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Multiply responsive protein nanoparticles are interesting for a variety of applications. Herein, we describe the synthesis of a vault nanoparticle that responds to both temperature and pH. Specifically, poly( N -isopropylacrylamide- co -acrylic acid) with a pyridyl disulfide end group was prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization. The polymer had a lower critical solution temperature (LCST) of 31.9 °C at pH 5, 44.0 °C at pH 6 and above 60 °C at pH 7. The polymer was conjugated to human major vault protein (hMVP), and the resulting nanoparticle was analyzed by UV–Vis, dynamic light scattering (DLS) and electron microscopy. The data demonstrated that the poly( N -isopropylacrylamide- co -acrylic acid)-vault conjugate did not respond to temperatures below 60 °C at pH 7, while the nanoparticles reversibly aggregated at pH 6. Furthermore, it was shown that the vault nanoparticle structure remained intact for at least three heat and cooling cycles. Thus, these dually responsive nanoparticles may serve as a platform for drug delivery and other applications. [ABSTRACT FROM AUTHOR]

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]

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]

Microbial enzymes catalyze various reactions inside and outside living cells. Among the widely studied enzymes, fungal enzymes have been used for some of the most diverse purposes, especially in bioremediation, biosynthesis, and many nature-inspired commercial applications. To improve their stability and catalytic ability, fungal enzymes are often immobilized on assorted materials, conventional as well as nanoscale. Recent advances in fungal enzyme immobilization provide effective and sustainable approaches to achieve improved environmental and commercial outcomes. This review aims to provide a comprehensive overview of commonly studied fungal enzymes and immobilization technologies. It also summarizes recent advances involving immobilized fungal enzymes for the degradation or assembly of compounds used in the manufacture of products, such as detergents, food additives, and fossil fuel alternatives. Furthermore, challenges and future directions are highlighted to offer new perspectives on improving existing technologies and addressing unexplored fields of applications. • Immobilized enzymes are emerging as effective biodegradation and biosynthesis agents. • Summary of representative fungal enzymes that were used in immobilized forms. • Thorough overview of industrial and environmental uses of immobilized fungal enzymes. • Future perspectives and challenges of design and applications of immobilized enzymes. [ABSTRACT FROM AUTHOR]

[Display omitted] • Fungal laccases were characterized, produced, and encapsulated in vault nanocages. • Thermal and pH stability of laccase notably improved by vault packaging (vlaccase). • vLaccase was superior to natural laccase in decolorizing and detoxifying dyes. • vLaccase treatment greatly reduced toxicity of synthetic dyes degradation products. This study presents an eco-friendly and efficient technology, using immobilized enzymes, vault-encapsulated laccases (vlaccase), for decolorization and detoxification of dyes. Vault encapsulation remarkably improved the performance of laccase at industrially relevant conditions, including neutral to alkaline pH and relatively high temperatures. Two representative anthraquinone and azo dyes, Reactive Blue 19 and Acid Orange 7, respectively, were rapidly decolorized (72% and 80%) by vlaccase treatment while natural laccase (nlaccase) achieved 40% and 32% decolorization. The toxicity of treated and untreated dyes was tested on model bacterial, algal, and insect cells. The inhibitory effects of dyes towards selected bacteria were reduced in vlaccase-treated samples. The chlorophyll synthesis in algae was less inhibited by dyes after vlaccase treatment. Furthermore, the toxicity of dye degradation products to insect cells was significantly mitigated in the vlaccase group. Collectively, these results indicate that vlaccase is a stable and strong enzymatic system for removing dyes from waters. [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]

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]

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]