title: Calpastatin exon 1B-derived peptide, a selective inhibitor of calpain: enhancing cell permeability by conjugation with penetratin.
abstract: The ubiquitous calpains, mu- and m-calpain, have been implicated in essential physiological 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 addition 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 versus 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 concentration 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 1) which inhibit the lysosomal cathepsins and partially the proteasome as well or even better than the calpains.
authors.affiliation: Abteilung für Klinische Chemie und Klinische Biochemie, Chirurgische Klinik Innenstadt, Klinikum der Ludwig-Maximilians-Universität, Nussbaumstr. 20, D-80336 München, Germany.
authors.forename: Shirley
authors.initials: S
authors.lastname: Gil-Parrado
authors.forename: Irmgard
authors.initials: I
authors.lastname: Assfalg-Machleidt
authors.forename: Ferdinando
authors.initials: F
authors.lastname: Fiorino
authors.forename: Dominga
authors.initials: D
authors.lastname: Deluca
authors.forename: Dietmar
authors.initials: D
authors.lastname: Pfeiler
authors.forename: Norbert
authors.initials: N
authors.lastname: Schaschke
authors.forename: Luis
authors.initials: L
authors.lastname: Moroder
authors.forename: Werner
authors.initials: W
authors.lastname: Machleidt
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000494:pharmacology*; D002154:Calpain / Q000037:antagonists & inhibitors*; D002352:Carrier Proteins / Q000737:chemistry / Q000494:pharmacology*; D002403:Cathepsins / Q000037:antagonists & inhibitors; D002460:Cell Line; D002463:Cell Membrane Permeability; D057846:Cell-Penetrating Peptides; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D004912:Erythrocytes / Q000201:enzymology; D005091:Exons; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D057846:Cell-Penetrating Peptides; D015853:Cysteine Proteinase Inhibitors; D010446:Peptide Fragments; D015759:Ionomycin; C033668:calpastatin; C414409:penetratin; D002403:Cathepsins; D002154:Calpain; C449002:mu-calpain