Identification of a novel calpain inhibitor using phage display.
Calpains are calcium- and thiol-dependent proteases that cleave a variety of intracellular substrates. Overactivation of the calpains has been implicated in a number of diseases and conditions such as ischemic stroke indicating a need for the development of calpain inhibitors. A major problem with current calpain inhibitors has been specific targeting to calpain. To identify highly specific calpain interacting peptides, we developed a peptide-phage library screening method based on the calcium-dependent conformation change associated with calpain activation. A phage-peptide library representing greater than 2 billion expressed 12-mers was incubated with calpain I in the presence of calcium. The calcium-dependent bound phage was then eluted by addition of EGTA. After four rounds of selection we found a conserved 5-mer sequence represented by LSEAL. Synthetic LSEAL inhibited tau-calpain interaction and in vitro proteolysis of tau- and alpha-synuclein by calpains. Deletion of the portion of the tau protein containing a homologous sequence to LSEAL resulted in decreased calpain-mediated tau degradation. These data suggest that these peptides may represent novel calpastatin mimetics.
Guttmann
Rodney P
RP
Department of Physiology, University of Kentucky, Lexington, KY 40536, USA. rodneyg@uky.edu
Day
George A
GA
Wang
Xiaohong
X
Bottiggi
Kara A
KA
D000595:Amino Acid Sequence; D001665:Binding Sites; D002154:Calpain / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D019151:Peptide Library; D010455:Peptides / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding
C503415:Leu-Ser-Glu-Ala-Leu; D009842:Oligopeptides; D019151:Peptide Library; D010455:Peptides; D002154:Calpain