Search Results (6)

Vault particles (VPs) are highly conserved large ribonucleoprotein complexes found exclusively in eukaryotes. They play critical roles in various cellular processes, but their involvement in cancer progression and multidrug resistance (MDR) is the most extensively studied. VPs are composed of the major vault protein (MVP), vault RNAs (vtRNAs), vault poly (ADP-ribose) polymerase, and telomerase-associated protein-1. These components are involved in the regulation of signaling pathways that affect tumor survival, proliferation, and metastasis. MVP has been associated with aggressive tumor phenotypes, while vtRNAs modulate cell proliferation, apoptosis, and autophagy. VPs also contribute to MDR by sequestering chemotherapeutic agents, altering their accumulation in the nucleus, and regulating lysosomal dynamics. Furthermore, small vault RNA-derived fragments participate in gene silencing and intercellular communication, reinforcing the role of precursors of vtRNAs in cancer development. Beyond their biological roles, VPs present a promising platform for drug delivery, due to their unique ability to encapsulate a wide range of biomolecules and therapeutic agents, followed by controlled release. This review compiles data from PubMed and Scopus, with a literature search conducted up until December 2024, highlighting current knowledge regarding VPs and their crucial involvement in cancer-related mechanisms and their applications in overcoming cancer drug resistance. Full article

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. Full article

The vault nanoparticle is a eukaryotic assembly consisting of 78 copies of the 99-kDa major vault protein. They generate two cup-shaped symmetrical halves, which in vivo enclose protein and RNA molecules. Overall, this assembly is mainly involved in pro-survival and cytoprotective functions. It also holds a remarkable biotechnological potential for drug/gene delivery, thanks to its huge internal cavity and the absence of toxicity/immunogenicity. The available purification protocols are complex, partly because they use higher eukaryotes as expression systems. Here, we report a simplified procedure that combines human vault expression in the yeast Komagataella phaffii, as described in a recent report, and a purification process we have developed. This consists of RNase pretreatment followed by size-exclusion chromatography, which is far simpler than any other reported to date. Protein identity and purity was confirmed by SDS-PAGE, Western blot and transmission electron microscopy. We also found that the protein displayed a significant propensity to aggregate. We thus investigated this phenomenon and the related structural changes by Fourier-transform spectroscopy and dynamic light scattering, which led us to determine the most suitable storage conditions. In particular, the addition of either trehalose or Tween-20 ensured the best preservation of the protein in native, soluble form. Full article

Vaults are protein nanoparticles that are found in almost all eukaryotic cells but are absent in prokaryotic ones. Due to their properties (nanometric size, biodegradability, biocompatibility, and lack of immunogenicity), vaults show enormous potential as a bio-inspired, self-assembled drug-delivery system (DDS). Vault architecture is directed by self-assembly of the “major vault protein” (MVP), the main component of this nanoparticle. Recombinant expression (in different eukaryotic systems) of the MVP resulted in the formation of nanoparticles that were indistinguishable from native vaults. Nowadays, recombinant vaults for different applications are routinely produced in insect cells and purified by successive ultracentrifugations, which are both tedious and time-consuming strategies. To offer cost-efficient and faster protocols for nanoparticle production, we propose the production of vault-like nanoparticles in Escherichia coli cells, which are still one of the most widely used prokaryotic cell factories for recombinant protein production. The strategy proposed allowed for the spontaneous encapsulation of the engineered cargo protein within the self-assembled vault-like nanoparticles by simply mixing the clarified lysates of the producing cells. Combined with well-established affinity chromatography purification methods, our approach contains faster, cost-efficient procedures for biofabrication in a well-known microbial cell factory and the purification of “ready-to-use” loaded protein nanoparticles, thereby opening the way to faster and easier engineering and production of vault-based DDSs. Full article

The vault nanoparticle is a eukaryotic ribonucleoprotein complex consisting of 78 individual 97 kDa-“major vault protein” (MVP) molecules that form two symmetrical, cup-shaped, hollow halves. It has a huge size (72.5 × 41 × 41 nm) and an internal cavity, wherein the vault poly(ADP-ribose) polymerase (vPARP), telomerase-associated protein-1 (TEP1), and some small untranslated RNAs are accommodated. Plenty of literature reports on the biological role(s) of this nanocomplex, as well as its involvement in diseases, mostly oncological ones. Nevertheless, much has still to be understood as to how vault participates in normal and pathological mechanisms. In this comprehensive review, current understanding of its biological roles is discussed. By different mechanisms, vault’s individual components are involved in major cellular phenomena, which result in protection against cellular stresses, such as DNA-damaging agents, irradiation, hypoxia, hyperosmotic, and oxidative conditions. These diverse cellular functions are accomplished by different mechanisms, mainly gene expression reprogramming, activation of proliferative/prosurvival signaling pathways, export from the nucleus of DNA-damaging drugs, and import of specific proteins. The cellular functions of this nanocomplex may also result in the onset of pathological conditions, mainly (but not exclusively) tumor proliferation and multidrug resistance. The current understanding of its biological roles in physiological and pathological processes should also provide new hints to extend the scope of its exploitation as a nanocarrier for drug delivery. Full article

The use of smart drug delivery systems (DDSs) is one of the most promising approaches to overcome some of the drawbacks of drug-based therapies, such as improper biodistribution and lack of specific targeting. Some of the most attractive candidates as DDSs are naturally occurring, self-assembling protein nanoparticles, such as viruses, virus-like particles, ferritin cages, bacterial microcompartments, or eukaryotic vaults. Vaults are large ribonucleoprotein nanoparticles present in almost all eukaryotic cells. Expression in different cell factories of recombinant versions of the “major vault protein” (MVP) results in the production of recombinant vaults indistinguishable from native counterparts. Such recombinant vaults can encapsulate virtually any cargo protein, and they can be specifically targeted by engineering the C-terminus of MVP monomer. These properties, together with nanometric size, a lumen large enough to accommodate cargo molecules, biodegradability, biocompatibility and no immunogenicity, has raised the interest in vaults as smart DDSs. In this work we provide an overview of eukaryotic vaults as a new, self-assembling protein-based DDS, focusing in the latest advances in the production and purification of this platform, its application in nanomedicine, and the current preclinical and clinical assays going on based on this nanovehicle. Full article