[Calpain super family and its interacting-proteins].
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Amino Acid Sequence↗
Biomedical subjects
H Sorimachi
Publications and source records attributed to H Sorimachi.
[Interaction trap using yeast two-hybrid system].
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Animals↗
Mutation of potential N-linked glycosylation sites in the Alzheimer's disease amyloid precursor protein (APP).
In order to study the mechanism of intracellular sorting and processing of the Alzheimer's disease amyloid precursor protein (APP), we deleted two potential N-linked glycosylation sites of APP by site-directed mutagenesis. Substitution of alanines for the critical asparagine residues Asn467 and Asn496 was performed. Wild-type and mutant APPs were expressed in COS-1 cells by cDNA transfection and the expressed of the protein and secretion of N-terminal large fragment was observed. The initial secretion of the mutant APP appeared to be slow compared with wild-type. In addition, we found that a distinct APP fragment, the cytosolic form, is transiently increased in the cytosol fraction of COS-1 cells. These results suggest that aberrant processing occurs following the expression of a mutant APP with Ala substituted for Asn, and that glycosylation may modulate the intracellular sorting of APP.
Alzheimer Disease↗
Primary sequences of rat mu-calpain large and small subunits are, respectively, moderately and highly similar to those of human.
cDNAs for rat mu-calpain large subunit and the calpain small subunit were cloned and sequenced. The large subunit encodes 713 amino-acid residues, which includes one deletion compared to that of human. The overall similarity is 89% to human mu-type, which is slightly lower than those compared between other types of calpain large subunits of rat and human (93-94%). On the other hand, the small subunit showed high conservation, being 94.0% identical to that of human. With these sequences, primary structures of all rat calpain subunits that have been considered to exist were completely elucidated.
Amino Acid Sequence↗
Immunocytochemical localization of a full-length myotonin protein kinase in rat L6 myoblasts.
Expansion mutation of CTG-repeat motifs within myotonin protein kinase (MtPK) gene is responsible for pathological changes in myotonic dystrophy (DM). To explore its pathological role in skeletal muscle, a full-length human MtPK cDNA was transfected into rat L6 myogenic cell line. Recombinantly expressed human MtPK protein in L6 cell line has a predicted molecular mass of 70 kDa. We have raised a polyclonal antibody against a synthetic peptide in the deduced sequence of the C-terminal portion of MtPK. MtPK in L6 cell is localized to perinuclear region, that resembles with sarcoplasmic reticulum. The MtPK-transfected myoblast cells established in this study will allow us to elucidate the molecular pathomechanism of muscle manifestations in DM.
Animals↗
Effect of artificial (CTG) repeat expansion on the expression of myotonin protein kinase (MtPK) in COS-1 cells.
A major challenge in the study of a new genetic entity called triplet-repeat disease is to identify the role of triplet repeats in the pathogenesis of the disease. We have developed a strategy to demonstrate the effect in the 3'-untranslated end of the (CTG) repeats in myotonic dystrophy gene (MtPK) and found that repeat expansion (CTG46) causes a slight decrease in the translation rate of MtPK cDNA which correlates with the finding in patients with myotonic dystrophy of a low amount of MtPK protein in muscle. These results provide an important clue for characterizing the genetic abnormality in other triplet-repeat diseases.
Animals↗
Structure and physiological functions of ubiquitous and tissue-specific calpain species. Muscle-specific calpain, p94, interacts with connectin/titin.
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Animals↗
Highly conserved structure in the promoter region of the gene for muscle-specific calpain, p94.
p94 belongs to the calcium-dependent cysteine protease (calpain) family which has been detected from human to mold. In contrast to the conventional m- and mu-calpains which are expressed ubiquitously, expression of p94 predominates in skeletal muscle, and the mRNA for p94 is at least 10-times more abundant than mRNAs encoding in the m- and mu-types. The unique feature of p94 is that it undergoes rapid and exhaustive autolysis with a half-life of less than half an hour. To elucidate the nature of specific and abundant expression in skeletal muscle, and to proceed toward gene targeting p94, we have cloned and characterized mouse and rat genes for p94, and compared them with that of the human sequence. The sequence comparison among three mammalian species revealed several conserved regions including possible transcription factor binding sites. Furthermore, mouse and rat upstream regions of p94 are conserved over 3 kb suggesting that expression of p94 in skeletal muscle of both rodents is similarly regulated.
Animals↗
Muscle-specific calpain, p94, responsible for limb girdle muscular dystrophy type 2A, associates with connectin through IS2, a p94-specific sequence.
p94, a muscle-specific member of calpain family, is unique in that it undergoes rapid and exhaustive autolysis with a half-life of less than 1 h resulting in its disappearance from muscle. Recently, p94 was shown to be responsible for limb girdle muscular dystrophy type 2A. To elucidate the muscular proteolytic system mediated by p94 and to solve the mystery of its unusually rapid autolysis, we searched for p94-binding proteins by the two-hybrid system. Although calpain small subunit plays a crucial role for regulation of ubiquitous calpains, it did not associate with p94. After a screening of skeletal muscle library, connectin (or titin), a gigantic filamentous protein spanning the M- to Z-lines of muscle sarcomere, was found to bind to p94 through a p94-specific region, IS2. The connectin-insoluble fraction of washed myofibrils contained full-length intact p94, suggesting that connectin regulates p94 activity.
Amino Acid Sequence↗
Up-regulation of dystrophin mRNA by exposure to dibutyryl cAMP in the C2C12 muscle cell line.
The effect of dibutyryl cAMP on dystrophin mRNA expression was investigated in C2C12 myogenic cells by a semi-quantitative RT-PCR method. The dystrophin mRNA level was enhanced by the addition of low concentrations of dibutyryl cAMP for 16h in the growth medium. The relative amounts of dystrophin/G3PDH mRNA at 10(-4) M dibutyryl cAMP were about 2.6-fold higher compared to control. We examined MLC1a mRNA expression in order to know whether the induction of dystrophin mRNA is due to the facilitation of myogenic differentiation by dibutyryl cAMP. MLC1a mRNA expression at 10(-4) M dibutyryl cAMP was similar to that of control. Thus we conclude that the dibutyryl cAMP enhancement of dystrophin mRNA expression is not due to a secondary event involving the formation of differentiated myotubes. The induction of CREB mRNA by dibutyryl cAMP suggests that the up-regulation of dystrophin mRNA expression might occur via transcriptional activation.
Animals↗
Conventional protein kinase C (PKC)-alpha and novel PKC epsilon, but not -delta, increase the secretion of an N-terminal fragment of Alzheimer's disease amyloid precursor protein from PKC cDNA transfected 3Y1 fibroblasts.
A large soluble N-terminal fragment of Alzheimer's disease amyloid precursor protein (secreted form of APP: APPs) is produced by constitutive processing in the middle of the amyloid beta-protein portion of APP. Recent studies indicate that the activation of endogenous protein kinase C (PKC) with phorbol ester raises the rate of secretion of APPs. We constructed rat fibroblast 3Y1 cells that stably overexpress PKC isoenzymes alpha, delta, or epsilon, and analyzed the amount of APPs released from these PKC transfectants. The levels of APPs released from 3Y1 cells overexpressing PKC alpha and -epsilon were higher than those from PKC delta-transfected and control cells expressing vector only. These results suggest that specific isoforms of PKC regulate the secretion of APPs through a signaling pathway.
Alzheimer Disease↗
Arachidonate metabolites affect the secretion of an N-terminal fragment of Alzheimer's disease amyloid precursor protein.
Amyloid precursor protein (APP) is degraded within the amyloid beta-protein (A beta) domain and its large soluble N-terminal fragment (secreted form of APP: APPs) is secreted into the culture media of many kinds of cells. We report here a quantitative increase in APPs secretion in the medium of human glioblastoma A172 cells grown under serum-free conditions. When A172 cells were treated with inhibitors of the arachidonate cascade, a modulation of APPs secretion was observed; the addition of small amounts of indomethacin increased secretory cleavage, but higher doses suppressed it. Nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenases, also inhibited APPs secretion. These results suggest that arachidonate metabolites of the leukotriene pathway may promote APPs release upon extracellular signaling via a signal transduction pathway, while metabolites of the prostaglandin pathway inhibit APPs secretion.
Amyloid beta-Peptides↗
Identification of a third ubiquitous calpain species--chicken muscle expresses four distinct calpains.
In the mammalian calpain system, two isozymes, mu- and m-types, have been well-characterized, and are considered to be conserved in the avian system as well. Thus, chicken calpain, whose large subunit was cloned in 1984, has long been regarded as 'm-type', since chicken also possesses 'mu-type' activity, although its structure has not yet been elucidated. In this study, we identified three kinds of cDNAs encoding distinct chicken calpain large subunits. Two of the three were highly similar to the mammalian mu-type and p94, respectively. The third shows a much higher similarity to mammalian m-type than the first identified chicken calpain, indicating that this molecule, which has been considered as 'm-type', should be renamed. We, therefore, designated it 'mu/m-calpain', because its sequence and Ca(2+)-sensitivity lie between mu- and m-types. Northern blot analyses revealed that chicken mCL and muCL, as well as mu/mCL, show ubiquitous expression, while p94 was detected predominantly in skeletal muscle, as previously reported. Chicken skeletal muscle, therefore, expresses at least four types of calpain, three ubiquitous and one tissue-specific.
Amino Acid Sequence↗
A novel ligand for CD44 is serglycin, a hematopoietic cell lineage-specific proteoglycan. Possible involvement in lymphoid cell adherence and activation.
The lymphocyte adhesion molecule CD44 recognizes a non-hyaluronate proteoglycan, gp600, secreted by mouse T cell line CTLL2. We now demonstrate that gp600 is identical to serglycin, a member of the small proteoglycan family stored in intracellular secretory granules of lymphoid, myeloid, and some tumor cells. Purified gp600 has the ability to bind specifically to CD44, and the binding is dependent on activation of CD44. The CD44-binding elements on gp600 or serglycin are glycosaminoglycans consisting of chondroitin 4-sulfate. Serglycin is readily exocytosed, and its interaction with active form CD44 augments the CD3-dependent degranulation of CD44 positive CTL clones. We conclude that the serglycin secreted from secretory granules of hematopoietic cells is a novel ligand for CD44, and could regulate lymphoid cell adherence and activation.
Amino Acid Sequence↗
Calpain dissociates into subunits in the presence of calcium ions.
Calpain is a calcium dependent cysteine protease consisting of a catalytic 80K subunit and a regulatory 30K subunit. It has therefore been believed that calpain functions as a dimer. Here we have found that calpain dissociates into subunits in the presence of the Ca2+ required for the expression of activity and that the dissociated 80K subunit is enzymatically fully active. Moreover, the 80K subunit shows a calcium sensitivity identical to the activated form of calpain but not to the original control calpain. The results suggest that the activation of calpain corresponds to the dissociation into subunits in the presence of Ca2+ and that calpain functions as a monomer of the 80K subunit in vivo.
Amino Acid Sequence↗
A catalytic subunit of calpain possesses full proteolytic activity.
Previous studies on the refolding of calpain, a heterodimer comprising a catalytic 80 kDa subunit and a regulatory 30 kDa subunit, indicate that both subunits are required for the expression of full protease activity. We reexamined the conditions for refolding of calpain and found that under optimized conditions the renatured 80 kDa subunit has full enzyme activity even in the absence of the 30 kDa subunit. The 30 kDa subunit stabilizes the 80 kDa subunit rather than enhancing its activity. The theory that calpain functions as a dimer requires reexamination.
Bacterial Proteins↗
Calpain: novel family members, activation, and physiologic function.
The current status of calpain research is summarized on the basis of the most recent results. The main points are as follows. (i) Calpain constitutes a large family. (ii) Ca2+ ions cause the dissociation of calpain into subunits and the resulting free 80 kDa subunit is the active form of the enzyme. This dissociation corresponds to the activation of calpain. (iii) Some powerful clues have been obtained that will be helpful for analyzing the physiological function.
Animals↗
[Myotonin protein kinase].
The mutation underlying myotonic dystrophy is the expansion of polymorphic CTG repeat in the 3'-noncoding region of the myotonin protein kinase (MtPK) gene mapping to chromosome 19q13.3. A full-length cDNA of human MtPK was cloned and expressed in COS-1 cells. MtPK is recovered from the COS cell extract as a 70 kDa protein, which coincides with the size deduced from the predicted amino acid sequence. Biochemical characteristics of MtPK expressed in COS cells and its expression are investigated.
Chromosomes, Human, Pair 19↗