RESUMEN
Vault ribonucleoprotein particles are naturally designed nanocages, widely found in the eukaryotic kingdom. Vaults consist of 78 copies of the major vault protein (MVP) that are organized in 2 symmetrical cup-shaped halves, of an approximate size of 70x40x40 nm, leaving a huge internal cavity which accommodates the vault poly(ADP-ribose) polymerase (vPARP), the telomerase-associated protein-1 (TEP1) and some small untranslated RNAs. Diverse hypotheses have been developed on possible functions of vaults, based on their unique capsular structure, their rapid movements and the distinct subcellular localization of the particles, implicating transport of cargo, but they are all pending confirmation. Vault particles also possess many attributes that can be exploited in nanobiotechnology, particularly in the creation of vehicles for the delivery of multiple molecular cargoes. Here we review what is known about the structure and dynamics of the vault complex and discuss a possible mechanism for the vault opening process. The recent findings in the characterization of the vaults in cells and in its natural microenvironment will be also discussed.
Asunto(s)
Partículas Ribonucleoproteicas en Bóveda , Partículas Ribonucleoproteicas en Bóveda/metabolismo , Partículas Ribonucleoproteicas en Bóveda/química , Partículas Ribonucleoproteicas en Bóveda/genética , Humanos , Animales , Poli(ADP-Ribosa) Polimerasas/metabolismo , Poli(ADP-Ribosa) Polimerasas/químicaRESUMEN
Vaults are eukaryotic ribonucleoproteins consisting of 78 copies of the major vault protein (MVP), which assemble into a nanoparticle with an about 60 nm volume-based size, enclosing other proteins and RNAs. Regardless of their physiological role(s), vaults represent ideal, natural hollow nanoparticles, which are produced by the assembly of the sole MVP. Here, we have expressed in Komagataella phaffi and purified an MVP variant carrying a C-terminal Z peptide (vault-Z), which can tightly bind an antibody's Fc portion, in view of targeted delivery. Via surface plasmon resonance analysis, we could determine a 2.5 nM affinity to the monoclonal antibody Trastuzumab (Tz)/vault-Z 1:1 interaction. Then, we characterized the in-solution interaction via co-incubation, ultracentrifugation, and analysis of the pelleted proteins. This showed virtually irreversible binding up to an at least 10:1 Tz/vault-Z ratio. As a proof of concept, we labeled the Fc portion of Tz with a fluorophore and conjugated it with the nanoparticle, along with either Tz or Cetuximab, another monoclonal antibody. Thus, we could demonstrate antibody-dependent, selective uptake by the SKBR3 and MDA-MB 231 breast cancer cell lines. These investigations provide a novel, flexible technological platform that significantly extends vault-Z's applications, in that it can be stably conjugated with finely adjusted amounts of antibodies as well as of other molecules, such as fluorophores, cell-targeting peptides, or drugs, using the Fc portion as a scaffold.
Asunto(s)
Nanopartículas , Trastuzumab , Partículas Ribonucleoproteicas en Bóveda , Humanos , Partículas Ribonucleoproteicas en Bóveda/metabolismo , Partículas Ribonucleoproteicas en Bóveda/química , Nanopartículas/química , Trastuzumab/química , Línea Celular Tumoral , Cetuximab/química , Anticuerpos Monoclonales/química , Inmunoconjugados/químicaRESUMEN
The small noncoding vault RNA (vtRNA) is a component of the vault complex, a ribonucleoprotein complex found in most eukaryotes. Emerging evidence suggests that vtRNAs may be involved in the regulation of a variety of cellular functions when unassociated with the vault complex. Here, we demonstrate a novel role for vtRNA in synaptogenesis. Using an in vitro synapse formation model, we show that murine vtRNA (mvtRNA) promotes synapse formation by modulating the MAPK signaling pathway. mvtRNA is transported to the distal region of neurites as part of the vault complex. Interestingly, mvtRNA is released from the vault complex in the neurite by a mitotic kinase Aurora-A-dependent phosphorylation of MVP, a major protein component of the vault complex. mvtRNA binds to and activates MEK1 and thereby enhances MEK1-mediated ERK activation in neurites. These results suggest the existence of a regulatory mechanism of the MAPK signaling pathway by vtRNAs as a new molecular basis for synapse formation.
Asunto(s)
Sistema de Señalización de MAP Quinasas , ARN Pequeño no Traducido/metabolismo , Sinapsis/metabolismo , Secuencia de Aminoácidos , Animales , Aurora Quinasa A/metabolismo , Línea Celular , Regulación hacia Abajo/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Cinesinas/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Neuritas/metabolismo , Oligonucleótidos Antisentido/farmacología , Densidad Postsináptica/efectos de los fármacos , Densidad Postsináptica/metabolismo , Unión Proteica/efectos de los fármacos , ARN Interferente Pequeño/metabolismo , Sinapsis/efectos de los fármacos , Partículas Ribonucleoproteicas en Bóveda/química , Partículas Ribonucleoproteicas en Bóveda/metabolismoRESUMEN
Vault particles are the largest naturally occurring ribonucleoprotein complexes found in the cytoplasm. In all 78 copies of major vault protein (MVP) assemble on polyribosome templates, forming recombinant vault particles, which are of great interest as encapsulation carriers for therapeutics delivery and enzyme stabilization. Baculovirus-insect cell expression is the only system that has been developed for recombinant vault synthesis, but it has low scalability and slow production rate. In this study, we demonstrated the first use of yeast cells for the production of vault particles with full integrity and functionality solely by expressing the complementary DNA (cDNA) encoding MVP. Vaults synthesized in Pichia pastoris yeast cells are morphologically indistinguishable from recombinant vault particles produced in insect cells, and are able to package and stabilize enzymes resulting in improved longevity and catalytic efficiency. Thus, our results imply that the yeast system is an economical alternative to insect cells for the production of recombinant vaults. The consistency of vault morphology between yeast and insect cell systems also underlines that polyribosome templating may be conserved among eukaryotes, which promises the synthesis and assembly of recombinant human vault particles in other eukaryotic organisms.
Asunto(s)
Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Partículas Ribonucleoproteicas en Bóveda/metabolismo , Animales , Humanos , Nanopartículas/química , Nanopartículas/metabolismo , Ingeniería de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Saccharomyces cerevisiae/genética , Partículas Ribonucleoproteicas en Bóveda/química , Partículas Ribonucleoproteicas en Bóveda/genética , Partículas Ribonucleoproteicas en Bóveda/aislamiento & purificaciónRESUMEN
BACKGROUND: Vaults are eukaryotic ribonucleoprotein particles composed of up 78 copies of the 97â¯kDa major vault protein that assembles into a barrel-like, "nanocapsule" enclosing poly(ADP-ribose) polymerase, telomerase-associated protein-1 and small untranslated RNAs. Overall, the molecular mass of vault particles amounts to about 13â¯MDa. Although it has been implicated in several cellular functions, its physiological roles remain poorly understood. Also, the possibility to exploit it as a nanovector for drug delivery is currently being explored in several laboratories. METHODS: Using the baculovirus expression system, vaults were expressed and purified by a dialysis step using a 1â¯MDa molecular weight cutoff membrane and a subsequent size exclusion chromatography. Purity was assessed by SDS-PAGE, transmission electron microscopy and dynamic light scattering. Particle's endocytic uptake was monitored by flow cytometry and confocal microscopy. RESULTS: The purification protocol here reported is far simpler and faster than those currently available and lead to the production of authentic vault. We then demonstrated its clathrin-mediated endocytic uptake by normal fibroblast and glioblastoma, but not carcinoma cell lines. In contrast, no significant caveolin-mediated endocytosis was detected. CONCLUSIONS: These results provide the first evidence for an intrinsic propensity of the vault complex to undergo endocytic uptake cultured eukaryotic cells. GENERAL SIGNIFICANCE: The newly developed purification procedure will greatly facilitate any investigation based on the use of the vault particle as a natural nanocarrier. Its clathrin-mediated endocytic uptake observed in normal and in some tumor cell lines sheds light on its physiological role.
Asunto(s)
Endocitosis/fisiología , Fibroblastos/citología , Glioblastoma/metabolismo , Nanopartículas/administración & dosificación , Partículas Ribonucleoproteicas en Bóveda/química , Partículas Ribonucleoproteicas en Bóveda/metabolismo , Animales , Células Cultivadas , Sistemas de Liberación de Medicamentos , Endocitosis/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Glioblastoma/patología , Humanos , Nanopartículas/química , Transducción de Señal , SpodopteraRESUMEN
Natural vault nanoparticles are ribonucleoprotein particles with a mass of 13 MDa that have been found in a wide variety of eukaryotes. Empty recombinant vaults are assembled from heterologously expressed Major Vault Protein (MVP), forming the barrel-shaped vault shell. These structures are morphologically indistinguishable from natural vault particles. Here, we describe the packaging and purification of exogenous proteins into these recombinant vault particles by mixing with proteins attached to the INT domain that binds to MVP.
Asunto(s)
Nanopartículas , Partículas Ribonucleoproteicas en Bóveda , Animales , Enzimas Inmovilizadas , Expresión Génica , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Nanopartículas/química , Nanopartículas/ultraestructura , Nanotecnología , Ratas , Proteínas Recombinantes de Fusión/química , Células Sf9 , Partículas Ribonucleoproteicas en Bóveda/químicaRESUMEN
Prior crystal structures of the vault have provided clues of its structural variability but are non-conclusive due to crystal packing. Here, we obtained vaults by engineering at the N terminus of rat major vault protein (MVP) an HIV-1 Gag protein segment and determined their near-atomic resolution (â¼4.8 Å) structures in a solution/non-crystalline environment. The barrel-shaped vaults in solution adopt two conformations, 1 and 2, both with D39 symmetry. From the N to C termini, each MVP monomer has three regions: body, shoulder, and cap. While conformation 1 is identical to one of the crystal structures, the shoulder in conformation 2 is translocated longitudinally up to 10 Å, resulting in an outward-projected cap. Our structures clarify the structural discrepancies in the body region in the prior crystallography models. The vault's drug-delivery potential is highlighted by the internal disposition and structural flexibility of its Gag-loaded N-terminal extension at the barrel waist of the engineered vault.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Ingeniería de Proteínas/métodos , Partículas Ribonucleoproteicas en Bóveda/química , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/química , Secuencia de Aminoácidos , Animales , Baculoviridae/genética , Baculoviridae/metabolismo , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Partículas Ribonucleoproteicas en Bóveda/genética , Partículas Ribonucleoproteicas en Bóveda/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Vaults are naturally occurring ovoid nanoparticles constructed from a protein shell that is composed of multiple copies of major vault protein (MVP). The vault-interacting domain of vault poly(ADP-ribose)-polymerase (INT) has been used as a shuttle to pack biomolecular cargo in the vault lumen. However, the interaction between INT and MVP is poorly understood. It is hypothesized that the release rate of biomolecular cargo from the vault lumen is related to the interaction between MVP and INT. To tune the release of molecular cargos from the vault nanoparticles, we determined the interactions between the isolated INT-interacting MVP domains (iMVP) and wild-type INT and compared them to two structurally modified INT: 15-amino acid deletion at the C terminus (INTΔC15) and histidine substituted at the interaction surface (INT/DSA/3 H) to impart a pH-sensitive response. The apparent affinity constants determined using surface plasmon resonance (SPR) biosensor technology are 262 ± 4 nM for iMVP/INT, 1800 ± 160 nM for iMVP/INTΔC15 at pH 7.4. The INT/DSA/3 H exhibits stronger affinity to iMVP (K Dapp = 24 nM) and dissociates at a slower rate than wild-type INT at pH 6.0.
Asunto(s)
Poli(ADP-Ribosa) Polimerasas/metabolismo , Mapas de Interacción de Proteínas , Partículas Ribonucleoproteicas en Bóveda/metabolismo , Animales , Concentración de Iones de Hidrógeno , Modelos Moleculares , Poli(ADP-Ribosa) Polimerasas/química , Dominios y Motivos de Interacción de Proteínas , Ratas , Partículas Ribonucleoproteicas en Bóveda/químicaRESUMEN
Vault nanoparticles represent promising vehicles for drug and probe delivery. Innately found within human cells, vaults are stable, biocompatible nanocapsules possessing an internal volume that can encapsulate hundreds to thousands of molecules. They can also be targeted. Unlike most nanoparticles, vaults are nonimmunogenic and monodispersed and can be rapidly produced in insect cells. Efforts to create vaults with modified properties have been, to date, almost entirely limited to recombinant bioengineering approaches. Here we report a systematic chemical study of covalent vault modifications, directed at tuning vault properties for research and clinical applications, such as imaging, targeted delivery, and enhanced cellular uptake. As supra-macromolecular structures, vaults contain thousands of derivatizable amino acid side chains. This study is focused on establishing the comparative selectivity and efficiency of chemically modifying vault lysine and cysteine residues, using Michael additions, nucleophilic substitutions, and disulfide exchange reactions. We also report a strategy that converts the more abundant vault lysine residues to readily functionalizable thiol terminated side chains through treatment with 2-iminothiolane (Traut's reagent). These studies provide a method to doubly modify vaults with cell penetrating peptides and imaging agents, allowing for in vitro studies on their enhanced uptake into cells.
Asunto(s)
Sistemas de Liberación de Medicamentos , Colorantes Fluorescentes/química , Nanopartículas/química , Imagen Óptica , Partículas Ribonucleoproteicas en Bóveda/química , Animales , Células CHO , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cricetulus , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/farmacología , Células HeLa , Humanos , Ratones , Microscopía Confocal , Estructura Molecular , Células RAW 264.7 , Relación Estructura-Actividad , Partículas Ribonucleoproteicas en Bóveda/síntesis química , Partículas Ribonucleoproteicas en Bóveda/farmacologíaRESUMEN
Vault particles are naturally occurring proteinaceous cages with promising application as molecular containers. The use of vaults as functional transporters requires a profound understanding of their structural stability to guarantee the protection and controlled payload delivery. Previous results performed with bulk techniques or at non-physiological conditions have suggested pH as a parameter to control vault dynamics. Here we use Atomic Force Microscopy (AFM) to monitor the structural evolution of individual vault particles while changing the pH in real time. Our experiments show that decreasing the pH of the solution destabilize the barrel region, the central part of vault particles, and leads to the aggregation of the cages. Additional analyses using Quartz-Crystal Microbalance (QCM) and Differential Scanning Fluorimetry (DSF) are consistent with our single molecule AFM experiments. The observed topographical defects suggest that low pH weakens the bonds between adjacent proteins. We hypothesize that the observed effects are related to the strong polar character of the protein-protein lateral interactions. Overall, our study unveils the mechanism for the influence of a biologically relevant range of pHs on the stability and dynamics of vault particles.
Asunto(s)
Microscopía de Fuerza Atómica/métodos , Tecnicas de Microbalanza del Cristal de Cuarzo/métodos , Partículas Ribonucleoproteicas en Bóveda/química , Concentración de Iones de Hidrógeno , Modelos Moleculares , Nanotecnología , Estabilidad ProteicaRESUMEN
Vault nanoparticles packaged with enzymes were synthesized as agents for efficiently degrading environmental contaminants. Enzymatic biodegradation is an attractive technology for in situ cleanup of contaminated environments because enzyme-catalyzed reactions are not constrained by nutrient requirements for microbial growth and often have higher biodegradation rates. However, the limited stability of extracellular enzymes remains a major challenge for practical applications. Encapsulation is a recognized method to enhance enzymatic stability, but it can increase substrate diffusion resistance, lower catalytic rates, and increase the apparent half-saturation constants. Here, we report an effective approach for boosting enzymatic stability by single-step packaging into vault nanoparticles. With hollow core structures, assembled vault nanoparticles can simultaneously contain multiple enzymes. Manganese peroxidase (MnP), which is widely used in biodegradation of organic contaminants, was chosen as a model enzyme in the present study. MnP was incorporated into vaults via fusion to a packaging domain called INT, which strongly interacts with vaults' interior surface. MnP fused to INT and vaults packaged with the MnP-INT fusion protein maintained peroxidase activity. Furthermore, MnP-INT packaged in vaults displayed stability significantly higher than that of free MnP-INT, with slightly increased Km value. Additionally, vault-packaged MnP-INT exhibited 3 times higher phenol biodegradation in 24 h than did unpackaged MnP-INT. These results indicate that the packaging of MnP enzymes in vault nanoparticles extends their stability without compromising catalytic activity. This research will serve as the foundation for the development of efficient and sustainable vault-based bioremediation approaches for removing multiple contaminants from drinking water and groundwater.
Asunto(s)
Contaminantes Ambientales/aislamiento & purificación , Nanopartículas/química , Nanotecnología/métodos , Peroxidasas/metabolismo , Partículas Ribonucleoproteicas en Bóveda/química , Biocatálisis , Biodegradación Ambiental , Estabilidad de Enzimas , Cinética , Nanopartículas/ultraestructura , Peroxidasas/química , Phanerochaete/enzimología , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismoRESUMEN
The high proliferation rate of cancer cells, together with environmental factors such as hypoxia and nutrient deprivation can cause Endoplasmic Reticulum (ER) stress. The protein kinase PERK is an essential mediator in one of the three ER stress response pathways. Genetic and pharmacological inhibition of PERK has been reported to limit tumor growth in xenograft models. Here we provide evidence that inactive PERK interacts with the nuclear pore-associated Vault complex protein and that this compromises Vault-mediated nuclear transport of PTEN. Pharmacological inhibition of PERK under ER stress results is abnormal sequestration of the Vault complex, leading to increased cytoplasmic PTEN activity and lower AKT activation. As the PI3K/PTEN/AKT pathway is crucial for many aspects of cell growth and survival, this unexpected effect of PERK inhibitors on AKT activity may have implications for their potential use as therapeutic agents.
Asunto(s)
Fosfohidrolasa PTEN/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Partículas Ribonucleoproteicas en Bóveda/metabolismo , eIF-2 Quinasa/metabolismo , Sustitución de Aminoácidos , Línea Celular Tumoral , Estrés del Retículo Endoplásmico/efectos de los fármacos , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/metabolismo , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Proteínas Nucleares/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , Partículas Ribonucleoproteicas en Bóveda/química , eIF-2 Quinasa/antagonistas & inhibidores , eIF-2 Quinasa/genéticaRESUMEN
We report a novel approach to a new class of bioengineered, monodispersed, self-assembling vault nanoparticles consisting of a protein shell exterior with a lipophilic core interior designed for drug and probe delivery. Recombinant vaults were engineered to contain a small amphipathic α-helix derived from the nonstructural protein 5A of hepatitis C virus, thereby creating within the vault lumen a lipophilic microenvironment into which lipophilic compounds could be reversibly encapsulated. Multiple types of electron microscopy showed that attachment of this peptide resulted in larger than expected additional mass internalized within the vault lumen attributable to incorporation of host lipid membrane constituents spanning the vault waist (>35 nm). These bioengineered lipophilic vaults reversibly associate with a sample set of therapeutic compounds, including all-trans retinoic acid, amphotericin B, and bryostatin 1, incorporating hundreds to thousands of drug molecules per vault nanoparticle. Bryostatin 1 is of particular therapeutic interest because of its ability to potently induce expression of latent HIV, thus representing a preclinical lead in efforts to eradicate HIV/AIDS. Vaults loaded with bryostatin 1 released free drug, resulting in activation of HIV from provirus latency in vitro and induction of CD69 biomarker expression following intravenous injection into mice. The ability to preferentially and reversibly encapsulate lipophilic compounds into these novel bioengineered vault nanoparticles greatly advances their potential use as drug delivery systems.
Asunto(s)
Bioingeniería , Portadores de Fármacos/química , Interacciones Hidrofóbicas e Hidrofílicas , Nanopartículas/química , Partículas Ribonucleoproteicas en Bóveda/química , Animales , Brioestatinas/química , Línea Celular , Humanos , Ratones , Modelos Moleculares , Estructura Secundaria de ProteínaRESUMEN
Vaults are the largest ribonucleoprotein particles found in eukaryotic cells, with an unclear cellular function and promising applications as vehicles for drug delivery. In this article, we examine the local stiffness of individual vaults and probe their structural stability with atomic force microscopy under physiological conditions. Our data show that the barrel, the central part of the vault, governs both the stiffness and mechanical strength of these particles. In addition, we induce single-protein fractures in the barrel shell and monitor their temporal evolution. Our high-resolution atomic force microscopy topographies show that these fractures occur along the contacts between two major vault proteins and disappear over time. This unprecedented systematic self-healing mechanism, which enables these particles to reversibly adapt to certain geometric constraints, might help vaults safely pass through the nuclear pore complex and potentiate their role as self-reparable nanocontainers.
Asunto(s)
Elasticidad , Partículas Ribonucleoproteicas en Bóveda/química , Estrés MecánicoRESUMEN
Vaults are very large oligomeric ribonucleoproteins conserved among a variety of species. The rat vault 3D structure shows an ovoid oligomeric particle, consisting of 78 major vault protein monomers, each of approximately 861 amino acids. Vaults are probably the largest ribonucleoprotein structures in eukaryote cells, being approximately 70 nm in length with a diameter of 40 nm--the size of three ribosomes and with a lumen capacity of 50 million Å(3). We use both protein sequences and inferred ancestral sequences for in silico virtual resurrection of tertiary and quaternary structures to search for vaults in a wide variety of eukaryotes. We find that the vault's phylogenetic distribution is widespread in eukaryotes, but is apparently absent in some notable model organisms. Our conclusion from the distribution of vaults is that they were present in the last eukaryote common ancestor but they have apparently been lost from a number of groups including fungi, insects, and probably plants. Our approach of inferring ancestral 3D and quaternary structures is expected to be useful generally.
Asunto(s)
Simulación por Computador , Eucariontes/genética , Filogenia , Partículas Ribonucleoproteicas en Bóveda/química , Alveolados/química , Secuencia de Aminoácidos , Amebozoos/química , Animales , Proteínas Bacterianas/química , Coanoflagelados/química , Eucariontes/química , Hongos/química , Genes de Plantas , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Plantas/química , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Ratas , Alineación de Secuencia , Partículas Ribonucleoproteicas en Bóveda/genéticaRESUMEN
Our previous behavioural, biochemical and immunohistochemical analyses conducted in selected regions (supra/sub oesophageal masses) of the Octopus vulgaris brain detected a cytoplasmic poly-ADP-ribose polymerase (more than 90% of total enzyme activity). The protein was identified as the vault-free form of vault-poly-ADP-ribose polymerase. The present research extends and integrates the biochemical characterization of poly-ADP-ribosylation system, namely, reaction product, i.e., poly-ADP-ribose, and acceptor proteins, in the O. vulgaris brain. Immunochemical analyses evidenced that the sole poly-ADP-ribose acceptor was the octopus cytoskeleton 50-kDa actin. It was present in both free, endogenously poly-ADP-ribosylated form (70kDa) and in complex with V-poly-ADP-ribose polymerase and poly-ADP-ribose (260kDa). The components of this complex, alkali and high salt sensitive, were purified and characterized. The kind and the length of poly-ADP-ribose corresponded to linear chains of 30-35 ADP-ribose units, in accordance with the features of the polymer synthesized by the known vault-poly-ADP-ribose polymerase. In vitro experiments showed that V-poly-ADP-ribose polymerase activity of brain cytoplasmic fraction containing endogenous actin increased upon the addition of commercial actin and was highly reduced by ATP. Anti-actin immunoblot of the mixture in the presence and absence of ATP showed that the poly-ADP-ribosylation of octopus actin is a dynamic process balanced by the ATP-dependent polymerization of the cytoskeleton protein, a fundamental mechanism for synaptic plasticity.
Asunto(s)
Actinas/metabolismo , Encéfalo/enzimología , Octopodiformes/enzimología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Partículas Ribonucleoproteicas en Bóveda/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/química , Animales , Plasticidad Neuronal/genética , Poli Adenosina Difosfato Ribosa/metabolismo , Poli(ADP-Ribosa) Polimerasas/química , Polimerizacion , Sinapsis/metabolismo , Sinapsis/fisiología , Partículas Ribonucleoproteicas en Bóveda/químicaRESUMEN
The vault particle, with a molecular weight of about 10 MDa, is the largest ribonucleoprotein that has been described. The X-ray structure of intact rat vault has been solved at a resolution of 3.5 Å [Tanaka et al. (2009), Science, 323, 384-388], showing an overall barrel-shaped architecture organized into two identical moieties, each consisting of 39 copies of the major vault protein (MVP). The model deposited in the PDB includes 39 MVP copies (half a vault) in the crystal asymmetric unit. A 2.1 Å resolution structure of the seven N-terminal repeats (R1-7) of MVP has also been determined [Querol-Audí et al. (2009), EMBO J. 28, 3450-3457], revealing important discrepancies with respect to the MVP models for repeats R1 and R2. Here, the re-refinement of the vault structure by incorporating the high-resolution information available for the R1-7 domains, using the deformable elastic network (DEN) approach and maintaining strict 39-fold noncrystallographic symmetry is reported. The new refinement indicates that at the resolution presently available the MVP shell can be described well as only one independent subunit organized with perfect D39 molecular symmetry. This refinement reveals that significant rearrangements occur in the N-terminus of MVP during the closing of the two vault halves and that the 39-fold symmetry breaks in the cap region. These results reflect the highly dynamic nature of the vault structure and represent a necessary step towards a better understanding of the biology and regulation of this particle.
Asunto(s)
Partículas Ribonucleoproteicas en Bóveda/química , Animales , Modelos Moleculares , Conformación Proteica , Ratas , Difracción de Rayos XRESUMEN
The cDNA clone coding a major vault protein (MVP)-like protein was derived from Echinococcus multilocularis cysts. MVP is a main component of vault particles, which are the largest cytoplasmic ribonucleoprotein particles in eukaryotic cells. We sequenced and characterized E. multilocularis MVP (EmMVP). The nucleotide sequence of the emmvp cDNA clone was 2607 bp in the full length open reading frame and its deduced amino acid sequence had several signature motifs which were specific to MVP families. Immunoblot analysis with mouse anti-EmMVP antiserum revealed that crude antigens of E. multilocularis included EmMVP protein. Furthermore, our results showed that the expression of EmMVP protein in an Sf9 insect cell line using a baculovirus vector directed the formation of particles that shared similar biochemical characteristics with other vault proteins and the distinct vault-like morphology when negatively stained and examined by electron microscopy.
Asunto(s)
Echinococcus multilocularis/química , Proteínas del Helminto/genética , Partículas Ribonucleoproteicas en Bóveda/genética , Secuencia de Aminoácidos , Animales , Antígenos Helmínticos/química , Antígenos Helmínticos/genética , Antígenos Helmínticos/inmunología , Secuencia de Bases , Clonación Molecular , ADN Complementario/química , ADN de Helmintos/química , Equinococosis/inmunología , Echinococcus multilocularis/genética , Echinococcus multilocularis/inmunología , Femenino , Proteínas del Helminto/química , Proteínas del Helminto/ultraestructura , Humanos , Sueros Inmunes/inmunología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Alineación de Secuencia , Análisis de Secuencia de ADN , Células Sf9 , Partículas Ribonucleoproteicas en Bóveda/química , Partículas Ribonucleoproteicas en Bóveda/ultraestructuraRESUMEN
We examine the advantages of going beyond sequence similarity and use both protein three-dimensional (3D) structure prediction and then quaternary structure (docking) of inferred 3D structures to help evaluate whether comparable sequences can fold into homologous structures with sufficient lateral associations for quaternary structure formation. Our test case is the major vault protein (MVP) that oligomerizes in multiple copies to form barrel-like vault particles and is relatively widespread among eukaryotes. We used the iterative threading assembly refinement server (I-TASSER) to predict whether putative MVP sequences identified by BLASTp and PSI Basic Local Alignment Search Tool are structurally similar to the experimentally determined rodent MVP tertiary structures. Then two identical predicted quaternary structures from I-TASSER are analyzed by RosettaDock to test whether a pair-wise association occurs, and hence whether the oligomeric vault complex is likely to form for a given MVP sequence. Positive controls for the method are the experimentally determined rat (Rattus norvegicus) vault X-ray crystal structure and the purple sea urchin (Strongylocentrotus purpuratus) MVP sequence that forms experimentally observed vaults. These and two kinetoplast MVP structural homologs were predicted with high confidence value, and RosettaDock predicted that these MVP sequences would dock laterally and therefore could form oligomeric vaults. As the negative control, I-TASSER did not predict an MVP-like structure from a randomized rat MVP sequence, even when constrained to the rat MVP crystal structure (PDB:2ZUO), thus further validating the method. The protocol identified six putative homologous MVP sequences in the heterobolosean Naegleria gruberi within the excavate kingdom. Two of these sequences are predicted to be structurally similar to rat MVP, despite being in excess of 300 residues shorter. The method can be used generally to help test predictions of homology via structural analysis.
Asunto(s)
Homología Estructural de Proteína , Partículas Ribonucleoproteicas en Bóveda/química , Secuencia de Aminoácidos , Animales , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Conformación Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Ratas , Erizos de Mar , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Vaults are naturally-occurring ribonucleoprotein particles found in nearly all eukaryotic cells. They were named for their morphological resemblance to the vaulted ceilings of gothic cathedrals. These ubiquitous nanoparticles are quite abundant with 10(4)-10(6) copies found in the cytoplasm depending on cell type. The structural shell of the particle can self-assemble from 78 copies of a single protein, the major vault protein. This finding has allowed vaults to be bioengineered, resulting in a variety of new functions and capabilities directed toward overcoming many limitations posed by current gene and drug delivery systems. In this study, we demonstrate that recombinant vaults, with the addition of a cell penetration peptide, TAT, can be rapidly delivered to cells in vitro with significantly elevated binding and uptake efficiency. This TAT-vault nanoparticle could be a valuable tool for improving the retention and penetration of therapeutic drugs at tumor sites.