EC:3.4.22.54 - FACTA Search

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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)

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

The ketomethylene phenylalanal and alanal analogues of Cbz-Val-Phe-H and Cbz-Val-Ala-H have been prepared and the Ki values versus chicken gizzard smooth muscle calpain were determined. The ketomethylene isosteres were significantly less potent than the corresponding dipeptide aldehydes, and this loss in activity is attributed to the absence of a critical interaction between the P1-P2 amide bond and the peptide binding region of calpain.
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PMID:The synthesis of ketomethylene pseudopeptide analogues of dipeptide aldehyde inhibitors of calpain. 1002 15

Limb-girdle muscular dystrophy type 2a arises from mutations in the Ca²⁺-activated intracellular cysteine protease calpain-3. This calpain isoform is abundant in skeletal muscle and differs from the main isoforms, calpain-1 and -2, in being a homodimer and having two short insertion sequences. The first of these, IS1, interrupts the protease core and must be cleaved for activation and substrate binding. Here, to learn how calpain-3 can be regulated and inhibited, we determined the structures of the calpain-3 protease core with IS1 present or proteolytically excised. To prevent intramolecular IS1 autoproteolysis, we converted the active-site Cys to Ala. Small-angle X-ray scattering (SAXS) analysis of the C129A mutant suggested that IS1 is disordered and mobile enough to occupy several locations. Surprisingly, this was also true for the apo version of this mutant. We therefore concluded that IS1 might have a binding partner in the sarcomere and is unstructured in its absence. After autoproteolytic IS1 removal from the active Cys¹²⁹ calpain-3 protease core, we could solve its crystal structures with and without the cysteine protease inhibitors E-64 and leupeptin covalently bound to the active-site cysteine. In each structure, the active state of the protease core was assembled by the cooperative binding of two Ca²⁺ ions to the equivalent sites used in calpain-1 and -2. These structures of the calpain-3 active site with residual IS1 and with bound E-64 and leupeptin may help guide the design of calpain-3-specific inhibitors.
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PMID:Structures of human calpain-3 protease core with and without bound inhibitor reveal mechanisms of calpain activation. 2938 17