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The objective of this study was to study the postmortem calpain change in ostrich muscle. Iliotibialis cranialis and Obturatorius medialis muscles were removed from the both sides of carcasses (n=8). The muscles from the left side were sampled after 0, 1, 2, 3, and 7days of storage at 5°C, while the right-side muscles were taken at 1-, 3-, and 7-day postmortem for shear force measurements. The results showed that the calpain-1 activity was not detected in ostrich muscle during the entire 7-day postmortem storage period, while the calpain-11 was. The unautolyzed calpain-11 activity decreased and the autolyzed calpain-11 activity increased with time postmortem. Desmin content and shear force did not change during postmortem storage although a minor degradation of desmin was observed. Therefore, our results suggest that limited postmortem proteolysis (as suggested by the limited degradation of desmin) and tenderization might be due to the lack of calpain-1 and/or insufficient calpain-11 activity present in ostrich muscle., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The cysteine protease family members play important roles in various pivotal cellular processes. The difficulty in the analysis of the effects of cysteine protease aberrations in cancer comes as a result of the fact that they take part in complex proteolytic pathways. Nevertheless, there is a vast amount of data regarding the involvement of distinct members of this family in divergent types of cancer. Cysteine proteases assist migration and development of the disease, as well as increase the invasiveness of particular kinds of tumors. They are designated as both drug targets, as well as cancer susceptibility biomarkers. This implies that the abnormalities in their activity and expression patterns may be associated with the hallmarks of cancer. This review demonstrates that the influence of cysteine proteases on different mechanisms underlying cancer is undisputable. Thus, they are potent targets for future study and should be recognized as key players in the fight against cancer.

Background: Calpains are Ca2+-dependent cysteine proteases that participate in a range of crucial cellular processes. Dysfunction of these enzymes may cause, for instance, life-threatening diseases in humans, the loss of sex determination in nematodes and embryo lethality in plants. Although the calpain family is well characterized in animal and plant model organisms, there is a great lack of knowledge about these genes in unicellular eukaryote species (i.e. protists). Here, we study the distribution and evolution of calpain genes in a wide range of eukaryote genomes from major branches in the tree of life., Results: Our investigations reveal 24 types of protein domains that are combined with the calpain-specific catalytic domain CysPc. In total we identify 41 different calpain domain architectures, 28 of these domain combinations have not been previously described. Based on our phylogenetic inferences, we propose that at least four calpain variants were established in the early evolution of eukaryotes, most likely before the radiation of all the major supergroups of eukaryotes. Many domains associated with eukaryotic calpain genes can be found among eubacteria or archaebacteria but never in combination with the CysPc domain., Conclusions: The analyses presented here show that ancient modules present in prokaryotes, and a few de novo eukaryote domains, have been assembled into many novel domain combinations along the evolutionary history of eukaryotes. Some of the new calpain genes show a narrow distribution in a few branches in the tree of life, likely representing lineage-specific innovations. Hence, the functionally important classical calpain genes found among humans and vertebrates make up only a tiny fraction of the calpain family. In fact, a massive expansion of the calpain family occurred by domain shuffling among unicellular eukaryotes and contributed to a wealth of functionally different genes.

Introduction: Previous studies have tested the hypothesis that calpain and/or proteasome inhibition is beneficial in Duchenne muscular dystrophy, based largely on evidence that calpain and proteasome activities are enhanced in the mdx mouse., Methods: mRNA expression of ubiquitin-proteasome and calpain system components were determined using real-time polymerase chain reaction in skeletal muscle and heart in the golden retriever muscular dystrophy model. Similarly, calpain 1 and 2 and proteasome activities were determined using fluorometric activity assays., Results: We found that less than half of the muscles tested had increases in proteasome activity, and only half had increased calpain activity. In addition, transcriptional regulation of the ubiquitin-proteasome system was most pronounced in the heart, where numerous components were significantly decreased., Conclusion: This study illustrates the diversity of expression and activities of the ubiquitin-proteasome and calpain systems, which may lead to unexpected consequences in response to pharmacological inhibition., (Copyright © 2011 Wiley Periodicals, Inc.)

Calpain has long been an enigmatic enzyme, although it is involved in a variety of biological phenomena. Recent progress in calpain genetics has highlighted numerous physiological contexts in which the functions of calpain are of great significance. This review focuses on recent findings in the field of calpain genetics and the importance of calpain function. Calpain is an intracellular Ca(2+)-dependent cysteine protease (EC 3.4.22.17; Clan CA, family C02) found in almost all eukaryotes. It is also present in a few bacteria, but not in archaebacteria. Calpain has limited proteolytic activity; rather, it transforms or modulates the structure and/or activity of its substrates. It is, therefore, referred to as a 'modulator protease'. Within the human genome, 15 genes (CAPN1-3, CAPN5-16) encode a calpain-like protease (CysPc) domain along with several different functional domains. Thus, calpains can be regarded as a distinct family of versatile enzymes that fulfil numerous tasks in vivo. Genetic studies show that a variety of defects in many different organisms, including lethality, muscular dystrophies and gastropathy, actually stem from calpain deficiencies. The cause-effect relationships identified by these studies form the basis for ongoing and future studies regarding the physiological role of calpains.

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.

The most studied members of the calpain protease superfamily are CAPN1 and 2, which are conserved across vertebrates. Another similar family member called mu/m-CAPN has been identified in birds alone. Here, we establish that mu/m-CAPN shares one-to-one orthology with CAPN11, previously described only in eutherians (placental mammals). We use the name CAPN11 for this family member and identify orthologues across vertebrate lineages, which form a monophyletic phylogenetic clade directly ancestral to CAPN1 and 2. In lineages branching before therians (live-bearing mammals), the CAPN11 coding region has evolved under strong purifying selection, with low nonsynonymous (d(N)) versus synonymous (d(S)) substitution rates (d(N)/d(S) = 0.076 across pretherians), and its transcripts were detected widely across different tissues. These characteristics are present in CAPN1 and 2 across vertebrate lineages and indicate that pretherian CAPN11 likewise has conserved a wide physiological function. However, an approximately 7-fold elevation in d(N)/d(S) is evident along the CAPN11 branch splitting eutherians from platypus, paralleled by a shift to "testis-specific" gene regulation. Estimates of d(N)/d(S) in eutherians were approximately 3-fold elevated compared with pretherians and coding and transcriptional-level evidence suggests that CAPN11 is functionally absent in marsupials. Many CAPN11 sites are functionally constrained in eutherians to conserve a residue with radically different biochemical properties to a fixed state shared between pretherian CAPN11 and CAPN1 and 2. Protein homology modeling demonstrated that many such eutherian-specific residue replacements modify or ablate interactions with the calpain inhibitor calpastatin that are observed in both pretherian orthologues and CAPN1/2. We propose a model akin to the Dykhuizen-Hartl effect, where inefficient purifying selection and increased genetic drift associated with a reduction in effective population size, drove the fixation of mutations in regulatory and coding regions of CAPN11 of a common marsupial-eutherian ancestor. A subset of these changes had a cumulative adaptive advantage in a eutherian ancestor because of lineage-specific aspects of sperm physiology, whereas in marsupials, no advantage was realized and the gene was disabled. This work supports that functional divergence among gene family member orthologues is possible in the absence of widespread positive selection.

Plasmodium falciparum encodes a single calpain that has a distinct domain composition restricted to alveolates. To evaluate the potential of this protein as a drug target, we assessed its essentiality. Both gene disruption by double cross-over and gene truncation by single cross-over recombination failed. We were also unable to achieve allelic replacement by using a missense mutation at the catalytic cysteine codon, although we could obtain synonymous allelic replacement parasites. These results suggested that the calpain gene and its proteolytic activity are important for optimal parasite growth. To gain further insight into its biological role, we used the FKBP degradation domain system to generate a fusion protein whose stability in transfected parasites could be modulated by a small FKBP ligand, Shield1 (Shld1). We made a calpain-GFP-FKBP fusion through single cross-over integration at the endogenous calpain locus. Calpain levels were knocked down and parasite growth was greatly impaired in the absence of Shld1. Parasites were delayed in their ability to transition out of the ring stage and in their ability to progress to the S phase. Calpain is required for cell cycle progression in Plasmodium parasites and appears to be an attractive drug target. We have shown that regulated knockdowns are possible in P. falciparum and can be useful for evaluating essentiality and function.

The structural features and evolutionary interrelationships of the intracellular Ca2+-dependent cysteine enzymes calpains, proteases of the family C2 (EC 3.4.22.17), are considered. A variety of identified sequences of calpains and calpain-like polypeptides found in organisms of different taxons, from the simplest to mammals, are described. Calpains of the major evolutionary groups, typical and atypical, are classified by the analysis of their phylogenetic tree and are differentiated due to the presence of the calmodulin-like Ca2+-binding domain. It is shown that, along with enzymes having "advanced" characteristics (heterodimeric structure, presence of tissue-specific isoforms and splice variants, regulation by the endogenous inhibitor calpastatin, and others), higher organisms contain homologues of calpains of lower eukaryotes. A high degree of homology of the catalytic domain of calpains and the variable structure of other functional domains indicate that calpains are implicated in various physiological processes with the retention of their regulatory role.

Calpain is a Ca(2+)-activated proteolytic enzyme involved in neurodegeneration in a variety of injuries and diseases of the central nervous system (CNS). Many calpain homologs have been discovered. Depending on the tissue distribution, calpains are broadly classified as ubiquitous and tissue-specific. Ubiquitous calpain isoforms, -calpain and m-calpain, are abundantly expressed in the CNS. Calpastatin, an endogenous protein inhibitor, regulates the activity of ubiquitous calpain. Overactivation of calpain may degrade calpastatin, limiting its regulatory efficiency. Molecular structures of calpain and calpastatin have been deduced from cDNA cloning. The precise physiological function of calpain remains elusive. However, experimental evidence strongly suggests an important role for calpain in causing neurodegeneration in various injuries and diseases of the CNS. The increase in intracellular free Ca(2+) levels in the course of injuries and diseases in the CNS causes overactivation of calpain, promoting degradation of key cytoskeletal and membrane proteins. Cleavage of these key proteins by calpain is an irreversible process that perturbs the integrity and stability of CNS cells, leading to programmed cell death or apoptosis. Calpain in conjunction with caspases can cause apoptosis of the CNS cells. An aberrant Ca(2+) homeostasis inevitably activates calpain, which plays a crucial role in the pathophysiology of the CNS injuries and diseases. Therefore, calpain is a potential therapeutic target to prevent neurodegeneration. To this end, various cell-permeable calpain inhibitors have been synthesized for pharmacological inhibition of calpain activity. Some calpain inhibitors have shown significant neuroprotection in animal models of the CNS injuries and diseases, indicating their therapeutic potential.

Calpains are cysteine proteases first identified 50 years ago. Because they are present in the cytosol of mammalian cells and because they are activated in response to Ca2+ mobilization, they are thought to be involved mainly in cell signalling pathways. They could participate in cellular responses such as apoptosis, proliferation, extracellular matrix adhesion and motility, that have relevance to pathophysiological issues in ischemia, inflammation, repair and tumor progression. Here we consider calpain functions in inflammatory reaction. We report the recent observation that calpain inhibitors reduce the development of acute and chronic inflammation. This has opened the door for understanding how these enzymes are effective in inflammation. We present data suggesting that calpains are primarily responsible for the activation of nuclear factor-kappa B, a transcription factor with a pivotal role in inflammation. They are involved in inflammatory cell adhesion and migration, pro-inflammatory mediator release and anti-inflammatory hormone resistance as well. In addition, we emphasize the intriguing possibility that calpains are externalized during inflammatory process and that they play a role in the microenvironment of inflammatory cells. Thus, both intracellular and extracellular calpains would offer novel therapeutic targets in inflammation.

Three distinct Ca(2+)-activated proteolytic activities could be proven in rat liver mitochondria. The proteolytic activities detected in the presence of Ca2+ are different in the two mitochondrial soluble compartments, the matrix and the intermembrane space. Strikingly three Ca(2+)-activated proteolytic activities (M1, M2 and M3) appear in the matrix whereas the intermembrane space contains only two such activities. These proteolytic activities are similar to the calpains already described in the cellular cytosol with regard to their optimal pH and inhibition profiles. The Ca2+ requirements for activation of M1 and M2 correspond to those of the micromolar and millimolar Ca(2+)-requiring proteinases, whereas the concentration of Ca2+ required to activate M3 is an intermediate value.

The effect of acidic phospholipids on proteolysis of protein kinase C (PKC) by mu-calpain was examined at Ca++ concentrations ranging from 10(-7) to 10(-4) M. The gamma species, among the molecular species of PKC, was more susceptible to calpain than the alpha and beta (beta I/beta II) and was hydrolysed at Ca++ concentrations greater than or equal to 10(-6) M. Acidic phospholipids enhanced proteolysis of PKC gamma and lowered Ca++ concentrations required for it to the level below 10(-6) M. Among the phospholipids tested, phosphatidylinositol-bisphosphate showed the most prominent effect; phosphatidylinositol and phosphatidylserine were less effective. Polyphosphoinositides, hence, may constitute an essential structure in cell membranes for positive regulation of calpain activity.

Previously we reported results of an incubation experiment with neurofilaments that supported the existence of a mu-type of Ca2+-activated neutral protease in the rat peripheral nerve; it was active with microM order Ca2+ (mu-CANP). This time, we partially purified the mu-CANP from a crude CANP fraction of rat peripheral nerve by using a DE52 column and a Phenyl-Sepharose column followed again by DE52 column chromatography. The presence of mu-CANP was verified by an immunoblotting technique. The mu-CANP degraded the neurofilament triplet as previously reported; i.e., among the neurofilament triplet, the 160K component was most sensitive, and in the order of the 160K, 68K, and 200K components, respectively.

The purposes of the current study were to: determine if human lenses contain calpain II (EC.34.22.17) activity, measure the effect of aging and anatomical location on lens calpain II activity, and determine if human lenses contain the endogenous calpain inhibitor calpastatin. Both enzymatic and immunologic assays indicated that human lenses contained calpain II activity. Calpain II activity was highest in the cortex of lenses from young donors, and lowest in the nucleus of aged lenses, where it was sometimes nondetectable. In some cases, calpain II activity persisted in the nucleus of lenses from donors greater than 70 years of age. Human lenses also contained endogenous calpain inhibitor (calpastatin) in excess over calpain enzymatic activity. Calpastatin activity did not decrease during aging. Although human lenses contained approximately 3% of the calpain activity found in rat lenses, calpain II may still be a major endopeptidase in human lenses. Demonstration of calpain II in human lenses suggested that calpain II could be involved in both lens maturation and cataract formation.