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)

The typical calpain proteases are a subset of a wider superfamily and regulate a broad spectrum of physiological processes. Here we characterised Atlantic halibut complete-coding orthologues of calpain-1, calpain-2-like, "muscle-specific" calpain-3, plus calpain-11, a recently recognised vertebrate-wide family member. Phylogenetic analysis established the relationship of each sequence within a comprehensive framework of vertebrate calpains, including teleost paralogues. This approach provided significant insight into the evolution of teleost calpains. For example, teleost sequences considered calpain-2 orthologues formed a monophyletic clade external to sister clades for tetrapod calpain-2 and vertebrate calpain-8. Thus, teleost "calpain-2" is likely not directly orthologous to tetrapod calpain-2 and represents a calpain-2-like protein. The characteristic domain structure of typical calpains was observed in each halibut sequence, although calpain-3, as for other teleosts, retained only one (IS2) of three further domains found in human calpain-3 (NS, IS1 and IS2). Transcripts for capn1, capn2-like and capn11 were widely detected across eleven halibut tissues, whereas capn3 was detected in striated muscles, spleen and ovary, but absent or relatively less abundant in other tissues. We assessed the transcript expression of each calpain gene in fast-twitch skeletal muscle where nutritional state was altered with 60days feed restriction, followed by 60days satiation refeeding. Measured by quantitative real-time PCR, capn1 transcript levels were highest during maximal fasting and then steadily decreased with refeeding, where muscle was in net positive protein balance. Conversely capn2-like showed little response, whereas capn3 and capn11 transcript levels were lowest at maximal fasting before being strongly constitutively upregulated with subsequent refeeding. Halibut capn3 transcript abundance was on average 6.5, 23.7 and 5.9 fold greater than capn1, capn2-like and capn11 respectively in skeletal muscle across nutritional states. In turn, transcript levels of capn1 and capn11 were invariably higher than capn2-like, but were dependent on nutritional state compared to each other. The differential regulation of these genes in response to nutritional status suggests distinct roles for typical calpain family members in regulating the balance between catabolism and growth in teleost skeletal muscle.
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PMID:Characterisation of capn1, capn2-like, capn3 and capn11 genes in Atlantic halibut (Hippoglossus hippoglossus L.): Transcriptional regulation across tissues and in skeletal muscle at distinct nutritional states. 2009 71

An immunofluorescence microscopy method for following changes in myofibrillar-bound calpain 3 was developed. Afterward, proteolytic changes in calpain 3(p94), calpain 1, titin, and nebulin were examined in myofibrils prepared from ovine longissimusthoracis et lumborum (LTL) stored for 0, 1, 2, and 3 days postmortem. Western blot analysis revealed that the levels of intact calpain 3 (expressed as percentage of the level immediately postmortem) were 80%, 10% and not detectable in myofibrils prepared at 1, 2, and 3 days, respectively. Western blots for calpain 1 also indicated conversion of the intact protein (80 kDa) to a 76 kDa fragment during the same time period. Thus calpains 1 and 3 appear to be activated during postmortem storage. Immunofluorescence microscopy using an IS1 region specific antibody revealed that calpain 3 staining was most intense at the sarcomere Z- and M-lines. The fluorescence intensity declined significantly during storage, paralleling changes in the proteolytic breakdown of titin and nebulin associated with these structures.
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PMID:Postmortem changes in myofibrillar-bound calpain 3 revealed by immunofluorescence microscopy. 2206 53

CAPN3 is a calpain superfamily member that is predominantly expressed in skeletal muscle. So far, clear CAPN3 orthologs were found only in vertebrates. CAPN3 is a unique protease in that it undergoes extremely rapid and exhaustive autolysis and that autolyzed fragments spontaneously associate each other to reconstitute the proteolytic activity. These unique properties of CAPN3 are dependent on IS1 and IS2, two CAPN3-characterizing sequences that do not exist in other calpains or any other proteases. To understand how IS1 and IS2 are conserved among vertebrates, this data article provides amino acid sequence alignment of representative vertebrate CAPN3s. For further analysis and discussion, see Ono et al. [1].
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PMID:Amino acid sequence alignment of vertebrate CAPN3/calpain-3/p94. 2695 93

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

Calpains are intracellular, calcium-activated cysteine proteases. Calpain-3 is abundant in skeletal muscle, where its mutation-induced loss of function causes limb-girdle muscular dystrophy type 2A. Unlike the small subunit-containing calpain-1 and -2, the calpain-3 isoform homodimerizes through pairing of its C-terminal penta-EF-hand domain. It also has two unique insertion sequences (ISs) not found in the other calpains: IS1 within calpain-3's protease core and IS2 just prior to the penta-EF-hand domain. Production of either native or recombinant full-length calpain-3 to characterize the function of these ISs is challenging. Therefore, here we used recombinant rat calpain-2 as a stable surrogate and inserted IS1 into its equivalent position in the protease core. As it does in calpain-3, IS1 occupied the catalytic cleft and restricted the enzyme's access to substrate and inhibitors. Following activation by Ca²⁺, IS1 was rapidly cleaved by intramolecular autolysis, permitting the enzyme to freely accept substrate and inhibitors. The surrogate remained functional until extensive intermolecular autoproteolysis inactivated the enzyme, as is typical of calpain-2. Although the small-molecule inhibitors E-64 and leupeptin limited intermolecular autolysis of the surrogate, they did not block the initial intramolecular cleavage of IS1, establishing its role as a propeptide. Surprisingly, the large-molecule calpain inhibitor, calpastatin, completely blocked enzyme activity, even with IS1 intact. We suggest that calpastatin is large enough to oust IS1 from the catalytic cleft and take its place. We propose an explanation for why calpastatin can inhibit calpain-2 bearing the IS1 insertion but cannot inhibit WT calpain-3.
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PMID:Insertion sequence 1 from calpain-3 is functional in calpain-2 as an internal propeptide. 3025 72

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