RESUMEN
Pulchellin is a type 2 ribosome-inactivating protein isolated from seeds of the Abrus pulchellus tenuiflorus plant. This study aims to obtain active and homogeneous protein for structural and biological studies that will clarify the functional aspects of this toxin. The DNA fragment encoding pulchellin A-chain was cloned and inserted into pGEX-5X to express the recombinant pulchellin A-chain (rPAC) as a fusion protein in Escherichia coli. The deduced amino acid sequence analyses of the rPAC presented a high sequential identity (> 86%) with the A-chain of abrin-c. The ability of the rPAC to depurinate rRNA in yeast ribosome was also demonstrated in vitro. In order to validate the toxic activity we promoted the in vitro association of the rPAC with the recombinant pulchellin binding chain (rPBC). Both chains were incubated in the presence of a reduced/oxidized system, yielding an active heterodimer (rPAB). The rPAB showed an apparent molecular mass of approximately 60 kDa, similar to the native pulchellin. The toxic activities of the rPAB and native pulchellin were compared by intraperitoneal injection of different dilutions into mice. The rPAB was able to kill 50% of the tested mice with doses of 45 microg x kg(-1). Our results indicated that the heterodimer showed toxic activity and a conformational pattern similar to pulchellin. In addition, rPAC produced in this heterologous system might be useful for the preparation of immunoconjugates with potential as a therapeutic agent.
Asunto(s)
Abrus/genética , Proteínas de Plantas/metabolismo , Subunidades de Proteína/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Ribosomas/metabolismo , Abrus/química , Secuencia de Aminoácidos , Animales , Dicroismo Circular , Clonación Molecular , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , ADN de Plantas/genética , ADN de Plantas/metabolismo , Inyecciones Intraperitoneales , Ratones , Datos de Secuencia Molecular , N-Glicosil Hidrolasas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/toxicidad , Conformación Proteica , Subunidades de Proteína/química , Subunidades de Proteína/toxicidad , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN de Planta/genética , ARN de Planta/metabolismo , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/toxicidad , Saccharomyces cerevisiae/metabolismo , Semillas/química , Homología de Secuencia de AminoácidoRESUMEN
Using 2-DE of total cell protein extracts, we compared soluble proteins from murine melanoma lines Tm1 and Tm5 with proteins from the nontumoral cell melan-a from which they were derived. Seventy-one of the 452 spots (average) detected with CBB were differentially accumulated, i.e., increased or decreased twofold. Forty-four spots were identified by PMF/MALDI-TOF, 15 with increased and 29 with decreased protein levels. SAGE showed that 17/34 (50%) of the differentially accumulated proteins, pI range 4-7, presented similar differences at the mRNA level. Major reductions in protein were observed in tumor cells of proteins that degrade reactive oxygen species (ROS). Decreases of > or = twofold in GST, superoxide dismutase, aldehyde dehydrogenase, thioredoxin, peroxiredoxin 2, and peroxiredoxin 6 protein were observed. SAGE indicated the reduction of other proteins involved in ROS degradation. As expected, the accumulation of exogenous peroxides was significantly higher in the tumor cells while the levels of glutathionylation were two times lower in the tumor cells compared to melan-a. The differential accumulation of proteins involved in oncogene/tumor suppressor pathways was observed. Melanoma cells can favor survival pathways activated by ROS by inhibiting p53 pathways and activation of Ras and c-myc pathways.