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Calpains are intracellular Ca2+-regulated cysteine proteases that are essential for various cellular functions. Mammalian conventional calpains (calpain-1 and calpain-2) modulate the structure and function of their substrates by limited proteolysis. Thus, it is critically important to determine the site(s) in proteins at which calpains cleave. However, the calpains' substrate specificity remains unclear, because the amino acid (aa) sequences around their cleavage sites are very diverse. To clarify calpains' substrate specificities, 84 20-mer oligopeptides, corresponding to P10-P10′ of reported cleavage site sequences, were proteolyzed by calpains, and the catalytic efficiencies (kcat/Km) were globally determined by LC/MS. This analysis revealed 483 cleavage site sequences, including 360 novel ones. The kcat/Kms for 119 sites ranged from 12.5–1,710 M−1s−1. Although most sites were cleaved by both calpain-1 and −2 with a similar kcat/Km, sequence comparisons revealed distinct aa preferences at P9-P7/P2/P5′. The aa compositions of the novel sites were not statistically different from those of previously reported sites as a whole, suggesting calpains have a strict implicit rule for sequence specificity, and that the limited proteolysis of intact substrates is because of substrates' higher-order structures. Cleavage position frequencies indicated that longer sequences N-terminal to the cleavage site (P-sites) were preferred for proteolysis over C-terminal (P′-sites). Quantitative structure-activity relationship (QSAR) analyses using partial least-squares regression and >1,300 aa descriptors achieved kcat/Km prediction with r = 0.834, and binary-QSAR modeling attained an 87.5% positive prediction value for 132 reported calpain cleavage sites independent of our model construction. These results outperformed previous calpain cleavage predictors, and revealed the importance of the P2, P3′, and P4′ sites, and P1-P2 cooperativity. Furthermore, using our binary-QSAR model, novel cleavage sites in myoglobin were identified, verifying our predictor. This study increases our understanding of calpain substrate specificities, and opens calpains to “next-generation,” i.e. activity-related quantitative and cooperativity-dependent analyses.

Calpains are intracellular Ca2+ -requiring 'modulator proteases', which modulate cellular functions by limited and specific proteolysis. p94/calpain3, a skeletal-muscle specific calpain, has been one of the representative calpain species which indicates physiological importance of calpain proteolytic system; a defect of proteolytic activity of p94 causes limb girdle muscular dystrophy type2A (LGMD2A, also called 'calpainopathy'). Immunohistochemical studies on myofibrils showed that p94 localizes at the Z- and N2-line regions of sarcomeres. It was also identified by the yeast two hybrid studies that p94 binds to the N2A and M-line regions of connectin. Furthermore, genetic studies indicate that p94 is indispensable for skeletal muscles, although its precise functions are still unclear. Interestingly, connectin provides sarcomere not only with elasticity but also with binding sites to various multi-functional proteins such as muscle ankyrin repeat proteins (MARPs), muscle RING finger proteins (MURFs), titin-capping protein (T-cap/telethonin), sarcomeric-alpha-actinin, p94 etc. Binding sites for these proteins are not randomly placed along connectin but rather accumulated in the Z-, N2-, and/or M-line regions, indicating the existence of 'signal complexes' unique to each regions. The concept of these complexes are strongly supported by the facts that mutations of connectin or its binding proteins in these regions severely perturb muscle functions, as in the case of LGMD2A caused by mutations in the p94 gene. Therefore, it is hypothesized that the 'signal complexes' in the Z-, N2-, and M-lines modulate muscle cell homeostasis by transducing signals of external stimulations/stresses to trigger appropriate response at various different cellular events such as protein modification and gene expressions. In this article, we performed detailed immunohistochemical analyses of p94 on isolated single myofibers. Together with recent findings about p94, it is suggested that sarcomeric localization of p94, especially its M-line localization, is affected by the combination of cellular contexts such as contractile status of myofibrils, fiber type compositions, sarcomeric maturation, and the composition of the 'signal complexes' in each region.

Calpain is a Ca2؉ -regulated cytosolic protease. Mammals have 14 calpain genes, half of which are predominantly expressed in specific organ(s); the rest are expressed ubiquitously. A defect in calpains causes lethality/pathogenicity, indicating their physiological indispensability. nCL-2/calpain-8a was identified as a stomach-specific calpain, whose physiological functions are unclear. To elucidate these, we characterized nCL-2 in detail. Unexpectedly, nCL-2 was localized strictly to the surface mucus cells in the gastric epithelium and the mucus-secreting goblet cells in the duodenum. Yeast twohybrid screening identified several nCL-2-intracting molecules. Of these, the ␤-subunit of coatomer complex (␤-COP) occurs in the stomach pit cells and is proteolyzed by nCL-2 in vitro. Furthermore, ␤-COP and nCL-2 co-expressed in COS7 cells co-localized in the Golgi, and Ca 2؉ -ionophore stimulation caused the proteolysis of ␤-COP near the linker region, resulting in the dissociation of ␤-COP from the Golgi. These results strongly suggest novel functions for nCL-2 that involve the membrane trafficking of mucus cells via interactions with coat protein.Calpain (EC 3.4.22.17) is an intracellular Ca 2ϩ -regulated cysteine protease, comprising a superfamily in most organisms, including some bacteria, budding yeasts, fungi, plants, and animals. Calpains proteolyze specific substrates at very limited sites to irreversibly modify/modulate their functions, structures, and activities, and are thus called "modulator proteases." Fourteen calpain genes have been identified in humans and mice and can be classified into two categories according to their expression, i.e. ubiquitous or tissue-specific (1-3). Ubiquitous -and m-calpains, two major calpains in mammals, form a heterodimer composed of a distinct 80-kDa catalytic large subunit (abbreviated to "CL" and "mCL," respectively) 2 and a common 30-kDa regulatory small subunit (30K). Calpain three-dimensional structures define four (I-IV) and two (V and VI) domains in the large and small subunits, respectively: the regulatory N-terminal domain (I), the protease domain (II), the C2-domain-like Ca 2ϩ /phospholipid-binding domain (III), the penta-EF-hand domains (IV and VI), and the Glyclustering domain (V) (4, 5). In the absence of Ca 2ϩ , the protease domain (II) is further divided into two subdomains, IIa and IIb, which form a single active domain upon binding to Ca 2ϩ (6, 7). Genetic studies in mammals and other organisms have clearly shown the physiological indispensability of the calpains. A gene knock-out mouse with a mutation in the gene for 30K, Capn4, exhibits embryonic lethality (8); mutations in the human gene for skeletal-muscle-specific p94/calpain 3, CAPN3, are responsible for limb-girdle muscular dystrophy type 2A (9); and a single nucleotide polymorphism in intron 3 of the human gene for calpain 10, CAPN10, is associated with type 2 diabetes (10). Mutations in the calpain genes of various other organisms, including yeasts, plants, nematodes, and Drosophila, result in sign...