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H Sorimachi

Publications and source records attributed to H Sorimachi.

At least 55 records · Page 3Linked to original sources
PubMed1999

Expression and functional characteristics of calpain 3 isoforms generated through tissue-specific transcriptional and posttranscriptional events.

Calpain 3 is a nonlysosomal cysteine protease whose biological functions remain unknown. We previously demonstrated that this protease is altered in limb girdle muscular dystrophy type 2A patients. Preliminary observations suggested that its gene is subjected to alternative splicing. In this paper, we characterize transcriptional and posttranscriptional events leading to alterations involving the NS, IS1, and IS2 regions and/or the calcium binding domains of the mouse calpain 3 gene (capn3). These events can be divided into three groups: (i) splicing of exons that preserve the translation frame, (ii) inclusion of two distinct intronic sequences between exons 16 and 17 that disrupt the frame and would lead, if translated, to a truncated protein lacking domain IV, and (iii) use of an alternative first exon specific to lens tissue. In addition, expression of these isoforms seems to be regulated. Investigation of the proteolytic activities and titin binding abilities of the translation products of some of these isoforms clearly indicated that removal of these different protein segments affects differentially the biochemical properties examined. In particular, removal of exon 6 impaired the autolytic but not fodrinolytic activity and loss of exon 16 led to an increased titin binding and a loss of fodrinolytic activity. These results are likely to impact our understanding of the pathophysiology of calpainopathies and the development of therapeutic strategies.

Alternative Splicing↗
PubMed1998

The deletion of the C-terminal tail and addition of an endoplasmic reticulum targeting signal to Alzheimer's amyloid precursor protein change its localization, secretion, and intracellular proteolysis.

The metabolic pathway of Alzheimer's amyloid precursor protein (APP) involves restricted intracellular proteolysis by secretases, which leads to the secretion of the N-terminal soluble APP (sAPP) and the generation of a cell-associated C-terminal fragment. The precise cellular sites at which these processes occur remain unknown. In this report, we describe the route of APP sorting and the processing site using novel systems with and without sorting signals on the APP molecule. One system involves the replacement of the C-terminal ten amino acids of APP with Adenoviral E19 protein containing an endoplasmic reticulum (ER) retrieval signal (APPE19); the other involves deleting the last ten amino acids corresponding to the replaced site (APPdeltaC10). APPE19 localized mainly within the cis/medial Golgi compartment and exclusively suppresses the secretion of APP. In contrast, deletion of the C-terminal tail promotes sAPP secretion by a constitutive secretion pathway. Metabolic labeling followed by immunoprecipitation with anti-APP antibody revealed that APPE19 is rapidly degraded within 30 min and that the subsequent intracellular turnover rate is decreased with 40% of the protein retained within the cells even after a chase period a 3 h. In contrast, APPdeltaC10 is rapidly eliminated from the intracellular compartments and secreted into the culture medium. The surface internalization and recycling processes of this protein are relatively impaired compared with wild-type APP. The ratios of the levels of production to secretion of sAPP alpha, the N-terminal, soluble APP fragment released by alpha-secretase, are proportional to the secretion efficiencies among APP species, suggesting the localization of alpha-secretase within a compartment late in the constitutive secretion pathway. These secretion mutants which utilize ER targeting signals are useful tools for analyzing the location of secretases and the intracellular degradation system within a constitutive secretion pathway such as ER quality control.

Adenovirus Early Proteins↗
PubMed1998

The NH2 terminus of titin spans the Z-disc: its interaction with a novel 19-kD ligand (T-cap) is required for sarcomeric integrity.

Titin is a giant elastic protein in vertebrate striated muscles with an unprecedented molecular mass of 3-4 megadaltons. Single molecules of titin extend from the Z-line to the M-line. Here, we define the molecular layout of titin within the Z-line; the most NH2-terminal 30 kD of titin is located at the periphery of the Z-line at the border of the adjacent sarcomere, whereas the subsequent 60 kD of titin spans the entire width of the Z-line. In vitro binding studies reveal that mammalian titins have at least four potential binding sites for alpha-actinin within their Z-line spanning region. Titin filaments may specify Z-line width and internal structure by varying the length of their NH2-terminal overlap and number of alpha-actinin binding sites that serve to cross-link the titin and thin filaments. Furthermore, we demonstrate that the NH2-terminal titin Ig repeats Z1 and Z2 in the periphery of the Z-line bind to a novel 19-kD protein, referred to as titin-cap. Using dominant-negative approaches in cardiac myocytes, both the titin Z1-Z2 domains and titin-cap are shown to be required for the structural integrity of sarcomeres, suggesting that their interaction is critical in titin filament-regulated sarcomeric assembly.

Actinin↗
PubMed1998

Purification of native p94, a muscle-specific calpain, and characterization of its autolysis.

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.

Amino Acid Sequence↗
PubMed1998

Skeletal muscle-specific calpain, p49: structure and physiological function.

Recent studies indicate that calpain, a cytosolic Ca2+-dependent protease, constitutes a large family comprising ubiquitous, tissue-specific, and atypical calpains. p94 is a homologue of the catalytic large subunit of calpain, expressed predominantly in skeletal muscle. Recently, p94 has been found to interact with connectin/titin, a muscle elastic protein, and its gene has been identified as being responsible for limb-girdle muscular dystrophy type 2A. The loss of function of a calpain species eventually leads to the activation of proteases including other calpain species responsible for muscle degradation. p94 does not form a complex with the small subunit of calpain (30K), but exists as a homodimer. This, together with other results, led us to consider a novel mechanism for the activation of calpain, a Ca2+-induced subunit rearrangement.

Animals↗
PubMed1998

A novel aspect of calpain activation.

Calpain, a Ca2+-dependent biomodulator, alters the properties of substrate proteins by cleaving them at a limited number of specific sites. Recent studies of the structure-function relationship of calpain and X-ray analysis of its Ca2+-binding domain have revealed hitherto unknown features of the regulation of calpain activity. A novel dissociation/autolysis mechanism for the activation of calpain at the membrane is proposed, which incorporates recent findings from structure-function studies of calpain, and its implications are discussed.

Animals↗
PubMed1998

Functional defects of a muscle-specific calpain, p94, caused by mutations associated with limb-girdle muscular dystrophy type 2A.

p94 (calpain3), a muscle-specific member of the calpain family, has been shown to be responsible for limb-girdle muscular dystrophy type 2A (LGMD2A), a form of autosomal recessive and progressive neuromuscular disorder. To elucidate the molecular mechanism of LGMD2A, we constructed nine p94 missense point mutants found in LGMD2A and analyzed their p94 unique properties. All mutants completely or almost completely lose the proteolytic activity against a potential substrate, fodrin. However, some of the mutants still possess autolytic activity and/or connectin/titin binding ability, indicating these properties are not necessary for the LGMD2A phenotypes. These results provide strong evidence that LGMD2A results from the loss of proteolysis of substrates by p94, suggesting a novel molecular mechanism leading to muscular dystrophies.

Animals↗
PubMed1998

Structure and physiology of calpain, an enigmatic protease.

Calpain is one of the most extensively studied proteinases (1). Although its enzymatic and structural properties have been well characterized, neither the structure-function relationship nor physiological functions are completely understood. In recent years, increasing numbers of molecules showing sequence similarity to calpain have been identified and the concept of a "calpain super family" has become general (2, 3). The term "calpain" originally meant a Ca(2+)-activated, neutral, and intracellular cysteine proteinase, although a proteinase domain similar to that of calpain is a prerequisite for a member of the "calpain super family" (4, 5). The molecular diversity of calpain has attracted interest to its structural and functional transition during evolution. Here we describe the state of current knowledge, progress, and clues to the next phase of calpain research.

Calcium-Binding Proteins↗
PubMed1998

Overexpression, purification, and characterization of human m-calpain and its active site mutant, m-C105S-calpain, using a baculovirus expression system.

Recombinant human m-calpain was produced in a soluble form at a level of 20 mg/liter of Sf-9 cell culture by the coexpression of recombinant human m-calpain large (m80K) and small (30K) subunits using a baculovirus expression system. The expressed m-calpain was purified by sequential column chromatographies on DEAE-Toyopearl, gel-filtration, and Mono Q by the same method used to purify native m-calpain. The recombinant m-calpain had a specific activity of 691 U/mg and a Ka value (Ca2+ requirement for 50% caseinolysis activity) of 0.4 mM, which are essentially identical to those of native rabbit m-calpain. A mutant m-calpain large subunit, m-C105S-80K, where the active-site cysteine-105 is converted to serine by site-directed mutagenesis, was coexpressed with 30K in Sf-9 cells, purified, and characterized. m-C105S-calpain does not degrade casein nor an artificial tetra-peptide substrate, succinyl-Leu-Leu-Val-Tyr-MCA. Further, it shows no autolytic activity with Ca2+. This is the first report of the large-scale production of a fully active m-calpain species in the baculovirus system.

Amino Acid Sequence↗
PubMed1998

Molecular cloning and characterization of a novel tissue-specific calpain predominantly expressed in the digestive tract.

In the course of the genomic cloning of nCL-2, a stomach-specific calpain, we identified a genomic clone encoding a novel member of the calpain large subunit family and designated it 'nCL-4'. First, using exon sequences, we cloned the cDNA for mouse nCL-4. Based on this sequence, we also cloned the cDNAs for rat and human nCL-4. In the case of human nCL-4, the longest open reading frame encodes 690 amino acid residues (Mr 79095) with equal sequence similarities (50-55%) to both ubiquitous and organ-specific calpain large subunits from mammals. The deduced amino acid sequence revealed that nCL-4 is highly conserved among mammals. nCL-4 can be aligned without significant deletions or insertions, and, thus, like other calpains, can be divided into four domains (I-IV). The significant similarity of domains II and IV to those in conventional calpain large subunits suggests the potential protease activity and Ca2+-binding ability of nCL-4. Northern blot analysis revealed that the mRNA for nCL-4 is expressed predominantly in stomach and small intestine but not in uterus, suggesting specialized functions of nCL-4 in the digestive tract. When overexpressed in COS-7 cells, a specific band for nCL-4 was detected. In addition, the gene coding for nCL-4 was localized on human chromosome 1.

Amino Acid Sequence↗
PubMed1997

Structure and physiological function of calpains.

For a long time now, two ubiquitously expressed mammalian calpain isoenzymes have been used to explore the structure and function of calpain. Although these two calpains, mu- and m-calpains, still attract intensive interest because of their unique characteristics, various distinct homologues to the protease domain of mu- and m-calpains have been identified in a variety of organisms. Some of these 'novel' calpain homologues are involved in important biological functions. For example, p94 (also called calpain 3), a mammalian calpain homologue predominantly expressed in skeletal muscle, is genetically proved to be responsible for limb-girdle muscular dystrophy type 2A. Tra-3, a calpain homologue in nematodes, is involved in the sex determination cascade during early development. PalB, a key gene product involved in the alkaline adaptation of Aspergillus nidulans, is the first example of a calpain homologue present in fungi. These findings indicate various important functional roles for intracellular proteases belonging to the calpain superfamily.

Amino Acid Sequence↗
PubMed1997

Expanded CTG repeats in myotonin protein kinase suppresses myogenic differentiation.

A full-length mutant human myotonin protein kinase (MtPK) cDNA having a 46 expanded CTG-repeat size or a wild type containing 5 CTG repeats was stably transfected into mouse C2C12 cell line in order to explore the effects of the expansion mutation of trinucleotide repeats in the 3' untranslated region on developing myogenic cells. Each established clone expressed a human 70 kDa MtPK protein without proteolytic processing. Differentiation experiments indicated that stable mutant MtPK cDNA-transformants suppressed myogenic differentiation, whereas wild-type transformants exhibited almost normal myogenesis. The disturbance of the expression of neuronal nitric oxide synthase, a mediator of myoblast fusion, suggests that signal transduction system might be involved in the muscle manifestations of mutant MtPK.

Animals↗
PubMed1997

Tissue-specific expression and alpha-actinin binding properties of the Z-disc titin: implications for the nature of vertebrate Z-discs.

Titins are giant filamentous proteins which connect Z-discs and M-lines in the sarcomeres of vertebrate striated muscles. Comparison of the N-terminal region of titin (Z-disc region) from different skeletal and cardiac muscles reveals a 900-residue segment which is expressed in different length variants, dependent on tissue type. When searching for ligands of this differentially expressed domain by a yeast-two hybrid approach, we detected binding to alpha-actinin. The isolated alpha-actinin cDNAs were derived from the C-terminal region of the alpha-actinin isoform (alpha-actinin-2) encoded by the ACTN2 gene. Therefore, the two antiparallel subunits of an alpha-actinin-2 homodimer will attach to actin at their respective C termini, whereas they will bind to the Z-disc titin at their N termini. This may thus explain how alpha-actinins can cross-link antiparallel titin and thin filaments from opposing sarcomeres. The alpha-actinin-2 binding site of the Z-disc titin is located within a sequence of 45-residue repeats, referred to as Z-repeat region. Both the N-terminal and C-terminal Z-repeats have alpha-actinin binding properties and are expressed in all striated muscles. By contrast, the more central Z-repeats are expressed in slow and fast skeletal muscles, as well as embryonic and adult cardiac muscles, in different copy numbers. Such alternative splicing of the Z-disc titin appears to be important for the tissue and fibre type diversity of the Z-disc lattice.

Actinin↗
PubMed1997

Muscle-specific calpain, p94, interacts with the extreme C-terminal region of connectin, a unique region flanked by two immunoglobulin C2 motifs.

Using the yeast two-hybrid system, we have recently reported that skeletal muscle-specific calpain, p94, binds specifically to connectin (or titin), a gigantic muscle elastic protein. Connectin has at least two binding sites for p94; one is at the N2-line region and the other is at the extreme C-terminus. In order to analyze the interaction between p94 and the C-terminus of connectin, we examined the C-terminal sequence of human skeletal muscle connectin. The sequence was essentially identical to that of heart muscle reported by Labeit and Kolmerer (1995, Science 270, 293-296), and the minimal binding site for p94 contained two IgC2 motifs and the intervening sequence called "M-is7." The exon encoding M-is7 is reported to be alternatively spliced depending on muscle tissues, resulting in the existence of both types of connectin with and without M-is7. However, the C-terminal region of connectin bound to p94 through M-is7. Our results suggest that the interaction between p94 and the C-terminus of skeletal muscle-type connectin is involved in tissue-specific myofibriogenesis.

Amino Acid Sequence↗
PubMed1997

Molecular cloning and characterization of cDNAs for the mu-type large subunit and the small subunit of chicken calpain.

In chicken, three calpain isozymes expressed ubiquitously, mu-, mu/m- and m-types, have been identified at the cDNA and/or protein level, but the complete sequence of the mu-calpain large subunit (microCL) and its small subunit (30K) remain to be determined. In this report, we isolated and identified cDNA clones for chicken microCL and 30K, uncovering all molecules of the chicken ubiquitous calpain system. The longest open reading frame of microCL encodes 715 amino acid residues (M(r) = 81,410). The deduced amino acid sequence is more similar to that of human microCL (82%) than that of chicken mCL (61.6%) and mu/mCL (70.6%). As for 30K, several cDNA clones were isolated, but a full length cDNA are not isolated, presumably because of a GC-rich sequence of the 5'-terminus. The longest open reading frame encodes 214 amino acid residues, showing significant similarity to mammalian 30Ks. Northern blot analysis of microCL and 30K mRNA shows ubiquitous expression in all chicken tissues examined. The results indicate that microCL and 30K are well conserved in both chicken and mammal, confirming that at least three independent ubiquitous calpain species exist in chicken.

Amino Acid Sequence↗
PubMed1997

Deletion of an endosomal/lysosomal targeting signal promotes the secretion of Alzheimer's disease amyloid precursor protein (APP).

Alzheimer's disease amyloid precursor protein (APP) generates a beta-amyloid protein (A beta) that is a main component of the senile plaques found in the brains of Alzheimer's disease patients. APP is thought to undergo proteolysis via two different pathways, the amyloidogenic pathway which produces A beta, and the non-amyloidogenic pathway which releases a large N-terminal fragment into the medium. The proteases that mediate these processes remain unidentified. The physiological function of APP is not clear yet. Therefore, the cytoplasmic region of APP has attracted much interest, because this region is highly conserved among species, and members of the amyloid precursor-like protein (APLP) family. Several potentially functional sequences exist in the region, including signal sequences for protein sorting and a G0-protein binding sequence. We constructed two mutants, 695 deltaNPTY and 695 deltaGYEN. They lack potential endosome/lysosome targeting signals, NPTY and GY, in the cytoplasmic domain of APP695, respectively. The mutant APPs had longer half-lives and were secreted more easily into the medium than the wild type, suggesting that these sequences are important for the secretion and metabolism of APP.

Alzheimer Disease↗
PubMed1997

Novel isoform of myotonin protein kinase: gene product of myotonic dystrophy is localized in the sarcoplasmic reticulum of skeletal muscle.

It is quite important to know the exact localization and function of myotonin protein kinase (MtPK), identified as the gene product of myotonic dystrophy, the most prevalent disease with multisystem disorders among muscular dystrophies. To investigate the localization of MtPK, we raised a polyclonal antibody against a synthetic peptide chosen within the deduced sequence of MtPK. This antibody detected both a membrane-bound 70-kd protein and a soluble 55-kd protein on Western blots of human muscles. By using this antibody for immunohistochemical studies of both biopsied human skeletal muscle fibers and mature innervated cultured muscle fibers, we can now demonstrate by confocal laser scanning microscopy that MtPK is localized mainly in the I-band. By immunoelectron microscopy, it was determined that MtPK is a membrane-bound protein localized mainly in the terminal cisternae of the sarcoplasmic reticulum. To our knowledge, this is the first documentation of the ultrastructural localization of MtPK. This finding is quite important for clarifying the pathophysiological basis of myotonic dystrophy, which might be due to a dysregulation of calcium metabolism.

Actins↗