RESUMO
The plant cytotoxin ricin enters mammalian cells by receptor-mediated endocytosis, undergoing retrograde transport to the ER (endoplasmic reticulum) where its catalytic A chain (RTA) is reductively separated from the holotoxin to enter the cytosol and inactivate ribosomes. The currently accepted model is that the bulk of ER-dislocated RTA is degraded by proteasomes. We show in the present study that the proteasome has a more complex role in ricin intoxication than previously recognized, that the previously reported increase in sensitivity of mammalian cells to ricin in the presence of proteasome inhibitors simply reflects toxicity of the inhibitors themselves, and that RTA is a very poor substrate for proteasomal degradation. Denatured RTA and casein compete for a binding site on the regulatory particle of the 26S proteasome, but their fates differ. Casein is degraded, but the mammalian 26S proteasome AAA (ATPase associated with various cellular activities)-ATPase subunit RPT5 acts as a chaperone that prevents aggregation of denatured RTA and stimulates recovery of catalytic RTA activity in vitro. Furthermore, in vivo, the ATPase activity of Rpt5p is required for maximal toxicity of RTA dislocated from the Saccharomyces cerevisiae ER. The results of the present study implicate RPT5/Rpt5p in the triage of substrates in which either activation (folding) or inactivation (degradation) pathways may be initiated.
Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Ricina/metabolismo , Animais , Caseínas/química , Caseínas/metabolismo , Bovinos , Retículo Endoplasmático/metabolismo , Células HeLa , Humanos , Ricina/química , Saccharomyces cerevisiae/metabolismo , Transdução de SinaisRESUMO
Ricin is a potent A-B toxin that is transported from the cell surface to the cytosol, where it inactivates ribosomes, leading to cell death. Ricin enters cells via endocytosis, where only a minute number of ricin molecules reach the endoplasmic reticulum (ER) lumen. Subsequently, the ricin A chain traverses the ER bilayer by a process referred to as dislocation or retrograde translocation to gain access to the cytosol. To study the molecular processes of ricin A chain dislocation, we have established, for the first time, a human cell system in which enzymatically attenuated ricin toxin A chains (RTA(E177D) and RTA(Δ177-181)) are expressed in the cell and directed to the ER. Using this human cell-based system, we found that ricin A chains underwent a rapid dislocation event that was quite distinct from the dislocation of a canonical ER soluble misfolded protein, null Hong Kong variant of α(1)-antitrypsin. Remarkably, ricin A chain dislocation occurred via a membrane-integrated intermediate and utilized the ER protein SEL1L for transport across the ER bilayer to inhibit protein synthesis. The data support a model in which ricin A chain dislocation occurs via a novel strategy of utilizing the hydrophobic nature of the ER membrane and selective ER components to gain access to the cytosol.
Assuntos
Retículo Endoplasmático/metabolismo , Bicamadas Lipídicas/química , Ricina/química , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Citosol/metabolismo , Epitopos/química , Glicosídeo Hidrolases/química , Humanos , Focalização Isoelétrica , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/química , Dobramento de Proteína , alfa 1-Antitripsina/químicaRESUMO
The plant cytotoxin ricin enters target mammalian cells by receptor-mediated endocytosis and undergoes retrograde transport to the endoplasmic reticulum (ER). Here, its catalytic A chain (RTA) is reductively separated from the cell-binding B chain, and free RTA enters the cytosol where it inactivates ribosomes. Cytosolic entry requires unfolding of RTA and dislocation across the ER membrane such that it arrives in the cytosol in a vulnerable, nonnative conformation. Clearly, for such a dislocated toxin to become active, it must avoid degradation and fold to a catalytic conformation. Here, we show that, in vitro, Hsc70 prevents aggregation of heat-treated RTA, and that RTA catalytic activity is recovered after chaperone treatment. A combination of pharmacological inhibition and cochaperone expression reveals that, in vivo, cytosolic RTA is scrutinized sequentially by the Hsc70 and Hsp90 cytosolic chaperone machineries, and that its eventual fate is determined by the balance of activities of cochaperones that regulate Hsc70 and Hsp90 functions. Cytotoxic activity follows Hsc70-mediated escape of RTA from an otherwise destructive pathway facilitated by Hsp90. We demonstrate a role for cytosolic chaperones, proteins typically associated with folding nascent proteins, assembling multimolecular protein complexes and degrading cytosolic and stalled, cotranslocational clients, in a toxin triage, in which both toxin folding and degradation are initiated from chaperone-bound states.
Assuntos
Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico HSC70/metabolismo , Chaperonas Moleculares/metabolismo , Ricina/metabolismo , Eletroforese em Gel de Poliacrilamida , Proteínas de Choque Térmico HSP90/metabolismo , Células HeLa , Humanos , Conformação Proteica , Ribossomos/metabolismo , Ricina/toxicidade , UbiquitinaçãoRESUMO
The hepatitis C virus RNA genome possesses a variety of conserved structural elements, in both coding and non-coding regions, that are important for viral replication. These elements are known or predicted to modulate key life cycle events, such as translation and genome replication, some involving conformational changes induced by long-range RNA-RNA interactions. One such element is SLVI, a stem-loop (SL) structure located towards the 5' end of the core protein-coding region. This element forms an alternative RNA-RNA interaction with complementary sequences in the 5' untranslated regions that are independently involved in the binding of the cellular microRNA 122 (miR122). The switch between 'open' and 'closed' structures involving SLVI has previously been proposed to modulate translation, with lower translation efficiency associated with the 'closed' conformation. In the current study, we have used selective 2'-hydroxyl acylation analysed by primer extension to validate this RNA-RNA interaction in the absence and presence of miR122. We show that the long-range association (LRA) only forms in the absence of miR122, or otherwise requires the blocking of miR122 binding combined with substantial disruption of SLVI. Using site-directed mutations introduced to promote open or closed conformations of the LRA we demonstrate no correlation between the conformation and the translation phenotype. In addition, we observed no influence on virus replication compared to unmodified genomes. The presence of SLVI is well-documented to suppress translation, but these studies demonstrate that this is not due to its contribution to the LRA. We conclude that, although there are roles for SLVI in translation, the LRA is not a riboswitch regulating the translation and replication phenotypes of the virus.
RESUMO
After endocytic uptake by mammalian cells, the heterodimeric plant toxin ricin is transported to the endoplasmic reticulum (ER), where the ricin A chain (RTA) must cross the ER membrane to reach its ribosomal substrates. Here, using gel filtration chromatography, sedimentation, fluorescence, fluorescence resonance energy transfer, and circular dichroism, we show that both fluorescently labeled and unlabeled RTA bind both to ER microsomal membranes and to negatively charged liposomes. The binding of RTA to the membrane at 0-30 degrees C exposes certain RTA residues to the nonpolar lipid core of the bilayer with little change in the secondary structure of the protein. However, major structural rearrangements in RTA occur when the temperature is increased. At 37 degrees C, membrane-bound toxin loses some of its helical content, and its C terminus moves closer to the membrane surface where it inserts into the bilayer. RTA is then stably bound to the membrane because it is nonextractable with carbonate. The sharp temperature dependence of the structural changes does not coincide with a lipid phase change because little change in fluorescence-detected membrane mobility occurred between 30 and 37 degrees C. Instead, the structural rearrangements may precede or initiate toxin retrotranslocation through the ER membrane to the cytosol. The sharp temperature dependence of these changes in RTA further suggests that they occur optimally in mammalian targets of the plant toxin.
Assuntos
Retículo Endoplasmático/metabolismo , Microssomos/metabolismo , Ricina/química , Membrana Celular/metabolismo , Citosol/metabolismo , Conformação Molecular , Fosfolipídeos/química , Conformação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Espectrometria de Fluorescência/métodos , Propriedades de Superfície , TemperaturaRESUMO
The RNA segment encoding the guanylyltransferase (VP3) from 12 group A rotavirus isolates has been sequenced following RT-PCR and molecular cloning of the full-length amplicons produced. Alignment of the derived amino acid sequences including those of the four VP3 sequences available from GenBank revealed two levels of sequence divergence. Virus isolates from humans showed greater than 94% sequence identity, whereas those isolated from different mammalian species showed as low as 79% sequence identity. The exceptions were avian virus isolates, which diverged approximately 45% from those of mammalian origin, and the human virus isolates DS1 and 69M, which showed much closer (over 90%) identity to viruses of bovine origin, suggesting that these human isolates may have undergone recent reassortment events with a bovine virus. Analysis of the sequences for a putative enzymic active site has revealed that the KXTAMDXEXP and KXXGNNH motifs around amino acids 385 and 545, respectively, are conserved across both group A and C rotaviruses.
Assuntos
Proteínas do Capsídeo/genética , Nucleotidiltransferases/genética , Rotavirus/enzimologia , Rotavirus/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Aves , Bovinos , Sequência Conservada , DNA Viral/genética , Humanos , Mamíferos , Dados de Sequência Molecular , Filogenia , Rotavirus/classificação , Rotavirus/isolamento & purificação , Homologia de Sequência de AminoácidosRESUMO
Several protein toxins, including the A chain of ricin (RTA), enter mammalian cells by endocytosis and subsequently reach their cytosolic substrates by translocation across the endoplasmic reticulum (ER) membrane. To achieve this export, such toxins exploit the ER-associated protein degradation (ERAD) pathway but must escape, at least in part, the normal degradative fate of ERAD substrates. Toxins that translocate from the ER have an unusually low lysine content. Since lysyl residues are potential ubiquitination sites, it has been proposed that this paucity of lysines reduces the chance of ubiquitination and subsequent ubiquitin-mediated proteasomal degradation [Hazes, B., and Read, R. J. (1997) Biochemistry 36, 11051-11054]. Here we provide experimental support for this hypothesis. The two lysyl residues within RTA were changed to arginyl residues. Their replacement in RTA did not have a significant stabilizing effect, suggesting that the endogenous lysyl residues are not the usual sites for ubiquitin attachment. However, when four additional lysines were introduced into RTA in a way that did not compromise the activity, structure, or stability of the toxin, degradation was significantly enhanced. Enhanced degradation resulted from ubiquitination that predisposed the toxin to proteasomal degradation. Treatment with the proteasome inhibitor clasto-lactacystin beta-lactone increased the cytotoxicity of the lysine-rich RTA to a level approaching that of wild-type ricin. The introduction of four additional lysyl residues into a second ribosome-inactivating protein, abrin A chain, also dramatically decreased the cytotoxicity of the holotoxin compared to wild-type abrin. This effect could also be reversed by proteasomal inhibition. Our data support the hypothesis that the evolution of a low lysine content is a degradation-avoidance strategy for toxins that retrotranslocate from the ER.