The Experts below are selected from a list of 3234 Experts worldwide ranked by ideXlab platform
Isabelle Richard - One of the best experts on this subject based on the ideXlab platform.
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A single c.1715G>C calpain 3 gene variant causes dominant calpainopathy with loss of calpain 3 expression and activity.
Human mutation, 2020Co-Authors: John Vissing, Isabelle Richard, Carinne Roudaut, Morten Duno, Julia Dahlqvist, Jerome Poupiot, Thomas O. KragAbstract:Recessively inherited limb girdle muscular dystrophy (LGMD) type 2A is the most common LGMD worldwide. Here, we report the first single missense variant in CAPN3 causing dominantly inherited calpainopathy. A 43-year-old proband, his father and two sons were heterozygous for a c.1715G>C p.(Arg572Pro) variant in CAPN3. Affected family members had at least three of the following; muscle pain, a LGMD2A pattern of muscle weakness and wasting, muscle fat replacement on MRI, myopathic muscle biopsy, and elevated creatine kinase. Total calpain 3 protein expression was 4 ± 3% of normal. In vitro analysis of c.1715G>C and the previously described c.643_663del variant indicated that the mutant proteins lack autolytic and proteolytic activity and decrease the quantity of wild-type CAPN3 protein. Our findings suggest that dominantly inherited calpainopathy is not unique to the previously reported c.643_663del mutation of CAPN3, and that dominantly inherited calpainopathy should be considered for other single variations in CAPN3. This article is protected by copyright. All rights reserved.
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A single c.1715G>C calpain 3 gene variant causes dominant calpainopathy with loss of calpain 3 expression and activity
Human Mutation, 2020Co-Authors: John Vissing, Isabelle Richard, Carinne Roudaut, Morten Duno, Julia Dahlqvist, Jerome Poupiot, Thomas KragAbstract:Recessively inherited limb girdle muscular dystrophy (LGMD) type 2A is the most common LGMD worldwide. Here, we report the first single missense variant in CAPN3 causing dominantly inherited calpainopathy. A 43-year-old proband, his father and two sons were heterozygous for a c.1715G>C p.(Arg572Pro) variant in CAPN3. Affected family members had at least three of the following; muscle pain, a LGMD2A pattern of muscle weakness and wasting, muscle fat replacement on magnetic resonance imaging, myopathic muscle biopsy, and elevated creatine kinase. Total calpain 3 protein expression was 4 ± 3% of normal. In vitro analysis of c.1715G>C and the previously described c.643_663del variant indicated that the mutant proteins lack autolytic and proteolytic activity and decrease the quantity of wild-type CAPN3 protein. Our findings suggest that dominantly inherited calpainopathy is not unique to the previously reported c.643_663del mutation of CAPN3, and that dominantly inherited calpainopathy should be considered for other single variations in CAPN3.
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CAPN3-mediated processing of C-terminal titin replaced by pathological cleavage in titinopathy
Human molecular genetics, 2015Co-Authors: Karine Charton, Isabelle Richard, J. Sarparanta, Anna Vihola, Astrid Milic, P. Jonson, Laurence Suel, H. Luque, Imène Boumela, Bjarne UddAbstract:Mutations in the extreme C-terminus of titin (TTN), situated in the sarcomeric M-band, cause tibial muscular dystrophy (TMD) and limb-girdle muscular dystrophy 2J (LGMD2J). The mutations ultimately cause a loss of C-terminal titin, including a binding site for the protease calpain 3 (CAPN3), and lead to a secondary CAPN3 deficiency in LGMD2J muscle. CAPN3 has been previously shown to bind C-terminal titin and to use it as a substrate in vitro. Interestingly, mutations in CAPN3 underlie limb-girdle muscular dystrophy 2A (LGMD2A). Here, we aimed to clarify the relationship of CAPN3 and M-band titin in normal and pathological muscle. In vitro analyses identified several CAPN3 cleavage sites in C-terminal titin that were defined by protein sequencing. Furthermore, cleavage products were detected in normal muscle extracts by western blotting and in situ by immunofluorescence microscopy. The TMD/LGMD2J mutation FINmaj proved to alter this processing in vitro, while binding of CAPN3 to mutant titin was preserved. Unexpectedly, the pathological loss of M-band titin due to TMD/LGMD2J mutations was found to be independent of CAPN3, whereas the involvement of ubiquitous calpains is likely. We conclude that proteolytic processing of C-terminal titin by CAPN3 may have an important role in normal muscle, and that this process is disrupted in LGMD2A and in TMD/LGMD2J due to CAPN3 deficiency and to the loss of C-terminal titin, respectively.
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Restriction of Calpain3 Expression to the Skeletal Muscle Prevents Cardiac Toxicity and Corrects Pathology in a Murine Model of Limb-Girdle Muscular Dystrophy
Circulation, 2013Co-Authors: Carinne Roudaut, Marc Bartoli, Karine Charton, Laurence Suel, Jerome Poupiot, Florence Le Roy, Isabelle RichardAbstract:Background Genetic defects in calpain3 (CAPN3) lead to limb-girdle muscular dystrophy type 2A, a disease of the skeletal muscle that affects predominantly the proximal limb muscles. We previously demonstrated the potential of adeno-associated virus-mediated transfer of the CAPN3 gene to correct the pathological signs in a murine model for limb-girdle muscular dystrophy type 2A after intramuscular and locoregional administrations. Methods and Results Here, we showed that intravenous injection of calpain3-expressing vector in mice can induce mortality in a dose-dependent manner. An anatomopathological investigation revealed large areas of fibrosis in the heart that we related to unregulated proteolytic activity of calpain3. To circumvent this toxicity, we developed new adeno-associated virus vectors with skeletal muscle-restricted expression by using new muscle-specific promoters that include the CAPN3 promoter itself and by introducing a target sequence of the cardiac-specific microRNA-208a in the cassette. Our results show that CAPN3 transgene expression can be successfully suppressed in the cardiac tissue, preventing the cardiac toxicity, whereas expression of the transgene in skeletal muscle reverts the pathological signs of calpain3 deficiency. Conclusions The molecular strategies used in this study may be useful for any gene transfer strategy with potential toxicity in the heart.
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P.3.12 Characterization of CAPN3-dependent proteolysis of C-terminal titin
Neuromuscular Disorders, 2013Co-Authors: J. Sarparanta, Isabelle Richard, Karine Charton, P. Jonson, H. Luque, Bjarne UddAbstract:The titinopathies tibial muscular dystrophy (TMD) and limb-girdle muscular dystrophy 2J (LGMD2J) are caused by mutations in the C-terminus of titin, residing in the sarcomeric M-band. Mutations identified so far affect the last Ig domain M10 or the preceding is7 region. The FINmaj mutation, underlying TMD/LGMD2J in patients of Finnish origin, causes the exchange of four amino acids in M10 and presumably leads to domain unfolding. The other known mutations cause missense changes of single amino acids or truncation of the ultimate C-terminus. Loss of C-terminal titin epitopes in IF microscopy and reduced amount of C-terminal titin fragments in western blotting suggest that increased or abnormal proteolytic turnover of mutant titin may contribute to the pathomechanism. The muscle-specific protease calpain 3 (CAPN3) binds M-band titin at the is7 region; the interaction is thought to regulate the autolytic activation of CAPN3. LGMD2J patients and FINmaj knock-in mice show secondary CAPN3 deficiency, reflecting loss of the binding site and consequent dysregulation of CAPN3 activity. To elucidate the proteolytic events involved in the pathogenesis of M-band titinopathies, we investigated the cleavage of C-terminal titin by CAPN3. Cleavage fragments generated by CAPN3 were identified by coexpressing various wild-type and mutant titin constructs with active or inactive CAPN3 in cell culture, and comparing the resulting fragment patterns. Active CAPN3 produced several titin fragments, which were characterized by western blotting, protein sequencing and mass spectrometry for identification of cleavage sites. Furthermore, targeted mutagenesis of predicted CAPN3 recognition sites was utilized for understanding the sequence determinants of CAPN3 cleavage and studying the order of cleavage events.
Hiroyuki Sorimachi - One of the best experts on this subject based on the ideXlab platform.
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Developing fluorescence sensor probe to capture activated muscle-specific calpain-3 (CAPN3) in living muscle cells.
Biology open, 2020Co-Authors: Koichi Ojima, Shoji Hata, Hiroyuki Sorimachi, Fumiko Shinkai-ouchi, Susumu Muroya, Yasuko OnoAbstract:ABSTRACT Calpain-3 (CAPN3) is a muscle-specific type of calpain whose protease activity is triggered by Ca2+. Here, we developed CAPN3 sensor probes (SPs) to detect activated-CAPN3 using a fluorescence/Forster resonance energy transfer (FRET) technique. In our SPs, partial amino acid sequence of calpastatin, endogenous CAPN inhibitor but CAPN3 substrate, is inserted between two different fluorescence proteins that cause FRET. Biochemical and spectral studies revealed that CAPN3 cleaved SPs and changed emission wavelengths of SPs. Importantly, SPs were scarcely cleaved by CAPN1 and CAPN2. Furthermore, our SP successfully captured the activation of endogenous CAPN3 in living myotubes treated with ouabain. Our SPs would become a promising tool to detect the dynamics of CAPN3 protease activity in living cells.
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an eccentric calpain CAPN3 p94 calpain 3
Biochimie, 2016Co-Authors: Yasuko Ono, Shoji Hata, Koichi Ojima, Fumiko Shinkaiouchi, Hiroyuki SorimachiAbstract:Calpains are Ca(2+)-regulated proteolytic enzymes that are involved in a variety of biological phenomena. Calpains process substrates by limited proteolysis to modulate various protein functions in the cell, and are thus called "modulator proteases." CAPN3, previously called p94 or calpain-3, has unique features that are not found in any of the other 14 human calpains, or even in other proteases. For instance, CAPN3 undergoes extremely rapid and exhaustive autodegradation. CAPN3 is also the first (and so far, the only) intracellular enzyme found to depend on Na(+) for its activation. CAPN3 has both proteolytic and non-proteolytic functions. It has the interesting distinction of being the only protease, other than a few virus proteases, with the ability to regain protease function after its autolytic dissociation; this occurs through a process known as intermolecular complementation (iMOC). Gene mutations causing CAPN3 defects are responsible for limb-girdle muscular dystrophy type 2A (LGMD2A). Unusual characteristics of CAPN3 have fascinated researchers, but have also hampered conventional biochemical analysis. In this review, we describe significant findings about CAPN3 from its discovery to the present, and suggest promising avenues for future CAPN3 research.
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amino acid sequence alignment of vertebrate CAPN3 calpain 3 p94
Data in Brief, 2015Co-Authors: Yasuko Ono, Hiroyuki SorimachiAbstract: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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Amino acid sequence alignment of vertebrate CAPN3/calpain-3/p94.
Data in brief, 2015Co-Authors: Yasuko Ono, Hiroyuki SorimachiAbstract: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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An eccentric calpain, CAPN3/p94/calpain-3.
Biochimie, 2015Co-Authors: Yasuko Ono, Shoji Hata, Fumiko Shinkai-ouchi, Koichi Ojima, Hiroyuki SorimachiAbstract:Calpains are Ca(2+)-regulated proteolytic enzymes that are involved in a variety of biological phenomena. Calpains process substrates by limited proteolysis to modulate various protein functions in the cell, and are thus called "modulator proteases." CAPN3, previously called p94 or calpain-3, has unique features that are not found in any of the other 14 human calpains, or even in other proteases. For instance, CAPN3 undergoes extremely rapid and exhaustive autodegradation. CAPN3 is also the first (and so far, the only) intracellular enzyme found to depend on Na(+) for its activation. CAPN3 has both proteolytic and non-proteolytic functions. It has the interesting distinction of being the only protease, other than a few virus proteases, with the ability to regain protease function after its autolytic dissociation; this occurs through a process known as intermolecular complementation (iMOC). Gene mutations causing CAPN3 defects are responsible for limb-girdle muscular dystrophy type 2A (LGMD2A). Unusual characteristics of CAPN3 have fascinated researchers, but have also hampered conventional biochemical analysis. In this review, we describe significant findings about CAPN3 from its discovery to the present, and suggest promising avenues for future CAPN3 research.
Yasuko Ono - One of the best experts on this subject based on the ideXlab platform.
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Developing fluorescence sensor probe to capture activated muscle-specific calpain-3 (CAPN3) in living muscle cells.
Biology open, 2020Co-Authors: Koichi Ojima, Shoji Hata, Hiroyuki Sorimachi, Fumiko Shinkai-ouchi, Susumu Muroya, Yasuko OnoAbstract:ABSTRACT Calpain-3 (CAPN3) is a muscle-specific type of calpain whose protease activity is triggered by Ca2+. Here, we developed CAPN3 sensor probes (SPs) to detect activated-CAPN3 using a fluorescence/Forster resonance energy transfer (FRET) technique. In our SPs, partial amino acid sequence of calpastatin, endogenous CAPN inhibitor but CAPN3 substrate, is inserted between two different fluorescence proteins that cause FRET. Biochemical and spectral studies revealed that CAPN3 cleaved SPs and changed emission wavelengths of SPs. Importantly, SPs were scarcely cleaved by CAPN1 and CAPN2. Furthermore, our SP successfully captured the activation of endogenous CAPN3 in living myotubes treated with ouabain. Our SPs would become a promising tool to detect the dynamics of CAPN3 protease activity in living cells.
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A muscle-specific calpain, CAPN3, forms a homotrimer.
Biochimica et biophysica acta. Proteins and proteomics, 2020Co-Authors: Shoji Hata, Naoko Doi, Fumiko Shinkai-ouchi, Yasuko OnoAbstract:Abstract Calpain-3 (CAPN3), a 94-kDa member of the calpain protease family, is abundant in skeletal muscle. Mutations in the CAPN3 gene cause limb girdle muscular dystrophy type 2A, indicating that CAPN3 plays important roles in muscle physiology. CAPN3 has several unique features. A crystallographic study revealed that its C-terminal penta–EF-hand domains form a homodimer, suggesting that CAPN3 functions as a homodimeric protease. To analyze complex formation of CAPN3 in a more convenient manner, we performed blue native polyacrylamide gel electrophoresis and found that the observed molecular weight of native CAPN3, as well as recombinant CAPN3, was larger than 240 kDa. Further analysis by cross-linking and sequential immunoprecipitation revealed that CAPN3 in fact forms a homotrimer. Trimer formation was abolished by the deletion of the PEF domain, but not the CAPN3-specific insertion sequences NS, IS1, and IS2. The PEF domain alone formed a homodimer, as reported, but addition of the adjacent CBSW domain to its N-terminus reinforced the trimer-forming property. Collectively, these results suggest that CAPN3 forms a homotrimer in which the PEF domain's dimer-forming ability is influenced by other domains.
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an eccentric calpain CAPN3 p94 calpain 3
Biochimie, 2016Co-Authors: Yasuko Ono, Shoji Hata, Koichi Ojima, Fumiko Shinkaiouchi, Hiroyuki SorimachiAbstract:Calpains are Ca(2+)-regulated proteolytic enzymes that are involved in a variety of biological phenomena. Calpains process substrates by limited proteolysis to modulate various protein functions in the cell, and are thus called "modulator proteases." CAPN3, previously called p94 or calpain-3, has unique features that are not found in any of the other 14 human calpains, or even in other proteases. For instance, CAPN3 undergoes extremely rapid and exhaustive autodegradation. CAPN3 is also the first (and so far, the only) intracellular enzyme found to depend on Na(+) for its activation. CAPN3 has both proteolytic and non-proteolytic functions. It has the interesting distinction of being the only protease, other than a few virus proteases, with the ability to regain protease function after its autolytic dissociation; this occurs through a process known as intermolecular complementation (iMOC). Gene mutations causing CAPN3 defects are responsible for limb-girdle muscular dystrophy type 2A (LGMD2A). Unusual characteristics of CAPN3 have fascinated researchers, but have also hampered conventional biochemical analysis. In this review, we describe significant findings about CAPN3 from its discovery to the present, and suggest promising avenues for future CAPN3 research.
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amino acid sequence alignment of vertebrate CAPN3 calpain 3 p94
Data in Brief, 2015Co-Authors: Yasuko Ono, Hiroyuki SorimachiAbstract: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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Amino acid sequence alignment of vertebrate CAPN3/calpain-3/p94.
Data in brief, 2015Co-Authors: Yasuko Ono, Hiroyuki SorimachiAbstract: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]
Carinne Roudaut - One of the best experts on this subject based on the ideXlab platform.
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A single c.1715G>C calpain 3 gene variant causes dominant calpainopathy with loss of calpain 3 expression and activity.
Human mutation, 2020Co-Authors: John Vissing, Isabelle Richard, Carinne Roudaut, Morten Duno, Julia Dahlqvist, Jerome Poupiot, Thomas O. KragAbstract:Recessively inherited limb girdle muscular dystrophy (LGMD) type 2A is the most common LGMD worldwide. Here, we report the first single missense variant in CAPN3 causing dominantly inherited calpainopathy. A 43-year-old proband, his father and two sons were heterozygous for a c.1715G>C p.(Arg572Pro) variant in CAPN3. Affected family members had at least three of the following; muscle pain, a LGMD2A pattern of muscle weakness and wasting, muscle fat replacement on MRI, myopathic muscle biopsy, and elevated creatine kinase. Total calpain 3 protein expression was 4 ± 3% of normal. In vitro analysis of c.1715G>C and the previously described c.643_663del variant indicated that the mutant proteins lack autolytic and proteolytic activity and decrease the quantity of wild-type CAPN3 protein. Our findings suggest that dominantly inherited calpainopathy is not unique to the previously reported c.643_663del mutation of CAPN3, and that dominantly inherited calpainopathy should be considered for other single variations in CAPN3. This article is protected by copyright. All rights reserved.
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A single c.1715G>C calpain 3 gene variant causes dominant calpainopathy with loss of calpain 3 expression and activity
Human Mutation, 2020Co-Authors: John Vissing, Isabelle Richard, Carinne Roudaut, Morten Duno, Julia Dahlqvist, Jerome Poupiot, Thomas KragAbstract:Recessively inherited limb girdle muscular dystrophy (LGMD) type 2A is the most common LGMD worldwide. Here, we report the first single missense variant in CAPN3 causing dominantly inherited calpainopathy. A 43-year-old proband, his father and two sons were heterozygous for a c.1715G>C p.(Arg572Pro) variant in CAPN3. Affected family members had at least three of the following; muscle pain, a LGMD2A pattern of muscle weakness and wasting, muscle fat replacement on magnetic resonance imaging, myopathic muscle biopsy, and elevated creatine kinase. Total calpain 3 protein expression was 4 ± 3% of normal. In vitro analysis of c.1715G>C and the previously described c.643_663del variant indicated that the mutant proteins lack autolytic and proteolytic activity and decrease the quantity of wild-type CAPN3 protein. Our findings suggest that dominantly inherited calpainopathy is not unique to the previously reported c.643_663del mutation of CAPN3, and that dominantly inherited calpainopathy should be considered for other single variations in CAPN3.
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Species-specific titin splicing regulates cardiotoxicity associated with calpain 3 gene therapy for limb-girdle muscular dystrophy 2A
Science Translational Medicine, 2019Co-Authors: William Lostal, Nathalie Bourg, Carinne Roudaut, Karine Charton, Laurence Suel, Marine Faivre, Heather Best, John Edward Smith, Jochen Gohlke, Guillaume CorreAbstract:Limb-girdle muscular dystrophy type 2A (LGMD2A or LGMDR1) is a neuromuscular disorder caused by mutations in the calpain 3 gene (CAPN3). Previous experiments using adeno-associated viral (AAV) vector-mediated calpain 3 gene transfer in mice indicated cardiac toxicity associated with the ectopic expression of the calpain 3 transgene. Here, we performed a preliminary dose study in a severe double-knockout mouse model deficient in calpain 3 and dysferlin. We evaluated safety and biodistribution of AAV9-desmin-hCAPN3 vector administration to nonhuman primates (NHPs) with a dose of 3 × 1013 viral genomes/kg. Vector administration did not lead to observable adverse effects or to detectable toxicity in NHP. Of note, the transgene expression did not produce any abnormal changes in cardiac morphology or function of injected animals while reaching therapeutic expression in skeletal muscle. Additional investigation on the underlying causes of cardiac toxicity observed after gene transfer in mice and the role of titin in this phenomenon suggest species-specific titin splicing. Mice have a reduced capacity for buffering calpain 3 activity compared to NHPs and humans. Our studies highlight a complex interplay between calpain 3 and titin binding sites and demonstrate an effective and safe profile for systemic calpain 3 vector delivery in NHP, providing critical support for the clinical potential of calpain 3 gene therapy in humans.
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Restriction of Calpain3 Expression to the Skeletal Muscle Prevents Cardiac Toxicity and Corrects Pathology in a Murine Model of Limb-Girdle Muscular Dystrophy
Circulation, 2013Co-Authors: Carinne Roudaut, Marc Bartoli, Karine Charton, Laurence Suel, Jerome Poupiot, Florence Le Roy, Isabelle RichardAbstract:Background Genetic defects in calpain3 (CAPN3) lead to limb-girdle muscular dystrophy type 2A, a disease of the skeletal muscle that affects predominantly the proximal limb muscles. We previously demonstrated the potential of adeno-associated virus-mediated transfer of the CAPN3 gene to correct the pathological signs in a murine model for limb-girdle muscular dystrophy type 2A after intramuscular and locoregional administrations. Methods and Results Here, we showed that intravenous injection of calpain3-expressing vector in mice can induce mortality in a dose-dependent manner. An anatomopathological investigation revealed large areas of fibrosis in the heart that we related to unregulated proteolytic activity of calpain3. To circumvent this toxicity, we developed new adeno-associated virus vectors with skeletal muscle-restricted expression by using new muscle-specific promoters that include the CAPN3 promoter itself and by introducing a target sequence of the cardiac-specific microRNA-208a in the cassette. Our results show that CAPN3 transgene expression can be successfully suppressed in the cardiac tissue, preventing the cardiac toxicity, whereas expression of the transgene in skeletal muscle reverts the pathological signs of calpain3 deficiency. Conclusions The molecular strategies used in this study may be useful for any gene transfer strategy with potential toxicity in the heart.
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Restriction of Calpain3 Expression to the Skeletal Muscle Prevents Cardiac Toxicity and Corrects Pathology in a Murine Model of Limb-Girdle Muscular Dystrophy
Circulation, 2013Co-Authors: Carinne Roudaut, Marc Bartoli, Karine Charton, Laurence Suel, Jerome Poupiot, Florence Le Roy, Isabelle RichardAbstract:Background—Genetic defects in calpain3 (CAPN3) lead to limb-girdle muscular dystrophy type 2A, a disease of the skeletal muscle that affects predominantly the proximal limb muscles. We previously demonstrated the potential of adeno-associated virus–mediated transfer of the CAPN3 gene to correct the pathological signs in a murine model for limb-girdle muscular dystrophy type 2A after intramuscular and locoregional administrations. Methods and Results—Here, we showed that intravenous injection of calpain3-expressing vector in mice can induce mortality in a dose-dependent manner. An anatomopathological investigation revealed large areas of fibrosis in the heart that we related to unregulated proteolytic activity of calpain3. To circumvent this toxicity, we developed new adeno-associated virus vectors with skeletal muscle–restricted expression by using new muscle-specific promoters that include the CAPN3 promoter itself and by introducing a target sequence of the cardiac-specific microRNA-208a in the cassette...
Shoji Hata - One of the best experts on this subject based on the ideXlab platform.
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Developing fluorescence sensor probe to capture activated muscle-specific calpain-3 (CAPN3) in living muscle cells.
Biology open, 2020Co-Authors: Koichi Ojima, Shoji Hata, Hiroyuki Sorimachi, Fumiko Shinkai-ouchi, Susumu Muroya, Yasuko OnoAbstract:ABSTRACT Calpain-3 (CAPN3) is a muscle-specific type of calpain whose protease activity is triggered by Ca2+. Here, we developed CAPN3 sensor probes (SPs) to detect activated-CAPN3 using a fluorescence/Forster resonance energy transfer (FRET) technique. In our SPs, partial amino acid sequence of calpastatin, endogenous CAPN inhibitor but CAPN3 substrate, is inserted between two different fluorescence proteins that cause FRET. Biochemical and spectral studies revealed that CAPN3 cleaved SPs and changed emission wavelengths of SPs. Importantly, SPs were scarcely cleaved by CAPN1 and CAPN2. Furthermore, our SP successfully captured the activation of endogenous CAPN3 in living myotubes treated with ouabain. Our SPs would become a promising tool to detect the dynamics of CAPN3 protease activity in living cells.
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A muscle-specific calpain, CAPN3, forms a homotrimer.
Biochimica et biophysica acta. Proteins and proteomics, 2020Co-Authors: Shoji Hata, Naoko Doi, Fumiko Shinkai-ouchi, Yasuko OnoAbstract:Abstract Calpain-3 (CAPN3), a 94-kDa member of the calpain protease family, is abundant in skeletal muscle. Mutations in the CAPN3 gene cause limb girdle muscular dystrophy type 2A, indicating that CAPN3 plays important roles in muscle physiology. CAPN3 has several unique features. A crystallographic study revealed that its C-terminal penta–EF-hand domains form a homodimer, suggesting that CAPN3 functions as a homodimeric protease. To analyze complex formation of CAPN3 in a more convenient manner, we performed blue native polyacrylamide gel electrophoresis and found that the observed molecular weight of native CAPN3, as well as recombinant CAPN3, was larger than 240 kDa. Further analysis by cross-linking and sequential immunoprecipitation revealed that CAPN3 in fact forms a homotrimer. Trimer formation was abolished by the deletion of the PEF domain, but not the CAPN3-specific insertion sequences NS, IS1, and IS2. The PEF domain alone formed a homodimer, as reported, but addition of the adjacent CBSW domain to its N-terminus reinforced the trimer-forming property. Collectively, these results suggest that CAPN3 forms a homotrimer in which the PEF domain's dimer-forming ability is influenced by other domains.
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an eccentric calpain CAPN3 p94 calpain 3
Biochimie, 2016Co-Authors: Yasuko Ono, Shoji Hata, Koichi Ojima, Fumiko Shinkaiouchi, Hiroyuki SorimachiAbstract:Calpains are Ca(2+)-regulated proteolytic enzymes that are involved in a variety of biological phenomena. Calpains process substrates by limited proteolysis to modulate various protein functions in the cell, and are thus called "modulator proteases." CAPN3, previously called p94 or calpain-3, has unique features that are not found in any of the other 14 human calpains, or even in other proteases. For instance, CAPN3 undergoes extremely rapid and exhaustive autodegradation. CAPN3 is also the first (and so far, the only) intracellular enzyme found to depend on Na(+) for its activation. CAPN3 has both proteolytic and non-proteolytic functions. It has the interesting distinction of being the only protease, other than a few virus proteases, with the ability to regain protease function after its autolytic dissociation; this occurs through a process known as intermolecular complementation (iMOC). Gene mutations causing CAPN3 defects are responsible for limb-girdle muscular dystrophy type 2A (LGMD2A). Unusual characteristics of CAPN3 have fascinated researchers, but have also hampered conventional biochemical analysis. In this review, we describe significant findings about CAPN3 from its discovery to the present, and suggest promising avenues for future CAPN3 research.
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An eccentric calpain, CAPN3/p94/calpain-3.
Biochimie, 2015Co-Authors: Yasuko Ono, Shoji Hata, Fumiko Shinkai-ouchi, Koichi Ojima, Hiroyuki SorimachiAbstract:Calpains are Ca(2+)-regulated proteolytic enzymes that are involved in a variety of biological phenomena. Calpains process substrates by limited proteolysis to modulate various protein functions in the cell, and are thus called "modulator proteases." CAPN3, previously called p94 or calpain-3, has unique features that are not found in any of the other 14 human calpains, or even in other proteases. For instance, CAPN3 undergoes extremely rapid and exhaustive autodegradation. CAPN3 is also the first (and so far, the only) intracellular enzyme found to depend on Na(+) for its activation. CAPN3 has both proteolytic and non-proteolytic functions. It has the interesting distinction of being the only protease, other than a few virus proteases, with the ability to regain protease function after its autolytic dissociation; this occurs through a process known as intermolecular complementation (iMOC). Gene mutations causing CAPN3 defects are responsible for limb-girdle muscular dystrophy type 2A (LGMD2A). Unusual characteristics of CAPN3 have fascinated researchers, but have also hampered conventional biochemical analysis. In this review, we describe significant findings about CAPN3 from its discovery to the present, and suggest promising avenues for future CAPN3 research.
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Muscle-specific calpain-3 is phosphorylated in its unique insertion region for enrichment in a myofibril fraction.
Genes to cells : devoted to molecular & cellular mechanisms, 2014Co-Authors: Koichi Ojima, Shoji Hata, Yasuko Ono, Satoru Noguchi, Ichizo Nishino, Hiroyuki SorimachiAbstract:CAPN3 (also called p94/calpain-3) is a skeletal muscle-specific calpain, an intracellular cysteine protease. Loss of CAPN3 protease activity and/or structural functions cause limb-girdle muscular dystrophy type 2A (LGMD2A). However, the precise mechanism of action of CAPN3 in skeletal muscles in vivo remains largely elusive. By studying the protein modifications that regulate CAPN3 activity, we found that CAPN3 was phosphorylated. By performing mutagenesis and mass spectrometry analyses, we identified two Ser residues at positions 629 and 636 in human CAPN3 that are phosphorylated and showed that S629 is a major phosphorylation site. Intriguingly, rapid and exhaustive autolysis of CAPN3 was slightly attenuated by the substitution of S629. In skeletal muscles, phosphorylated CAPN3 was enriched in the myofibril fraction. These results imply that phosphorylated CAPN3 is a myofibril structural component and/or participates in myofibril-based signaling pathways, rather than functions as a protease. We evaluated the relationship between phosphorylated CAPN3 and the pathology of LGMD2A. The level of phosphorylated CAPN3 was greatly reduced in LGMD2A muscles. Our findings suggest that phosphorylated CAPN3 is involved in the pathology of LGMD2A through defects in myofibril integrity and/or signaling pathways. This is the first report that phosphorylation of CAPN3 may be involved in its physiological function.
