RESUMO
Vaults are ribonucleoprotein complexes comprised of the 100 kDa major vault protein (MVP), the 2 high m.w. vault proteins p193 (VPARP) and p240 (TEP1) and an untranslated small RNA (vRNA). Increased levels of MVP, vault-associated vRNA and vaults have been linked directly to non-P-glycoprotein-mediated multidrug resistance (MDR). To further characterize the putative role of vaults in MDR, expression levels of all of the vault proteins were examined in various MDR cell lines. Subcellular fractionation of vault particles revealed that all 3 vault proteins are increased in MDR cells compared to the parental, drug-sensitive cells. Furthermore, protein analysis of subcellular fractions of the drug-sensitive, MVP-transfected AC16 cancer cell line indicated that vault levels are increased, in this stable line. Since TEP1 is shared by both vaults and the telomerase complex, TEP1 protein (and vault) levels were compared with telomerase activity in a variety of cell lines, including various MDR lines. Our studies demonstrate that while vault levels may be a good predictor of drug resistance, their up-regulation alone is not sufficient to confer the drug-resistant phenotype. This implies a requirement of an additional factor(s) for vault-mediated MDR.
Assuntos
Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Neoplasias/genética , Partículas de Ribonucleoproteínas em Forma de Abóbada/biossíntese , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/etiologia , Neoplasias/metabolismo , RNA Mensageiro/biossíntese , Proteínas de Ligação a RNA , Telomerase/metabolismo , Transfecção , Células Tumorais Cultivadas , Regulação para CimaRESUMO
The vault complex is a ubiquitous 13-MDa ribonucleoprotein assembly, composed of three proteins (TEP1, 240 kDa; VPARP, 193 kDa; and MVP, 100 kDa) that are highly conserved in eukaryotes and an untranslated RNA (vRNA). The vault has been shown to affect multidrug resistance in cancer cells, and one particular component, MVP, is thought to play a role in the transport of drug from the nucleus. To locate the position of the vRNA, vaults were treated with RNases, and cryo-electron microscopy (cryo-EM) was performed on the resulting complexes. Using single-particle reconstruction techniques, 3,476 particle images were combined to generate a 22-A-resolution structure. Difference mapping between the RNase-treated vault and the previously calculated intact vault reconstructions reveals the vRNA to be at the ends of the vault caps. In this position, the vRNA may interact with both the interior and exterior environments of the vault. The finding of a 16-fold density ring at the top of the cap has allowed modeling of the WD40 repeat domain of the vault TEP1 protein within the cryo-EM vault density. Both stoichiometric considerations and the finding of higher resolution for the computationally selected and refined "barrel only" images indicate a possible symmetry mismatch between the barrel and the caps. The molecular architecture of the complex is emerging, with 96 copies of MVP composing the eightfold symmetric barrel, and the vRNA together with one copy of TEP1 and four predicted copies of VPARP comprising each cap.
Assuntos
Modelos Moleculares , RNA/química , RNA/isolamento & purificação , Sequências Repetitivas de Aminoácidos , Partículas de Ribonucleoproteínas em Forma de Abóbada/química , Partículas de Ribonucleoproteínas em Forma de Abóbada/isolamento & purificação , Animais , Proteínas de Transporte/química , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/ultraestrutura , Simulação por Computador , Microscopia Crioeletrônica , Proteínas de Ligação a Fosfato , Estrutura Terciária de Proteína , RNA/ultraestrutura , Ratos , Ribonucleases/metabolismo , Partículas de Ribonucleoproteínas em Forma de Abóbada/ultraestruturaRESUMO
Vaults are 13 megadalton ribonucleoprotein particles composed largely of the major vault protein (MVP) and two high molecular weight proteins, p240 and p193, and a small vault RNA (vRNA). Increased levels of MVP expression, vault-associated vRNA, and vaults have been linked directly to multidrug resistance (MDR). To further define the putative role of vaults in MDR, we produced monoclonal antibodies against the Mr 193,000 vault protein and studied its expression levels in various multidrug-resistant cell lines. We find that, like MVP, p193 mRNA and protein levels are increased in various multidrug-resistant cell lines. Subcellular fractionation of vault particles revealed that vault-associated p193 levels are increased in multidrug-resistant cells as compared with the parental, drug-sensitive cells. Furthermore, protein analysis of postnuclear supernatants and co-immunoprecipitation studies show that drug-sensitive MVP-transfected tumor cells lack this up-regulation in vault-associated p193. Our observations indicate that vault formation is limited not only by the expression of the MVP but also by the expression or assembly of at least one of the other vault proteins.
Assuntos
Neoplasias/metabolismo , Partículas de Ribonucleoproteínas em Forma de Abóbada/biossíntese , Anticorpos Monoclonais/imunologia , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Humanos , Peso Molecular , Células Tumorais Cultivadas , Regulação para CimaRESUMO
Mammalian vaults are ribonucleoprotein (RNP) complexes, composed of a small ribonucleic acid and three proteins of 100, 193, and 240 kD in size. The 100-kD major vault protein (MVP) accounts for >70% of the particle mass. We have identified the 193-kD vault protein by its interaction with the MVP in a yeast two-hybrid screen and confirmed its identity by peptide sequence analysis. Analysis of the protein sequence revealed a region of approximately 350 amino acids that shares 28% identity with the catalytic domain of poly(ADP-ribose) polymerase (PARP). PARP is a nuclear protein that catalyzes the formation of ADP-ribose polymers in response to DNA damage. The catalytic domain of p193 was expressed and purified from bacterial extracts. Like PARP, this domain is capable of catalyzing a poly(ADP-ribosyl)ation reaction; thus, the 193-kD protein is a new PARP. Purified vaults also contain the poly(ADP-ribosyl)ation activity, indicating that the assembled particle retains enzymatic activity. Furthermore, we show that one substrate for this vault-associated PARP activity is the MVP. Immunofluorescence and biochemical data reveal that p193 protein is not entirely associated with the vault particle, suggesting that it may interact with other protein(s). A portion of p193 is nuclear and localizes to the mitotic spindle.
Assuntos
Poli(ADP-Ribose) Polimerases/metabolismo , alfa-Globulinas/química , alfa-Globulinas/genética , Sequência de Aminoácidos , Animais , Proteína BRCA1/química , Proteína BRCA1/genética , Células COS , Domínio Catalítico/genética , Domínio Catalítico/fisiologia , Núcleo Celular/enzimologia , Clonagem Molecular , Citoplasma/enzimologia , Fibroblastos , Células HeLa , Humanos , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Homologia de Sequência de Aminoácidos , Fuso Acromático/enzimologia , Partículas de Ribonucleoproteínas em Forma de Abóbada/química , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Partículas de Ribonucleoproteínas em Forma de Abóbada/isolamento & purificação , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Leveduras/genéticaRESUMO
BACKGROUND: The vault is a ubiquitous and highly conserved ribonucleoprotein particle of approximately 13 MDa. This particle has been shown to be upregulated in certain multidrug-resistant cancer cell lines and to share a protein component with the telomerase complex. Determination of the structure of the vault was undertaken to provide a first step towards understanding the role of this cellular component in normal metabolism and perhaps to shed some light on its role in mediating drug resistance. RESULTS: Over 1300 particle images were combined to calculate an approximately 31 A resolution structure of the vault. Rotational power spectra did not yield a clear symmetry peak, either because of the thin, smooth walls or inherent flexibility of the vault. Although cyclic eightfold (C8) symmetry was imposed, the resulting reconstruction may be partially cylindrically averaged about the eightfold axis. Our results reveal the vault to be a hollow, barrel-like structure with two protruding caps and an invaginated waist. CONCLUSIONS: Although the normal cellular function of the vault is as yet undetermined, the structure of the vault is consistent with either a role in subcellular transport, as previously suggested, or in sequestering macromolecular assemblies.