Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.22.54 (calpain 3)
 430 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Limb girdle muscular dystrophy type 2A is linked to a skeletal muscle-specific calpain isoform known as p94. Isolation of the intact 94-kDa enzyme has been difficult to achieve due to its rapid autolysis, and uncertainty has arisen over its Ca2+-dependence for activity. We have expressed a C-terminally truncated form of the enzyme that comprises the protease core (domains I and II) along with its insertion sequence, IS1, and N-terminal leader sequence, NS. This 47-kDa p94I-II mini-calpain was stable during purification. In the presence of Ca2+, p94I-II cleaved itself within the NS and IS1 sequences. Mapping of the autolysis sites showed that NS and IS1 have the potential to be removed without damage to the protease core. Ca2+-dependent autolysis must be an intramolecular event because the inactive p94I-II C129S mutant was not cleaved by incubation with wild-type p94I-II. In addition, the rate of autolysis of p94I-II was independent of the concentration of the enzyme.
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PMID:The protease core of the muscle-specific calpain, p94, undergoes Ca2+-dependent intramolecular autolysis. 1248

The calcium-dependent protease calpain is involved in numerous functions, including the control of cell survival, plasticity and motility. Whereas the isoforms calpain 1 and 2 have been described as ubiquitously expressed enzymes, calpain 3 has been called "muscle-specific", although trace amounts of calpain 3 mRNA have been detected by Northern blot in brain homogenates. In this study, we validated antibodies raised either against the peptides that were specific for a given isoform or the peptides present in all the three isoforms. We then used the anti-calpain 3 antibodies together with antibodies directed against cell-type-specific proteins to determine by double- and triple-labelling immunocytochemistry if the protease is expressed in specific cell populations of rat as well as lesser mouse lemur (Microcebus murinus) brain. Calpain 3 was almost exclusively found in cells displaying astrocyte morphology. These cells, most of which co-expressed glial fibrillary acidic protein, were particularly numerous close to the striatal subventricular zone (where numerous neurones forming the rostral migratory stream (RMS) towards the olfactory bulbs are generated) and the RMS itself. Other immunoreactive cells were found close to the pial surface of the forebrain, in the corpus callosum and in the dentate gyrus. Calpain 3 may be involved in astrocyte plasticity and/or motility.
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PMID:Calpain 3 is expressed in astrocytes of rat and Microcebus brain. 1266 60

Human circulating PBMC (peripheral blood mononuclear cells) contain three calpain isoforms distinguishable on the basis of their chromatographic properties. Two of these proteases belong to the ubiquitous calpain subfamily, corresponding to the classical mu- and m-calpain forms. The third, which shows peculiar activating and regulatory properties, is an alternatively spliced calpain 3 (p94) form. This new calpain differs from calpain 3 in that it has lost IS1 insertion and exon 15, a lysine-rich sequence regarded as a nuclear translocation signal. PBMC p94-calpain undergoes activation and inactivation without the accumulation of a low-Ca2+-requiring form that is typical of the classical activation processes of mu- and m-calpain. Furthermore, it differs from the ubiquitous forms in that it displays a lower sensitivity to calpastatin. On the basis of these selective properties, it can be postulated that PBMC p94-calpain can be activated in response to specific stimuli that are not effective on the other calpain isoenzymes. The enzyme is preferentially expressed in B- and T-lymphocytes, whereas it is poorly expressed in natural killer cells and almost undetectable in polymorphonuclear cells. This distribution might reflect the specific function of this protease, which is preferentially present in cells devoted to the production of the humoral, rather than to the cellular, immune response.
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PMID:Characterization of a new p94-like calpain form in human lymphocytes. 1288 47

In this study, we generated three SAGE libraries from melanoma tissues. Using bioinformatics tools usually applied to microarray data, we identified several genes, including novel transcripts, which are preferentially expressed in melanoma. SAGE results converged with previous microarray analysis on the importance of intracellular calcium and G-protein signaling, and the Wnt/Frizzled family. We also examined the expression of CD74, which was specifically, albeit not abundantly, expressed in the melanoma libraries using a melanoma progression tissue microarray, and demonstrate that this protein is expressed by melanoma cells but not by benign melanocytes. Many genes involved in intracellular calcium and G-protein signaling were highly expressed in melanoma, results we had observed earlier from microarray studies (Bittner et al., 2000). One of the genes most highly expressed in our melanoma SAGE libraries was a calcium-regulated gene, calpain 3 (p94). Immunohistochemical analysis demonstrated that calpain 3 moves from the nuclei of non-neoplastic cells to the cytoplasm of malignant cells, suggesting activation of this intracellular proteinase. Our SAGE results and the clinical validation data demonstrate how SAGE profiles can highlight specific links between signaling pathways as well as associations with tumor progression. This may provide insights into new genes that may be useful for the diagnosis and therapy of melanoma.
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PMID:Generation and analysis of melanoma SAGE libraries: SAGE advice on the melanoma transcriptome. 1475 46

The physiological role of the skeletal muscle-specific calpain 3, p94, is presently unknown, but defects in its gene cause limb girdle muscular dystrophy type 2A. This calcium-dependent cysteine protease resembles the large subunit of m-calpain but with three unique additional sequences: an N-terminal region (NS), and two insertions (IS1 and IS2). The latter two insertions have been linked to the chronic instability of the whole enzyme both in vivo and in vitro. We have shown previously that the core of p94 comprising NS, domains I and II, and IS1 is stable as a recombinant protein in the absence of Ca(2+) and undergoes autolysis in its presence. Here we show that p94I-II cannot hydrolyze an exogenous substrate before autolysis but is increasingly able to do so when autolysis proceeds for several hours. This gain in activity is caused by cleavage of IS1 during autolysis because a deletion mutant lacking the NS region (p94I-II DeltaNS) shows the same activation profile. Similarly, the calpain inhibitors E-64 and leupeptin have almost no inhibitory effect on substrate hydrolysis by p94I-II soon after calcium addition but cause complete inhibition when autolysis progresses for several hours. As autolysis proceeds, there is release of the internal IS1 peptide, but the two portions of the core remain tightly associated. Modeling of p94I-II suggests that IS1 contains an amphipathic alpha-helix flanked by extended loops. The latter are the targets of autolysis and limited digestion by exogenous proteases. The presence and location of the alpha-helix in recombinant IS1 were confirmed by circular dichroism and by the introduction of a L286P helix-disrupting mutation. Within p94I-II, L286P caused premature autoproteolysis of the enzyme. IS1 is an elaboration of a loop in domain II near the active site, and it acts as an internal autoinhibitory propeptide, blocking the active site of p94 from substrates and inhibitors.
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PMID:Insertion sequence 1 of muscle-specific calpain, p94, acts as an internal propeptide. 1507 71

Calpains are calcium-modulated proteases which respond to Ca2+ signals by removing limited portions of protein substrates, thereby irreversibly modifying their function(s). Members of this protease family are present in a variety of organisms ranging from mammals to plants; some of them are ubiquitously expressed, while others are tissue specific. Although calpains are apparently involved in a multitude of physiological and pathological events, their functions are still poorly understood. In two cases, however, the alteration of a member of the calpain family has been clearly identified as being responsible for a human disease: the loss of function of calpain 3 causes limb girdle muscular dystrophy type 2A, and mutations in the gene coding for calpain 10 have been shown to correlate with non-insulin-dependent diabetes.
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PMID:Calpain-related diseases. 1533 56

Premature visual impairment due to lens opacification is a debilitating characteristic of untreated diabetes. Lens opacification is primarily due to the insolubilization of crystallins, proteins essential for lens optical properties, and recent studies have suggested that a major cause of this insolubilization may be the unregulated proteolysis of crystallins by calpains. These are intracellular cysteine proteases whose activation requires the presence of calcium (Ca2+) and elevated levels of lens Ca2+ is a condition associated with both diabetic cataractogenesis and other forms of the disorder. A number of calpains have been identified in the lens, including calpain 2, calpain 10 and two isozymes of calpain 3: Lp82 and Lp85. The use of animal hereditary cataract models have suggested that calpain 2 and/or Lp82 may be the major calpains involved in murine cataractogenesis with contributions from calpain 10 and Lp85. However, calpain 2 appears to be the major calpain involved in murine diabetic cataractogenesis and the strongest candidate of the calpains for a role in human types of cataractogenesis. Here, we present an overview of recent evidence on which these observations are based with an emphasis on the ability of calpains to proteolyse lens crystallins and calpain structural features, which appear to be involved in the Ca2+-mediated activation of these enzymes.
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PMID:Role of calpains in diabetes mellitus-induced cataractogenesis: a mini review. 1536 98

We examined the influence of sepsis on the expression and activity of the calpain and caspase systems in skeletal muscle. Sepsis was induced in rats by cecal ligation and puncture (CLP). Control rats were sham operated. Calpain activity was determined by measuring the calcium-dependent hydrolysis of casein and by casein zymography. The activity of the endogenous calpain inhibitor calpastatin was measured by determining the inhibitory effect on calpain activity in muscle extracts. Protein levels of mu- and m-calpain and calpastatin were determined by Western blotting, and calpastatin mRNA was measured by real-time PCR. Caspase-3 activity was determined by measuring the hydrolysis of the fluorogenic caspase-3 substrate Ac-DEVD-AMC and by determining protein and mRNA expression for caspase-3 by Western blotting and real-time PCR, respectively. In addition, the role of calpains and caspase-3 in sepsis-induced muscle protein breakdown was determined by measuring protein breakdown rates in the presence of specific inhibitors. Sepsis resulted in increased muscle calpain activity caused by reduced calpastatin activity. In contrast, caspase-3 activity, mRNA levels, and activated caspase-3 29-kDa fragment were not altered in muscle from septic rats. Sepsis-induced muscle proteolysis was blocked by the calpain inhibitor calpeptin but was not influenced by the caspase-3 inhibitor Ac-DEVD-CHO. The results suggest that sepsis-induced muscle wasting is associated with increased calpain activity, secondary to reduced calpastatin activity, and that caspase-3 activity is not involved in the catabolic response to sepsis.
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PMID:Sepsis stimulates calpain activity in skeletal muscle by decreasing calpastatin activity but does not activate caspase-3. 1556 79

Previous family studies revealed a large number of calpain 3 ( CAPN3 ) mutations that cause recessive forms of limb girdle muscular dystrophy (LGMD2A) with selective atrophy of the proximal limb muscles. Correlations between the nature and site of a particular mutation and its corresponding phenotype, however, can only be established from homozygous mutations, which are particularly rare in the alternatively spliced NS, IS1 and IS2 regions of CAPN3. Here we identified a sibling pair with LGMD2A-type muscular dystrophy caused by a homozygous Ser606Leu (S606L) substitution in the IS2 linker domain. Normal protein levels, unaltered myofibrillar targeting and conserved calcium-induced autocatalytic activity of the mutated protein could be demonstrated in muscle biopsies from one patient. Despite this inconspicuous modification of the IS2 linker between domains III and IV, both patients developed signs and symptoms of the disease within their second decade of life. The unexpected severity of the clinical manifestation points to the high relevance of the calpain 3-specific IS2 segment between domains III and IV. We conclude that the structural motif around the Ser606 residue represents an important functional site that may regulate the transient activation and limited proteolysis of calpain 3.
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PMID:Limb girdle muscular dystrophy in a sibling pair with a homozygous Ser606Leu mutation in the alternatively spliced IS2 region of calpain 3. 1584 48

Chronic inflammation in tibialis anterior muscles of mdx mice was produced by a single injection of a recombinant adenovirus vector (AV) expressing an immunogenic beta-galactosidase (beta-gal). In regions of intense beta-gal staining, mononuclear infiltrates abounded, and muscle fibers showed strong extrasynaptic utrophin immunostaining, restoration of dystrophin-associated protein complex, and a marked reduction of the prevalence of centronucleation. Immunoblot analysis confirmed an increase of endogenous utrophin without an increase of the mRNA of the major muscle isoform utrA. Significantly better maximal tetanic force values were demonstrated in the inflammatory versus control mdx muscles. The resistance to lengthening contraction- induced damage was also significantly increased in the former. In muscles of mice lacking TNF-alpha gene, AV vector did not induce inflammation and extrajunctional utrophin increase did not occur. In the inflammatory mdx muscles, proteolytic activity of calcium-activated calpain was reduced, and in mdx myotubes in vitro, incubation with NO donors also reduced calpain-mediated utrophin proteolysis. Since utrophin was shown to be a natural substrate of calpain and known inhibitors of calpain in cultured mdx myotubes increased utrophin levels, the above results were consistent with the following conclusions: (1) extrasynaptic utrophin increase is mainly responsible for the antidystrophic effect; (2) extrasynaptic utrophin increase is a result of posttranscriptional mechanism(s) related to proinflammatory factors; and (3) reduction of endogenous muscle calpain activity by inflammatory cytokines has an important role in the stabilization and increase of the extrasynaptic utrophin.
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PMID:Factors associated with induced chronic inflammation in mdx skeletal muscle cause posttranslational stabilization and augmentation of extrasynaptic sarcolemmal utrophin. 1587 80


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