RESUMEN
Translocation of the catalytic domain of diphtheria toxin (DT) across the endosomal membrane to the cytoplasm of mammalian cells requires the low-pH-dependent insertion of a hydrophobic helical hairpin (TH8-TH9) that is buried within the T domain of the native protein. Mutations of Pro345, which terminates helix TH8, have been reported to block toxicity for Vero cells. We found that mutant toxins in which Pro345 had been replaced by Cys, Glu, or Gly were profoundly defective at low pH in forming channels in planar phospholipid bilayers and in permeabilizing phospholipid vesicles to entrapped fluorophores. Experiments with isolated T domain containing a polarity-sensitive fluorophore attached to Cys at position 332 suggest that the P345E mutation blocks membrane insertion. None of the Pro345 mutations shifted the pH-dependence of binding in solution of the hydrophobic fluorophore, 2-p-toluidinyl-naphthalene 7-sulfonate. The results indicate that proline at position 345 is required for the T domain to insert into phospholipid bilayers or to adopt a functional conformation within the bilayer.
Asunto(s)
Membrana Celular/química , Toxina Diftérica/química , Liposomas/química , Animales , Transporte Biológico/genética , Membrana Celular/metabolismo , Chlorocebus aethiops , Toxina Diftérica/genética , Toxina Diftérica/metabolismo , Liposomas/metabolismo , Mutación , Prolina/química , Prolina/genética , Células VeroRESUMEN
The fusion protein toxin DAB389IL-2 is composed of the catalytic and transmembrane domains of diphtheria toxin genetically linked to human interleukin 2 (IL-2). This fusion toxin is selectively toxic for eukaryotic cells which express the high-affinity form of the IL-2 receptor and the mechanism of intoxication parallels that of native diphtheria toxin. We used site-directed mutagenesis to introduce Pro residues into each of the three helical layers of the transmembrane domain. Although each of the mutations results in the complete loss of cytotoxic activity, individual mutants were found to vary with respect to channel formation in planar lipid bilayers, binding affinity and melting temperature. We propose that each of the three helix layers plays a critical role in the productive delivery of the catalytic domain to the cell cytosol.
Asunto(s)
Toxina Diftérica/metabolismo , Inmunotoxinas/metabolismo , Interleucina-2/metabolismo , Secuencia de Aminoácidos , Línea Celular , Toxina Diftérica/química , Toxina Diftérica/genética , Humanos , Inmunotoxinas/química , Inmunotoxinas/genética , Interleucina-2/química , Interleucina-2/genética , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de FluorescenciaRESUMEN
The T domain of diphtheria toxin, which extends from residue 202 to 378, causes the translocation of the catalytic A fragment (residues 1-201) across endosomal membranes and also forms ion-conducting channels in planar phospholipid bilayers. The carboxy terminal 57-amino acid segment (322-378) in the T domain is all that is required to form these channels, but its ability to do so is greatly augmented by the portion of the T domain upstream from this. In this work, we show that in association with channel formation by the T domain, its NH2 terminus, as well as some or all of the adjacent hydrophilic 63 amino acid segment, cross the lipid bilayer. The phenomenon that enabled us to demonstrate that the NH2-terminal region of the T domain was translocated across the membrane was the rapid closure of channels at cis negative voltages when the T domain contained a histidine tag at its NH2 terminus. The inhibition of this effect by trans nickel, and by trans streptavidin when the histidine tag sequence was biotinylated, clearly established that the histidine tag was present on the trans side of the membrane. Furthermore, the inhibition of rapid channel closure by trans trypsin, combined with mutagenesis to localize the trypsin site, indicated that some portion of the 63 amino acid NH2-terminal segment of the T domain was also translocated to the trans side of the membrane. If the NH2 terminus was forced to remain on the cis side, by streptavidin binding to the biotinylated histidine tag sequence, channel formation was severely disrupted. Thus, normal channel formation by the T domain requires that its NH2 terminus be translocated across the membrane from the cis to the trans side, even though the NH2 terminus is >100 residues removed from the channel-forming part of the molecule.
Asunto(s)
Activación del Canal Iónico/fisiología , Receptores de Superficie Celular/química , Receptores de Superficie Celular/fisiología , Secuencia de Aminoácidos , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Biotina , Estimulación Eléctrica , Electrofisiología , Endosomas/química , Endosomas/metabolismo , Factor de Crecimiento Similar a EGF de Unión a Heparina , Histidina/farmacocinética , Péptidos y Proteínas de Señalización Intercelular , Activación del Canal Iónico/efectos de los fármacos , Membrana Dobles de Lípidos/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/fisiología , Datos de Secuencia Molecular , Níquel/metabolismo , Níquel/farmacología , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Estructura Terciaria de Proteína , Receptores de Superficie Celular/genética , Estreptavidina , TripsinaRESUMEN
In its activated 63 kDa form, the protective antigen (PA) component of anthrax toxin forms a heptameric prepore, which converts to a pore (channel) in endosomal membranes at low pH and mediates translocation of the toxin's enzymic moieties to the cytosol. It has been proposed that the prepore-to-pore conversion involves a conformational rearrangement of a disordered amphipathic loop (D2L2; residues 302-325), in which loops from the 7 protomers combine to form a transmembrane 14-stranded beta barrel. To test this model, we generated Cys substitutions in 24 consecutive residues of the D2L2 loop, formed channels in artificial bilayers with each mutant, and examined changes in channel conductance after adding the thiol-reactive, bilayer-impermeant reagent methanethiosulfonate ethyltrimethylammonium (MTS-ET) to the trans compartment. The rationale for these experiments is that reaction of MTS-ET with a Cys residue adds a positively charged group and therefore would likely reduce channel conductance if the residue were in the ion-conducting pathway. We found alternating reduction and absence of reduction of conductance in consecutive residues over two stretches (residues 302-311 and 316-325). This pattern is consistent with alternating polar and apolar residues of the two stretches projecting into the pore lumen and into the bilayer, respectively. Residues connecting these two stretches (residues 312-315) were responsive to MTS-ET, consistent with their being in a turn region. Single channels formed by selected mutants (H304C and N306C) showed multiple conductance step changes in response to MTS-ET, consistent with an oligomeric pore. We also found that the binding site for the channel-blocking tetraalkylammonium ions is located cis relative to the inserted D2L2 loops. These findings constitute strong evidence in favor of the model of conversion of the prepore to a 14-stranded beta barrel pore and solidify the foundation for studies to understand the mechanism of translocation by anthrax toxin.
Asunto(s)
Antígenos Bacterianos/química , Toxinas Bacterianas/química , Canales Iónicos/química , Secuencia de Aminoácidos , Antígenos Bacterianos/efectos de los fármacos , Bacillus anthracis/química , Detergentes , Conductividad Eléctrica , Indicadores y Reactivos , Activación del Canal Iónico , Canales Iónicos/efectos de los fármacos , Mesilatos/farmacología , Datos de Secuencia Molecular , Compuestos de Amonio Cuaternario/farmacología , EstereoisomerismoRESUMEN
81 participants in a village in Guatemala were administered a translated version of the 1986 Love Attitudes Scale of Hendrick and Hendrick. Analysis showed that the men scored significantly higher on the Ludus love style than the women. Research among other Latinos outside the United States is suggested.
Asunto(s)
Actitud , Etnicidad/psicología , Amor , Población Rural , Adolescente , Adulto , Anciano , Comparación Transcultural , Femenino , Identidad de Género , Guatemala , Humanos , Masculino , Persona de Mediana Edad , Inventario de PersonalidadRESUMEN
Previous work has established that the 61 amino acid stretch from residue 322 to 382 in the T-domain of diphtheria toxin forms channels indistinguishable in ion-conducting properties from those formed by the entire T-domain. In the crystal structure of the toxin's water-soluble form, the bulk of this stretch is an alpha-helical hairpin, designated TH8-9. The present study was directed at determining which residues in TH8-9 line the ion-conducting pathway of the channel; i.e., its lumen or entrances. To this end, we singly mutated 49 of TH8-9's 51 residues (328-376) to cysteines, formed channels with the mutant T-domain proteins in planar lipid bilayers, and then determined whether they reacted with small, charged, lipid-insoluble, sulfhydryl-specific methanethiosulfonate (MTS) derivatives added to the bathing solutions. The indication of a reaction, and that the residue lined the ion-conducting pathway, was a sudden change in single-channel conductance and/or flickering behavior. The results of this study were surprising in two respects. First, of the 49 cysteine-substituted residues in TH8-9 tested, 23 reacted with MTS derivatives in a most unusual pattern consisting of two segments: one extending from 329 to 341 (11 of 13 reacted), and the other from 347 to 359 (12 of 13 reacted); none of the residues outside of these two segments appeared to react. Second, in every cysteine mutant channel manifesting an MTS effect, only one transition in single-channel conductance (or flickering behavior) occurred, not the several expected for a multimeric channel. Our results are not consistent with an alpha-helical or beta-strand model for the channel, but instead suggest an open, flexible structure. Moreover, contrary to common sense, they indicate that the channel is not multimeric but is formed from only one TH8-9 unit of the T-domain.
Asunto(s)
Cisteína/genética , Toxina Diftérica/metabolismo , Canales Iónicos/química , Canales Iónicos/genética , Mutación , Ácidos Tiosulfónicos/metabolismo , Secuencia de Aminoácidos , Canales Iónicos/metabolismo , Membrana Dobles de Lípidos , Mesilatos/metabolismo , EstereoisomerismoRESUMEN
Acidic conditions within the endosomal lumen induce the T domain of receptor-bound diphtheria toxin (DT) to insert into the endosomal membrane and mediate translocation of the toxin's catalytic domain to the cytosol. A conformational rearrangement in the toxin occurring near pH5 allows a buried apolar helical hairpin of the native T domain (helices TH8 and TH9) to undergo membrane insertion. If the inserted hairpin spans the bilayer, as hypothesized, then the two acidic residues within the TL5 interhelical loop, Glu 349 and Asp 352, should become exposed at the neutral cytosolic face of the membrane and reionize. To investigate the roles of these residues in toxin action, we characterized mutant toxins in which one or both acidic residues had been replaced with nonionizable ones. Each of two double mutants examined showed a several-fold reduction in cytotoxicity in 24-h Vero cell assays (sixfold for E349A + D352A and fourfold for E349Q + D352N), whereas the individual E349Q and D352N mutations caused smaller reductions in toxicity. The single and double mutations also attenuated the toxin's ability to permeabilize Vero cells to Rb+ at low pH and decreased channel formation by the toxin in artificial planar bilayers. Neither of the double mutations affected the pH-dependence profile of the toxin's conformational rearrangement in solution, as measured by binding of the hydrophobic fluorophore, 2-p-toluidinyl-naphthalene 6-sulfonate. The results demonstrate that, although there is no absolute requirement for an acidic residue within the TL5 loop for toxicity, Glu 349 and Asp 352 do significantly enhance the biological activity of the protein. The data are consistent with a model in which ionization of these residues at the cytosolic face of the endosomal membrane stabilizes the TH8/TH9 hairpin in a transmembrane configuration, thereby facilitating channel formation and translocation of the toxin's catalytic chain.
Asunto(s)
Ácido Aspártico/metabolismo , Membrana Celular/metabolismo , Toxina Diftérica/metabolismo , Glutatión/metabolismo , Animales , Transporte Biológico , Chlorocebus aethiops , Toxina Diftérica/química , Toxina Diftérica/genética , Concentración de Iones de Hidrógeno , Mutagénesis Sitio-Dirigida , Células VeroRESUMEN
The mCAT-2 gene encodes a Na(+)-independent cationic amino acid (AA) transporter that is inducibly expressed in a tissue-specific manner in various physiological conditions. When mCAT-2 protein is expressed in Xenopus oocytes, the elicited AA transport properties are similar to the biochemically defined transport system y+. The mCAT-2 protein sequence is closely related to another cationic AA transporter (mCAT-1); these related proteins elicit virtually identical cationic AA transport in Xenopus oocytes. The two genes differ in their tissue expression and induction patterns. Here we report the presence of diverse 5' untranslated region (UTR) sequences in mCAT-2 transcripts. Sequence analysis of 22 independent mCAT-2 cDNA clones reveals that the cDNA sequences converge precisely 16 bp 5' of the initiator AUG codon. Moreover, analysis of genomic clones shows that the mCAT-2 gene 5'UTR exons are dispersed over 18 kb. Classical promoter and enhancer elements are present in appropriate positions 5' of the exons and their utilization results in regulated mCAT-2 mRNA accumulation in skeletal muscle and liver following partial hepatectomy. The isoform adjacent to the most distal promoter is found in all tissues and cell types previously shown to express mCAT-2, while the other 5' UTR isoforms are more tissue specific in their expression. Utilization of some or all of five putative promoters was documented in lymphoma cell clones, liver, and skeletal muscle. TATA-containing and (G+C)-rich TATA-less promoters appear to control mCAT-2 gene expression. The data indicate that the several distinct 5' mCAT-2 mRNA isoforms result from transcriptional initiation at distinct promoters and permit flexible transcriptional regulation of this cationic AA transporter gene.
Asunto(s)
Proteínas Portadoras/biosíntesis , Regiones Promotoras Genéticas , Animales , Arginina/metabolismo , Secuencia de Bases , Proteínas Portadoras/genética , Línea Celular , Clonación Molecular , ADN Complementario , Exones , Femenino , Biblioteca Genómica , Hígado/metabolismo , Linfoma de Células T/metabolismo , Lisina/metabolismo , Mamíferos , Ratones , Ratones Endogámicos AKR , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Músculo Esquelético/metabolismo , Oligodesoxirribonucleótidos , Secuencias Repetitivas de Ácidos Nucleicos , Análisis de Secuencia de ADN , TATA Box , Transcripción Genética , Células Tumorales CultivadasRESUMEN
Translocation of the catalytic domain of diphtheria toxin across the endosomal membrane to the cytosolic compartment depends on low-pH-triggered insertion of the toxin's T (transmembrane) domain into the membrane. The T domain, consisting of nine alpha-helices arranged in three layers, was cloned and expressed as a discrete protein in Escherichia coli, and mutant forms were prepared and characterized. To investigate the relative movements of the three layers under various conditions, we generated two mutant forms of the domain, each containing an artificial intramolecular disulfide bridge linking the buried apolar hairpin (TH8-TH9) to one of the other two layers. Both disulfides inhibited exposure of the domain's apolar regions in solution at low pH, as determined by 2-p-toluidinylnaphthalene-6-sulfonate binding, and blocked its ability to form channels in artificial bilayers. Reduction of the bridges abolished these effects. Reduced forms of the mutant proteins were reacted with pyrenylmaleimide, a fluorescent probe, to monitor separation of the layers. Strong excimer bands seen in both mutants at neutral pH were undiminished at pH 5, indicating the retention of gross conformation in solution under acidic conditions. The addition of phospholipid vesicles at pH 5, but not at pH 7.5, quenched excimer fluorescence, reflecting the physical separation of the TH8-TH9 hairpin from the other layers upon the T domain's interaction with the bilayer. The results indicate that (i) the conformation of the isolated T domain closely resembles that seen in the whole toxin, (ii) the TH8-TH9 hairpin separates from both of the other layers of the domain as an essential step of membrane insertion, and (iii) this separation is triggered by contact of the domain with the membrane under acidic conditions.
Asunto(s)
Toxina Diftérica/química , Toxina Diftérica/metabolismo , Endosomas/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , Secuencia de Aminoácidos , Membrana Celular/metabolismo , Clonación Molecular , Citosol/metabolismo , Toxina Diftérica/aislamiento & purificación , Disulfuros , Escherichia coli , Membranas Intracelulares/metabolismo , Canales Iónicos/fisiología , Membrana Dobles de Lípidos , Modelos Moleculares , Modelos Estructurales , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosfatidilgliceroles , Desnaturalización Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Espectrometría de FluorescenciaRESUMEN
To investigate structure/function relationships involved in the delivery of the diphtheria toxin (DT) catalytic (C) domain to the cytosol of target cells, we have constructed and characterized internal in-frame deletion mutants in the transmembrane (T) domain of the fusion toxin DAB389IL-2. This fusion protein is composed of the C and T domains of DT to which human interleukin-2 (IL-2) has been genetically fused. The mutant fusion toxins were compared to DAB389IL-2 with respect to cytotoxic potency, receptor binding affinity, channel formation in planar lipid membranes, and sensitivity to proteolytic digestion. We demonstrate that genetic fusion of human IL-2 sequences to a diphtheria toxin-related fragment that contains less than full-length transmembrane helix 9 results in a fusion protein that binds to the high affinity IL-2 receptor, but has lost > or = 3 logs of cytotoxic potency and has decreased ability to insert into planar membranes and form stable channels. These observations are consistent with the hypothesis that an intact transmembrane helix 9 is essential for the formation of stable channels that are required for the efficient delivery of the C domain to the cytosol of target cells.
Asunto(s)
Antivirales/farmacología , Toxina Diftérica/farmacología , Inmunotoxinas/farmacología , Interleucina-2/farmacología , Poli(ADP-Ribosa) Polimerasas/farmacología , Secuencia de Aminoácidos , Transporte Biológico , Compartimento Celular , Citosol/metabolismo , Análisis Mutacional de ADN , Toxina Diftérica/genética , Sistemas de Liberación de Medicamentos , Humanos , Interleucina-2/genética , Datos de Secuencia Molecular , Poli(ADP-Ribosa) Polimerasas/genética , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/farmacología , Eliminación de Secuencia , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
The diphtheria toxin channel is believed to be a homooligomer of its T domain in which each subunit consists of two alpha-helices, lying within the membrane, connected by a short interhelical loop of four amino acids (residues 349-352). To investigate the validity and implications of this model, we singly mutated each of these amino acids to cysteines, formed channels with the mutant T-domain proteins in planar lipid bilayers, and added to the trans compartment sulfhydryl-specific reagents [methanethiosulfonate derivatives (MTS-ER)] that introduce a positive or negative charge to reacted cysteines. The introduction of a positive charge at residue 351 or 352 (through the MTS-ER reactions) resulted in a step decrease in single-channel conductance, whereas the introduction of a negative charge resulted in a step increase. The opposite sign of these effects indicates the predominantly electrostatic nature of the phenomenon and implies that residues 351 and 352 lie close to the channel entrance. The same reactions at residue 350 resulted in very little change in channel conductance but instead changed the character of the natural rapid flickering of the channel between open and closed states to one in which the channel spent more time in the closed state; this may have resulted from the group introduced at position 350 acting as a tethered channel blocker. The MTS derivatives had no effect on channels containing a cysteine at position 349, suggesting that this residue faces away from the channel entrance. We propose that the step changes in conductance or flickering pattern result from the chemical reaction of one MTS-ER molecule with one cysteine, and thus a bimolecular chemical reaction is being witnessed at the single molecule level. From the distribution of waiting times between the appearance (i.e., the opening) of a channel and the step change in its conductance or flickering pattern, we can calculate a pseudo-first-order rate constant, which can then be converted to a second-order rate constant, for the chemical reaction.