RESUMO
The TNF-Related Apoptosis Inducing Ligand (TRAIL) cytokine triggers apoptosis specifically in cancer cells. Susceptibility of a given cell to TRAIL depends on the activity of regulatory proteins, one of the most important of which is BID. The aim of this study was to increase the cytotoxic potential of TRAIL against cancer cells. TRAIL was fused to the BH3 domain of BID. Hence, TRAIL acted not only as an anticancer agent, but also as a specific carrier for the BID fragment. Two fusion protein variants were obtained by genetic engineering, harboring two different linker sequences. The short linker allowed both parts of the fusion protein to fold into their native structures. The long linker influenced the structure of the fused proteins but nonetheless resulted in their highest cytotoxic activity. Optimal buffer formulation was determined for all the analyzed TRAIL variants. Fusing the BH3 domain of BID to TRAIL improved the cytotoxic potential of TRAIL. Further, these findings may be useful for the optimization of other anticancer drugs based on TRAIL, since the appropriate formulation would secure their native structures during prolonged storage.
Assuntos
Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Citotoxinas , Proteínas Recombinantes de Fusão , Ligante Indutor de Apoptose Relacionado a TNF , Células A549 , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/biossíntese , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/química , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/isolamento & purificação , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/farmacologia , Citotoxinas/biossíntese , Citotoxinas/química , Citotoxinas/isolamento & purificação , Citotoxinas/farmacologia , Células Hep G2 , Humanos , Domínios Proteicos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Ligante Indutor de Apoptose Relacionado a TNF/química , Ligante Indutor de Apoptose Relacionado a TNF/isolamento & purificação , Ligante Indutor de Apoptose Relacionado a TNF/farmacologiaRESUMO
The Bcl-2 family of proteins regulates mitochondrial functions during cell death by modulating the efflux of death-promoting proteins such as cytochrome c and endonuclease G. Upon the binding of death ligands to their receptors, caspase-8 cleaves Bid, a BH3-only protein, into tBid that causes the mitochondrial damages resulting in the release of cytochrome c and endonuclease G. Also, another BH3-only protein, hNoxa, has been shown to induce the efflux of cytochrome c from the mitochondria. Whether the efflux proteins from the mitochondria in response to tBid or hNoxa are the same or different, however, has not been addressed. We have demonstrated that endonuclease G activities are not detectable among the proteins released from isolated mitochondria by hNoxa but are detectable in that by tBid. These results suggest that the efflux of proteins from the mitochondria are differentially modulated by tBid and hNoxa.