the flagellum in three bending states. The PFR lattice in straight flagella repeats every 56 nm along the length of the axoneme, matching the spacing of the connecting proteins. During flagellar bending, the PFR crystallographic unit cell lengths remain constant while the interaxial angles vary, similar to a jackscrew. The axoneme drives the expansion and compression of the PFR lattice. We propose that the PFR modifies the in-plane axoneme motion to produce the characteristic trypanosome bihelical motility as captured by high-speed light microscope videography.
Asunto(s)
Flagelos/química , Flagelos/fisiología , Trypanosoma brucei brucei/química , Trypanosoma brucei brucei/fisiología , Animales , Fenómenos Biofísicos , Microscopía por Crioelectrón , Flagelos/ultraestructura , Humanos , Modelos Biológicos , Modelos Moleculares , Movimiento/fisiología , Conformación Proteica , Proteínas Protozoarias/química , Proteínas Protozoarias/fisiología , Proteínas Protozoarias/ultraestructura , Trypanosoma brucei brucei/ultraestructuraRESUMEN
DNA double-strand breaks (DSBs) present a critical threat to genomic integrity, often precipitating genomic instability and oncogenesis. Repair of DSBs predominantly occurs through homologous recombination (HR) and non-homologous end joining (NHEJ). In HR-deficient cells, DNA polymerase theta (Polθ) becomes critical for DSB repair via microhomology-mediated end joining (MMEJ), also termed theta-mediated end joining (TMEJ). Thus, Polθ is synthetically lethal with BRCA1/2 and other HR factors, underscoring its potential as a therapeutic target in HR-deficient cancers. However, the molecular mechanisms governing Polθ-mediated MMEJ remain poorly understood. Here we present a series of cryo-electron microscopy structures of the Polθ helicase domain (Polθ-hel) in complex with DNA containing 3'-overhang. The structures reveal the sequential conformations adopted by Polθ-hel during the critical phases of DNA binding, microhomology searching, and microhomology annealing. The stepwise conformational changes within the Polθ-hel subdomains and its functional dimeric state are pivotal for aligning the 3'-overhangs, facilitating the microhomology search and subsequent annealing necessary for DSB repair via MMEJ. Our findings illustrate the essential molecular switches within Polθ-hel that orchestrate the MMEJ process in DSB repair, laying the groundwork for the development of targeted therapies against the Polθ-hel.
RESUMEN
Plant-derived vesicles (PDVs) are attractive for therapeutic applications, including as potential nanocarriers. However, a concern with oral delivery of PDVs is whether they would remain intact in the gastrointestinal tract. We found that 82% of cabbage PDVs were destroyed under conditions mimicking the upper digestive tract. To overcome this limitation, we developed a delivery method whereby lyophilized Eudragit S100-coated cabbage PDVs were packaged into a capsule (Cap-cPDVs). Lyophilization and suspension of PDVs did not have an appreciable impact on PDV structure, number, or therapeutic effect. Additionally, packaging the lyophilized Eudragit S100-coated PDVs into capsules allowed them to pass through the upper gastrointestinal tract for delivery into the colon better than did suspension of PDVs in phosphate-buffered saline. Cap-cPDVs showed robust therapeutic effect in a dextran sulfate sodium-induced colitis mouse model. These findings could have broad implications for the use of PDVs as orally delivered nanocarriers of natural therapeutic plant compounds or other therapeutics.
Asunto(s)
Colitis , Ratones , Animales , Concentración de Iones de Hidrógeno , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Ácidos Polimetacrílicos/química , Administración Oral , Sistemas de Liberación de MedicamentosRESUMEN
Vaccines based on recombinant proteins avoid the toxicity and antivector immunity associated with live vaccine (for example, viral) vectors, but their immunogenicity is poor, particularly for CD8(+) T-cell responses. Synthetic particles carrying antigens and adjuvant molecules have been developed to enhance subunit vaccines, but in general these materials have failed to elicit CD8(+) T-cell responses comparable to those for live vectors in preclinical animal models. Here, we describe interbilayer-crosslinked multilamellar vesicles formed by crosslinking headgroups of adjacent lipid bilayers within multilamellar vesicles. Interbilayer-crosslinked vesicles stably entrapped protein antigens in the vesicle core and lipid-based immunostimulatory molecules in the vesicle walls under extracellular conditions, but exhibited rapid release in the presence of endolysosomal lipases. We found that these antigen/adjuvant-carrying vesicles form an extremely potent whole-protein vaccine, eliciting endogenous T-cell and antibody responses comparable to those for the strongest vaccine vectors. These materials should enable a range of subunit vaccines and provide new possibilities for therapeutic protein delivery.
Asunto(s)
Inmunidad Celular , Inmunidad Humoral , Liposomas/química , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología , Adyuvantes Inmunológicos/química , Animales , Portadores de Fármacos , Diseño de Fármacos , Memoria Inmunológica , Membrana Dobles de Lípidos/química , Liposomas/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ovalbúmina/inmunología , Vacunas Sintéticas/química , Vacunas Virales/química , Vacunas Virales/inmunologíaRESUMEN
Polymeric micelles formed by the self-assembly of amphiphilic block copolymers can be used to encapsulate hydrophobic drugs for tumor-delivery applications. Filamentous carriers with high aspect ratios offer potential advantages over spherical carriers, including prolonged circulation times. In this work, mixed micelles composed of poly(ethylene oxide)-poly[(R)-3-hydroxybutyrate]-poly(ethylene oxide) (PEO-PHB-PEO) and Pluronic F-127 (PF-127) were used to encapsulate a near-infrared fluorophore. The micelle formulations were assessed for tumor accumulation after tail vein injection to xenograft tumor-bearing mice by noninvasive optical imaging. The mixed micelle formulation that facilitated the highest tumor accumulation was shown by cryo-electron microscopy to be filamentous in structure compared to spherical structures of pure PF-127 micelles. In addition, increased dye loading efficiency and dye stability were attained in this mixed micelle formulation compared to pure PEO-PHB-PEO micelles. Therefore, the optimized PEO-PHB-PEO/PF-127 mixed micelle formulation offers advantages for cancer delivery over micelles formed from the individual copolymer components.
Asunto(s)
Medios de Contraste/administración & dosificación , Portadores de Fármacos/administración & dosificación , Verde de Indocianina/administración & dosificación , Melanoma Experimental/diagnóstico , Poloxámero/química , Polietilenglicoles/química , Animales , Fenómenos Químicos , Medios de Contraste/análisis , Medios de Contraste/farmacocinética , Portadores de Fármacos/análisis , Portadores de Fármacos/farmacocinética , Composición de Medicamentos , Estabilidad de Medicamentos , Humanos , Verde de Indocianina/análisis , Verde de Indocianina/farmacocinética , Inyecciones Intravenosas , Masculino , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Ratones Endogámicos BALB C , Micelas , Prohibitinas , Organismos Libres de Patógenos Específicos , Espectroscopía Infrarroja Corta , Distribución Tisular , Imagen de Cuerpo EnteroRESUMEN
Alzheimer's disease is a neurodegenerative disorder characterized by the accumulation of amyloid plaques in the brain. This amyloid primarily contains amyloid-beta (Abeta), a 39- to 43-aa peptide derived from the proteolytic cleavage of the endogenous amyloid precursor protein. The 42-residue-length Abeta peptide (Abeta(1-42)), the most abundant Abeta peptide found in plaques, has a much greater propensity to self-aggregate into fibrils than the other peptides and is believed to be more pathogenic. Synthetic human Abeta(1-42) peptides self-aggregate into stable but poorly-ordered helical filaments. We determined their structure to approximately 10-A resolution by using cryoEM and the iterative real-space reconstruction method. This structure reveals 2 protofilaments winding around a hollow core. Previous hairpin-like NMR models for Abeta(17-42) fit well in the cryoEM density map and reveal that the juxtaposed protofilaments are joined via the N terminus of the peptide from 1 protofilament connecting to the loop region of the peptide in the opposite protofilament. This model of mature Abeta(1-42) fibrils is markedly different from previous cryoEM models of Abeta(1-40) fibrils. In our model, the C terminus of Abeta forms the inside wall of the hollow core, which is supported by partial proteolysis analysis.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/ultraestructura , Amiloide/metabolismo , Amiloide/ultraestructura , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/ultraestructura , Péptidos/metabolismo , Secuencia de Aminoácidos , Péptidos beta-Amiloides/química , Microscopía por Crioelectrón , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Procesamiento Proteico-Postraduccional , Electricidad EstáticaRESUMEN
Understanding maturation pathways of broadly neutralizing antibodies (bnAbs) against HIV-1 can be highly informative for HIV-1 vaccine development. A lineage of J038 bnAbs is now obtained from a long-term SHIV-infected macaque. J038 neutralizes 54% of global circulating HIV-1 strains. Its binding induces a unique "up" conformation for one of the V2 loops in the trimeric envelope glycoprotein and is heavily dependent on glycan, which provides nearly half of the binding surface. Their unmutated common ancestor neutralizes the autologous virus. Continuous maturation enhances neutralization potency and breadth of J038 lineage antibodies via expanding antibody-Env contact areas surrounding the core region contacted by germline-encoded residues. Developmental details and recognition features of J038 lineage antibodies revealed here provide a new pathway for elicitation and maturation of V2-targeting bnAbs.
Asunto(s)
Infecciones por VIH , Seropositividad para VIH , VIH-1 , Anticuerpos Neutralizantes , Anticuerpos ampliamente neutralizantes , Epítopos , Anticuerpos Anti-VIH , Humanos , Productos del Gen env del Virus de la Inmunodeficiencia HumanaRESUMEN
Bottom-up fabrication of self-assembled nanomaterials requires control over forces and interactions between building blocks. We report here on the formation and architecture of supramolecular structures constructed from two different peptide amphiphiles. Inclusion of four alanines between a 16-mer peptide and a 16 carbon long aliphatic tail resulted in a secondary structure shift of the peptide headgroups from α helices to ß sheets. A concomitant shift in self-assembled morphology from nanoribbons to core-shell worm-like micelles was observed by cryogenic transmission electron microscopy (cryo-TEM) and atomic force microscopy (AFM). In the presence of divalent magnesium ions, these a priori formed supramolecular structures interacted in distinct manners, highlighting the importance of peptide amphiphile design in self-assembly.
Asunto(s)
Interacciones Hidrofóbicas e Hidrofílicas , Péptidos/química , Péptidos/metabolismo , Secuencia de Aminoácidos , Magnesio/farmacología , Imagen Molecular , Datos de Secuencia Molecular , Unión Proteica/efectos de los fármacos , Pliegue de Proteína/efectos de los fármacos , Estructura Secundaria de Proteína , SolucionesRESUMEN
Synaptotagmin acts as a Ca(2+) sensor in neurotransmitter release through its two C(2) domains. Ca(2+)-dependent phospholipid binding is key for synaptotagmin function, but it is unclear how this activity cooperates with the SNARE complex involved in release or why Ca(2+) binding to the C(2)B domain is more crucial for release than Ca(2+) binding to the C(2)A domain. Here we show that Ca(2+) induces high-affinity simultaneous binding of synaptotagmin to two membranes, bringing them into close proximity. The synaptotagmin C(2)B domain is sufficient for this ability, which arises from the abundance of basic residues around its surface. We propose a model wherein synaptotagmin cooperates with the SNAREs in bringing the synaptic vesicle and plasma membranes together and accelerates membrane fusion through the highly positive electrostatic potential of its C(2)B domain.
Asunto(s)
Calcio/farmacología , Fusión de Membrana/efectos de los fármacos , Fosfolípidos/metabolismo , Sinaptotagminas/metabolismo , Animales , Sitios de Unión , Calcio/metabolismo , Liposomas/química , Liposomas/metabolismo , Modelos Biológicos , Modelos Moleculares , Docilidad , Unión Proteica/efectos de los fármacos , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ratas , Espectrometría de Fluorescencia , Vesículas Sinápticas/efectos de los fármacos , Vesículas Sinápticas/metabolismo , Sinaptotagminas/químicaRESUMEN
A critical issue for current liposomal carriers in clinical applications is their leakage of the encapsulated drugs that are cytotoxic to non-target tissues. We have developed partially polymerized liposomes composed of polydiacetylene lipids and saturated lipids. Cross-linking of the diacetylene lipids prevents the drug leakage even at 40 °C for days. These inactivated drug carriers are non-cytotoxic. Significantly, more than 70% of the encapsulated drug can be instantaneously released by a laser that matches the plasmon resonance of the tethered gold nanoparticles on the liposomes, and the therapeutic effect was observed in cancer cells. The remote activation feature of this novel drug delivery system allows for precise temporal and spatial control of drug release.
Asunto(s)
Preparaciones de Acción Retardada/química , Liposomas/química , Nanopartículas del Metal/química , 1,2-Dipalmitoilfosfatidilcolina/química , Compuestos de Anilina , Disponibilidad Biológica , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Microscopía por Crioelectrón , Preparaciones de Acción Retardada/síntesis química , Preparaciones de Acción Retardada/efectos de la radiación , Diinos/química , Doxorrubicina/administración & dosificación , Doxorrubicina/farmacología , Estabilidad de Medicamentos , Endocitosis , Femenino , Fluoresceínas/administración & dosificación , Fluoresceínas/farmacocinética , Glicina , Oro/química , Humanos , Iminoácidos/administración & dosificación , Iminoácidos/farmacocinética , Rayos Láser , Liposomas/síntesis química , Liposomas/efectos de la radiación , Lisofosfolípidos/química , Nanopartículas del Metal/efectos de la radiación , Compuestos de Organotecnecio/administración & dosificación , Compuestos de Organotecnecio/farmacocinética , Tamaño de la Partícula , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Polímeros/síntesis química , Polímeros/química , Resonancia por Plasmón de SuperficieRESUMEN
Phenoloxidases (POs) occur in all organisms and are involved in skin and hair coloring in mammals, and initiating melanization in wound healing. Mutation or overexpression of PO can cause albinism or melanoma, respectively. SDS can convert inactive PO and the oxygen carrier hemocyanin (Hc) into enzymatically active PO. Here we present single-particle cryo-EM maps at subnanometer resolution and pseudoatomic models of the 24-oligomeric Hc from scorpion Pandinus imperator in resting and SDS-activated states. Our structural analyses led to a plausible mechanism of Hc enzyme PO activation: upon SDS activation, the intrinsically flexible Hc domain I twists away from domains II and III in each subunit, exposing the entrance to the active site; this movement is stabilized by enhanced interhexamer and interdodecamer interactions, particularly in the central linker subunits. This mechanism could be applicable to other type 3 copper proteins, as the active site is highly conserved.
Asunto(s)
Hemocianinas/química , Monofenol Monooxigenasa/química , Monofenol Monooxigenasa/metabolismo , Escorpiones/metabolismo , Dodecil Sulfato de Sodio/farmacología , Tensoactivos/farmacología , Animales , Sitios de Unión , Dominio Catalítico , Microscopía por Crioelectrón , Activación Enzimática , Hemocianinas/metabolismo , Modelos Moleculares , Conformación Proteica , Subunidades de Proteína/química , Subunidades de Proteína/metabolismoRESUMEN
RNA folding occurs via a series of transitions between metastable intermediate states. It is unknown whether folding intermediates are discrete structures folding along defined pathways or heterogeneous ensembles folding along broad landscapes. We use cryo-electron microscopy and single-particle image reconstruction to determine the structure of the major folding intermediate of the specificity domain of a ribonuclease P ribozyme. Our results support the existence of a discrete conformation for this folding intermediate.
Asunto(s)
Microscopía por Crioelectrón , Ribonucleasa P/química , Ribonucleasa P/metabolismo , Secuencia de Aminoácidos , Bacillus/enzimología , Dicroismo Circular , Modelos Moleculares , Datos de Secuencia MolecularRESUMEN
Using RNA as a material for nanoparticle construction provides control over particle size and shape at the nano-scale. RNA nano-architectures have shown promise as delivery vehicles for RNA interference (RNAi) substrates, allowing multiple functional entities to be combined on a single particle in a programmable fashion. Rather than employing a completely bottom-up approach to scaffold design, here multiple copies of an existing synthetic supramolecular RNA nano-architecture serve as building blocks along with additional motifs for the design of a novel truncated tetrahedral RNA scaffold, demonstrating that rationally designed RNA assemblies can themselves serve as modular pieces in the construction of larger rationally designed structures. The resulting tetrahedral scaffold displays enhanced characteristics for RNAi-substrate delivery in comparison to similar RNA-based scaffolds, as evidenced by its increased functional capacity, increased cellular uptake and ultimately an increased RNAi efficacy of its adorned Dicer substrate siRNAs. The unique truncated tetrahedral shape of the nanoparticle core appears to contribute to this particle's enhanced function, indicating the physical characteristics of RNA scaffolds merit significant consideration when designing platforms for delivery of functional RNAs via RNA nanoparticles.
Asunto(s)
ARN Helicasas DEAD-box/química , Nanoestructuras/química , Interferencia de ARN , ARN/química , Ribonucleasa III/química , Proteínas de Ciclo Celular/química , Línea Celular Tumoral , Microscopía por Crioelectrón , Proteínas Fluorescentes Verdes/química , Humanos , Luz , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , Tamaño de la Partícula , Reacción en Cadena de la Polimerasa , Conformación Proteica , Proteínas Serina-Treonina Quinasas/química , Proteínas Proto-Oncogénicas/química , ARN Interferente Pequeño , Dispersión de Radiación , Programas Informáticos , Termodinámica , Quinasa Tipo Polo 1RESUMEN
The 26S proteasome is specialized for regulated protein degradation and formed by a dynamic regulatory particle (RP) that caps a hollow cylindrical core particle (CP) where substrates are proteolyzed. Its diverse substrates unify as proteasome targets by ubiquitination. We used cryogenic electron microscopy (cryo-EM) to study how human 26S proteasome interacts with M1-linked hexaubiquitin (M1-Ub6) unanchored to a substrate and E3 ubiquitin ligase E6AP/UBE3A. Proteasome structures are available with model substrates extending through the RP ATPase ring and substrate-conjugated K63-linked ubiquitin chains present at inhibited deubiquitinating enzyme hRpn11 and the nearby ATPase hRpt4/hRpt5 coiled coil. In this study, we find M1-Ub6 at the hRpn11 site despite the absence of conjugated substrate, indicating that ubiquitin binding at this location does not require substrate interaction with the RP. Moreover, unanchored M1-Ub6 binds to this hRpn11 site of the proteasome with the CP gating residues in both the closed and opened conformational states.
Asunto(s)
Adenosina Trifosfatasas/química , Poliubiquitina/química , Complejo de la Endopetidasa Proteasomal/química , Transactivadores/química , Ubiquitina-Proteína Ligasas/química , Ubiquitina/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Sitios de Unión , Microscopía por Crioelectrón , Expresión Génica , Humanos , Simulación del Acoplamiento Molecular , Poliubiquitina/genética , Poliubiquitina/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteolisis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Transactivadores/genética , Transactivadores/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Magainin, a 23-residue antibiotic peptide, interacts directly with the lipid bilayer leading to cell lysis in a strongly concentration-dependent fashion. Utilizing cryo-electron microscopy, we have directly observed magainin interacting with synthetic DMPC/DMPG membranes. Visual examination shows that visibly unperturbed vesicles are often found adjacent to vesicles that are lysed or porous, demonstrating that magainin disruption is a highly stochastic process. Quantitatively, power spectra of large numbers of porous vesicles can be averaged together to produce the equivalent of an electron scattering curve, which can be related to theory, simulation, and published neutron scattering experiments. We demonstrate that magainin-induced pores in lipid vesicles have a mean diameter of approximately 80 A, compatible with earlier reported results in multilayer stacks. In addition to establishing a connection between experiments in multilayer stacks and vesicles, this also demonstrates that computed power spectra from windowed-out regions of cryo-EM images can be compared to neutron scattering data in a meaningful way, even though the pores of interest cannot yet be individually identified in images. Cryo-EM offers direct imaging of systems in configurations closely related to in vivo conditions, whereas neutron scattering has a greater variety of mechanisms for specific contrast variation via D2O and deuterated lipids. Combined, the two mechanisms support each other, and provide a clearer picture of such 'soft' systems than either could provide alone.
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Péptidos Catiónicos Antimicrobianos/química , Liposomas/química , Proteínas de Xenopus/química , Algoritmos , Animales , Simulación por Computador , Dimiristoilfosfatidilcolina/química , Magaininas , Microscopía Electrónica/métodos , Modelos Teóricos , Neutrones , Fosfatidilgliceroles/química , Dispersión de Radiación , Procesos Estocásticos , Temperatura , XenopusRESUMEN
Self-assembly of peptide amphiphiles into nanostructures makes them attractive for a variety of applications in drug and peptide delivery. We here report on the interactions of micelles composed of a palmitoylated, pro-apoptotic peptide derived from p53 tumor suppressor protein with a human cancer cell line. Characterization of self-assembly in aqueous buffered solutions revealed formation of elongated rod-like micelles above a critical micelle concentration. Our results however demonstrate that monomers instead of micelles are internalized, a finding that correlates with the dynamic nature of the assemblies and the noncovalent interactions that hold them together. Internalization is shown to occur via adsorption-mediated, energy-dependent pathways, resulting in accumulation of the material in endocytic vesicles. We conclude that palmitoylation of peptides is an efficient way to increase peptide permeability inside SJSA-1 cells and that increased micelle stability would be required for intact micelle internalization.