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)

Calpain 3 is a member of the calpain family of calcium-dependent intracellular proteases. Thirteen years ago it was discovered that mutations in calpain 3 (CAPN3) result in an autosomal recessive and progressive form of limb girdle muscular dystrophy called limb girdle muscular dystrophy type 2A. While calpain 3 mRNA is expressed at high levels in muscle and appears to have some role in developmental processes, muscles of patients and mice lacking calpain 3 still form apparently normal muscle during prenatal development; thus, a functional calpain 3 protease is not mandatory for muscle to form in vivo but it is a pre-requisite for muscle to remain healthy. Despite intensive research in this field, the physiological substrates of the calpain 3 protein (hereafter referred to as CAPN3) and its alternatively spliced isoforms remain elusive. The existence of these multiple isoforms complicates the search for the physiological functions of CAPN3 and its pathophysiological role. In this review, we summarize the genetic and biochemical evidence that point to loss of function of the full-length isoform of CAPN3, also known as p94, as the pathogenic isoform. We also argue that its natural substrates must reside in its proximity within the sarcomere where it is stored in an inactive state anchored to titin. We further propose that CAPN3 has many attributes that make it ideally suited as a sensor of sarcomeric integrity and function, involved in its repair and maintenance. Loss of these CAPN3-mediated activities can explain the "progressive" development of muscular dystrophy.
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PMID:Calpain 3, the "gatekeeper" of proper sarcomere assembly, turnover and maintenance. 1897 5

Limb-girdle muscular dystrophy type 2A (LGMD2A) is a recessive genetic disorder caused by mutations in calpain 3 (CAPN3). Calpain 3 plays different roles in muscular cells, but little is known about its functions or in vivo substrates. The aim of this study was to identify the genes showing an altered expression in LGMD2A patients and the possible pathways they are implicated in. Ten muscle samples from LGMD2A patients with in which molecular diagnosis was ascertained were investigated using array technology to analyze gene expression profiling as compared to ten normal muscle samples. Upregulated genes were mostly those related to extracellular matrix (different collagens), cell adhesion (fibronectin), muscle development (myosins and melusin) and signal transduction. It is therefore suggested that different proteins located or participating in the costameric region are implicated in processes regulated by calpain 3 during skeletal muscle development. Genes participating in the ubiquitin proteasome degradation pathway were found to be deregulated in LGMD2A patients, suggesting that regulation of this pathway may be under the control of calpain 3 activity. As frizzled-related protein (FRZB) is upregulated in LGMD2A muscle samples, it could be hypothesized that beta-catenin regulation is also altered at the Wnt signaling pathway, leading to an incorrect myogenesis. Conversely, expression of most transcription factor genes was downregulated (MYC, FOS and EGR1). Finally, the upregulation of IL-32 and immunoglobulin genes may induce the eosinophil chemoattraction explaining the inflammatory findings observed in presymptomatic stages. The obtained results try to shed some light on identification of novel therapeutic targets for limb-girdle muscular dystrophies.
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PMID:Gene expression profiling in limb-girdle muscular dystrophy 2A. 1901 33

In an attempt to identify potential therapeutic targets for the correction of muscle wasting, the gene expression of several pivotal proteins involved in protein metabolism was investigated in experimental atrophy induced by transient or definitive denervation, as well as in four animal models of muscular dystrophies (deficient for calpain 3, dysferlin, alpha-sarcoglycan and dystrophin, respectively). The results showed that: (a) the components of the ubiquitin-proteasome pathway are upregulated during the very early phases of atrophy but do not greatly increase in the muscular dystrophy models; (b) forkhead box protein O1 mRNA expression is augmented in the muscles of a limb girdle muscular dystrophy 2A murine model; and (c) the expression of cardiac ankyrin repeat protein (CARP), a regulator of transcription factors, appears to be persistently upregulated in every condition, suggesting that CARP could be a hub protein participating in common pathological molecular pathway(s). Interestingly, the mRNA level of a cell cycle inhibitor known to be upregulated by CARP in other tissues, p21(WAF1/CIP1), is consistently increased whenever CARP is upregulated. CARP overexpression in muscle fibres fails to affect their calibre, indicating that CARP per se cannot initiate atrophy. However, a switch towards fast-twitch fibres is observed, suggesting that CARP plays a role in skeletal muscle plasticity. The observation that p21(WAF1/CIP1) is upregulated, put in perspective with the effects of CARP on the fibre type, fits well with the idea that the mechanisms at stake might be required to oppose muscle remodelling in skeletal muscle.
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PMID:Cardiac ankyrin repeat protein is a marker of skeletal muscle pathological remodelling. 1914 34

Proteolytically active calpain-3/p94 is clearly vital for normal muscle function, since its absence leads to limb girdle muscular dystrophy 2A, but its function and regulatory control are poorly understood. Here we use single muscle fibers, individually skinned by microdissection, to investigate the diffusibility and autolytic activation of calpain-3 in situ. Virtually all calpain-3 present in mature muscle fibers is tightly bound in the vicinity of the titin N2A line and triad junctions and remains so irrespective of fiber stretching or raised [Ca(2+)]. Most calpain-3 is evidently bound within the contractile filament lattice, because (i) its slow diffusional loss is slowed further by locking myosin and actin into rigor and (ii) detergent dispersion of membranes causes rapid washout of most ryanodine receptors and sarcoplasmic reticulum Ca(2+) pumps with little accompanying washout of calpain-3. Calpain-3 autolyzes (becoming proteolytically active) in a tightly calcium-dependent manner. It remains in its nonactivated full-length form if [Ca(2+)] is maintained at < or = 50 nm, the normal resting level, even with brief increases to 2-20 mum during repeated tetanic contractions, but it becomes active (though still bound) if [Ca(2+)] is kept slightly elevated at 200 nm ( approximately 20% autolysis in 1 h). Calpain-3 did not spontaneously autolyze even when free in solution with 200 nm Ca(2+) for up to 60 min. These findings explain why calpain-3 remains quiescent with normal exercise but is activated following eccentric (stretching) contractions, when resting [Ca(2+)] is elevated, and how a protease such as calpain-3 can be very Ca(2+)-sensitive yet highly specific in its actions.
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PMID:Endogenous calpain-3 activation is primarily governed by small increases in resting cytoplasmic [Ca2+] and is not dependent on stretch. 1914 34

Loss-of-function mutations in calpain 3 have been shown to cause limb-girdle muscular dystrophy type 2A (LGMD2A), an autosomal recessive disorder that results in gradual wasting of the muscles of the hip and shoulder areas. Due to the inherent instability of calpain 3, recombinant expression of the full-length enzyme has not been possible, making in vitro analysis of specific LGMD2A-causing mutations difficult. However, because calpain 3 is highly similar in amino acid sequence to calpain 2, the recently solved crystal structure of full-length, Ca2+-bound, calpastatin-inhibited rat calpain 2 has allowed us to model calpain 3 as a Ca2+-bound homodimer. The model revealed three distinct areas of the enzyme that undergo a large conformational change upon Ca2+ binding. Located in these areas are several residues that undergo mutation to cause LGMD2A. We investigated the in vitro effects of six of these mutations by making the corresponding mutations in rat calpain 2. All six mutations examined in this study resulted in a decrease in enzyme activity. All but one of the mutations caused an increased rate of autoproteolytic degradation of the enzyme as witnessed by SDS-PAGE, indicating the decrease in enzyme activity is caused, at least in part, by an increase in the rate of autoproteolytic degradation. The putative in vivo effects of these mutations on calpain 3 activity are discussed with respect to their ability to cause LGMD2A.
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PMID:Limb-girdle muscular dystrophy type 2A can result from accelerated autoproteolytic inactivation of calpain 3. 1922 46

Calpains are cysteine proteases comprising members ubiquitously expressed in human tissues and other tissue-specific isoforms. Alterations of calpain 3 (p94), the muscle-specific isoform that contains three peculiar sequences (NS, IS1 and IS2), are strictly associated to the limb-girdle muscular dystrophy type 2A, in which a myonuclear apoptosis has been documented. Our recent demonstration of a proapoptotic role of ubiquitous calpains in drug-induced apoptosis of melanoma cells prompted us to investigate the expression of calpain 3 in human melanoma cell lines undergoing apoptosis and in melanocytic lesions. In melanoma cell lines, we have identified two novel splicing variants of calpain 3 (hMp78 and hMp84): they have an atypical initiation exon and a putative nuclear localization signal, the shorter one lacks IS1 inset and both proteins are extremely unstable. Virtually, both isoforms (prevalently as cleavage forms) are localized in cytoplasm and in nucleoli. In cisplatin-treated preapoptotic cells, an increase of both transcription and autoproteolytic cleavage of the novel variants is observed; the latter event is prevented by the inhibitor of ubiquitous calpains, calpeptin, which is also able to protect from apoptosis. Interestingly, among melanocytic lesions, the expression of these novel variants is significantly downregulated, compared with benign nevi, in the most aggressive ones, i.e. in vertical growth phase melanoma and, even more, in metastatic melanoma cells, characterized by invasiveness properties and usually highly resistant to apoptosis. On the whole, our observations suggest that calpain 3 variants can play a proapoptotic role in melanoma cells and its downregulation, as observed in highly aggressive lesions, could contribute to melanoma progression.
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PMID:Novel variants of muscle calpain 3 identified in human melanoma cells: cisplatin-induced changes in vitro and differential expression in melanocytic lesions. 1938 80

Mutations in the non-lysosomal cysteine protease calpain-3 cause autosomal recessive limb girdle muscular dystrophy. Pathological mechanisms occurring in this disease have not yet been elucidated. Here, we report both morphological and biochemical evidence of mitochondrial abnormalities in calpain-3 knockout (C3KO) muscles, including irregular ultrastructure and distribution of mitochondria. The morphological abnormalities in C3KO muscles are associated with reduced in vivo mitochondrial ATP production as measured by (31)P magnetic resonance spectroscopy. Mitochondrial abnormalities in C3KO muscles also correlate with the presence of oxidative stress; increased protein modification by oxygen free radicals and an elevated concentration of the anti-oxidative enzyme Mn-superoxide dismutase were observed in C3KO muscles. Previously we identified a number of mitochondrial proteins involved in beta-oxidation of fatty acids as potential substrates for calpain-3. In order to determine if the mitochondrial abnormalities resulted from the loss of direct regulation of mitochondrial proteins by calpain-3, we validated the potential substrates that were identified in previous proteomic studies. This analysis showed that the beta-oxidation enzyme, VLCAD, is cleaved by calpain-3 in vitro, but we were not able to confirm that VLCAD is an in vivo substrate for calpain-3. However, the activity of VLCAD was decreased in C3KO mitochondrial fractions compared with wild type, a finding that likely reflects a general mitochondrial dysfunction. Taken together, these data suggest that mitochondrial abnormalities leading to oxidative stress and energy deficit are important pathological features of calpainopathy and possibly represent secondary effects of the absence of calpain-3.
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PMID:Mitochondrial abnormalities, energy deficit and oxidative stress are features of calpain 3 deficiency in skeletal muscle. 1948 97

Immunoblot is currently the preferred laboratory test to assist the diagnosis of limb-girdle muscular dystrophy (LGMD) 2A (calpainopathy). To assess whether immunohistochemistry may offer a reliable alternative screening we used two antibodies, Calp3-2C4 (exon 1) and Calp3-12A2 (exon 8), to label blots and sections of skeletal muscle from controls and patients with LGMD2A and other muscle diseases. In LGMD2A muscle biopsies a high degree of concordance was found with Calp3-2C4: labelling on sections was absent in patients with no bands on immunoblot and detected in those where CAPN3 bands were seen. Calp3-12A2 results were less consistent, with most samples retaining labelling. Interestingly, CAPN3 was found in all muscle sections from disease control patients irrespective of its detection on immunoblot. Our results show that immunohistochemistry with Calp3-2C4 has a similar pickup rate of LGMD2A as immunoblot and it may therefore be useful for distinguishing the majority of genuine CAPN3 defects from secondary protein reduction. However immunoblot is still needed when CAPN3 is present on sections to show secondary CAPN3 reduction and to identify LGMD2A with variable reduction of CAPN3 bands.
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PMID:Immunohistochemical analysis of calpain 3: advantages and limitations in diagnosing LGMD2A. 1955 29

1. Skeletal muscle fibres contain ubiquitous (mu-calpain and m-calpain) and muscle-specific (calpain-3) Ca(2+)-dependent proteases. The physiological roles of the calpains are not well understood, although ubiquitous calpains have been associated with apoptosis and myogenesis and calpain-3 is likely involved in sarcomeric remodelling. A defect in the expression of calpain-3 results in limb-girdle muscular dystrophy Type 2A. 2. At resting [Ca(2+)](i), calpains are present predominantly in their full-length, unautolysed/unactivated forms. Once activated, mu-calpain and calpain-3 appear in their autolysed forms and this measurement can be used to determine when in vivo activation occurs. Endogenously expressed mu-calpain and calpain-3 are activated within a physiological [Ca(2+)] range in a Ca(2+)- and time-dependent manner. 3. In skeletal muscle, mu-calpain is a freely diffusible protein that binds rapidly when [Ca(2+)](i) is increased. Calpain-3 is tightly bound in skeletal muscle fibres at the N2A line of the large elastic protein titin. 4. Overall, neither mu-calpain nor calpain-3 are activated immediately following sprint, endurance or eccentric exercise, despite the frequent episodes of high cytoplasmic [Ca(2+)] that would occur during these types of muscle contractions. Importantly, however, a substantial proportion of calpain-3, but not mu-calpain, is activated 24 h after a single bout of eccentric exercise. 5. In vitro studies have shown that calpain-3 becomes activated if exposed for a prolonged period of time (> 1 h) to resting cytoplasmic [Ca(2+)] that are approximately two- to fourfold higher than normal. This suggests that the small but sustained increase in [Ca(2+)](i) that likely occurs after eccentric contractions is both high and long enough to result in calpain-3 activation and supports the role for calpain-3 in sarcomeric remodelling.
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PMID:Calpains, skeletal muscle function and exercise. 1979 1

Limb-girdle muscular dystrophy type 2A is an autosomal recessive disorder generated by inactivating mutations in the gene coding for the muscle specific protease calpain-3. It is mainly expressed in skeletal muscle as a monomeric multidomain protein characterized by three unique insertion sequences (NS, IS1, IS2). It is unstable, and undergoes very rapid autolysis in solution, therefore, its heterologous expression and purification have been difficult. So far, calpain-3 substrates have been only identified in vitro and with indirect approaches. We have therefore decided to perform a comprehensive study of the substrates of the protease by comparing the 2D electrophoretic profile of myotubes from obtained from calpain-3 knockout and wild type mice. Digestion of differentially expressed spots was followed by mass spectrometry analysis. We could identify 16 proteins which differed in knockout and wild type mice. Among them: desmin, nestin, spectrin and PDLIM1 were of particular interest. In vitro experiments have then revealed that only PDLIM1 is cleaved directly by the protease, and that a fragment of about 8 kDa is released from the C-terminal portion of the protein.
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PMID:A proteomic study of calpain-3 and its involvement in limb girdle muscular dystrophy type 2a. 1992 29


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