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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.

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., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

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., (© 2021 Wakatsuki et al.)

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., (© 2018 Wiley Periodicals, Inc.)

Background: Vaults are eukaryotic ribonucleoprotein particles composed of up 78 copies of the 97 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 13 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 1 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., (Copyright © 2018. Published by Elsevier B.V.)

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 (∼4.8 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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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.

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.

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.

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.

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.

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., (Copyright © 2014. Published by Elsevier Inc.)

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.

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., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

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., (© 2013 Elsevier Inc. All rights reserved.)

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.

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Vaults are naturally occurring nanoparticles found widely in eukaryotes. The particles can be produced in large quantities and are assembled in situ from multiple copies of the single structural protein following expression. Using molecular engineering, recombinant vaults can be functionally modified and targeted, and their contents can be controlled by packaging. Here, we review the development of engineered vaults as a platform for a wide variety of therapeutic applications and we examine future directions for this unique nanoparticle system.

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.

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.

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.

Vault particles possess many attributes that can be exploited in nanobiotechnology, particularly in the creation of drug delivery nanodevices. These include self-assembly, 100 nm size range, a dynamic structure that may be controlled for manipulation of drug release kinetics and natural presence in humans ensuring biocompatibility. The flexibility and the adaptability of this system have been greatly enhanced by the emerging atomic-level information and improved comprehension of vault structure and dynamics. It seems likely that this information will allow their specific tailoring to the individual requirements of each drug and target tissue. These properties provide vaults with an enormous potential as a versatile delivery platform., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Vault is the largest nonicosahedral cytosolic nucleoprotein particle ever described. The widespread presence and evolutionary conservation of vaults suggest important biologic roles, although their functions have not been fully elucidated. X-ray structure of vault from rat liver was determined at 3.5 Å resolution. It exhibits an ovoid shape with a size of 40 × 40 × 67 nm(3). The cage structure of vault consists of a dimer of half-vaults, with each half-vault comprising 39 identical major vault protein (MVP) chains. Each MVP monomer folds into 12 domains: nine structural repeat domains, a shoulder domain, a cap-helix domain and a cap-ring domain. Interactions between the 42-turn-long cap-helix domains are key to stabilizing the particle. The other components of vaults, telomerase-associated proteins, poly(ADP-ribose) polymerases and small RNAs, are in location in the vault particle by electron microscopy.

Vault nanoparticles were engineered to enhance their escape from the endosomal compartment by fusing a membrane lytic peptide derived from adenovirus protein VI (pVI) to the N-terminus of the major vault protein to form pVI-vaults. We demonstrate that these pVI-vaults disrupt the endosomal membrane using three different experimental protocols including (1) enhancement of DNA transfection, (2) co-delivery of a cytosolic ribotoxin, and (3) direct visualization by fluorescence. Furthermore, direct targeting of vaults to specific cell surface epidermal growth factor receptors led to enhanced cellular uptake and efficient delivery of vaults to the cytoplasm. This process was monitored with fluorescent vaults, and morphological changes in the endosomal compartment were observed. By combining targeting and endosomal escape into a single recombinant vault, high levels of transfection efficiency were achieved using low numbers of vault particles. These results demonstrate that engineered vaults are effective, efficient, and nontoxic nanoparticles for targeted delivery of biomaterials to the cell cytoplasm., (© 2011 American Chemical Society)

The vault nanoparticle is one of the largest known ribonucleoprotein complexes in the sub-100 nm range. Highly conserved and almost ubiquitously expressed in eukaryotes, vaults form a large nanocapsule with a barrel-shaped morphology surrounding a large hollow interior. These properties make vaults an ideal candidate for development into a drug delivery vehicle. In this study, the first example of using vaults towards this goal is reported. Recombinant vaults are engineered to encapsulate the highly insoluble and toxic hydrophobic compound all-trans retinoic acid (ATRA) using a vault-binding lipoprotein complex that forms a lipid bilayer nanodisk. These recombinant vaults offer protection to the encapsulated ATRA from external elements. Furthermore, a cryo-electron tomography (cryo-ET) reconstruction shows the vault-binding lipoprotein complex sequestered within the vault lumen. Finally, these ATRA-loaded vaults show enhanced cytotoxicity against the hepatocellular carcinoma cell line HepG2. The ability to package therapeutic compounds into the vault is an important achievement toward their development into a viable and versatile platform for drug delivery., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The cellular vaults have been described for the first time in 1986 as ribonucleoprotein complexes composed of three proteins, MVP, TEP1 and vPARP and several vRNA strains. Biochemical and structural studies revealed their ubiquitous existence in the cytoplasm of many eukaryotic cells and their barrel-like structure indicating their engagement in the intracellular transport. Furthermore, the high homology between MVP and LRP which was already known to be involved in multidrug resistance mechanism opened a discussion about the role of vaults in both normal and cancer cells. The histopathology research demonstrated an increased amount of MVP/LRP proteins in the cancer as well as showed translocation possibility between cytoplasm and nuclear envelope, which can be of crucial point in the prevention of nucleus against anticancer drugs.

Vaults are naturally occurring ribonucleoprotein particles with an enormous interior volume, large enough to encapsulate hundreds of proteins. They are highly conserved and are present in nearly all eukaryotic cells ranging from 10(4) to 10(7) particles per cell. Recombinant vaults can be produced in vitro and engineered to allow cell targeting and protein packaging. These nanometer-sized particles have many desirable characteristics that may give them advantages for use as drug delivery vehicles. Using photoactivatable green fluorescent protein (PAGFP) labeled vaults, we demonstrate that the particles rapidly diffuse throughout the cytoplasm following single pixel photoactivation in live cells. Their in vivo movement remained relatively unchanged despite exposure to a variety of cellular stresses, suggesting that vaults are largely unconstrained in the cytoplasm. Fluorescence resonance energy transfer (FRET) was observed from polyethylene glycol (PEG) fused hybrid cells that expressed either CFP or YFP labeled vaults, indicating that vaults can exchange major vault protein (MVP) subunits in vivo. Investigation into the mechanism of this exchange in vitro using recombinant vaults demonstrated that they were capable of rapidly separating at the particle waist and reassembling back into whole vaults, supporting a half vault exchange mechanism. This data suggests a means whereby vaults can functionally interact with their cellular environment and deliver materials packaged within their interior.

Native vaults are nanoscale particles found abundantly in the cytoplasm of most eukaryotic cells. They have a capsule-like structure with a thin shell surrounding a "hollow" interior compartment. Recombinant vault particles were found to self-assemble following expression of the major vault protein (MVP) in a baculovirus expression system, and these particles are virtually identical to native vaults. Such particles have been recently studied as potential delivery vehicles. In this study, we focus on immobilization of vault particles on a solid substrate, such as glass, as a first step to study their interactions with cells. To this end, we first engineered the recombinant vaults by fusing two different tags to the C-terminus of MVP, a 3 amino acid RGD peptide and a 12 amino acid RGD-strep-tag peptide. We have demonstrated two strategies for immobilizing vaults on solid substrates. The barrel-and-cap structure of vault particles was observed for the first time, by atomic force microscopy (AFM), in a dry condition. This work proved the feasibility of immobilizing vault nanoparticles on a material surface, and the possibility of using vault nanoparticles as localized and sustainable drug carriers as well as a biocompatible surface moiety.

Vaults are ubiquitous ribonucleoprotein complexes involved in a diversity of cellular processes, including multidrug resistance, transport mechanisms and signal transmission. The vault particle shows a barrel-shaped structure organized in two identical moieties, each consisting of 39 copies of the major vault protein MVP. Earlier data indicated that vault halves can dissociate at acidic pH. The crystal structure of the vault particle solved at 8 A resolution, together with the 2.1-A structure of the seven N-terminal domains (R1-R7) of MVP, reveal the interactions governing vault association and provide an explanation for a reversible dissociation induced by low pH. The structural comparison with the recently published 3.5 A model shows major discrepancies, both in the main chain tracing and in the side chain assignment of the two terminal domains R1 and R2.

Vaults are large protein nanocapsules that may be useful as drug delivery vehicles due to their normal presence in humans, their large interior volume, their simple structural composition consisting of multiple copies of one protein, and a recombinant production system that also provides a means to tailor their structure. However, for vaults to be effective in such applications, efficient means to load the interiors of the capsules must be demonstrated. Here we describe the use of a domain derived from a vault lumen-associated protein as a carrier to target both gold nanoclusters and heterologous His-tagged proteins to specific binding sites on the vault interior wall.

Vaults, at 13 MDa, are the largest ribonucleoprotein particles known. In vitro, expression of the major vault protein (MVP) alone in Sf9 insect cells results in the production of recombinant particles with characteristic vault structure. With the ultimate goal of using recombinant vaults as nanocapsules for the delivery of biomolecules, we have employed a variety of spectroscopic techniques (i.e., circular dichroism, fluorescence spectroscopy, and light scattering) along with electron microscopy, to characterize the structural stability of vaults over a wide range of pH (3-8) and temperature (10-90 degrees C). Ten different conformational states of the vaults were identified over the pH and temperature range studied with the most stable region at pH 6-8 below 40 degrees C and least stable at pH 4-6 above 60 degrees C. A unique intermediate molten globulelike state was also identified at pH 6 and approximately 55 degrees C. EM imaging showed the opening of intact vaults into flowerlike structures when transitioning from neutral to acidic pH. This information has potential use in the development of recombinant vaults into nanocapsules for drug delivery since one mechanism by which therapeutic agents entrapped in vaults could be released is through an opening of the intact vault structure.

Nonviral methods of gene delivery possess several advantages over that of viral-based vectors, including having increased safety. However, the ability to achieve effective transport of therapeutic molecules across host cell membranes via nonviral methods remains a significant goal. Cell-derived nanoparticles known as vaults have been proposed as novel candidate transfer vehicles for various foreign molecules. Recombinant vault particles enter cells via macropinocytosis or phagocytosis but lack demonstrable membrane penetrating activity. To explore the feasibility of improving vault penetration into target cells, we incorporated the membrane lytic domain of adenovirus protein VI (pVI) into the interior of recombinant vault particles via fusion to the vault poly(ADP-ribose) polymerase (VPARP) interaction domain. The membrane lytic activity of the pVI domain was retained upon incorporation into vault particles. Moreover, internalization of vault-pVI complexes into murine macrophages promoted co-delivery of a soluble ribotoxin or a cDNA plasmid encoding GFP. These findings indicate that vault particles can be modified to enhance cell transfer of selected biomolecules.

Vaults are among the largest cytoplasmic ribonucleoprotein particles and are found in numerous eukaryotic species. Roles in multidrug resistance and innate immunity have been suggested, but the cellular function remains unclear. We have determined the x-ray structure of rat liver vault at 3.5 angstrom resolution and show that the cage structure consists of a dimer of half-vaults, with each half-vault comprising 39 identical major vault protein (MVP) chains. Each MVP monomer folds into 12 domains: nine structural repeat domains, a shoulder domain, a cap-helix domain, and a cap-ring domain. Interactions between the 42-turn-long cap-helix domains are key to stabilizing the particle. The shoulder domain is structurally similar to a core domain of stomatin, a lipid-raft component in erythrocytes and epithelial cells.

The unique and evolutionary highly conserved major vault protein (MVP) is the main component of ubiquitous, large cellular ribonucleoparticles termed vaults. The 100 kDa MVP represents more than 70% of the vault mass which contains two additional proteins, the vault poly (ADP-ribose) polymerase (vPARP) and the telomerase-associated protein 1 (TEP1), as well as several short untranslated RNAs (vRNA). Vaults are almost ubiquitously expressed and, besides chemotherapy resistance, have been implicated in the regulation of several cellular processes including transport mechanisms, signal transmissions and immune responses. Despite a growing amount of data from diverse species and systems, the definition of precise vault functions is still highly complex and challenging. Here we review the current knowledge on MVP and vaults with focus on regulatory functions in intracellular signal transduction and immune defence.

Vault is a 12.9 MDa ribonucleoprotein particle with a barrel-like shape, two protruding caps and an invaginated waist structure that is highly conserved in a wide variety of eukaryotes. Multimerization of the major vault protein (MVP) is sufficient to assemble the entire exterior shell of the barrel-shaped vault particle. Multiple copies of two additional proteins, vault poly(ADP-ribose) polymerase (VPARP) and telomerase-associated protein 1 (TEP1), as well as a small vault RNA (vRNA), are also associated with vault. Here, the crystallization of vault particles is reported. The crystals belong to space group C2, with unit-cell parameters a = 708.0, b = 385.0, c = 602.9 angstroms, beta = 124.8 degrees . Rotational symmetry searches based on the R factor and correlation coefficient from noncrystallographic symmetry (NCS) averaging indicated that the particle has 39-fold dihedral symmetry.

It is supposed that ribonucleoprotein particle vault is involved in detoxification processes and thus is related to multidrug resistance. The vault is composed of three proteins and three vault RNAs, hvg-1, -2 and -3. The direct interactions between vault components and drugs were not reported. Recently, we revealed the interactions between vault RNAs and mitoxantrone. Here, we examined the interactions between hvg-2 and six antitumor drugs by a chemical shift perturbation method of NMR. It was found that in addition to mitoxantrone, hvg-2 can interact with two drugs basically in the same way using the same site. The difference in the affinity was also noticed among three drugs. Hvg-2 did not bind to the other three drugs. It is suggested that the common or closely related chemical structure of the positive three drugs is recognized by vault RNA.

Vaults are the largest known cytoplasmic ribonucleoprotein structures and may function in innate immunity. The vault shell self-assembles from 96 copies of major vault protein and encapsulates two other proteins and a small RNA. We crystallized rat liver vaults and several recombinant vaults, all among the largest non-icosahedral particles to have been crystallized. The best crystals thus far were formed from empty vaults built from a cysteine-tag construct of major vault protein (termed cpMVP vaults), diffracting to about 9-A resolution. The asymmetric unit contains a half vault of molecular mass 4.65 MDa. X-ray phasing was initiated by molecular replacement, using density from cryo-electron microscopy (cryo-EM). Phases were improved by density modification, including concentric 24- and 48-fold rotational symmetry averaging. From this, the continuous cryo-EM electron density separated into domain-like blocks. A draft atomic model of cpMVP was fit to this improved density from 15 domain models. Three domains were adapted from a nuclear magnetic resonance substructure. Nine domain models originated in ab initio tertiary structure prediction. Three C-terminal domains were built by fitting poly-alanine to the electron density. Locations of loops in this model provide sites to test vault functions and to exploit vaults as nanocapsules.

Vaults are self-assembled ribonucleoprotein nanocapsules that consist of multiple copies of three proteins (major vault protein, VPARP, and TEP1) and an untranslated RNA. Although their function has not been determined, vaults are found in nearly all eukaryotic cells. This study describes the use of fluorescence spectroscopy, multiangle laser light scattering (MALLS), and the quartz crystal microbalance (QCM) as tools in investigating recombinant vault conformational change in response to a varied solution pH. Identification of conditions for reversible vault disassembly and reassembly could enable application of these nanocapsules in drug delivery and in nanomaterials synthesis. Initial monitoring of changes in the intrinsic fluorescence intensity of vaults showed a 60% increase at pH 3.4 compared to that at pH 6.5, suggesting vaults exhibit a more open conformation at low pH. Fluorescence quenching studies provided further evidence of a vault structural change at low pH. MALLS data suggested a decrease in molecular mass accompanied by a clear increase in the radius of gyration as the solution pH was shifted from 6.5 to 3.4. This result prompted the hypothesis that vaults dissociate at least partially at low pH. Using the QCM to study adsorption of the vault onto self-assembled monolayers, data that suggest vault dissociation at low pH, even when the vault is in an adsorbed state, were also obtained. Finally, transmission electron microscopy (TEM) of negatively stained vaults at pH 6.5 and 3.4 confirmed the fluorescence spectroscopy, MALLS, and QCM findings by providing visual evidence that vaults disassemble into halves as the solution pH is lowered from 6.5 to 3.4.

Vaults are barrel-shaped ribonucleoprotein particles that are abundant in certain tumors and multidrug resistant cancer cells. Prokaryotic relatives of the major vault protein, MVP, have not been identified. We used sequence analysis and molecular modeling to show that MVP and the toxic anion resistance protein, TelA of Rhodobacter sphaeroides strain 2.4.1, share a novel fold that consists of a three-stranded antiparallel beta-sheet. Because of this strong structural correspondence, we examined whether mammalian cell vaults respond to tellurite treatment. In the presence of the oxyanion tellurite, large vault aggregates, or vaultosomes, appear at the cell periphery in 15 min or less. Vaultosome formation is temperature-dependent, reversible, and occurs in normal human umbilical vein endothelial cells as well as transformed HeLa cervical cancer cells. Vaultosome formation is not restricted to tellurite and occurs in the presence of other toxic oxyanions (selenate, selinite, arsenate, arsenite, vanadate). In addition, vaultosomes form independently from other stress-induced ribonucleoprotein complexes, stress granules and aggresomes. Vaultosome formation is therefore a unique cellular response to an environmental toxin.

We have recently shown in MCF-7 cells that nuclear phosphatase and tensin homologue deleted on chromosome 10 (PTEN) down-regulates phosphorylation of p44/42 and cyclin D1 and induces G(1) cell cycle arrest, whereas cytoplasmic PTEN down-regulates phosphorylation of Akt, up-regulates p27, and induces apoptosis. In this manner, nucleocytoplasmic partitioning of PTEN seems to differentially regulate the cell cycle and apoptosis. We have also reported that PTEN has nuclear localization signal-like sequences required for major vault protein (MVP)-mediated nuclear translocation. To date, several other proteins are reported to interact with MVP, including extracellular signal-regulated kinases and steroid receptors, suggesting that MVP is likely to be involved in signal transduction through nucleocytoplasmic transport. However, the exact mechanism of MVP-mediated nucleocytoplasmic shuttling remains elusive. PTEN reportedly interacts in vitro with the EF hand-like motif of MVP in a Ca(2+)-dependent manner. The current study shows that small interfering RNA-mediated MVP silencing decreases the nuclear localization of PTEN and increases phosphorylation of nuclear p44/42. We show in situ that PTEN-MVP interaction is Ca(2+) dependent and is abolished by Mg(2+). Nuclear localization of PTEN is decreased by increasing Ca(2+) levels in culture medium in a dose-dependent manner. Ca(2+) ionophore A23187 increases nuclear localization of PTEN and decreases phosphorylation of nuclear p44/42. Finally, we show that Ca(2+)-dependent PTEN-MVP interaction is not related to MVP's tyrosil phosphorylation but rather due to its conformational modification. Our observations suggest that Ca(2+) regulates PTEN's nuclear entry through a tyrosil phosphorylation-independent conformational change in MVP. Collectively, our data present evidence of a novel crosstalk between the Ca(2+) signaling-mediated regulation of the cell cycle and MVP-mediated nuclear PTEN localization and function.

Vaults are 13 million Da ribonucleoprotein particles with a highly conserved structure. Expression and assembly by multimerization of an estimated 96 copies of a single protein, termed the major vault protein (MVP), is sufficient to form the minimal structure and entire exterior shell of the barrel-shaped vault particle. Multiple copies of two additional proteins, VPARP and TEP1, and a small untranslated vault RNA are also associated with vaults. We used the Sf9 insect cell expression system to form MVP-only recombinant vaults and performed a series of protein-mixing experiments to test whether this particle shell is able to exclude exogenous proteins from interacting with the vault interior. Surprisingly, we found that VPARP and TEP1 are able to incorporate into vaults even after the formation of the MVP vault particle shell is complete. Electrospray molecular mobility analysis and spectroscopic studies of vault-interacting proteins were used to confirm this result. Our results demonstrate that the protein shell of the recombinant vault particle is a dynamic structure and suggest a possible mechanism for in vivo assembly of vault-interacting proteins into preformed vaults. Finally, this study suggests that the vault interior may functionally interact with the cellular milieu.

Molecular cloning of feline lung resistance-related protein (LRP) was performed to evaluate the relationship between its expression level and drug resistance against chemotherapeutics. The nucleotide sequence of the coding region of feline LRP cDNA was found to be 2670-bp long and to show 84.2-92.6% homology to its human, mouse, and rat counterparts. The expression level of feline LRP mRNA was relatively high in lung, jejunum, and colon. An adriamycin (ADM)-resistant feline lymphoma subline, FT-1/ADM, showed a high level of MDR1 mRNA expression compared with parental FT-1 cells. However, no relationship was observed between the drug-resistant phenotype and the LRP mRNA expression level. Although no direct contribution of LRP to the development of the drug-resistant phenotype was observed, further investigation is advisable.

Major vault protein (MVP) is the main constituent of vaults, large ribonucleoprotein particles implicated in resistance to cancer therapy and correlated with poor survival prognosis. Here, we report the structure of the main repeat element in human MVP. The approximately 55 amino acid residue MVP domain has a unique, novel fold that consists of a three-stranded antiparallel beta-sheet. The solution NMR structure of a two-domain fragment reveals the interdomain contacts and relative orientations of the two MVP domains. We use these results to model the assembly of 672 MVP domains from 96 MVP molecules into the ribs of the 13MDa vault structure. The unique features include a thin, skin-like structure with polar residues on both the cytoplasmic and internal surface, and a pole-to-pole arrangement of MVP molecules. These studies provide a starting point for understanding the self-assembly of MVP into vaults and their interactions with other proteins. Chemical shift perturbation studies identified the binding site of vault poly(ADP-ribose) polymerase, another component of vault particles, indicating that MVP domains form a new class of interaction-mediating modules.

Human vaults are the largest cytoplasmic ribonucleoprotein and are overexpressed in cancer cells. Vaults reportedly function in the extrusion of xenobiotics from the nuclei of resistant cells, but the interactions of xenobiotics with the vault-associated proteins or non-coding RNAs have never been directly observed. In the present study, we show that vault RNAs (vRNAs), specifically the hvg-1 and hvg-2 RNAs, bind to a chemotherapeutic compound, mitoxantrone. Using an in-line probing assay (spontaneous transesterification of RNA linkages), we have identified the mitoxantrone binding region within the vRNAs. In addition, we analyzed the interactions between vRNAs and mitoxantrone in the cellular milieu, using an in vitro translation inhibition assay. Taken together, our results clearly suggest that vRNAs have the ability to bind certain chemotherapeutic compounds and these interactions may play an important role in vault function, by participating in the export of toxic compounds.

Vaults are large ribonucleoprotein particles found in a wide variety of eukaryotes. When imaged by electron-microscopy vaults present a strikingly conserved barrel-shaped structure with an invaginated waist and two protruding caps. In this work, we present two dimensional (2D) and three dimensional (3D) crystals of naturally produced vaults in murine and monkey cells, respectively. The 2D-crystals presented a hexagonal packing with the lattice parameter defined by the diameter of the vault barrel. Fourier transforms from images of the negatively stained 2D-crystals showed spots till about 45 A resolution. The 3D-crystals reached about 0.15 x 0.15 x 0.02 mm3 in size and presented a flat triangular morphology with well-developed faces. The preliminary characterization of these 3D-crystals, which diffract very weakly to approximately 10 A resolution, suggests a trigonal packing with the R32 space group symmetry. The 3D-crystals appear to be formed by adding layers of vaults, which retain the hexagonal organization seen in the 2D-crystals, with relative shifts that maximize the interdigitation of particles in adjacent layers. Accurate crystal symmetry in the 2D- and 3D-crystals requires neighbor particles interacting according to a 6-fold and a 3-fold dihedral symmetry, respectively. Compatibility with the reported 8-fold symmetry would imply multiples of 24-fold rotational symmetry, in agreement with the recently proposed 48-fold dihedral symmetry for reconstituted recombinant vaults.

One of the central issues facing the emerging field of nanotechnology is cellular compatibility. Nanoparticles have been proposed for diagnostic and therapeutic applications, including drug delivery, gene therapy, biological sensors, and controlled catalysis. Viruses, liposomes, peptides, and synthetic and natural polymers have been engineered for these applications, yet significant limitations continue to prevent their use. Avoidance of the body's natural immune system, lack of targeting specificity, and the inability to control packaging and release are remaining obstacles. We have explored the use of a naturally occurring cellular nanoparticle known as the vault, which is named for its morphology with multiple arches reminiscent of cathedral ceilings. Vaults are 13-MDa ribonucleoprotein particles with an internal cavity large enough to sequester hundreds of proteins. Here, we report a strategy to target and sequester biologically active materials within the vault cavity. Attachment of a vault-targeting peptide to two proteins, luciferase and a variant of GFP, resulted in their sequestration within the vault cavity. The targeted proteins confer enzymatic and fluorescent properties on the recombinant vaults, both of which can be detected by their emission of light. The modified vaults are compatible with living cells. The ability to engineer vault particles with designed properties and functionalities represents an important step toward development of a biocompatible nanocapsule.

Background: Vaults are intriguing ribonucleoprotein assemblies with an unknown function that are conserved among higher eukaryotes. The Pacific coast sea urchin, Strongylocentrotus purpuratus, is an invertebrate model organism that is evolutionarily closer to humans than Drosophila and C. elegans, neither of which possesses vaults. Here we compare the structures of sea urchin and mammalian vaults and analyze the subcellular distribution of vaults during sea urchin embryogenesis., Results: The sequence of the sea urchin major vault protein (MVP) was assembled from expressed sequence tags and genome traces, and the predicted protein was found to have 64% identity and 81% similarity to rat MVP. Sea urchin MVP includes seven approximately 50 residue repeats in the N-terminal half of the protein and a predicted coiled coil domain in the C-terminus, as does rat MVP. A cryoelectron microscopy (cryoEM) reconstruction of isolated sea urchin vaults reveals the assembly to have a barrel-shaped external structure that is nearly identical to the rat vault structure. Analysis of the molecular composition of the sea urchin vault indicates that it contains components that may be homologs of the mammalian vault RNA component (vRNA) and protein components (VPARP and TEP1). The sea urchin vault appears to have additional protein components in the molecular weight range of 14-55 kDa that might correspond to molecular contents. Confocal experiments indicate a dramatic relocalization of MVP from the cytoplasm to the nucleus during sea urchin embryogenesis., Conclusions: These results are suggestive of a role for the vault in delivering macromolecules to the nucleus during development.