Predictions of Cleavability of Calpain Proteolysis by Quantitative Structure-Activity Relationship Analysis Using Newly Determined Cleavage Sites and Catalytic Efficiencies of an Oligopeptide Array.

Calpains are intracellular Ca(2+)-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. Thekcat/Kms for 119 sites ranged from 12.5-1,710 M(-1)s(-1) Although most sites were cleaved by both calpain-1 and -2 with a similarkcat/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 achievedkcat/Kmprediction withr= 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.

A Gastrointestinal Calpain Complex, G-calpain, Is a Heterodimer of CAPN8 and CAPN9 Calpain Isoforms, Which Play Catalytic and Regulatory Roles, Respectively.

Calpains (CAPN) are a family of Ca2+-dependent cysteine proteases that regulate various cellular functions by cleaving diverse substrates. Of the 15 mammalian calpains, CAPN8 and CAPN9 are two that are expressed predominantly in the gastrointestinal tract, where they interact to form a protease complex, termed G-calpain. However, because native G-calpain exhibits a highly restricted expression pattern, it has never been purified, and the interactions between CAPN8 and CAPN9 have not been characterized. Here, we clarified the molecular nature of G-calpain by using recombinant proteins and transgenic mice expressing FLAG-tagged CAPN8 (CAPN8-FLAG). Recombinant mouse CAPN8 and CAPN9 co-expressed in eukaryotic expression systems exhibited the same mobility as native mouse G-calpain in Blue Native-PAGE gels, and CAPN8-FLAG immunoprecipitation from stomach homogenates of the transgenic mice showed that CAPN9 was the only protein that associated with CAPN8-FLAG. These results indicated that G-calpain is a heterodimer of CAPN8 and CAPN9. In addition, active recombinant G-calpain was expressed and purified using an in vitro translation system, and the purified protease exhibited enzymatic properties that were comparable with that of calpain-2. We found that an active-site mutant of CAPN8, but not CAPN9, compromised G-calpain's substrate cleavage activity, and that the N-terminal helix region of CAPN8 and the C-terminal EF-hands of CAPN8 and CAPN9 were involved in CAPN8/9 dimerization. Furthermore, CAPN8 protein in Capn9-/- mice was almost completely lost, whereas CAPN9 was only partially lost in Capn8-/- mice. Collectively, these results demonstrated that CAPN8 and CAPN9 function as catalytic and chaperone-like subunits, respectively, in G-calpain.

The N- and C-terminal autolytic fragments of CAPN3/p94/calpain-3 restore proteolytic activity by intermolecular complementation.

CAPN3/p94/calpain-3, a calpain protease family member predominantly expressed in skeletal muscle, possesses unusually rapid and exhaustive autolytic activity. Mutations in the human CAPN3 gene impairing its protease functions cause limb-girdle muscular dystrophy type 2A (LGMD2A); yet, the connection between CAPN3's autolytic activity and the enzyme's function in vivo remain unclear. Here, we demonstrated that CAPN3 protease activity was reconstituted by intermolecular complementation (iMOC) between its two autolytic fragments. Furthermore, the activity of full-length CAPN3 active-site mutants was surprisingly rescued through iMOC with autolytic fragments containing WT amino acid sequences. These results provide evidence that WT CAPN3 can be formed by the iMOC of two different complementary CAPN3 mutants. The finding of iMOC-mediated restoration of calpain activity indicates a novel mechanism for the genotype-phenotype links in LGMD2A.

Calpain-6 deficiency promotes skeletal muscle development and regeneration.

Calpains are Ca(2+)-dependent modulator Cys proteases that have a variety of functions in almost all eukaryotes. There are more than 10 well-conserved mammalian calpains, among which eutherian calpain-6 (CAPN6) is unique in that it has amino acid substitutions at the active-site Cys residue (to Lys in humans), strongly suggesting a loss of proteolytic activity. CAPN6 is expressed predominantly in embryonic muscles, placenta, and several cultured cell lines. We previously reported that CAPN6 is involved in regulating microtubule dynamics and actin reorganization in cultured cells. The physiological functions of CAPN6, however, are still unclear. Here, to elucidate CAPN6's in vivo roles, we generated Capn6-deficient mice, in which a lacZ expression cassette was integrated into the Capn6 gene. These Capn6-deficient mouse embryos expressed lacZ predominantly in skeletal muscles, as well as in cartilage and the heart. Histological and biochemical analyses showed that the CAPN6 deficiency promoted the development of embryonic skeletal muscle. In primary cultured skeletal muscle cells that were induced to differentiate into myotubes, Capn6 expression was detected in skeletal myocytes, and Capn6-deficient cultures showed increased differentiation. Furthermore, we found that CAPN6 was expressed in the regenerating skeletal muscles of adult mice after cardiotoxin-induced degeneration. In this experimental system, Capn6-deficient mice exhibited more advanced skeletal-muscle regeneration than heterozygotes or wild-type mice at the same time point. These results collectively showed that a loss of CAPN6 promotes skeletal muscle differentiation during both development and regeneration, suggesting a novel physiological function of CAPN6 as a suppressor of skeletal muscle differentiation.

Muscle-specific calpain-3 is phosphorylated in its unique insertion region for enrichment in a myofibril fraction.

CAPN3 (also called p94/calpain-3) is a skeletal muscle-specific calpain, an intracellular cysteine protease. Loss of CAPN3 protease activity and/or structural functions cause limb-girdle muscular dystrophy type 2A (LGMD2A). However, the precise mechanism of action of CAPN3 in skeletal muscles in vivo remains largely elusive. By studying the protein modifications that regulate CAPN3 activity, we found that CAPN3 was phosphorylated. By performing mutagenesis and mass spectrometry analyses, we identified two Ser residues at positions 629 and 636 in human CAPN3 that are phosphorylated and showed that S629 is a major phosphorylation site. Intriguingly, rapid and exhaustive autolysis of CAPN3 was slightly attenuated by the substitution of S629. In skeletal muscles, phosphorylated CAPN3 was enriched in the myofibril fraction. These results imply that phosphorylated CAPN3 is a myofibril structural component and/or participates in myofibril-based signaling pathways, rather than functions as a protease. We evaluated the relationship between phosphorylated CAPN3 and the pathology of LGMD2A. The level of phosphorylated CAPN3 was greatly reduced in LGMD2A muscles. Our findings suggest that phosphorylated CAPN3 is involved in the pathology of LGMD2A through defects in myofibril integrity and/or signaling pathways. This is the first report that phosphorylation of CAPN3 may be involved in its physiological function.

Efficient expression and purification of recombinant human µ-calpain using an Escherichia coli expression system.

Calpains comprise a superfamily of Ca(2+) -regulated cysteine proteases that are indispensable for the regulation of various cellular functions. Of these, the mammalian µ- and m-calpains are the best characterized isoforms. They are ubiquitously expressed and form heterodimers consisting of a distinct 80-kDa catalytic subunit (CAPN1 for µ-calpain and CAPN2 for m-calpain) and a common 30-kDa regulatory subunit (CAPNS1). To date, various expression systems have been developed for producing recombinant calpains for structural and functional studies; however, no low-cost, simple and efficient bacterial expression system for µ-calpain has been available, because the protein forms aggregates. Here, we established an efficient method for producing active recombinant human µ-calpain using an Escherichia coli expression system. This was achieved by co-expressing CAPN1 and CAPNS1 lacking the N-terminal Gly-rich domain (CAPNS1ΔGR) in the SoluBL21 strain. From 1 L of E. coli culture, over 2 and 6 mg, respectively, of µ-calpain and its active-site mutant µ-calpain:C115S (CAPN1:C115S+CAPNS1ΔGR) were purified by two successive column chromatographies. Compared to the native enzyme, the purified µ-calpain showed almost identical properties, demonstrating its suitability for use in structural and functional studies. This is the first report of the bacterial expression and the simple and efficient purification of active recombinant µ-calpain.

Collaboration between a cis-interacting natural killer cell receptor and membrane sphingolipid is critical for the phagocyte function.

Inhibitory natural killer (NK) cell receptors recognize MHC class I (MHC-I) in trans on target cells and suppress cytotoxicity. Some NK cell receptors recognize MHC-I in cis, but the role of this interaction is uncertain. Ly49Q, an atypical Ly49 receptor expressed in non-NK cells, binds MHC-I in cis and mediates chemotaxis of neutrophils and type I interferon production by plasmacytoid dendritic cells. We identified a lipid-binding motif in the juxtamembrane region of Ly49Q and found that Ly49Q organized functional membrane domains comprising sphingolipids via sulfatide binding. Ly49Q recruited actin-remodeling molecules to an immunoreceptor tyrosine-based inhibitory motif, which enabled the sphingolipid-enriched membrane domain to mediate complicated actin remodeling at the lamellipodia and phagosome membranes during phagocytosis. Thus, Ly49Q facilitates integrative regulation of proteins and lipid species to construct a cell type-specific membrane platform. Other Ly49 members possess lipid binding motifs; therefore, membrane platform organization may be a primary role of some NK cell receptors.

Calpains: an elaborate proteolytic system.

Calpain is an intracellular Ca(2+)-dependent cysteine protease (EC 3.4.22.17; Clan CA, family C02). Recent expansion of sequence data across the species definitively shows that calpain has been present throughout evolution; calpains are found in almost all eukaryotes and some bacteria, but not in archaebacteria. Fifteen genes within the human genome encode a calpain-like protease domain. Interestingly, some human calpains, particularly those with non-classical domain structures, are very similar to calpain homologs identified in evolutionarily distant organisms. Three-dimensional structural analyses have helped to identify calpain's unique mechanism of activation; the calpain protease domain comprises two core domains that fuse to form a functional protease only when bound to Ca(2+)via well-conserved amino acids. This finding highlights the mechanistic characteristics shared by the numerous calpain homologs, despite the fact that they have divergent domain structures. In other words, calpains function through the same mechanism but are regulated independently. This article reviews the recent progress in calpain research, focusing on those studies that have helped to elucidate its mechanism of action. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.

Intravitreal injection or topical eye-drop application of a µ-calpain C2L domain peptide protects against photoreceptor cell death in Royal College of Surgeons' rats, a model of retinitis pigmentosa.

Mitochondrial µ-calpain initiates apoptosis-inducing factor (AIF)-dependent apoptosis in retinal photoreceptor degeneration. Mitochondrial µ-calpain inhibitors may represent therapeutic targets for the disease. Therefore, we sought to identify inhibitors of mitochondrial calpains and determine their effects in Royal College of Surgeons' (RCS) rats, an animal model of retinitis pigmentosa (RP). We synthesized 20-mer peptides of the C2-like (C2L) domain of µ-calpain. Two µ-calpain peptides N2 and N9 inhibited mitochondrial µ-calpain activity (IC(50); 892 and 498nM, respectively), but not other proteases. Western blotting showed that 50µM of both µ-calpain peptides caused specific degradation of mitochondrial µ-calpain. Three-dimensional structure of calpains suggested that the peptides N2 and N9 corresponded to the regions forming salt bridges between the protease core domain 2 and the C2L domain. We determined the inhibitory regions of µ-calpain peptides N2 and N9 using 10-mers, and one peptide, N2-10-2, inhibited the activity of mitochondrial µ-calpain (IC(50); 112nM). We next conjugated the peptide N2-10-2 to the C-terminal of HIV-1 tat (HIV), a cell-penetrating peptide. Using isolated rat liver mitochondria, 50µM HIV-conjugated µ-calpain N2-10-2 peptide (HIV-Nµ, IC(50); 285nM) significantly inhibited AIF truncation. The intravitreal injection of 20mM HIV-Nµ also prevented retinal photoreceptor apoptosis determined by TUNEL staining, and preserved retinal function assessed by electroretinography in RCS rats. Topical application of 40mM HIV-Nµ also prevented apoptosis of retinal photoreceptors in RCS rats. Our results demonstrate that HIV-Nµ, a peptide inhibitor of mitochondrial µ-calpain, offers a new modality for treating RP.

Calpain-6, a microtubule-stabilizing protein, regulates Rac1 activity and cell motility through interaction with GEF-H1.

Crosstalk between microtubules and actin filaments is crucial for various cellular functions, including cell migration, spreading and cytokinesis. The Rac1 GTPase plays a key role in such crosstalk at the leading edge of migrating cells in order to promote lamellipodial formation. However, the mechanism underlying the link between microtubules and Rac1 activation remains unclear. Here, we show that calpain-6 (CAPN6), a non-proteolytic calpain with microtubule-binding and -stabilizing activity, might participate in this crosstalk. Small interfering RNA (siRNA)-induced knockdown of Capn6 in NIH 3T3 cells resulted in Rac1 activation, which promoted cell migration, spreading and lamellipodial protrusion. This increase in Rac1 activity was abolished by knockdown of the Rho guanine nucleotide exchange factor GEF-H1 (officially known as Arhgef2). CAPN6 and GEF-H1 colocalized with microtubules and also interacted with each other through specific domains. Upon knockdown of Capn6, GEF-H1 was shown to translocate from microtubules to the lamellipodial region and to interact with Rac1. By contrast, RhoA activity was decreased upon knockdown of Capn6, although low levels of active RhoA or the presence of RhoA molecules appeared to be required for the Capn6-knockdown-induced Rac1 activation. We suggest that CAPN6 acts as a potential regulator of Rac1 activity, through a mechanism involving interaction with GEF-H1, to control lamellipodial formation and cell motility.

Calpain 8/nCL-2 and calpain 9/nCL-4 constitute an active protease complex, G-calpain, involved in gastric mucosal defense.

Calpains constitute a superfamily of Ca2+-dependent cysteine proteases, indispensable for various cellular processes. Among the 15 mammalian calpains, calpain 8/nCL-2 and calpain 9/nCL-4 are predominantly expressed in the gastrointestinal tract and are restricted to the gastric surface mucus (pit) cells in the stomach. Possible functions reported for calpain 8 are in vesicle trafficking between ER and Golgi, and calpain 9 are implicated in suppressing tumorigenesis. These highlight that calpains 8 and 9 are regulated differently from each other and from conventional calpains and, thus, have potentially important, specific functions in the gastrointestinal tract. However, there is no direct evidence implicating calpain 8 or 9 in human disease, and their properties and physiological functions are currently unknown. To address their physiological roles, we analyzed mice with mutations in the genes for these calpains, Capn8 and Capn9. Capn8(-/-) and Capn9(-/-) mice were fertile, and their gastric mucosae appeared normal. However, both mice were susceptible to gastric mucosal injury induced by ethanol administration. Moreover, the Capn8(-/-) stomach showed significant decreases in both calpains 9 and 8, and the same was true for Capn9(-/-). Consistent with this finding, in the wild-type stomach, calpains 8 and 9 formed a complex we termed "G-calpain," in which both were essential for activity. This is the first example of a "hybrid" calpain complex. To address the physiological relevance of the calpain 8 proteolytic activity, we generated calpain 8:C105S "knock-in" (Capn8(CS/CS)) mice, which expressed a proteolytically inactive, but structurally intact, calpain 8. Although, unlike the Capn8(-/-) stomach, that of the Capn8(CS/CS) mice expressed a stable and active calpain 9, the mice were susceptible to ethanol-induced gastric injury. These results provide the first evidence that both of the gastrointestinal-tract-specific calpains are essential for gastric mucosal defense, and they point to G-calpain as a potential target for gastropathies caused by external stresses.

Understanding the substrate specificity of conventional calpains.

Calpains are intracellular Ca(2+)-dependent Cys proteases that play important roles in a wide range of biological phenomena via the limited proteolysis of their substrates. Genetic defects in calpain genes cause lethality and/or functional deficits in many organisms, including humans. Despite their biological importance, the mechanisms underlying the action of calpains, particularly of their substrate specificities, remain largely unknown. Studies show that certain sequence preferences influence calpain substrate recognition, and some properties of amino acids have been related successfully to substrate specificity and to the calpains' 3D structure. The full spectrum of this substrate specificity, however, has not been clarified using standard sequence analysis algorithms, e.g., the position-specific scoring-matrix method. More advanced bioinformatics techniques were used recently to identify the substrate specificities of calpains and to develop a predictor for calpain cleavage sites, demonstrating the potential of combining empirical data acquisition and machine learning. This review discusses the calpains' substrate specificities, introducing the benefits of bioinformatics applications. In conclusion, machine learning has led to the development of useful predictors for calpain cleavage sites, although the accuracy of the predictions still needs improvement. Machine learning has also elucidated information about the properties of calpains' substrate specificities, including a preference for sequences over secondary structures and the existence of a substrate specificity difference between two similar conventional calpains, which has never been indicated biochemically.

The UFM1 system regulates ER-phagy through the ufmylation of CYB5R3.

Protein modification by ubiquitin-like proteins (UBLs) amplifies limited genome information and regulates diverse cellular processes, including translation, autophagy and antiviral pathways. Ubiquitin-fold modifier 1 (UFM1) is a UBL covalently conjugated with intracellular proteins through ufmylation, a reaction analogous to ubiquitylation. Ufmylation is involved in processes such as endoplasmic reticulum (ER)-associated protein degradation, ribosome-associated protein quality control at the ER and ER-phagy. However, it remains unclear how ufmylation regulates such distinct ER-related functions. Here we identify a UFM1 substrate, NADH-cytochrome b5 reductase 3 (CYB5R3), that localizes on the ER membrane. Ufmylation of CYB5R3 depends on the E3 components UFL1 and UFBP1 on the ER, and converts CYB5R3 into its inactive form. Ufmylated CYB5R3 is recognized by UFBP1 through the UFM1-interacting motif, which plays an important role in the further uyfmylation of CYB5R3. Ufmylated CYB5R3 is degraded in lysosomes, which depends on the autophagy-related protein Atg7- and the autophagy-adaptor protein CDK5RAP3. Mutations of CYB5R3 and genes involved in the UFM1 system cause hereditary developmental disorders, and ufmylation-defective Cyb5r3 knock-in mice exhibit microcephaly. Our results indicate that CYB5R3 ufmylation induces ER-phagy, which is indispensable for brain development.

Skeletal muscle-specific calpain is an intracellular Na+-dependent protease.

Because intracellular [Na(+)] is kept low by Na(+)/K(+)-ATPase, Na(+) dependence is generally considered a property of extracellular enzymes. However, we found that p94/calpain 3, a skeletal-muscle-specific member of the Ca(2+)-activated intracellular "modulator proteases" that is responsible for a limb-girdle muscular dystrophy ("calpainopathy"), underwent Na(+)-dependent, but not Cs(+)-dependent, autolysis in the absence of Ca(2+). Furthermore, Na(+) and Ca(2+) complementarily activated autolysis of p94 at physiological concentrations. By blocking Na(+)/K(+)-ATPase, we confirmed intracellular autolysis of p94 in cultured cells. This was further confirmed using inactive p94:C129S knock-in (p94CS-KI) mice as negative controls. Mutagenesis studies showed that much of the p94 molecule contributed to its Na(+)/Ca(2+)-dependent autolysis, which is consistent with the scattered location of calpainopathy-associated mutations, and that a conserved Ca(2+)-binding sequence in the protease acted as a Na(+) sensor. Proteomic analyses using Cs(+)/Mg(2+) and p94CS-KI mice as negative controls revealed that Na(+) and Ca(2+) direct p94 to proteolyze different substrates. We propose three roles for Na(+) dependence of p94; 1) to increase sensitivity of p94 to changes in physiological [Ca(2+)], 2) to regulate substrate specificity of p94, and 3) to regulate contribution of p94 as a structural component in muscle cells. Finally, this is the first example of an intracellular Na(+)-dependent enzyme.

MURF1 deficiency suppresses unloading-induced effects on osteoblasts and osteoclasts to lead to bone loss.

Loss of mechanical stress or unloading causes disuse osteoporosis that leads to fractures and deteriorates body function and affects mortality rate in aged population. This bone loss is due to reduction in osteoblastic bone formation and increase in osteoclastic bone resorption. MuRF1 is a muscle RING finger protein which is involved in muscle wasting and its expression is enhanced in the muscle of mice subjected to disuse condition such as hind limb unloading (HU). However, whether MuRF1 is involved in bone loss due to unloading is not known. We therefore examined the effects of MuRF1 deficiency on unloading-induced bone loss. We conducted hind limb unloading of MuRF1 KO mice and wild-type control mice. Unloading induced about 60% reduction in cancellous bone volume (BV/TV) in WT mice. In contrast, MuRF1 deficiency suppressed unloading-induced cancellous bone loss. The cortical bone mass was also reduced by unloading in WT mice. In contrast, MuRF1 deficiency suppressed this reduction in cortical bone mass. To understand whether the effects of MuRF1 deficiency suppress bone loss is on the side of bone formation or bone resorption, histomorphometry was conducted. Unloading reduced bone osteoblastic formation rate (BFR) in WT. In contrast, MuRF1 deficiency suppressed this reduction. Regarding bone resorption, unloading increased osteoclast number in WT. In contrast, MURF1 deficiency suppressed this osteoclast increase. These data indicated that the ring finger protein, MURF1 is involved in disuse-induced bone loss in both of the two major bone remodeling activities, osteoblastic bone formation and osteoclastic bone resorption.

Does the severity of the LGMD2A phenotype in compound heterozygotes depend on the combination of mutations?

INTRODUCTION: Limb-girdle muscular dystrophy type 2A (LGMD2A) is caused by a deficiency of calpain-3/p94. Although the symptoms in most LGMD2A patients are generally homogeneous, some variation in the severity and progression of the disease has been reported. METHODS: We describe 2 patients who carry the same combination of compound heterozygous mutations (pG222R/pR748Q) and whose symptoms are exceptionally benign compared to homozygotes with each missense mutation. RESULTS: The benign phenotype observed in association with the combined pG222R and pR748Q mutations suggested that it may result from a compensatory effect of compound heterozygosity rather than the individual mutations themselves. Our analyses revealed that these two mutations exert different effects on the protease activity of calpain-3, suggesting "molecular complementation" in these patients. CONCLUSION: We propose several hypotheses to explain how this specific combination of mutations may rescue the normal proteolytic activity of calpain-3, resulting in an exceptionally benign phenotype.

Calpain chronicle--an enzyme family under multidisciplinary characterization.

Calpain is an intracellular Ca2+-dependent cysteine protease (EC 3.4.22.17; Clan CA, family C02) discovered in 1964. It was also called CANP (Ca2+-activated neutral protease) as well as CASF, CDP, KAF, etc. until 1990. Calpains are found in almost all eukaryotes and a few bacteria, but not in archaebacteria. Calpains have a limited proteolytic activity, and function to transform or modulate their substrates' structures and activities; they are therefore called, "modulator proteases." In the human genome, 15 genes--CAPN1, CAPN2, etc.--encode a calpain-like protease domain. Their products are calpain homologs with divergent structures and various combinations of functional domains, including Ca2+-binding and microtubule-interaction domains. Genetic studies have linked calpain deficiencies to a variety of defects in many different organisms, including lethality, muscular dystrophies, gastropathy, and diabetes. This review of the study of calpains focuses especially on recent findings about their structure-function relationships. These discoveries have been greatly aided by the development of 3D structural studies and genetic models.

[Physiological importance of calpains in gastric mucosal defense].

The continuous and/or improper ingestion of irritants, including alcohol, NSAIDs, and Helicobacter pylori, often leads to serious gastropathies, affecting a wide range of people. A complex gastric defense system works to protect against these threats, for example by secreting mucus. Recently, by analysis of gene targeting mice for two gastrointestinal-tract-specific calpains, calpain-8 and calpain-9, we have demonstrated that they are cooperatively involved in the mucosal defense against stress-induced gastropathies. Calpains-8 and -9 are members of Ca2+ -dependent intracellular proteases comprising a superfamily in almost all eukaryotes, and form a functional complex, "G-calpain", expressed specifically in the mucus-producing cells. In this review, we show our recent results on calpains -8 and -9, and discuss gastric mucosal defense mechanisms involving them.

CaMPDB: a resource for calpain and modulatory proteolysis.

While the importance of modulatory proteolysis in research has steadily increased, knowledge on this process has remained largely disorganized, with the nature and role of entities composing modulatory proteolysis still uncertain. We built CaMPDB, a resource on modulatory proteolysis, with a focus on calpain, a well-studied intracellular protease which regulates substrate functions by proteolytic processing. CaMPDB contains sequences of calpains, substrates and inhibitors as well as substrate cleavage sites, collected from the literature. Some cleavage efficiencies were evaluated by biochemical experiments and a cleavage site prediction tool is provided to assist biologists in understanding calpain-mediated cellular processes. CaMPDB is freely accessible at http://calpain.org.