RESUMEN
The formation of perfectly aligned myofibrils in striated muscle represents a dramatic example of supramolecular assembly in eukaryotic cells. Recently, considerable progress has been made in deciphering the roles that titin, the third most abundant protein in muscle, has in this process. An increasing number of sarcomeric proteins (ligands) are being identified that bind to specific titin domains. Titin may serve as a molecular blueprint for sarcomere assembly and turnover by specifying the precise position of its ligands within each half-sarcomere in addition to functioning as a molecular spring that maintains the structural integrity of the contracting myofibrils.
Asunto(s)
Proteínas Musculares/fisiología , Miofibrillas/fisiología , Proteínas Quinasas/fisiología , Animales , Conectina , Ligandos , Modelos Biológicos , Morfogénesis , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Miofibrillas/química , Isoformas de Proteínas/metabolismo , Sarcómeros/metabolismoRESUMEN
Calpain 3 is known as the skeletal muscle-specific member of the calpains, a family of intracellular nonlysosomal cysteine proteases. It was previously shown that defects in the human calpain 3 gene are responsible for limb girdle muscular dystrophy type 2A (LGMD2A), an inherited disease affecting predominantly the proximal limb muscles. To better understand the function of calpain 3 and the pathophysiological mechanisms of LGMD2A and also to develop an adequate model for therapy research, we generated capn3-deficient mice by gene targeting. capn3-deficient mice are fully fertile and viable. Allele transmission in intercross progeny demonstrated a statistically significant departure from Mendel's law. capn3-deficient mice show a mild progressive muscular dystrophy that affects a specific group of muscles. The age of appearance of myopathic features varies with the genetic background, suggesting the involvement of modifier genes. Affected muscles manifest a similar apoptosis-associated perturbation of the IkappaBalpha/nuclear factor kappaB pathway as seen in LGMD2A patients. In addition, Evans blue staining of muscle fibers reveals that the pathological process due to calpain 3 deficiency is associated with membrane alterations.
Asunto(s)
Apoptosis , Calpaína/deficiencia , Proteínas de Unión al ADN/metabolismo , Proteínas I-kappa B , Distrofias Musculares/enzimología , Distrofias Musculares/metabolismo , FN-kappa B/metabolismo , Transducción de Señal , Animales , Calpaína/química , Calpaína/genética , Calpaína/metabolismo , Creatina Quinasa/metabolismo , Cruzamientos Genéticos , Azul de Evans , Femenino , Fertilidad , Eliminación de Gen , Marcación de Gen , Genotipo , Masculino , Ratones , Ratones Noqueados , Fibras Musculares Esqueléticas/enzimología , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Músculo Esquelético/enzimología , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Distrofias Musculares/genética , Distrofias Musculares/patología , Inhibidor NF-kappaB alfa , Fenotipo , ARN Mensajero/análisis , ARN Mensajero/genética , Sarcolema/patologíaRESUMEN
We describe here a novel sarcomeric 145-kD protein, myopalladin, which tethers together the COOH-terminal Src homology 3 domains of nebulin and nebulette with the EF hand motifs of alpha-actinin in vertebrate Z-lines. Myopalladin's nebulin/nebulette and alpha-actinin-binding sites are contained in two distinct regions within its COOH-terminal 90-kD domain. Both sites are highly homologous with those found in palladin, a protein described recently required for actin cytoskeletal assembly (Parast, M.M., and C.A. Otey. 2000. J. Cell Biol. 150:643-656). This suggests that palladin and myopalladin may have conserved roles in stress fiber and Z-line assembly. The NH(2)-terminal region of myopalladin specifically binds to the cardiac ankyrin repeat protein (CARP), a nuclear protein involved in control of muscle gene expression. Immunofluorescence and immunoelectron microscopy studies revealed that myopalladin also colocalized with CARP in the central I-band of striated muscle sarcomeres. Overexpression of myopalladin's NH(2)-terminal CARP-binding region in live cardiac myocytes resulted in severe disruption of all sarcomeric components studied, suggesting that the myopalladin-CARP complex in the central I-band may have an important regulatory role in maintaining sarcomeric integrity. Our data also suggest that myopalladin may link regulatory mechanisms involved in Z-line structure (via alpha-actinin and nebulin/nebulette) to those involved in muscle gene expression (via CARP).
Asunto(s)
Proteínas Musculares/metabolismo , Músculo Esquelético/fisiología , Sarcómeros/metabolismo , Actinina/metabolismo , Secuencia de Aminoácidos , Animales , Northern Blotting , Células Cultivadas , Motivos EF Hand/genética , Humanos , Ratones , Microscopía Fluorescente , Datos de Secuencia Molecular , Proteínas Musculares/química , Proteínas Musculares/genética , Músculo Esquelético/citología , Miocardio/citología , Proteínas Nucleares/metabolismo , Filogenia , Unión Proteica , Estructura Terciaria de Proteína , Conejos , Proteínas Represoras/metabolismo , Sarcómeros/ultraestructura , Alineación de Secuencia , Técnicas del Sistema de Dos HíbridosRESUMEN
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.
Asunto(s)
Proteínas Musculares/química , Proteínas Musculares/metabolismo , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Sarcómeros/química , Sarcómeros/metabolismo , Actinina/química , Actinina/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Embrión de Pollo , Conectina , Expresión Génica/fisiología , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/ultraestructura , Proteínas Musculares/genética , Músculo Esquelético/química , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Miocardio/química , Miocardio/citología , Miocardio/metabolismo , Miofibrillas/química , Miofibrillas/metabolismo , Miofibrillas/ultraestructura , Proteínas Quinasas/genética , Estructura Terciaria de Proteína , Sarcómeros/ultraestructura , Transcripción Genética/fisiologíaRESUMEN
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.
Asunto(s)
Calpaína/genética , Calpaína/metabolismo , Isoenzimas , Procesamiento Postranscripcional del ARN , Transcripción Genética , Empalme Alternativo , Animales , Encéfalo/metabolismo , Proteínas Portadoras/metabolismo , Células Cultivadas , Clonación Molecular , Conectina , Cartilla de ADN , Embrión de Mamíferos/anatomía & histología , Embrión de Mamíferos/metabolismo , Humanos , Hibridación in Situ , Intrones , Cristalino/anatomía & histología , Cristalino/metabolismo , Ratones , Ratones Endogámicos BALB C , Proteínas de Microfilamentos/metabolismo , Modelos Genéticos , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Músculo Liso/metabolismo , Miocardio/metabolismo , Fragmentos de Péptidos/metabolismo , Proteínas Quinasas/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Distribución TisularRESUMEN
While conventional calpains, m- and mu-calpains named according to their calcium-dependence, are expressed in almost every tissues, mRNA of newly identified p94, which has a significant sequence similarity to the conventional calpain large subunits, is abundantly expressed only in skeletal muscle. In addition to this specific expression, p94 is distinct from conventional calpains in that it contains three unique regions showing no similarity to conventional calpain subunits. When rat and human p94 are compared, overall sequence similarity is 94.0%, which is close to those for m- and mu-calpain large subunits; 93.1% and 95.4% between human and rabbit, respectively, suggesting the evolutionary importance of p94. These calpain large subunit proteins, p94, m- and mu-types, can be considered to constitute a super family, whose p94, m- and mu-types represent the three major types. Sequences of the calpain large-subunit family members, including the recently reported Schistosoma calpain, are compared. Their evolutionary correlation and function are discussed on the basis of the results thus far obtained.
Asunto(s)
Calpaína/química , Músculos/enzimología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Evolución Biológica , Pollos , Humanos , Datos de Secuencia Molecular , Estructura Molecular , Conejos , Ratas , Schistosoma mansoni/enzimologíaRESUMEN
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.
Asunto(s)
Calpaína/genética , Homología de Secuencia de Aminoácido , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Humanos , Datos de Secuencia Molecular , Ratas , Alineación de Secuencia , Análisis de Secuencia de ADN , EstómagoRESUMEN
A mammalian homologue of the Aspergillus atypical calpain PalB, PalBH, was identified and its cDNA sequences were determined in human and mouse. The PalBH mRNA was expressed nearly ubiquitously throughout mammalian tissues. When expressed in COS cells, PalBH was enriched in the nucleus, suggesting its role is distinct from that of conventional calpains.
Asunto(s)
Calpaína/genética , Proteínas Fúngicas , Secuencia de Aminoácidos , Animales , Aspergillus/enzimología , Aspergillus/genética , Secuencia de Bases , Calpaína/química , Clonación Molecular , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Humanos , Ratones , Datos de Secuencia Molecular , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
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.
Asunto(s)
Calpaína/química , Isoenzimas/química , Músculos/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Calpaína/genética , Pollos , ADN Complementario/química , Isoenzimas/genética , Datos de Secuencia Molecular , ARN Mensajero/análisisRESUMEN
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.
Asunto(s)
Enfermedades Genéticas Congénitas/genética , Distrofia Miotónica/genética , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas , Secuencias Repetitivas de Ácidos Nucleicos , Animales , Secuencia de Bases , Línea Celular , Chlorocebus aethiops , Clonación Molecular , ADN Complementario/aislamiento & purificación , Electroforesis en Gel de Agar , Escherichia coli , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Distrofia Miotónica/enzimología , Proteína Quinasa de Distrofia Miotónica , Oligodesoxirribonucleótidos/síntesis química , Proteínas Quinasas/biosíntesis , Proteínas Recombinantes/biosíntesis , Transcripción Genética , TransfecciónRESUMEN
We have shown that the mRNAs for apopolysialoglycoproteins (apoPSGP) of rainbow trout contain various numbers of a repetitive sequence of 39 base-pairs encoding mature apoPSGP, and that this sequence is bordered by highly homologous 5' and 3' regions encoding pre-, pro- and telopeptides. These mRNAs are thought to be transcribed from different genes that constitute a large multiple gene family (more than 100 members). Here, we have determined the structures of several members of the apoPSGP gene family. The results show that two of three genomic DNA fragments contain two independent apoPSGP genes in the same orientation with unrelated sequences intervening. Five characterized genes have essentially the same organization and sequence. Each gene has four exons, and CAAT and TATA sequences were found in the 5'-flanking regions. However, two noteworthy differences were observed among the five genes; a diversity in the number of the 39 base-pair repeats, also observed among the cDNA clones, and a one-base polymorphism in the 39 base-pair repeat, which causes an amino acid change. This polymorphism was not detected among the cDNA clones obtained. The boundary positions of the genes are various and contain no transposon-like structures. The variation in the number of repeats and the absence of a rule for bordering positions of the genes suggest that apoPSGP genes may have been amplified by gene duplications, unequal recombination, and selection of chromosomes having larger numbers of apoPSGP genes.
Asunto(s)
Genes , Familia de Multigenes , Sialoglicoproteínas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Clonación Molecular , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Regiones Promotoras Genéticas , ARN Mensajero/genética , Secuencias Repetitivas de Ácidos Nucleicos , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , TruchaRESUMEN
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.
Asunto(s)
Actinina/metabolismo , Expresión Génica , Proteínas Musculares/genética , Músculo Esquelético/fisiología , Proteínas Quinasas/genética , Secuencia de Aminoácidos , Animales , Conectina , Humanos , Datos de Secuencia Molecular , Proteínas Musculares/metabolismo , Proteínas Quinasas/metabolismo , Conejos , Secuencias Repetitivas de Ácidos Nucleicos , Homología de Secuencia de Aminoácido , VertebradosRESUMEN
The giant myofibrillar protein titin contains within its C-terminal region a serine-threonine kinase of unknown function. We have identified a novel muscle specific RING finger protein, referred to as MURF-1, that binds in vitro to the titin repeats A168/A169 adjacent to the titin kinase domain. In myofibrils, MURF-1 is present within the periphery of the M-line lattice in close proximity to titin's catalytic kinase domain, within the Z-line lattice, and also in soluble form within the cytoplasm. Yeast two-hybrid screens with MURF-1 as a bait identified two other highly homologous MURF proteins, MURF-2 and MURF-3. MURF-1,2,3 proteins are encoded by distinct genes, share highly conserved N-terminal RING domains and in vitro form dimers/heterodimers by shared coiled-coil motifs. Of the MURF family, only MURF-1 interacts with titin repeats A168/A169, whereas MURF-3 has been reported to affect microtubule stability. Association of MURF-1 with M-line titin may potentially modulate titin's kinase activity similar to other known kinase-associated proteins, whereas differential expression and heterodimerization of MURF1, 2 and 3 may link together titin kinase and microtubule-dependent signal pathways in striated muscles.
Asunto(s)
Proteínas Musculares/química , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculos/química , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Dedos de Zinc/fisiología , Secuencia de Aminoácidos , Animales , Conectina , Dimerización , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Humanos , Ratones , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Músculos/citología , Músculos/metabolismo , Especificidad de Órganos , Filogenia , Mapeo Físico de Cromosoma , Unión Proteica , Estructura Terciaria de Proteína , ARN Mensajero/análisis , ARN Mensajero/genética , Ratas , Sarcómeros/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Técnicas del Sistema de Dos HíbridosRESUMEN
The calpains form a growing family of structurally related intracellular multidomain cysteine proteinases containing a papain-related catalytic domain, whose activity depends on calcium. The calpains are believed to play important roles in cytoskeletal remodeling processes, cell differentiation, apoptosis and signal transduction, but are also implicated in muscular dystrophy, cardiac and cerebral ischemia, platelet aggregation, restenosis, neurodegenerative diseases, rheumatoid arthritis and cataract formation. The best characterized calpains, the ubiquitously expressed mu- and m-calpains, are heterodimers consisting of a common 30-kDa small and a variable 80-kDa subunit. The recently determined crystal structures of human and rat m-calpain crystallized in the absence of calcium essentially explain the inactivity of the apoform by catalytic domain disruption, indicate several sites where calcium could bind causing reformation of a papain-like catalytic domain, and additionally reveal modes by which phospholipid membranes could reduce the calcium requirement. Current evidence points to a cooperative interaction of several sites, which, upon calcium binding, trigger the reformation of a papain-similar catalytic domain.
Asunto(s)
Calcio/fisiología , Calpaína/química , Animales , Calpaína/genética , Calpaína/fisiología , Humanos , RatasRESUMEN
p94, a muscle-specific member of the calpain family, also called calpain3 (CAPN3), has been identified as the gene product responsible for limb-girdle muscular dystrophy type 2A (LGMD2A). To elucidate the molecular mechanism of LGMD2A, the effects of missense point mutations found in LGMD2A on the unique properties of p94 were studied. All of the mutants examined to date lose their proteolytic activity against fodrin, a cytoskeletal protein, strongly suggesting that of the specific properties of p94, the loss of protease activity is the prime cause of LGMD2A. Studies of LGMD2A and p94 suggest a novel molecular mechanism for muscular dystrophy, showing that a combined pathologic and biochemical approach is effective.
Asunto(s)
Calpaína/fisiología , Distrofias Musculares/genética , Animales , Células COS , Calpaína/genética , Calpaína/metabolismo , Proteínas Portadoras/fisiología , Humanos , Hidrólisis , Proteínas de Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Distrofias Musculares/metabolismo , Mutación MissenseRESUMEN
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.
Asunto(s)
Calpaína/metabolismo , Animales , Sitios de Unión , Calcio/metabolismo , Calcio/farmacología , Calpaína/química , Activación Enzimática , Humanos , Relación Estructura-ActividadRESUMEN
The recent discovery of several new calpain species other than the two species thus far studied reveals that calpain, especially the calpain large subunit, constitutes a family comprising at least six members that can be classified into ubiquitous (mu, m- and mu/m-types) and tissue-specific (p94 or nCL-1 specific for skeletal muscle, and nCL-2 and -2' specific for stomach) calpains. The newly identified tissue-specific calpains have various characteristics distinct from conventional calpains in structure, manner of expression, and enzyme activity. Unique features of tissue specific calpains are discussed together with the evolutionary view of the calpain large subunit.
Asunto(s)
Calpaína/química , Secuencia de Aminoácidos , Animales , Humanos , Datos de Secuencia Molecular , Músculos/enzimología , Filogenia , Especificidad de la Especie , Estómago/enzimología , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Calpaína/metabolismo , Proteínas Bacterianas/farmacología , Calpaína/química , Chaperoninas , Proteínas de Escherichia coli , Proteínas de Choque Térmico/farmacología , Polietilenglicoles , Desnaturalización Proteica , Pliegue de ProteínaRESUMEN
A full-length cDNA of human myotonin protein kinase (MtPK) was cloned and expressed in COS-1 cells. MtPK is recovered from the cytosolic fraction of the COS extract as a 70 kDa protein, which coincides with the size deduced from the predicted amino acid sequence. The sequence has a significant homology to thymopoietin, a peptide hormone of the thymus. Biochemical characteristics of MtPK expressed in COS-1 cells and its expression in rat tissues are investigated.
Asunto(s)
Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas , Timopoyetinas/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Línea Celular , Clonación Molecular , ADN Complementario , Humanos , Datos de Secuencia Molecular , Proteína Quinasa de Distrofia Miotónica , Proteínas Quinasas/química , Ratas , Homología de Secuencia de AminoácidoRESUMEN
Calpain, a Ca(2+)-dependent cytosolic cysteine protease, proteolytically modulates specific substrates involved in Ca(2+)-mediated intracellular events, such as signal transduction, cell cycle, differentiation, and apoptosis. The 3D structure of m-calpain, in the absence of Ca(2+), revealed that the two subdomains (domains IIa and IIb) of the protease domain (II) have an 'open' conformation, probably due to interactions with other domains. Although the presence of an EF-hand structure was once predicted in the protease domain, no explicit Ca(2+)-binding structure was identified in the 3D structure. Therefore, it is predicted that if the protease domain is excised from the calpain molecule, it will have a Ca(2+)-independent protease activity. In this study, we have characterized a truncated human m-calpain that consists of only the protease domain. Unexpectedly, the proteolytic activity was Ca(2+)-dependent, very weak, and not effectively inhibited by calpastatin, a calpain inhibitor. Ca(2+)-dependent modification of the protease domain by the cysteine protease inhibitor, E-64c, was clearly observed as a SDS-PAGE migration change, indicating that the conformational changes of this domain are a result of Ca(2+) binding. These results suggest that the Ca(2+) binding to domain II, as well as to domains III, IV, and VI, is critical in the process of complete activation of calpain.