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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°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°C and least stable at pH 4–6 above 60°C. A unique intermediate molten globulelike state was also identified at pH 6 and ∼55°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. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:1376–1386, 2009 [ABSTRACT FROM AUTHOR]

Background: Generation of robust cell-mediated immune responses at mucosal surfaces while reducing overall inflammation is a primary goal for vaccination. Here we report the use of a recombinant nanoparticle as a vaccine delivery platform against mucosal infections requiring T cell-mediated immunity for eradication. Methodology/Principal Findings: We encapsulated an immunogenic protein, the major outer membrane protein (MOMP) of Chlamydia muridarum, within hollow, vault nanocapsules (MOMP-vaults) that were engineered to bind IgG for enhanced immunity. Intranasal immunization (i.n) with MOMP-vaults induced anti-chlamydial immunity plus significantly attenuated bacterial burden following challenge infection. Vault immunization induced anti-chlamydial immune responses and inflammasome formation but did not activate toll-like receptors. Moreover, MOMP-vault immunization enhanced microbial eradication without the inflammation usually associated with adjuvants. Conclusions/Significance: Vault nanoparticles containing immunogenic proteins delivered to the respiratory tract by the i.n. route can act as ''smart adjuvants'' for inducing protective immunity at distant mucosal surfaces while avoiding destructive inflammation. [ABSTRACT FROM AUTHOR]

A draft atomic structure has been determined for the 9.3-MDa protein shell of the vault cytoplasmic particle, revealing stave-like polypeptides forming the barrel-like structure of the vault. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 nano-particle 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. [ABSTRACT FROM AUTHOR]

Vault RNA (vRNA) genes have been cloned from several vertebrates including rat, mouse, and humans. Their copy numbers vary, as does the length of the encoded RNA. We have determined that the mouse genome contains two vRNA genes; one is expressed the other is a pseudogene. In vitro transcription of the rat vRNA gene by RNA polymerase III has previously been shown to be dependent on a combination of both external and internal promoter sequence elements. By comparing the upstream regions of the vertebrate vRNA genes, a 25 bp conserved sequence and a TATA box can be identified. Furthermore, the unique arrangement of the internal promoter boxes (one A and two B boxes) is conserved in the expressed human vRNA genes even though a new RNA polymerase III termination sequence has evolved between the two B boxes. [Copyright &y& Elsevier]

Background: Ribonucleoproteins carry out a variety of important tasks in the cell. In this study we show that a number of these contain a novel module, that we speculate mediates RNA-binding. Results: The TROVE module -- Telomerase, Ro and Vault module -- is found in TEP1 and Ro60 the protein components of three ribonucleoprotein particles. This novel module, consisting of one or more domains, may be involved in binding the RNA components of the three RNPs, which are telomerase RNA, Y RNA and vault RNA. A second conserved region in these proteins is shown to be a member of the vWA domain family. The vWA domain in TEP1 is closely related to the previously recognised vWA domain in VPARP a second component of the vault particle. This vWA domain may mediate interactions between these vault components or bind as yet unidentified components of the RNPs. Conclusions: This work suggests that a number of ribonucleoprotein components use a common RNA-binding module. The TROVE module is also found in bacterial ribonucleoproteins suggesting an ancient origin for these ribonucleoproteins. [ABSTRACT FROM AUTHOR]

Investigates the in vivo role of TEP1 protein in the stabilization and recruitment of the vault RNA to the vault particle. Role of TEP1 in the telomerase/vault complex; Effect of three-dimensional reconstruction cap density of mutant Tep1; Components of the cytoplasmic ribonucleoprotein complexes.

[Display omitted] • A novel approach to immobilize proteins in biomimetic silica was developed. • A bionanocapsule was used to encapsulate proteins and induce biosilicification. • The bionanocapsule protected proteins from inactivation during silica formation. • A nearly 100% immobilization efficiency was achieved for various proteins. • Immobilized proteins showed higher stability and remarkable reusability. Silica materials are attractive protein supports for applications in catalysis, sensing, water purification, and protein therapy. However, current approaches for loading proteins in silica are limited by inefficient protein immobilization, unintended leaching, the requirement for large quantities of silica precipitants, and associated protein inactivation. Here, we report a highly efficient and robust strategy that directly synthesizes protein-embedded silica via a vault protein nanocapsule-templated biomimetic silicification route. The vaults serve as nucleation sites for the deposition and condensation of silica precursors to form vault/silica composites. Using vaults encapsulated with proteins as the templating agent enables direct embedding of proteins in silica composites with nearly 100% immobilization efficiency. The vaults also act as protective shelters to preserve high protein activity during silica condensation. With the dual advantages of vault templating and protection, the proteins immobilized via this strategy retained over 90% activity after 30 reuse cycles and exhibited high activity, minimal leaching, and improved stability, which would be valuable in a broad variety of medical, industrial, and environmental applications. [ABSTRACT FROM AUTHOR]

Analyzes the interaction of the 193-kD vault protein, a nucleoprotein, with the 100-kD major vault proteins. Composition of mammalian vaults; Usage of peptide sequence analysis; Formation of adenosine diphosphate-ribose polymers in response to DNA damage; Catalytic domain of 193-kD; Immunofluorescence and biochemical data of 193-kD.

Vault RNAs, found in vault ribonucleoprotein complexes, are known to be one of many types of small noncoding RNAs (ncRNAs), but their specific function is not known. A new study identifies a small ncRNA from Trypanosome brucei as a vault RNA (vtRNA) based on sequence analysis and its association with the canonical vault component TEP1. Down-regulation of T. brucei vtRNA impairs mRNA splicing in a permeabilized cell system, suggesting new roles for these enigmatic biomolecules. [ABSTRACT FROM AUTHOR]

Vault poly(ADP-ribose) polymerase (VPARP) was originally identified as a minor protein component of the vault ribonucleoprotein particle, which may be involved in molecular assembly or subcellular transport. In addition to the association of VPARP with the cytoplasmic vault particle, subpopulations of VPARP localize to the nucleus and the mitotic spindle, indicating that VPARP may have other cellular functions. We found that VPARP was associated with telomerase activity and interacted with exogenously expressed telomerase-associated protein 1 (TEP1) in human cells. To study the possible role of VPARP in telomerase and vault complexes in vivo, mVparp-deficient mice were generated. Mice deficient in mVparp were viable and fertile for up to five generations, with no apparent changes in telomerase activity or telomere length. Vaults purified from mVparpdeficient mouse liver appeared intact, and no defect in association with other vault components was observed. Mice deficient in mTep1, whose disruption alone does not affect telomere function but does affect the stability of vault RNA, showed no additional telomerase or telomere-related phenotypes when the mTep1 deficiency was combined with an mVparp deficiency. These data suggest that murine mTep1 and mVparp, alone or in combination, are dispensable for normal development, telomerase catalysis, telomere length maintenance, and vault structure in vivo. [ABSTRACT FROM AUTHOR]

Multi-drug-resistant cancer cells frequently express elevated levels of ribonucleoprotein complexes termed vaults. The increased expression of vault proteins and their mRNAs has led to the suggestion that vaults may play a direct role in preventing drug toxicity. To further understand vault component up-regulation, the three proteins that comprise the vault, the major vault protein (MVP), vault poly(ADP-ribose) polymerase (VPARP), and telomerase-associated protein-1 (TEP1), were examined with respect to gene amplification and drug-induced chromatin remodeling. Gene amplification was not responsible for increased vault component levels in multi-drug-resistant cancer cell lines. The TATA-less murine MVP and human VPARP promoters were identified and functionally characterized. There was no significant activation of either the MVP or VPARP promoters in drug-resistant cell lines in comparison to their parental, drug-sensitive counterparts. Treatment of various cell lines with sodium butyrate, an inhibitor of histone deacetylase (HDAC), led to an increase in vault component protein levels. Furthermore, treatment with trichostatin A (TSA), a more specific inhibitor of HDAC, caused an increase in MVP protein, mRNA, and promoter activity. These results suggest that up-regulation of MVP in multi-drug resistance (MDR) may involve chromatin remodeling. [Copyright &y& Elsevier]

The major vault protein (MVP) mediates diverse cellular responses, including cancer cell resistance to chemotherapy and protection against inflammatory responses to Pseudomonas aeruginosa. Here, we report the use of photoactive probes to identify MVP as a target of the N-(3-oxo-dodecanoyl) homoserine lactone (C12), a quorum sensing signal of certain proteobacteria including P. aeruginosa. A treatment of normal and cancer cells with C12 or other N-acyl homoserine lactones (AHLs) results in rapid translocation of MVP into lipid raft (LR) membrane fractions. Like AHLs, inflammatory stimuli also induce LR-localization of MVP, but the C12 stimulation reprograms (functionalizes) bioactivity of the plasma membrane by recruiting death receptors, their apoptotic adaptors, and caspase-8 into LR. These functionalized membranes control AHL-induced signaling processes, in that MVP adjusts the protein kinase p38 pathway to attenuate programmed cell death. Since MVP is the structural core of large particles termed vaults, our findings suggest a mechanism in which MVP vaults act as sentinels that fine-tune inflammation-activated processes such as apoptotic signaling mediated by immunosurveillance cytokines including tumor necrosis factor-related apoptosis inducing ligand (TRAIL). [ABSTRACT FROM AUTHOR]

The full potential of vaccines relies on development of effective delivery systems and adjuvants and is critical for development of successful vaccine candidates. We have shown that recombinant vaults engineered to encapsulate microbial epitopes are highly stable structures and are an ideal vaccine vehicle for epitope delivery which does not require the inclusion of an adjuvant. We studied the ability of vaults which were engineered for use as a vaccine containing an immunogenic epitope of Chlamydia t rachomatis , polymorphic membrane protein G (PmpG), to be internalized into human monocytes and behave as a “natural adjuvant”. We here show that incubation of monocytes with the PmpG-1-vaults activates caspase-1 and stimulates IL-1β secretion through a process requiring the NLRP3 inflammasome and that cathepsin B and Syk are involved in the inflammasome activation. We also observed that the PmpG-1-vaults are internalized through a pathway that is transiently acidic and leads to destabilization of lysosomes. In addition, immunization of mice with PmpG-1-vaults induced PmpG-1 responsive CD4 + cells upon re-stimulation with PmpG peptide in vitro , suggesting that vault vaccines can be engineered for specific adaptive immune responses. We conclude that PmpG-1-vault vaccines can stimulate NLRP3 inflammasomes and induce PmpG-specific T cell responses. [ABSTRACT FROM AUTHOR]

Background: Modifications of adjuvants that induce cell-mediated over antibody-mediated immunity is desired for development of vaccines. Nanocapsules have been found to be viable adjuvants and are amenable to engineering for desired immune responses. We previously showed that natural nanocapsules called vaults can be genetically engineered to elicit Th1 immunity and protection from a mucosal bacterial infection. The purpose of our study was to characterize immunity produced in response to OVA within vault nanoparticles and compare it to another nanocarrier. Methodology and Principal Findings: We characterized immunity resulting from immunization with the model antigen, ovalbumin (OVA) encased in vault nanocapsules and liposomes. We measured OVA responsive CD8+ and CD4+ memory T cell responses, cytokine production and antibody titers in vitro and in vivo. We found that immunization with OVA contain in vaults induced a greater number of anti-OVA CD8+ memory T cells and production of IFNc plus CD4+ memory T cells. Also, modification of the vault body could change the immune response compared to OVA encased in liposomes. Conclusions/Significance: These experiments show that vault nanocapsules induced strong anti-OVA CD8+ and CD4+ T cell memory responses and modest antibody production, which markedly differed from the immune response induced by liposomes. We also found that the vault nanocapsule could be modified to change antibody isotypes in vivo. Thus it is possible to create a vault nanocapsule vaccine that can result in the unique combination of immunogen-responsive CD8+ and CD4+ T cell immunity coupled with an IgG1 response for future development of vault nanocapsule-based vaccines against antigens for human pathogens and cancer. [ABSTRACT FROM AUTHOR]

Background: Based on our preclinical findings, we are assessing the efficacy of intratumoral injection of dendritic cells (DC) transduced with an adenoviral vector expressing the secondary lymphoid chemokine (CCL21) gene (Ad-CCL21-DC) in a phase I trial in advanced non-small cell lung cancer (NSCLC). While this approach shows immune enhancement, the preparation of autologous DC for CCL21 genetic modification is cumbersome, expensive and time consuming. We are evaluating a non-DC based approach which utilizes vault nanoparticles for intratumoral CCL21 delivery to mediate antitumor activity in lung cancer. Principal Findings: Here we describe that vault nanocapsule platform for CCL21 delivery elicits antitumor activity with inhibition of lung cancer growth. Vault nanocapsule packaged CCL21 (CCL21-vaults) demonstrated functional activity in chemotactic and antigen presenting activity assays. Recombinant vaults impacted chemotactic migration of T cells and this effect was predominantly CCL21 dependent as CCL21 neutralization abrogated the CCL21 mediated enhancement in chemotaxis. Intratumoral administration of CCL21-vaults in mice bearing lung cancer enhanced leukocytic infiltrates (CXCR3+T, CCR7+T, IFNγ+T lymphocytes, DEC205+ DC), inhibited lung cancer tumor growth and reduced the frequencies of immune suppressive cells [myeloid derived suppressor cells (MDSC), T regulatory cells (Treg), IL-10 T cells]. CCL21-vaults induced systemic antitumor responses by augmenting splenic T cell lytic activity against parental tumor cells. Significance: This study demonstrates that the vault nanocapsule can efficiently deliver CCL21 to sustain antitumor activity and inhibit lung cancer growth. The vault nanocapsule can serve as an "off the shelf" approach to deliver antitumor cytokines to treat a broad range of malignancies. [ABSTRACT FROM AUTHOR]

Mass spectrometry (MS) and ion mobility with electrospray ionization (ESI) have the capability to measure and detect large noncovalent protein-ligand and protein-protein complexes. Using an ion mobility method of gas-phase electrophoretic mobility molecular analysis (GEMMA), protein particles representing a range of sizes can be separated by their electrophoretic mobility in air. Highly charged particles produced from a protein complex solution using electrospray can be manipulated to produce singly charged ions, which can be separated and quantified by their electrophoretic mobility. Results from ESI-GEMMA analysis from our laboratory and others were compared with other experimental and theoretically determined parameters, such as molecular mass and cryoelectron microscopy and X-ray crystal structure dimensions. There is a strong correlation between the electrophoretic mobility diameter determined from GEMMA analysis and the molecular mass for protein complexes up to 12 MDa, including the 93 kDa enolase dimer, the 480 kDa ferritin 24-mer complex, the 4.6 MDa cowpea chlorotic mottle virus (CCMV), and the 9 MDa MVP-vault assembly. ESI-GEMMA is used to differentiate a number of similarly sized vault complexes that are composed of different N-terminal protein tags on the MVP subunit. The average effective density of the proteins and protein complexes studied was 0.6 g/cm3. Moreover, there is evidence that proteins and protein complexes collapse or become more compact in the gas phase in the absence of water. [Copyright &y& Elsevier]

The vault is a highly conserved ribonucleoprotein particle found in all higher eukaryotes. It has a barrel-shaped structure and is composed of the major vault protein (MVP); vault poly(ADP-ribose) polymerase (VPARP); telomerase-associated protein 1 (TEP1); and small untranslated RNA (vRNA). Although its strong conservation and high abundance indicate an important cellular role, the function of the vault is unknown. In humans, vaults have been implicated in multidrug resistance during chemotherapy. Recently, assembly of recombinant vaults has been established in insect cells expressing only MVP. Here, we demonstrate that co-expression of MVP with one or both of the other two vault proteins results in their co-assembly into regularly shaped vaults. Particles assembled from MVP with N-terminal peptide tags of various length are compared. Cryoelectron microscopy (cryoEM) and single-particle image reconstruction methods were used to determine the structure of nine recombinant vaults of various composition, as well as wild-type and TEP1-deficient mouse vaults. Recombinant vaults with MVP N-terminal peptide tags showed internal density that varied in size with the length of the tag. Reconstruction of a recombinant vault with a cysteine-rich tag revealed 48-fold rotational symmetry for the vault. A model is proposed for the organization of MVP within the vault with all of the MVP N termini interacting non-covalently at the vault midsection and 48 copies of MVP forming each half vault. CryoEM difference mapping localized VPARP to three density bands lining the inner surface of the vault. Difference maps designed to localize TEP1 showed only weak density inside of the caps, suggesting that TEP1 may interact with MVP via a small interaction region. In the absence of atomic-resolution structures for either VPARP or TEP1, fold recognition methods were applied. A total of 21 repeats were predicted for the TEP1 WD-repeat domain, suggesting an unusually large β-propeller fold. [Copyright &y& Elsevier]

Amino-aromatic compounds, 2-amino-4-nitrotoluene (ANT), and 2,4-diaminotoluene (DAT) are carcinogens and environmentally persistent pollutants. In this study, we investigated their degradation by natural manganese peroxidase (nMnP) derived from Phanerochaete chrysosporium and recombinant manganese peroxidase packaged in vaults (vMnP). Encapsulation of manganese peroxidase (MnP) in ribonucleoprotein nanoparticle cages, called vaults, was achieved by creating recombinant vaults in yeast Pichia pastoris. Vault packaging increased the stability of MnP by locally sequestering multiple copies of the enzyme. Within 96 h, both vMnP and nMnP catalyzed over 72% removal of ANT in-vitro , which indicates that vault packaging did not limit substrate diffusion. It was observed that vMnP was more efficient than nMnP and P. chrysosporium for the catalysis of target contaminants. Only 57% of ANT was degraded by P. chrysosporium even when MnP activity reached about 480 U L−1 in cultures. At 1.5 U L−1 initial activity, vMnP achieved 38% of ANT and 51% of DAT degradation, whereas even 2.7 times higher activity of nMnP showed insignificant biodegradation of both compounds. These results imply that due to protection by vault cages, vMnP has lower inactivation rates. Thus, it works effectively at lower dosage for a longer duration compared to nMnP without requiring frequent replenishment. Collectively, these results indicate that fungal enzymes packaged in vault nanoparticles are more stable and active, and they would be effective in biodegradation of energetic compounds in industrial processes, waste treatment, and contaminated environments. Image 108 • Manganese peroxidase (MnP) was produced, purified, and packaged in vault nanocages. • Vault-MnP degraded 2-amino-4-nitrotoluene (ANT) and 2,4-diaminotoluene (DAT). • ANT and DAT catalysis rates for vault-MnP were higher than those for native MnP. • Vault packaging does not limit substrate diffusion to the enzymes' active sites. • Fungal enzymes packaged in vaults are more active, concentrated, and stable. [ABSTRACT FROM AUTHOR]

Summary 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. [ABSTRACT FROM AUTHOR]