Synthesis of cell-penetrating conjugates of calpain activator peptides.

TitleSynthesis of cell-penetrating conjugates of calpain activator peptides.
Publication TypeJournal Article
Year of Publication2007
AuthorsBánóczi, Z., Á. Tantos, A. Farkas, P. Tompa, P. Friedrich, and F. Hudecz
JournalBioconjug Chem
Date Published2007 Jan-Feb
KeywordsAmino Acid Sequence, Animals, Calpain, Cell Membrane Permeability, Cercopithecus aethiops, COS Cells, Membrane Proteins, Peptides

Calpains, the intracellular proteolytic enzymes, play important roles in various processes in cells. The lack of calpain or its overexpression is thought to be an underlying factor in some diseases. In this study, we report the synthesis of a new group of cell-penetrating calpastatin-peptide conjugates with the activating capacity of m-calpain intracellularly. In these constructs, peptides related to the calpastatin A or C subunit with the capabiliy of activation of isolated m-calpain was covalently conjugated to the C-terminal of penetratin via amide, thioether, or disulfide bond. These conjugates were prepared by solid-phase synthesis and/or by chemical ligation and properly characterized (MS, HPLC). Our results using isolated m-calpain suggest that conjugation does not interfere with the enzyme-activating effect of the calpastatin peptides; in fact, the efficiency of the conjugates was markedly higher. The conjugates with different bonds showed essentially the same level of activation. Internalization experiments with fluorophore (4-[7-hydroxycoumaryl] acetic acid (Hca) at the N-terminal of penetratin and/or 5(6)-carboxyfluorescein (cf)) labeled conjugates show that these constructs are taken up by COS-7 cells. Using cell lysates produced after incubation with the 1:1 (mol/mol) mixture of calpastatin A and C peptide conjugates, we found a significant calpain activating effect. We also noticed that the conjugate even with a disulfide bond between the components seems to be stable and activate m-calpain after intracellular translocation under the conditions studied. To the best of our knowledge, this is the first report to describe conjugates with an m-calpain activating effect on isolated enzymes and more importantly within living cells after transmembrane delivery. Thus, these conjugates seem to be appropriate as molecular tools to activate intracellular m-calpain and to study calpain functions in living cells.

Alternate JournalBioconjug. Chem.
PubMed ID17226965