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

We previously identified a third type of the calpain large subunit named p94 as a cDNA whose mRNA is expressed exclusively in skeletal muscle at levels approximately 10-fold more abundant than those of the conventional calpain subunit. Rat skeletal muscle fractions were screened by two anti-peptide antibodies raised against two specific sequences in p94, but the p94 protein could not be found. To examine this apparent discrepancy between the amounts of mRNA and protein, wild-type p94 was expressed in COS cells. Although p94 mRNA was expressed normally in COS cells, only very small amounts of the protein and its presumed degradation products were detected by the antibodies described above. A series of COOH-terminal deletion mutants was constructed and expressed in COS cells and L8 cells, a rat myoblast cell line. When IS2, one of the specific regions of p94, was completely eliminated, the truncated p94 proteins were expressed normally, and the amount of the expressed proteins was at least 100-fold higher than with wild-type p94. Moreover, when site-directed mutagenesis was introduced to change the presumed active-site cysteine of p94 to serine or alanine, the mutated p94 proteins were highly expressed like the IS2-deleted mutants. These results indicate the following. 1) The mRNA for p94 is normally transcribed in COS, L8, and muscle cells; 2) the p94 protein becomes active in the cytosol immediately after translation; 3) the p94 protein virtually disappears from cells by autocatalytic degradation; and 4) the p94-specific IS2 region plays an important role in this degradation. In vitro translation experiments support this idea. Furthermore, p94 shows nuclear localization when expressed in COS cells. The physiological function of p94 in muscle is discussed on the basis of the analysis of these transfectants.
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PMID:Muscle-specific calpain, p94, is degraded by autolysis immediately after translation, resulting in disappearance from muscle. 848 13

Hematopoiesis is tightly controlled by a family of cytokines that signal through a related set of receptors. The pleiotropic and overlapping response of a cell to different cytokines is reflected in the number and complex pattern of activated signal transducers. Of special interest is STAT5, which is stimulated by a large and diverse set of cytokines. In addition to the two highly homologous proteins, STAT5A and STAT5B, encoded by duplicated genes, expression and activation of a dominant-negative, carboxyl-truncated form has also been described in early hematopoietic progenitors. We show here that a protease expressed in early hematopoietic cells cleaves the alpha forms of STAT5A/5B (p96/p94) to generate carboxyl-truncated beta forms (p80/p77). Inhibition studies assigned this protease to the serine class of endopeptidases. Cell fractionation experiments showed that the protease is associated with the nucleus in a constitutively activated form and does not require an activated STAT5 substrate. The ability of a protease to modulate the specificity of an activated transcription factor is unprecedented and underlines the importance of proteases in regulation of cell functions.
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PMID:Carboxyl-truncated STAT5beta is generated by a nucleus-associated serine protease in early hematopoietic progenitors. 949 Jun 72

p94, a skeletal muscle-specific calpain, has attracted much attention because its gene is responsible for limb-girdle muscular dystrophy type 2A. p94, however, has not been characterized at the protein and enzyme levels, owing to its very rapid autolysis. In the present study, a purification procedure for p94 was first established by using a recombinant inactive p94 expressed in COS cells in which the active site cysteine residue was changed to serine [p94(C129S)]. The isolation of native p94 from rabbit skeletal muscle by the established method with conventional procedures was extremely difficult because p94 became highly unstable in a crude extract on the addition of NaCl for separation. Purification of native p94 was possible with an antibody-affinity column but only as an inactive enzyme; p94(C129S) was purified as a homodimer. Characterization of p94, especially autolysis, was performed with partly purified native p94 and p94(C129S). The autolysis of p94, which consisted at least partly of an intermolecular reaction, proceeded in three consecutive steps; 60 and 58 kDa fragments were produced as intermediates before a stable 55 kDa fragment appeared. Autolysis of p94 was regarded as a degradative step rather than for the activation of the enzyme. All the autolysis cleavage sites were located in the p94-specific insertion sequence 1 region, which explains why p94 is unstable compared with the other calpains. The autolysis sites in p94 clearly showed a different specificity relative to the autolytic and proteolytic cleavage sites of the ubiquitous mu- and m-calpains, in its preference for residues at the P3 to P1' sites, indicating a distinct substrate specificity and function for the muscle enzyme.
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PMID:Purification of native p94, a muscle-specific calpain, and characterization of its autolysis. 979 99

The ubiquitous calpain isoforms (mu- and m-calpain) are Ca(2+)-dependent cysteine proteases that require surprisingly high Ca(2+) concentrations for activation in vitro ( approximately 50 and approximately 300 microm, respectively). The molecular basis of such a high requirement for Ca(2+) in vitro is not known. In this study, we substantially reduced the concentration of Ca(2+) required for the activation of m-calpain in vitro through the specific disruption of interdomain interactions by structure-guided site-directed mutagenesis. Several interdomain electrostatic interactions involving lysine residues in domain II and acidic residues in the C(2)-like domain III were disrupted, and the effects of these mutations on activity and Ca(2+) sensitivity were analyzed. The mutation to serine of Glu-504, a residue that is conserved in both mu- and m-calpain and interacts most notably with Lys-234, reduced the in vitro Ca(2+) requirement for activity by almost 50%. The mutation of Lys-234 to serine or glutamic acid resulted in a similar reduction. These are the first reported cases in which point mutations have been able to reduce the Ca(2+) requirement of calpain. The structures of the mutants in the absence of Ca(2+) were shown by x-ray crystallography to be unchanged from the wild type, demonstrating that the increase in Ca(2+) sensitivity was not attributable to conformational change prior to activation. The conservation of sequence between mu-calpain, m-calpain, and calpain 3 in this region suggests that the results can be extended to all of these isoforms. Whereas the primary Ca(2+) binding is assumed to occur at EF-hands in domains IV and VI, these results show that domain II-domain III salt bridges are important in the process of the Ca(2+)-induced activation of calpain and that they influence the overall Ca(2+) requirement of the enzyme.
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PMID:Calpain mutants with increased Ca2+ sensitivity and implications for the role of the C(2)-like domain. 1110 42

Limb-girdle muscular dystrophy type 2A (LGMD2A) is the most frequent autosomal recessive muscular dystrophy. It is caused by mutations in the calpain-3 (CAPN3) gene. The majority of the mutations described to date are located in the coding sequence of the gene. However, it is estimated that 25% of the mutations are present at exon-intron boundaries and modify the pre-mRNA splicing of the CAPN3 transcript. We have previously described the first deep intronic mutation in the CAPN3 gene: c.1782+1072G>C mutation. This mutation causes the pseudoexonization of an intronic sequence of the CAPN3 gene in the mature mRNA. In the present work, we show that the point mutation generates the inclusion of the pseudoexon in the mRNA using a minigene assay. In search of a treatment that restores normal splicing, splicing modulation was induced by RNA-based strategies, which included antisense oligonucleotides and modified small-nuclear RNAs. The best effect was observed with antisense sequences, which induced pseudoexon skipping in both HeLa cells cotransfected with mutant minigene and in fibroblasts from patients. Finally, transfection of antisense sequences and siRNA downregulation of serine/arginine-rich splicing factor 1 (SRSF1) indicate that binding of this factor to splicing enhancer sequences is involved in pseudoexon activation.
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PMID:In vitro correction of a pseudoexon-generating deep intronic mutation in LGMD2A by antisense oligonucleotides and modified small nuclear RNAs. 2386 87

Digestive organ expansion factor (Def) is a nucleolar protein that plays dual functions: it serves as a component of the ribosomal small subunit processome for the biogenesis of ribosomes and also mediates p53 degradation through the cysteine proteinase calpain-3 (CAPN3). However, nothing is known about the exact relationship between Def and CAPN3 or the regulation of the Def function. In this report, we show that CAPN3 degrades p53 and its mutant proteins p53A138V, p53M237I, p53R248W, and p53R273P but not the p53R175H mutant protein. Importantly, we show that Def directly interacts with CAPN3 in the nucleoli and determines the nucleolar localisation of CAPN3, which is a prerequisite for the degradation of p53 in the nucleolus. Furthermore, we find that Def is modified by phosphorylation at five serine residues: S50, S58, S62, S87, and S92. We further show that simultaneous phosphorylations at S87 and S92 facilitate the nucleolar localisation of Capn3 that is not only essential for the degradation of p53 but is also important for regulating cell cycle progression. Hence, we propose that the Def-CAPN3 pathway serves as a nucleolar checkpoint for cell proliferation by selective inactivation of cell cycle-related substrates during organogenesis.
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PMID:Phosphorylation of Def Regulates Nucleolar p53 Turnover and Cell Cycle Progression through Def Recruitment of Calpain3. 2765 29