RESUMEN
The heterogeneity of small extracellular vesicles and presence of non-vesicular extracellular matter have led to debate about contents and functional properties of exosomes. Here, we employ high-resolution density gradient fractionation and direct immunoaffinity capture to precisely characterize the RNA, DNA, and protein constituents of exosomes and other non-vesicle material. Extracellular RNA, RNA-binding proteins, and other cellular proteins are differentially expressed in exosomes and non-vesicle compartments. Argonaute 1-4, glycolytic enzymes, and cytoskeletal proteins were not detected in exosomes. We identify annexin A1 as a specific marker for microvesicles that are shed directly from the plasma membrane. We further show that small extracellular vesicles are not vehicles of active DNA release. Instead, we propose a new model for active secretion of extracellular DNA through an autophagy- and multivesicular-endosome-dependent but exosome-independent mechanism. This study demonstrates the need for a reassessment of exosome composition and offers a framework for a clearer understanding of extracellular vesicle heterogeneity.
Asunto(s)
Exosomas/metabolismo , Exosomas/fisiología , Anexina A1/metabolismo , Proteínas Argonautas/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Micropartículas Derivadas de Células/metabolismo , ADN/metabolismo , Exosomas/química , Vesículas Extracelulares , Femenino , Humanos , Lisosomas/metabolismo , Masculino , Proteínas/metabolismo , ARN/metabolismoRESUMEN
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).
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Acil-Butirolactonas/metabolismo , Apoptosis , Bacterias/inmunología , Bacterias/metabolismo , Inmunomodulación , Transducción de Señal , Partículas Ribonucleoproteicas en Bóveda/metabolismo , Fenómenos Fisiológicos Bacterianos , Cromatografía Liquida , Humanos , Vigilancia Inmunológica , Espectrometría de Masas , Proteómica/métodosRESUMEN
PURPOSE: Glioblastoma prognosis remains grim despite maximal, multimodal management. Recent literature has demonstrated an increase in research devoted to experimental treatments, particularly those relying on the foundations of active immunotherapy with promising results. We hypothesize that the utilization of bioengineered recombinant vault nanoparticles coupled with glioma-associated antigens, such as the NY-ESO-1 peptide, may be capable of stimulating native dendritic cell (DC) maturation and inducing an anti-tumor response. METHODS: Immature DCs were cultured from the bone marrow of 4-6-week-old C57BL/6 mice. The three treatment groups consisted of: (1) DC and media, (2) DC with mCherry vault, and (3) DC with NYESO and vault. DC maturity was assessed via flow cytometric evaluation of CD11c, CD86, and MHC-II. Increase in CD86 Median Fluorescence Intensity (MFI) was analyzed in the CD11c+CD86+MHC-II+ population to determine the extent of maturation RESULTS: Our findings suggest that CP-MVP-NY-ESO-1-INT recombinant vault nanoparticles are efficiently bioengineered with exceptional integrity, are quickly internalized by immature DCs for antigen processing, and result in DC maturation. CONCLUSION: This study reports our preliminary results, which demonstrate the feasibility and progress regarding our immunotherapeutic technique utilizing NY-ESO-1 packaged vault nanoparticles to prime DCs for subsequent anti-cancer therapies.
Asunto(s)
Antígenos de Neoplasias/administración & dosificación , Antineoplásicos Inmunológicos/administración & dosificación , Células Dendríticas/inmunología , Glioma/inmunología , Glioma/terapia , Nanopartículas/administración & dosificación , Animales , Anticuerpos , Bioingeniería , Sistemas de Liberación de Medicamentos/métodos , Ratones Endogámicos C57BL , Proteínas Recombinantes/administración & dosificaciónRESUMEN
PURPOSE: Glioblastoma (GBM) is the most common and malignant primary adult brain tumor. Current care includes surgical resection, radiation, and chemotherapy. Recent clinical trials for GBM have demonstrated extended survival using interventions such as tumor vaccines or tumor-treating fields. However, prognosis generally remains poor, with expected survival of 20 months after randomization. Chemokine-based immunotherapy utilizing CCL21 locally recruits lymphocytes and dendritic cells to enhance host antitumor response. Here, we report a preliminary study utilizing CPZ-vault nanoparticles as a vehicle to package, protect, and steadily deliver therapy to optimize CCL21 therapy in a murine flank model of GBM. METHODS: GL261 cells were subcutaneously injected into the left flank of eight-week-old female C57BL/6 mice. Mice were treated with intratumoral injections of either: (1) CCL21-packaged vault nanoparticles (CPZ-CCL21), (2) free recombinant CCL21 chemokine empty vault nanoparticles, (3) empty vault nanoparticles, or 4) PBS. RESULTS: The results of this study showed that CCL21-packaged vault nanoparticle injections can decrease the tumor volume in vivo. Additionally, this study showed mice injected with CCL21-packaged vault nanoparticle had the smallest average tumor volume and remained the only treatment group with a negative percent change in tumor volume. CONCLUSIONS: This preliminary study establishes vault nanoparticles as a feasible vehicle to increase drug delivery and immune response in a flank murine model of GBM. Future animal studies involving an intracranial orthotopic tumor model are required to fully evaluate the potential for CCL21-packaged vault nanoparticles as a strategy to bypass the blood brain barrier, enhance intracranial immune activity, and improve intracranial tumor control and survival.
Asunto(s)
Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/patología , Quimiocina CCL21/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Glioblastoma/inmunología , Glioblastoma/patología , Inmunoterapia/métodos , Animales , Neoplasias Encefálicas/terapia , Línea Celular Tumoral , Quimiocina CCL21/inmunología , Femenino , Glioblastoma/terapia , Ratones Endogámicos C57BL , NanopartículasRESUMEN
"Vaults" are ubiquitously expressed endogenous ribonucleoprotein nanoparticles with potential utility for targeted drug delivery. Here, we show that recombinant human vault nanoparticles are readily engulfed by certain key human peripheral blood mononuclear cells (PBMC), predominately dendritic cells, monocytes/macrophages, and activated T cells. As these cell types are the primary targets for human immunodeficiency virus type 1 (HIV-1) infection, we examined the utility of recombinant human vaults for targeted delivery of antiretroviral drugs. We chemically modified three different antiretroviral drugs, zidovudine, tenofovir, and elvitegravir, for direct conjugation to vaults. Tested in infection assays, drug-conjugated vaults inhibited HIV-1 infection of PBMC with equivalent activity to free drugs, indicating vault delivery and drug release in the cytoplasm of HIV-1-susceptible cells. The ability to deliver functional drugs via vault nanoparticle conjugates suggests their potential utility for targeted drug delivery against HIV-1.
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Antirretrovirales/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Infecciones por VIH/tratamiento farmacológico , Nanopartículas/uso terapéutico , Antirretrovirales/química , Células Cultivadas , Citoplasma/metabolismo , Liberación de Fármacos , Infecciones por VIH/prevención & control , VIH-1 , Humanos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/virología , Nanopartículas/química , Nanopartículas/metabolismo , RibonucleoproteínasRESUMEN
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.
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Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Partículas Ribonucleoproteicas en Bóveda/metabolismo , Animales , Humanos , Nanopartículas/química , Nanopartículas/metabolismo , Ingeniería de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Saccharomyces cerevisiae/genética , Partículas Ribonucleoproteicas en Bóveda/química , Partículas Ribonucleoproteicas en Bóveda/genética , Partículas Ribonucleoproteicas en Bóveda/aislamiento & purificaciónRESUMEN
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 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.
RESUMEN
Major vault protein (MVP) is the main component of the vault complex, which is a highly conserved ribonucleoprotein complex found in most eukaryotic organisms. MVP or vaults have previously been found to be overexpressed in multidrug-resistant cancer cells and implicated in various cellular processes such as cell signaling and innate immunity. The precise function of MVP is, however, poorly understood and its expression and probable function in lower eukaryotes are not well characterized. In this study, we report that the Atlantic salmon louse expresses three full-length MVP paralogues (LsMVP1-3). Furthermore, we extended our search and identified MVP orthologues in several other ecdysozoan species. LsMVPs were shown to be expressed in various tissues at both transcript and protein levels. In addition, evidence for LsMVP to assemble into vaults was demonstrated by performing differential centrifugation. LsMVP was found to be highly expressed in cement, an extracellular material produced by a pair of cement glands in the adult female salmon louse. Cement is important for the formation of egg strings that serve as protective coats for developing embryos. Our results imply a possible novel function of LsMVP as a secretory cement protein. LsMVP may play a role in structural or reproductive functions, although this has to be further investigated.
Asunto(s)
Copépodos , Partículas Ribonucleoproteicas en Bóveda , Animales , Partículas Ribonucleoproteicas en Bóveda/metabolismo , Copépodos/metabolismo , Salmo salar/parasitología , Salmo salar/metabolismo , Femenino , Filogenia , Secuencia de AminoácidosRESUMEN
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.
Asunto(s)
Enzimas Inmovilizadas , Biocatálisis , Biodegradación Ambiental , Enzimas Inmovilizadas/metabolismoRESUMEN
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.
Asunto(s)
Lacasa , Nanopartículas , Compuestos Azo/química , Compuestos Azo/toxicidad , Biodegradación Ambiental , Colorantes/química , Enzimas Inmovilizadas , Lacasa/metabolismoRESUMEN
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.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Interacciones Hidrofóbicas e Hidrofílicas/efectos de los fármacos , Ingeniería de Proteínas , Tretinoina/administración & dosificación , Tretinoina/farmacología , Supervivencia Celular/efectos de los fármacos , Células Hep G2 , Humanos , Nanoestructuras/ultraestructura , Estructura Terciaria de Proteína , Espectrofotometría Ultravioleta , Tomografía , Partículas Ribonucleoproteicas en Bóveda/químicaRESUMEN
Extracellular vesicles and exomere nanoparticles are under intense investigation as sources of clinically relevant cargo. Here we report the discovery of a distinct extracellular nanoparticle, termed supermere. Supermeres are morphologically distinct from exomeres and display a markedly greater uptake in vivo compared with small extracellular vesicles and exomeres. The protein and RNA composition of supermeres differs from small extracellular vesicles and exomeres. Supermeres are highly enriched with cargo involved in multiple cancers (glycolytic enzymes, TGFBI, miR-1246, MET, GPC1 and AGO2), Alzheimer's disease (APP) and cardiovascular disease (ACE2, ACE and PCSK9). The majority of extracellular RNA is associated with supermeres rather than small extracellular vesicles and exomeres. Cancer-derived supermeres increase lactate secretion, transfer cetuximab resistance and decrease hepatic lipids and glycogen in vivo. This study identifies a distinct functional nanoparticle replete with potential circulating biomarkers and therapeutic targets for a host of human diseases.
Asunto(s)
Vesículas Extracelulares/metabolismo , MicroARNs/metabolismo , Nanopartículas/metabolismo , Enfermedad de Alzheimer/patología , Enzima Convertidora de Angiotensina 2/metabolismo , Transporte Biológico/fisiología , Biomarcadores/metabolismo , COVID-19/patología , Enfermedades Cardiovasculares/patología , Comunicación Celular/fisiología , Línea Celular Tumoral , Células HeLa , Humanos , Ácido Láctico/metabolismo , MicroARNs/genética , Nanopartículas/clasificación , Neoplasias/patología , Microambiente TumoralRESUMEN
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.
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Hígado/química , Partículas Ribonucleoproteicas en Bóveda/química , Animales , Secuencia de Bases , Microscopía por Crioelectrón , Cristalización , Modelos Moleculares , Datos de Secuencia Molecular , Ratas , Proteínas Recombinantes/químicaRESUMEN
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.
Asunto(s)
Biodegradación Ambiental , Contaminantes Ambientales/metabolismo , Nanopartículas/química , Compuestos Orgánicos/metabolismo , Peroxidasas , Phanerochaete/metabolismoRESUMEN
We report an effective and environmentally sustainable water treatment approach using enzymes encapsulated in biogenic vault nanoparticles. Manganese peroxidase (MnP), whose stability was remarkably extended by encapsulating into vaults, rapidly catalyzed the biotransformation of endocrine-disrupting compounds, including bisphenol A (BPA), bisphenol F (BPF), and bisphenol AP (BPAP). The vault-encapsulated MnP (vMnP) treatment removed 80-95% of each of the tested bisphenols (BPs) at lower enzyme dosage, while free native MnP (nMnP) only resulted in a 19-36% removal, over a 24-h period. Treatment by vMnP and nMnP resulted in considerable disparities in product species and abundance, which were consistent with the observed changes in the estrogenic activities of BPs. To test if vMnP-catalyzed transformations generated toxic intermediates, we assessed biological hallmarks of BP toxicity, namely, the ability to disrupt reproductive processes. The toxicity of vMnP-treated samples, as measured in the model organism, Caenorhabditis elegans, was dramatically reduced for all three BPs, including the reproductive indicators of BPA exposure such as reduced fertility and increased germ cell death. Collectively, our results indicate that the vMnP system represents an efficient and safe approach for the removal of BPs and promise the development of vault-encapsulated customized enzymes for treating other targeted organic compounds in contaminated waters.
RESUMEN
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.
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Nanopartículas , Partículas Ribonucleoproteicas en Bóveda , Animales , Enzimas Inmovilizadas , Expresión Génica , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Nanopartículas/química , Nanopartículas/ultraestructura , Nanotecnología , Ratas , Proteínas Recombinantes de Fusión/química , Células Sf9 , Partículas Ribonucleoproteicas en Bóveda/químicaRESUMEN
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 (â¼4.8 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.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Ingeniería de Proteínas/métodos , Partículas Ribonucleoproteicas en Bóveda/química , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/química , Secuencia de Aminoácidos , Animales , Baculoviridae/genética , Baculoviridae/metabolismo , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Partículas Ribonucleoproteicas en Bóveda/genética , Partículas Ribonucleoproteicas en Bóveda/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
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/cm(3). Moreover, there is evidence that proteins and protein complexes collapse or become more compact in the gas phase in the absence of water.
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Modelos Químicos , Modelos Moleculares , Fosfopiruvato Hidratasa/química , Fosfopiruvato Hidratasa/ultraestructura , Espectrometría de Masa por Ionización de Electrospray/métodos , Simulación por Computador , Dimerización , Electroforesis/métodos , Peso Molecular , Complejos Multiproteicos/química , Complejos Multiproteicos/ultraestructuraRESUMEN
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 mVparp-deficient 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.
Asunto(s)
Poli(ADP-Ribosa) Polimerasas/metabolismo , Telomerasa/metabolismo , Partículas Ribonucleoproteicas en Bóveda/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Células Clonales , Marcación de Gen , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Estructura Molecular , Poli(ADP-Ribosa) Polimerasas/deficiencia , Poli(ADP-Ribosa) Polimerasas/genética , Proteínas de Unión al ARN , Partículas Ribonucleoproteicas en Bóveda/química , Partículas Ribonucleoproteicas en Bóveda/deficiencia , Partículas Ribonucleoproteicas en Bóveda/genéticaRESUMEN
TEP1 is a protein component of two ribonucleoprotein complexes: vaults and telomerase. The vault-associated small RNA, termed vault RNA (VR), is dependent upon TEP1 for its stable association with vaults, while the association of telomerase RNA with the telomerase complex is independent of TEP1. Both of these small RNAs have been shown to interact with amino acids 1-871 of TEP1 in an indirect yeast three-hybrid assay. To understand the determinants of TEP1-RNA binding, we generated a series of TEP1 deletions and show by yeast three-hybrid assay that the entire Tetrahymena p80 homology region of TEP1 is required for its interaction with both telomerase and VRs. This region is also sufficient to target the protein to the vault particle. Electrophoretic mobility shift assays using the recombinant TEP1 RNA-binding domain (TEP1-RBD) demonstrate that it binds RNA directly, and that telomerase and VRs compete for binding. VR binds weakly to TEP1-RBD in vitro, but mutation of VR sequences predicted to disrupt helices near its central loop enhances binding. Antisense oligonucleotide-directed RNase H digestion of endogenous VR indicates that this region is largely single stranded, suggesting that TEP1 may require access to the VR central loop for efficient binding.