The ubiquitous calpains, m- and m-calpain, have been implicated in essential physiol. processes and various pathologies. Cell-permeable specific inhibitors are important tools to elucidate the roles of calpains in cultivated cells and animal models. The synthetic N-acetylated 27-mer peptide derived from exon B of the inhibitory domain 1 of human calpastatin (CP1B) is unique as a potent and highly selective reversible calpain inhibitor, but is poorly cell-permeant. By addn. of N-terminal cysteine residues we have generated a disulfide-conjugated CP1B with the cell-penetrating 16-mer peptide penetratin derived from the third helix of the Antennapedia homeodomain protein. The inhibitory potency and selectivity of CP1B for calpain vs. cathepsin B and L, caspase 3 and the proteasome was not affected by the conjugation with penetratin. The conjugate was shown to efficiently penetrate into living LCLC 103H cells, since it prevents ionomycin-induced calpain activation at 200-fold lower concn. than the non-conjugated inhibitor and is able to reduce calpain-triggered apoptosis of these cells. Penetratin-conjugated CP1B seems to be a promising alternative to the widely used cell-permeable peptide aldehydes (e.g. calpain inhibitor I) which inhibit the lysosomal cathepsins and partially the proteasome as well or even better than the calpains.
Calpastatin exon 1B-derived peptide, a selective inhibitor of calpain: Enhancing cell permeability by conjugation with penetratin / Shirley Gil, Parrado; Irmgard Assfalg, Machleidt; Fiorino, Ferdinando; Dominga, Deluca; Dietmar, Pfeiler; Norbert, Schaschke; Luis, Moroder; Werner, M. a. c. h. l. e. i. d. t.. - In: BIOLOGICAL CHEMISTRY. - ISSN 1431-6730. - STAMPA. - 384:3(2003), pp. 395-402.
Calpastatin exon 1B-derived peptide, a selective inhibitor of calpain: Enhancing cell permeability by conjugation with penetratin.
FIORINO, FERDINANDO;
2003
Abstract
The ubiquitous calpains, m- and m-calpain, have been implicated in essential physiol. processes and various pathologies. Cell-permeable specific inhibitors are important tools to elucidate the roles of calpains in cultivated cells and animal models. The synthetic N-acetylated 27-mer peptide derived from exon B of the inhibitory domain 1 of human calpastatin (CP1B) is unique as a potent and highly selective reversible calpain inhibitor, but is poorly cell-permeant. By addn. of N-terminal cysteine residues we have generated a disulfide-conjugated CP1B with the cell-penetrating 16-mer peptide penetratin derived from the third helix of the Antennapedia homeodomain protein. The inhibitory potency and selectivity of CP1B for calpain vs. cathepsin B and L, caspase 3 and the proteasome was not affected by the conjugation with penetratin. The conjugate was shown to efficiently penetrate into living LCLC 103H cells, since it prevents ionomycin-induced calpain activation at 200-fold lower concn. than the non-conjugated inhibitor and is able to reduce calpain-triggered apoptosis of these cells. Penetratin-conjugated CP1B seems to be a promising alternative to the widely used cell-permeable peptide aldehydes (e.g. calpain inhibitor I) which inhibit the lysosomal cathepsins and partially the proteasome as well or even better than the calpains.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.