RESUMEN
Trichosanthin is a ribosome-inactivating protein that possesses antitumor and antiviral activities. Clinical trials of trichosanthin on AIDS patients, however, elicit anaphylactic reactions. To reduce the antigenicity of trichosanthin as a drug while preserving its biological activity, the C-terminal domain (residues 203 to 247), which contains a putative antigenic site, was systemically deleted. We have found that the minimum length of trichosanthin that can fold into an active conformation is residue 1 to 240. The mini-trichosanthin (C7) generated by deleting the last seven C-terminal amino acid residues has 2.7-fold decrease in antigenicity, 10-fold reduction in in vitro ribosome-inactivation activity, and in vivo cytotoxicity toward K562 cells, and 2-fold reduction in abortificient activity. Structural analyses of C7 indicate decrease in the helix content, increased exposure of Trp192, and lower thermodynamic stability. The deletion of the C-terminal residues (Leu241 to Ala247) probably perturbs local structure of the C-terminal antigenic epitope that results in the decrease in antigenicity and activities of C7.
Asunto(s)
Abortivos no Esteroideos/inmunología , Fármacos Anti-VIH/inmunología , Antineoplásicos Fitogénicos/inmunología , Tricosantina/inmunología , Secuencia de Aminoácidos , Dicroismo Circular , Guanidina/farmacología , Modelos Moleculares , Datos de Secuencia Molecular , Desnaturalización Proteica , Ingeniería de Proteínas , Estructura Secundaria de Proteína , Ribosomas/efectos de los fármacos , Eliminación de Secuencia , Tricosantina/genéticaAsunto(s)
Enfermedad de Alzheimer/metabolismo , Encéfalo/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Fosfoproteínas/metabolismo , Proteínas tau/metabolismo , Western Blotting , Electroforesis en Gel de Poliacrilamida , Glucógeno Sintasa Quinasas , Humanos , Peso Molecular , Fosfoproteínas/aislamiento & purificación , Fosforilación , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas tau/aislamiento & purificaciónRESUMEN
Extensive in vitro phosphorylation of a purified preparation of control human brain tau consistently produces four rather than, as previously believed, three tau species on SDS-PAGE. The species thus generated are shifted on SDS-PAGE to positions that match those of PHF-tau isolated from Alzheimer's disease brain. A mixture of recombinant human tau isoforms phosphorylated by GSK-3 beta gave similar results to those obtained with control human brain tau. In vitro phosphorylation of the individual recombinant isoforms by GSK-3 beta showed that the four bands of PHF-tau are likely to consist of isoforms 3R,0 alone; 4R,0 with 3R,29; 4R,29 with 3R,58 and 4R,58 alone.