Determination of Peptide Substrate Specificity for μ-Calpain by a Peptide Library-based Approach

Abstract

Calpains are proteases that catalyze the limited cleavage of target proteins in response to Ca 2؉ signaling. Because of their involvement in pathological conditions such as post-ischemic injury and Alzheimer and Parkinson disease, calpains form a class of pharmacologically significant targets for inhibition. We have determined the sequence preference for the hydrolysis of peptide substrates of the ubiquitous -calpain isoform by a peptide library-based approach using the proteolytic core of -calpain (I-II). The approach, first described by Turk et al. (Turk, B. E., Huang, L. L., Piro, E. T., and Cantley, L. C. (2001) Nat. Biotechnol. 19, 661-667), involved the digestion of an N-terminally acetylated degenerate peptide library in conjunction with Edman sequencing to determine the specificity for residues found at primed positions. The cleavage consensus for these positions was then used to design a second, partially degenerate library, to determine specificity at unprimed positions. We have improved upon the original methodology by using a degenerate peptide dendrimer for determination of specificity at unprimed positions. By using this modified approach, the complete cleavage specificity profile for I-II was determined for all positions flanking the cleaved peptide. A previously known preference of calpains for hydrophobic amino acids at unprimed positions was confirmed. In addition, a novel residue specificity for primed positions was revealed to highlight the importance of these sites for substrate recognition. The optimal primed site motif (MER) was shown to be capable of directing cleavage to a specific peptide bond. Accordingly, we designed a fluorescent resonance energy transferbased substrate with optimal cleavage motifs on the primed and non-primed sides (PLFAER). The -calpain core shows a far greater turnover rate for our substrate than for those based on the cleavage site of ␣-spectrin or the proteolytic sequence consensus compiled from substrate alignments.Calpains (clan CA, family C2 in the MEROPS data base (1)), a family of calcium-activated intracellular proteases, are found in animals, plants, and possibly bacteria (2). They convert intracellular calcium signals (3) into a proteolytic signal by catalyzing the limited cleavage of target proteins (4, 5). Among the known cellular substrates of calpain are numerous cytoskeletal proteins, as well as some receptors and integral membrane proteins like the Na ϩ /Ca 2ϩ exchanger, NCX-3 (6). Calpains must be strictly regulated because they catalyze irreversible processing in the cell. In one scenario, calpains localize to the plasma membrane under activating conditions (7,8). This placement may act to position calpains where they can respond to brief calcium influxes from the opening of calcium channels, resulting in very localized and transient activity. Deactivation of calpain can come about in several ways: binding to the endogenous calpain inhibitor calpastatin (9); autoproteolytic inactivation; or simply the dissipation of local high calcium levels. Unr...

Mutant Huntingtin N-terminal Fragments of Specific Size Mediate Aggregation and Toxicity in Neuronal Cells

“…One possible explanation is that many proteases (e.g. calpains) recognize in their substrates a three-dimensional epitope composed of several amino acids that may be positioned apart from each other (38,39). Thus deletions of short linear stretches of 4 -5 residues may not be sufficient to prevent proteolysis by certain proteases.…”

Localization of low molecular weight crystallin peptides in the aging human lens using a MALDI mass spectrometry imaging approach

“…The dipeptides are His-Phe, Phe-His, and His-His, respectively, each motif then contains consecutive aromatic residues. As calpains, the primary proteases in the human lens, are known to cleave after consecutive aromatic residues (Cuerrier et al, 2005), this may suggest a role for lens-based calpains in these particular instances. Furthermore, tertiary structural elements rather than primary amino acid sequences are likely responsible for directing the cleavage of protein substrates by calpains (Cuerrier et al, 2005), which may explain the high degree of specificity that we have observed for the αA, αB and βA3 peptides.…”

“…As calpains, the primary proteases in the human lens, are known to cleave after consecutive aromatic residues (Cuerrier et al, 2005), this may suggest a role for lens-based calpains in these particular instances. Furthermore, tertiary structural elements rather than primary amino acid sequences are likely responsible for directing the cleavage of protein substrates by calpains (Cuerrier et al, 2005), which may explain the high degree of specificity that we have observed for the αA, αB and βA3 peptides. However, the activity of calpain in the primate lens has been reported to be largely inhibited by high levels of the endogenous calpain inhibitor -calpastatin Nakajima et al, 2006), indicating that other mechanisms may be responsible for these cleavages.…”

Mechanistic Role of Calpains in Postischemic Neurodegeneration

“…The various calpain isoforms are thought to have a common substrate profile. Despite multiple attempts at substrate sequence analysis Cuerrier et al, 2005), no definitive method exists to predict whether a given compound is a calpain substrate, or, if it is a substrate, to identify the cleavage site. Furthermore, there is evidence that cleavage can be regulated by modulation of secondary recognition sequences adjacent to the actual cleavage site (Wang and Yuen, 1999).…”