logo CaMPDB: Calpain for Modulatory Proteolysis Database

Calpain (EC, Clan CA, family C02) has a substrate specificity that is relatively restricted, and most of oligopeptides are not efficiently hydrolyzed. Casein is the most popular substrate for in vitro assays, and it is used either as the natural protein or modified with various chromophores, fluorescent reagents or isotopes. Calpain purified from skeletal muscle by standard methods has a specific activity of several hundred units per mg protein, where 1 unit corresponds to an increase of 1.0 absorbance unit at 280 nm per hour under the standard assay conditions (if 1 unit of calpain is incubated in 0.5 ml of 3 mg/ml casein, 0.1 M Tris-HCl (pH 7.5), 25 mM of 2-mercaptoethanol, and appropriate concentrations of CaCl2 at 30oC for 20 min, the acid-soluble supernatant made by adding 0.5 ml of 10% trichloroacetic acid and centrifugation shows an increase of 0.333 absorbance unit at 280 nm). Activity is dependent on the Ca2+ concentration, and that giving half maximal activity for μ- and m-calpains are around 50 μM and 0.3 mM, respectively.

The rules governing the specificity of the calpains remain unclear. This is the main motif for this Calpain for Modulatory Proteolysis Database (CaMPDB) project. It seems that calpain recognizes the overall 3-dimensional structure of its substrates more than the primary structure. Even so, hydrophobic (Tyr, Met, Leu, Val) and Arg residues tend to be preferred in position P2 (Brown & Crawford 1993; Stabach et al. 1997; Tompa et al. 2004). Protein kinases, phosphatases, phospholipases, cytoskeletal proteins, membrane proteins, cytokines, transcription factors, lens proteins, calmodulin-binding proteins and others have been suggested to be in vivo substrates, but clear evidence has not yet been obtained. Calpain proteolyzes these proteins in a limited manner rather than digesting them to small peptides, indicating its modulatory functions for the substrate proteins by cutting their interdomain regions (Saido et al. 1994b; Sorimachi et al. 1997) (Fig. 1-3). In the following sections, several representative examples are to be described.


Fig. 1. Modulation by "Eraser" Protease and "Modulator" Protease In contrast to the “Eraser” proteases such as proteasome and cathepsins, calpains very limitedly proteolyze substrates to modulate/change their activities, specificities, structures, intracellular localizations, and half-life. Therefore, calpain should be called “Modulator” protease.


Fig. 2. Different proteolytic modes of autophagic degradation, proteasome degradation, and calpain proteolysis There are three representative intracellular proteolytic systems, autophagy-lysosome system, ubiquitin-proteasome system, and calpain-calpastatin system. In each system, proteases functions rather differently: lysosome proteases such as cathepsins randomly and completely degrade substrates to amino acid levels; proteasomes regularly (but not specifically) degrade substrates to 8-12mer oligopeptides; whereas calpains proteolyze substrates mainly at the inter-domain polypeptide chain. Therefore, unlike substrates degraded by lysosomal proteases or proteasomes, those proteolyzed by calpains are functional in most cases.

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1. Activity of calpain

2. Substrates of calpains in brain function

3. p53 and calpain

4. Calpain and apoptosis

5. References