Mammalian vaults are ribonucleoprotein (RNP) complexes, composed of a small ribonucleic acid and three proteins 100, 193, 240 kD in size. The 100-kD major vault protein (MVP) accounts for >70% the particle mass. We have identified 193-kD by its interaction with MVP yeast two-hybrid screen confirmed identity peptide sequence analysis. Analysis revealed region ∼350 amino acids that shares 28% catalytic domain poly(ADP-ribose) polymerase (PARP). PARP is nuclear catalyzes formation...

Vaults are 13-MDa ribonucleoprotein particles composed largely of a 104-kDa protein, termed major vault protein or MVP, and small RNA, vRNA. While MVP levels have been found to increase up 15-fold in non-P-glycoprotein multidrug-resistant cell lines, the not investigated. As both function mechanism drug resistance cells unknown, we decided determine whether synthesis was coupled MDR. By cloning human gene for vRNA careful quantitation MDR cells, find that is considerable excess MVP....

Mass spectrometry (MS) and ion mobility with electrospray ionization (ESI) have the capability to measure detect large noncovalent protein-ligand protein-protein complexes. Using an method of gas-phase electrophoretic molecular analysis (GEMMA), protein particles representing a range sizes can be separated by their in air. Highly charged produced from complex solution using manipulated produce singly ions, which quantified mobility. Results ESI-GEMMA our laboratory others were compared other...

Vaults are large cytoplasmic ribonucleoprotein complexes of undetermined function. Mammalian vaults have two high molecular mass proteins 193 and 240 kDa. We identified a partial cDNA encoding the 240-kDa vault protein determined it is identical to mammalian telomerase-associated component, TEP1. TEP1 homolog Tetrahymena p80 telomerase has been shown interact specifically with RNA catalytic subunit hTERT. show that while component particle, no detectable activity. Using yeast three-hybrid...

Vaults are the largest (13 megadalton) cytoplasmic ribonucleoprotein particles known to exist in eukaryotic cells. They have a unique barrel-shaped structure with 8-fold symmetry. Although precise function of vaults is unknown, their wide distribution and highly conserved morphology eukaryotes suggests that essential must be important for function. The 100-kDa major vault protein (MVP) constitutes ∼75% particle mass predicted form central barrel portion vault. To gain insight into mechanisms...

Vaults are large cytoplasmic ribonucleoprotein particles with a sedimentation value of about 150 S. These contain unique small RNA (vault (vRNA)). We have determined the sequence associated vaults purified from both rat and bullfrog. The vRNA is 141 bases in length, whereas bullfrog present as two highly related species 89 94 bases. Despite differences length predicted secondary structures three vRNAs clearly related. All sequences to internal promoter elements necessary for transcription by...

One of the central issues facing emerging field nanotechnology is cellular compatibility. Nanoparticles have been proposed for diagnostic and therapeutic applications, including drug delivery, gene therapy, biological sensors, controlled catalysis. Viruses, liposomes, peptides, synthetic natural polymers engineered these yet significant limitations continue to prevent their use. Avoidance body's immune system, lack targeting specificity, inability control packaging release are remaining...

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 recombinant nanoparticle as vaccine delivery platform against infections requiring T immunity eradication.We encapsulated an immunogenic protein, major outer membrane protein (MOMP) Chlamydia muridarum, within hollow, vault nanocapsules (MOMP-vaults) that were engineered to bind IgG enhanced immunity. Intranasal immunization...

As a naturally occurring nanocapsule abundantly expressed in nearly all-eukaryotic cells, the barrel-shaped vault particle is perhaps an ideal structure to engineer for targeting specific cell types. Recombinant particles self-assemble from 96 copies of major protein (MVP), have dimensions 72.5 × 41 nm, and hollow interior large enough encapsulate hundreds proteins. In this study, three different tags were engineered onto C-terminus MVP: 11 amino acid epitope tag, 33 IgG-binding peptide, 55...

Background Based on our preclinical findings, we are assessing the efficacy of intratumoral injection dendritic cells (DC) transduced with an adenoviral vector expressing secondary lymphoid chemokine (CCL21) gene (Ad-CCL21-DC) in a phase I trial advanced non-small cell lung cancer (NSCLC). While this approach shows immune enhancement, preparation autologous DC for CCL21 genetic modification is cumbersome, expensive and time consuming. We evaluating non-DC based which utilizes vault...

We report a novel approach to new class of bioengineered, monodispersed, self-assembling vault nanoparticles consisting protein shell exterior with lipophilic core interior designed for drug and probe delivery. Recombinant vaults were engineered contain small amphipathic α-helix derived from the nonstructural 5A hepatitis C virus, thereby creating within lumen microenvironment into which compounds could be reversibly encapsulated. Multiple types electron microscopy showed that attachment...

Vault nanoparticles packaged with enzymes were synthesized as agents for efficiently degrading environmental contaminants. Enzymatic biodegradation is an attractive technology in situ cleanup of contaminated environments because enzyme-catalyzed reactions are not constrained by nutrient requirements microbial growth and often have higher rates. However, the limited stability extracellular remains a major challenge practical applications. Encapsulation recognized method to enhance enzymatic...

Vaults and telomerase are ribonucleoprotein (RNP) particles that share a common protein subunit, TEP1. Although its role in either complex has not yet been defined, TEP1 shown to interact with the mouse RNA several of human vault RNAs yeast three-hybrid assay. An mTep1(-/-) was previously generated which resulted no apparent change telomere length or activity six generations mTep1-deficient mice. Here we show levels association RNP also unaffected vaults purified from livers mice appear...

Vaults are ribonucleoprotein complexes comprised of the 100 kDa major vault protein (MVP), 2 high m.w. proteins p193 (VPARP) and p240 (TEP1) an untranslated small RNA (vRNA). Increased levels MVP, vault-associated vRNA vaults have been linked directly to non-P-glycoprotein–mediated multidrug resistance (MDR). To further characterize putative role in MDR, expression all were examined various MDR cell lines. Subcellular fractionation particles revealed that 3 increased cells compared parental,...

TEP1 is a mammalian telomerase-associated protein with similarity to the Tetrahymena telomerase p80. Like p80, associated activity and reverse transcriptase, it specifically interacts RNA. To determine role of mTep1 in function vivo, we generated mouse embryonic stem (ES) cells mice lacking mTep1. ThemTep1-deficient (mTep1−/−) were viable bred for seven successive generations no obvious phenotypic abnormalities. All murine tissues from mTep1−/− possessed level comparable that wild-type mice....

The vault complex is a ubiquitous 13-MDa ribonucleoprotein assembly, composed of three proteins (TEP1, 240 kDa; VPARP, 193 and MVP, 100 kDa) that are highly conserved in eukaryotes an untranslated RNA (vRNA). has been shown to affect multidrug resistance cancer cells, one particular component, thought play role the transport drug from nucleus. To locate position vRNA, vaults were treated with RNases, cryo-electron microscopy (cryo-EM) was performed on resulting complexes. Using...

Vaults are 13 million Da ribonucleoprotein particles with a highly conserved structure. Expression and assembly by multimerization of an estimated 96 copies single protein, termed the major vault protein (MVP), is sufficient to form minimal structure entire exterior shell barrel-shaped particle. Multiple two additional proteins, VPARP TEP1, small untranslated RNA also associated vaults. We used Sf9 insect cell expression system MVP-only recombinant vaults performed series protein-mixing...

Vault nanoparticles were engineered to enhance their escape from the endosomal compartment by fusing a membrane lytic peptide derived adenovirus protein VI (pVI) N-terminus of major vault form pVI-vaults. We demonstrate that these pVI-vaults disrupt using three different experimental protocols including (1) enhancement DNA transfection, (2) co-delivery cytosolic ribotoxin, and (3) direct visualization fluorescence. Furthermore, targeting vaults specific cell surface epidermal growth factor...

Abstract The vault nanoparticle is one of the largest known ribonucleoprotein complexes in sub‐100 nm range. Highly conserved and almost ubiquitously expressed eukaryotes, vaults form a large nanocapsule with barrel‐shaped morphology surrounding hollow interior. These properties make an ideal candidate for development into drug delivery vehicle. In this study, first example using towards goal reported. Recombinant are engineered to encapsulate highly insoluble toxic hydrophobic compound all‐...

Ribosomes are molecular machines that function in polyribosome complexes to translate genetic information, guide the synthesis of polypeptides, and modulate folding nascent proteins. Here, we report a surprising for polyribosomes as result systematic examination assembly large ribonucleoprotein complex, vault particle. Structural functional evidence points model whereby acts like 3D nanoprinter direct ordered translation multi-subunit homopolymer, process which refer templating....

Background Modifications of adjuvants that induce cell-mediated over antibody-mediated immunity is desired for development vaccines. Nanocapsules have been found to be viable and are amenable engineering immune responses. We previously showed natural nanocapsules called vaults can genetically engineered elicit Th1 protection from a mucosal bacterial infection. The purpose our study was characterize produced in response OVA within vault nanoparticles compare it another nanocarrier....