Calpains and Disease
http://www.100md.com
《新英格兰医药杂志》
Calpains are Ca2+-dependent cysteine proteases (proteolytic enzymes with cysteine in the catalytic site) that modulate cellular function. In humans, 14 independent genes encode members of the calpain superfamily. Some calpain proteases are confined to specific tissues, whereas others are ubiquitous. Tissue-specific calpains have been implicated in diabetes, cataracts, multiple sclerosis, cancer, Duchenne's muscular dystrophy, and Alzheimer's disease and are known to cause at least one disorder, autosomal recessive limb-girdle muscular dystrophy type 2A (LGMD2A). We will review recent findings on the role of the calpain superfamily in these disorders, focusing on LGMD2A, the most extensively characterized human calpainopathy.
Properties of Calpains
Two members of the calpain superfamily, calpains 1 (μ-calpain) and 2 (m-calpain), have been extensively studied.1 Each of these calpains, which differ in their sensitivity to Ca2+, consists of two different polypeptide subunits. The larger subunit (80 kD) has catalytic activity, whereas the smaller, 30-kD, subunit has a regulatory function (Figure 1).
Figure 1. Schematic Representation of the Domain Structure of the Large and Small Subunits of m-Calpain.
μ-Calpain has a similar heterodimeric structure, but it differs in its sensitivity to Ca2+. Adapted from a diagram provided by Dr. Hiroyuki Sorimachi, Laboratory of Biological Function, University of Tokyo, Tokyo.
The large subunits of μ-calpain and m-calpain are encoded by the CAPN1 and CAPN2 genes, respectively. Calpains 3, 8a, 9, 11, 12, and 13 also have 80-kD and 30-kD subunits. The 80-kD subunit has four domains: domain I is the N-terminal anchoring -helix domain and is important for regulating the activity and dissociation of the subunit2,3,4,5; domain II, a catalytic domain, has two subdomains in the absence of Ca2+6 7; domain III binds Ca2+ and phospholipids8; and domain IV, also called the penta–EF-hand domain (an EF-hand unit consists of two peptide helixes connected by a Ca2+-binding loop), is important for dimer formation.9 The 30-kD regulatory subunit of calpains 1, 2, and 9 consists of two domains: domain V is the N-terminal, glycine-rich, hydrophobic domain and domain VI the penta–EF-hand domain, which is similar to domain IV of the catalytic subunit.6,7 The 80-kD subunit of the other calpains (3, 8a, 11, 12, and 13) do not interact with the 30-kD subunit, although they do have domain IV. Calpains 5, 6, 7, 8b, 10, and 15 are atypical calpains in that some of their domains have been deleted or replaced. They lack domain IV and therefore presumably do not associate with the 30-kD subunit.
There are two classes of calpains: one (comprising calpains 1, 2, 5, 7, 10, 13, and 15) is ubiquitous in cytosol; the other (comprising calpains 3, 6, 8, 9, 11, and 12) occurs only or mainly in certain tissues.10 For example, calpain 8 is stomach-specific, and calpain 3 (CAPN3) is largely specific to skeletal muscle,1,11 although it is also present in cardiac muscle and the liver.12 The amino acid sequence of skeletal-muscle CAPN3 is similar to the sequences of the ubiquitous μ-calpain and m-calpain (which are structural variants that catalyze the same reaction), but it contains three specific insertion sequences.
The mechanisms by which calpains are activated and identify their protein targets are complex and poorly understood. Calpain activity is regulated by a ubiquitous specific inhibitor, calpastatin.13,14 The calpain–calpastatin interaction is important in regulation of the activity of μ-calpain and m-calpain,15 but the nature of this regulation in living cells is not understood. Studies using calpastatin have shown that calpains are clearly involved in some types of apoptosis in specific cell types and in response to certain apoptotic signals.12
An intricate strategy for the regulation of calpain activity seems necessary because calpain is an abundant cytoplasmic protease that can cleave many intracellular signaling and structural proteins. Membrane localization of calpains is an important mechanism for regulating their activity. In early work, it was thought that when a calpain binds to the plasma membrane, it is transformed from an inactive, proenzyme into an active, proteolytic enzyme by autolysis. However, other findings indicate that both μ-calpain and m-calpain are active proteolytic enzymes before autolysis and that interaction with a membrane may bind calpains to their substrates, rather than promote autolysis.16,17,18,19,20,21,22,23
Once activated on the membrane, the calpain presumably diffuses into the cytosol and becomes resistant to the inhibitory action of calpastatin.24 Substrate proteins are digested by the activated calpain on the membrane or in the cytosol.25 According to Gil-Parrado et al.,26 calpain activity is regulated not only by calpastatin but also by differential intracellular localization. In contrast, dissociation of the subunits that constitute a calpain appears to be less critical to its regulation. Ca2+ levels required to initiate autolysis are as high as or even slightly higher than the levels required for proteolytic activity and are much greater than the free Ca2+ levels in living cells.20,27,28 A solution to this paradox appeared when it was discovered that the presence of phospholipids, with phosphatidylinositol, lowered the Ca2+ levels required for autolysis of μ-calpain and m-calpain.29,30 Other studies have shown that autolysis of these two calpains is an intermolecular process,31 rather than an intramolecular process, as previously thought.16
μ-Calpain and m-calpain have diverse functions.11,32 They catalyze the proteolysis of proteins involved in cytoskeletal remodeling, cell-cycle regulation, signal transduction, cell differentiation, apoptosis and necrosis, embryonic development, and vesicular trafficking.3,4,32 For this reason, calpain activity has to be tightly regulated both temporarily and spatially to be effective and limited in scope.
Calpain-Associated Diseases
A number of pathologic conditions have been associated with disturbances of the calpain system. They include type 2 diabetes, cataracts, Duchenne's muscular dystrophy, Parkinson's disease, Alzheimer's disease, rheumatoid arthritis, ischemia, stroke and brain trauma, various platelet syndromes, hypertension, liver dysfunction, and some types of cancer (Table 1). Several oncogenes and tumor-suppressor gene products are substrates for members of the calpain family.48 Mutations in the CAPN3 gene cause LGMD2A, indicating that the protein that it encodes is important for muscle function.
Table 1. Examples of Pathologic Conditions That Have Been Associated with the Calpains.
Limb-Girdle Muscular Dystrophy Type 2A
LGMD2A (Online Mendelian Inheritance in Man number 253600 ) is one of the limb-girdle muscular dystrophies, a heterogeneous group of genetically determined progressive disorders of skeletal muscle. Seventeen genes responsible for various types of limb-girdle muscular dystrophy have been mapped,49,50 and the products of most of them have been identified (Figure 2). LGMD2A, the most prevalent form, accounts for at least 30 percent of all cases.
Figure 2. Schematic Representations of the Proteins Associated with Limb-Girdle Muscular Dystrophies (Top), of the Golgi Complex (Inset), and of the Structure of Muscle Fibers (Bottom).
LGMD2 denotes limb-girdle muscular dystrophy type 2, -DTN -dystrobrevin, FKRP fukutin-related protein, TRIM32 tripartite motif–containing protein 32, and T-cap titin-cap. The variants responsible for different subtypes of limb-girdle muscular dystrophy are shown in red. Adapted from a diagram provided by Peter Hackman and Bjarne Udd, Neurology Department, Vaasa Central Hospital, Vaasa, Finland.
Molecular Mechanisms
LGMD2A is caused by mutations in the CAPN3 (also called p94) gene,51 which encodes CAPN3, the largely skeletal-muscle–specific member of the calpain superfamily. CAPN3, located both in the cytosol and in the nucleus, requires extremely low levels of Ca2+ for activation. In skeletal-muscle cells, CAPN3 binds specifically to certain regions of titin, a giant structural muscle protein; this binding seems to stabilize CAPN3 and thereby prevent its autolysis. Mutations in the CAPN3 gene result in a cascade of events leading to CAPN3 deficiency and, eventually, to muscular dystrophy, but the underlying mechanisms are unknown.12,52 Several studies have suggested that the loss of CAPN3 processing activity, but not its hyperactivation or a defect in its structure, causes LGMD2A.53,54 According to other studies,55 CAPN3 deficiency causes myonuclear apoptosis (i.e., death of muscle-cell nuclei) and profound perturbation of the IB–nuclear factor (nuclear factor of kappa light-chain gene enhancer in B cells inhibitor ) pathway, such that a failure in CAPN3-dependent IB proteolysis results in accumulation of IB in the cytoplasm and nucleus and sequestration of nuclear factor-B in the cytoplasm, both of which culminate in the apoptosis of muscle cells.55
It is unlikely that an influx of extracellular Ca2+ into the sarcoplasm causes muscle fibers to degenerate, because CAPN3 is localized primarily in the myofibril and not on the sarcolemma.13,56 Animal models and findings before the onset of muscular dystrophy in young carriers of CAPN3 mutations suggest a role for CAPN3 in muscle maturation and sarcomere remodeling.57,58,59 In vitro, CAPN3 cleaves filamin C, which is specific to muscle, and regulates its ability to interact with - and -sarcoglycans, two proteins of the dystrophin–glycoprotein complex (dystrophin anchors muscle membranes to actin filaments in myofibrils).60 Since CAPN3 belongs to a large family of Ca2+-dependent proteases, at least seven of which are expressed in muscle, the calpain superfamily could form a proteolytic network in muscle and other cells. In this way, a defect in CAPN3 could distort the calpain system, leading to overactivation of other calpains.14
Clinical Features
Patients with LGMD2A have symmetrical and selective involvement of proximal limb-girdle muscles. They have normal intelligence and no cardiac or facial disturbances. The disease shows wide intrafamilial and interfamilial clinical variability.49,61,62,63,64,65 Findings on examination of muscle-biopsy specimens are consistent with a dystrophic or myopathic process (Figure 3). The serum level of creatine kinase is moderately or markedly increased, particularly in the active phase of the disorder.42,66,67 However, we have seen patients with a normal serum level of creatine kinase or a neurogenic pattern on electromyography,68 suggesting that the spectrum of variability in this calpainopathy might be broader than suspected and that a normal creatine kinase level should not be considered a reason to rule out a possible diagnosis of LGMD2A, even in ambulatory patients.
Figure 3. Histopathologic Features, Results of Muscle-Protein Analysis, and Clinical Characteristics in Limb-Girdle Muscular Dystrophy Type 2A.
Panel A shows the histologic features of normal muscle and muscle from a patient with limb-girdle muscular dystrophy type 2A (LGMD2A) and calpainopathy (hematoxylin–eosin stain). In the latter specimen, there is substantial variation in fiber size, with very large fibers surrounded by small groups of atrophic fibers, connective-tissue infiltration, and splitting. Panel B shows the results of multiplex Western blot analysis of the muscle proteins dystrophin, dysferlin, and calpain 3 from a normal control subject (lane C) and four unrelated patients with LGMD2A (lanes 1 through 4) (performed with monoclonal antibody Calp3C/12A2, which recognizes the 94-kD band of calpain 3). Myosin content served as a control for the amount of muscle protein loaded in the Ponceau-prestained blot. The blot shows almost complete absence of calpain 3 in Patient 1, partial deficiency in Patients 3 and 4, and a normal amount of calpain 3 in Patient 2; there was no clinical correlation with the severity of the phenotype. Image courtesy of Dr. Mariz Vainzof, Human Genome Center, Department of Biology, University of S?o Paulo, S?o Paulo. Panel C shows affected sisters homozygous for a mutation in CAPN3 resulting in the replacement of arginine with glutamine at position 769 of the CAPN3 protein. The intrafamilial variability of LGMD2A is evident: the older sister (left) is still ambulatory at the age of 47 years, whereas the younger sister (right), who is currently 33 years of age, has used a wheelchair since the age of 18.
The age at onset ranges from 2 to 40 years, but the disease usually first appears in the second or third decade of life, with the development of proximal weakness in the lower limbs. Although early-onset cases are usually more severe, cases with a later onset may occasionally involve rapid progression and inability to walk by the third decade. Use of a wheelchair may become necessary during the second through fifth decades, but some patients may remain ambulatory after the sixth decade.49,61,62,64 A more rapid progression in male patients than in female patients has been observed among Brazilian and Italian patients, an intriguing observation for which we have no explanation.62,69
CAPN3 Mutations, CAPN3 Protein Analysis, and Diagnostic Applications
The CAPN3 gene encompasses 24 exons and covers a 50-kb genomic region. It is expressed as a 3.5-kb transcript, which encodes a 94-kD translated protein (Figure 3). More than 130 CAPN3 mutations, with a distribution that varies according to ethnic group, have been identified to date (Leiden Database at http://www.dmd.nl/capn3_home.html)61 (Figure 4). Ten of 13 mutations found in Japanese patients were not found in other populations.63 Others are prevalent in isolated communities (such as the Basque mutation, which is also the most prevalent in Brazil) as a result of a founder effect.70,71 We also observed that 80 percent of the identified Brazilian mutations were concentrated in only six exons (Figure 4). A similar result was recently found in an Italian study.69
Figure 4. Distributions of CAPN3 Gene Mutations Worldwide and in the Brazilian and Italian Populations.
Small circles represent mutations identified only once within the given population and large circles mutations identified more than once, with numbers indicating the number of mutations identified. For example, the Basque mutation (the replacement of AG at positions 2362 and 2363 in exon 22 by TCATCT) was found in 19 of 100 alleles from 50 patients with limb-girdle muscular dystrophy type 2A in Brazil. The horizontal lines represent deletions and the vertical lines insertions. NS denotes N-terminal insertion sequence, and IS insertion sequence.
Western blots of muscle CAPN3 in LGMD2A can show a total or partial deficiency of CAPN3 or, more rarely, no detectable CAPN3 (Figure 3), with no direct correlation between the amount of CAPN3 and the severity of the phenotype. CAPN3 was detectable at very low levels or was undetectable in patients with LGMD2A whose disease ranged from mild to severe,64,72,73 whereas normal or almost normal 94-kD CAPN3 bands were found in 10 to 20 percent of patients with missense mutations in one or both alleles.62,64,72 CAPN3 levels in muscle are normal in some forms of limb-girdle muscular dystrophy, such as sarcoglycanopathy (in which sarcoglycan, part of the dystrophin–glycoprotein complex, is affected) and telethoninopathy (which is a disorder of the Z-disk protein of sarcomeres).73,74 However, a secondary reduction is seen in dysferlinopathy (in which dysferlin, which seems essential for maintaining the structural integrity of the muscle-fiber plasma membrane, is affected), suggesting a possible association between CAPN3 and dysferlin73,75,76 as well as between CAPN3 and muscle proteins related to some other forms of limb-girdle muscular dystrophy, such as LGMD2I (also known as fukutin-related protein limb-girdle dystrophy)77,78 and titinopathy (caused by mutations in the gene encoding titin).79,80,81,82 CAPN3 may influence the role of titin in sarcomere formation through proteolytic cleavage, and some mutations that reduce CAPN3–titin interactions might destabilize and inactivate CAPN3 or remove it from its endogenous substrates.59 In short, a normal amount of CAPN3 on Western blotting may be found in calpainopathies, whereas calpain may be reduced in amount or absent in other forms of LGMD, as a secondary effect. Therefore, for diagnostic purposes, muscle CAPN3 results should always be confirmed by mutation analysis, and screening for mutations should take into account the ethnic origin of affected patients.62,63,69
Genotype–Phenotype Correlation
Despite the marked clinical variability in LGMD2A, missense mutations are usually associated with a milder phenotype than are null mutations.61,64,66,68,72,83,84,85 Studies of patients with compound heterozygosity for missense or null mutations, however, showed that one null mutation is enough to result in a more severe phenotype.62 Null mutations are more often associated with absence of muscle CAPN3, and missense mutations are more often associated with a partial deficiency,61,62,64 although patients carrying the same null mutation may have total or partial CAPN3 deficiency.62 Moreover, the clinical course in patients with apparently normal CAPN3 levels in muscle is not mild, suggesting that although the CAPN3 protein is present, its normal autocatalytic activity is lost. Moreover, the observation that patients with LGMD2A may have an apparently normal amount of calpain suggests that the occurrence of this calpainopathy might be underestimated by protein analysis.
Location of the Second CAPN3 Mutation
In about 10 percent of patients with LGMD2A, only one mutated allele is detectable,61,62,64 suggesting that the second allele has mutations in noncoding regions. We recently identified a family with five affected siblings who carried an in-frame 3-bp deletion in one allele of CAPN3. The clinical course in these patients varied from mild to severe, but all of them had very high serum levels of creatine kinase (50 to 100 times the normal level). Muscle-biopsy specimens from two brothers had normal CAPN3 levels. Linkage analysis ruled out the possibility that the second mutation lay in the CAPN3 gene. Subsequently, we identified a pathogenic mutation in another gene (XK) responsible for their clinical course (unpublished data). We are not aware of other patients carrying one mutation in CAPN3 and a second nonallelic mutation, but the likelihood of finding a patient with two nonallelic or unrelated mutations (double heterozygotes) is at least of the same order of magnitude as the likelihood of finding compound heterozygotes, who are very frequent among patients with LGMD2A.61,62,64
Type 2 Diabetes
Type 2 diabetes affects more than 135 million people worldwide. Determining the genetic risk factors that increase susceptibility to type 2 diabetes will improve our understanding of the mechanism underlying this disorder and perhaps lead to better therapies. The association of the gene encoding calpain 10 (CAPN10), located at 2q37.3, with type 2 diabetes was initially reported by Horikawa and associates.35 Among the known calpains, calpain 10 is atypical, containing a domain III–like structure in place of the usual domain IV (Figure 1). So far, eight splice variants have been identified, among them three with no protease activity. The calpain 10 protein is ubiquitous in adult and fetal human tissues.35
A polymorphism within intron 3 of CAPN10 affects its translation to messenger RNA (mRNA).86 Decreased levels of calpain 10 mRNA were observed in association with the G/G genotype, leading to up-regulation of protein kinase C activity. Since down-regulation of this kinase is important for proper phosphorylation of insulin receptors,87 this polymorphism could cause insulin resistance. Elevated free fatty acid levels are also associated with some variants of CAPN10.88 Calpain 10 may have a role in the actin reorganization that is required for insulin-stimulated translocation of insulin-responsive glucose transporter 4 to the plasma membrane in adipocytes,89 suggesting that there is a link between calpain 10 activity and type 2 diabetes.
More recently, Johnson and associates90 investigated the role of calpain 10 in relation to the type 2 ryanodine receptor (RyR2), which is a Ca2+-release channel on the sarcoplasmic reticulum that may be a central molecule in the control of programmed cell death. They suggested that calpain 10 mediates the death pathway of pancreatic beta cells and that RyR2 plays an essential role in suppressing this pathway. According to these investigators, a novel apoptosis pathway is initiated when Ca2+ flux through RyR2 is blocked. This finding clearly defines a physiologic role for calpain 10 within a specific tissue. Sato and coauthors91 observed that the levels of CAPN10 transcripts in the white cells of rats before and after the onset of type 2 diabetes were significantly lower than those in control animals. They suggest that CAPN10 could be a potential candidate gene for predictive type 2 diabetes tests in humans.
Alzheimer's Disease
The typical brain lesions in Alzheimer's disease are deposits of -amyloid peptides in extracellular amyloid plaques and intracellular neurofibrillar tangles composed of the hyperphosphorylated microtubule-associated tau protein.12 Calpains are not directly related to the production of -amyloid peptides.92 However, Ca2+ and m-calpain levels are elevated,38,93,94 and autolysis of μ-calpain to its 76- and 78-kD forms is enhanced95 in brain tissue from patients with Alzheimer's disease. The regulatory protein p35, which aids in the development of neural tissue, is cleaved by calpains in brain tissue from patients with Alzheimer's disease into a 25-kD form (p25), activating cyclin-dependent kinase 5.96 According to Selkoe,97 p25 is responsible for the hyperphosphorylation of tau in the intracellular neurofibrillary tangles in the brain in Alzheimer's disease. This hyperphosphorylation makes tau highly resistant to degradation by μ-calpain.98 Finally, because the neurofilament proteins are excellent substrates for the calpains, the calpains have an important role in the necrotic neuronal death that accompanies Alzheimer's disease.12
Cataracts
More than 75 percent of cases of cataracts involve elevated levels of Ca2+. The activation of calpains triggered by these high levels of Ca2+ suggests a mechanism for the formation of cataracts. Currently, five calpains have been found in the lens: calpain 1 (μ-calpain), which is expressed only at low levels11; Lp85, which is not yet well characterized99; and the major enzymes — m-calpain,39 calpain 10,100 and Lp82.101 The role of the calpains in the development of cataracts in humans is not as well documented as it is in rats; in animal models of cataractogenesis, m-calpain appears to be the major calpain involved.102 It is the only calpain active in human lenses, and human crystallins are substrates for m-calpain in vitro.
Cataract formation in the lenses of young rats is probably one of the best-documented examples of a relationship between inappropriate calpain activity and tissue lesions.103 A wide variety of insults can cause a large influx of Ca2+ into the lens, with levels of Ca2+ high enough to trigger the proteolytic activity of m-calpains.104 The m-calpain in the lens cleaves the N-terminal region of the lens proteins -crystallin and -crystallin. The truncated crystallin aggregates resist additional proteolysis and form cataracts, which scatter light. The understanding that calpains are involved in some types of human cataracts and their underlying pathogenic mechanisms will be important in the search for calpain inhibitors as anticataract agents, as recently suggested by Nakamura et al.105
Calpain Inhibitors
More than 50 endogenous and exogenous inhibitors of the calpains have been described. They include cellular and extracellular proteins and drugs such as iodoacetate, iodoacetamide, and N-ethylmaleimide, which are inhibitors of cysteine proteases.12,106,107 The intracellular level of calpastatin correlates directly with calpain activation,108 and the affinity of calpastatin for the activated forms of the calpains is greater than its affinity for the proenzyme,109 indicating that both structural and conformational changes due to autolysis favor formation of the enzyme–inhibitor complex. The inhibitory efficiency of calpastatin and its level are important in the prevention of calpain-mediated lesions. Phosphorylation of calpastatin may alter its efficiency and specificity in isolated cells and skeletal muscle.110,111,112 This potential inhibitor has possible therapeutic applications in diseases involving the calpains.12,113
Intramuscular administration of the synthetic calpain inhibitor leupeptin to dystrophic mdx mice apparently prevents decreases in muscle-fiber diameter, suggesting that this protease inhibitor could prevent the loss of muscle mass in dystrophic mice.114 In rats, leupeptin, calpain inhibitor 1, and caspase 3 inhibitors reduce infarct size and postischemic apoptosis in hearts without modifying contractile recovery.115 Other studies suggest that treatment with leupeptin may rescue motor neurons from cell death and improve muscle function after nerve injury.116,117 The possibility that calpain inhibitors can restore cognition and synaptic transmission in transgenic models of Alzheimer's disease is being tested.113
Further elucidation of the roles of members of the calpain superfamily and of potential calpain inhibitors will be important for delineating the various approaches to be used in the treatment of diseases related to these proteins.
Supported by the Funda??o de Amparo à Pesquisa do Estado de S?o Paulo–Centro de Estudos do Genoma Humano and Conselho Nacional de Desenvolvimento Científico e Tecnológico.
We are indebted to Dr. Maria Rita Passos-Bueno, Dr. Mariz Vainzof, Dr. Eloisa S. Moreira, Dr. Rita C.M. Pavanello, Dr. Ivo Pavanello, Dr. Viviane Abreu N.C. Dantas, Marta Cánovas, Antonia Cerqueira, and Constancia Urbani for many years of invaluable collaboration.
Source Information
From the Human Genome Research Center, Departamento de Biologia, Instituto de Biociências; Universidade de S?o Paulo, S?o Paulo.
Address reprint requests to Dr. Zatz at the Departamento de Biologia, Instituto de Biociências, Universidade de S?o Paulo, Rua do Mat?o 277, Cidade Universitária, CEP 05508-900, S?o Paulo, Brazil, or at mayazatz@usp.br.
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Shields DC, Schaecher KE, Saido TC, Banik NL. A putative mechanism of demyelination in multiple sclerosis by a proteolytic enzyme, calpain. Proc Natl Acad Sci U S A 1999;96:11486-11491.
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Starling A, Kok F, Passos-Bueno MR, Vainzof M, Zatz M. A new form of autosomal dominant limb-girdle muscular dystrophy (LGMD1G) with progressive fingers and toes flexion limitation maps to chromosome 4p21. Eur J Hum Genet 2004;12:1033-1040.
Richard I, Broux O, Allamand V, et al. Mutations in the proteolytic enzyme calpain 3 cause limb-girdle muscular dystrophy type 2A. Cell 1995;81:27-40.
Sorimachi H, Beckmann JS. Defects of non-lysosomal proteolysis: calpain3 deficiency. In: Karpati G, ed. Structural and molecular basis of skeletal muscle diseases. Basel, Switzerland: ISN Neuropath Press, 2002:148-53.
Richard I, Roudaut C, Marchand S, et al. Loss of calpain 3 proteolytic activity leads to muscular dystrophy and to apoptosis-associated IkappaBalpha/nuclear factor kappaB pathway perturbation in mice. J Cell Biol 2000;151:1583-1590.
Tagawa K, Taya C, Hayashi Y, et al. Myopathy phenotype of transgenic mice expressing active site-mutated inactive p94 skeletal muscle-specific calpain, the gene product responsible for limb girdle muscular dystrophy type 2A. Hum Mol Genet 2000;9:1393-1402.
Baghdiguian S, Martin M, Richard I, et al. Calpain 3 deficiency is associated with myonuclear apoptosis and profound perturbation of the IkappaB alpha/NF-kappaB pathway in limb-girdle muscular dystrophy type 2A. Nat Med 1999;5:503-511.
Ozawa E, Noguchi S, Mizuno Y, Hagiwara Y, Yoshida M. From dystrophinopathy to sarcoglycanopathy: evolution of a concept of muscular dystrophy. Muscle Nerve 1998;21:421-438.
Spencer MJ, Guyon JR, Sorimachi H, et al. Stable expression of calpain 3 from a muscle transgene in vivo: immature muscle in transgenic mice suggests a role for calpain 3 in muscle maturation. Proc Natl Acad Sci U S A 2002;99:8874-8879.
Vainzof M, de Paula F, Tsanaclis AM, Zatz M. The effect of calpain-3 deficiency on the pattern of muscle degeneration in the earliest stages of LGMD2A. J Clin Pathol 2003;56:624-626.
Kramerova I, Kudryashova E, Tidball JG, Spencer MJ. Null mutations of calpain 3 (p94) in mice causes abnormal sarcomere formation in vivo and in vitro. Hum Mol Genet 2004;13:1373-1388.
Guyon JR, Kudryashova E, Potts A, et al. Calpain 3 cleaves filamin C and regulates its ability to interact with - and -sarcoglycans. Muscle Nerve 2003;28:472-483.
Richard I, Roudaut C, Saenz A, et al. Calpainopathy -- a survey of mutations and polymorphisms. Am J Hum Genet 1999;64:1524-1540.
de Paula F, Vainzof M, Passos-Bueno MR, et al. Clinical variability in calpainopathy: what makes the difference? Eur J Hum Genet 2002;10:825-832.
Chae J, Minami N, Jin Y, et al. Calpain 3 gene mutations: genetic and clinico-pathologic findings in limb-girdle muscular dystrophy. Neuromuscul Disord 2001;11:547-555.
Fanin M, Fulizio L, Nascimbeni AC, et al. Molecular diagnosis in LGMD2A: mutation analysis or protein testing? Hum Mutat 2004;24:52-62.
Fardeau M, Eymard B, Mignard C, Tome FM, Richard I, Beckmann JS. Chromosome 15-linked limb-girdle muscular dystrophy: clinical phenotypes in Reunion Island and French metropolitan communities. Neuromuscul Disord 1996;6:447-453.
Bushby KM. The limb-girdle muscular dystrophies -- multiple genes, multiple mechanisms. Hum Mol Genet 1999;8:1875-1882.
Bushby KM. Diagnostic criteria for the limb-girdle muscular dystrophies: report of the ENMC workshop on limb-girdle muscular dystrophies. Neuromuscul Disord 1995;5:71-74.
Starling A, de Paula F, Silva H, Vainzof M, Zatz M. Calpainopathy: how broad is the spectrum of clinical variability? J Mol Neurosci 2003;21:233-236.
Piluso G, Politano L, Aurino S, et al. The extensive scanning of the calpain-3 gene broadens the spectrum of LGMD2A phenotype. J Med Genet (in press).
Urtasun M, Saenz A, Roudaut C, et al. Limb-girdle muscular dystrophy in Guipuzcoa (Basque Country, Spain). Brain 1998;121:1735-1747.
Paula F, Saenz A, Cobo AM, Lopes de Munain A, Urtizberea A, Zatz M. The recurrent Basque mutation in Brazilian calpainopathy patients. Am J Hum Genet 2003;73:Suppl:580-580. abstract.
Anderson LV, Davison K, Moss JA, et al. Characterization of monoclonal antibodies to calpain 3 and protein expression in muscle from patients with limb-girdle muscular dystrophy type 2A. Am J Pathol 1998;153:1169-1179.
Vainzof M, Anderson LVB, Moreira ES, et al. Characterization of the primary defect in LGMD2A and analysis of its secondary effect in other LGMDs. Neurology 2000;54:Suppl:A436-A436. abstract.
Vainzof M, Moreira ES, Suzuki OT, et al. Telethonin protein expression in neuromuscular disorders. Biochim Biophys Acta 2002;1588:33-40.
Anderson LV, Harrison RM, Pogue R, et al. Secondary reduction in calpain 3 expression in patients with limb girdle muscular dystrophy type 2B and Miyoshi myopathy (primary dysferlinopathies). Neuromuscul Disord 2000;10:553-559.
Vainzof M, Anderson LVB, McNally EM, et al. Dysferlin protein analysis in limb-girdle muscular dystrophies. J Mol Neurosci 2001;17:71-80.
Bushby KM, Beckmann JS. The 105th ENMC sponsored workshop: pathogenesis in the non-sarcoglycan limb-girdle muscular dystrophies, Naarden, April 12-14, 2002. Neuromuscul Disord 2003;13:80-90.
Yamamoto LU, Paula F, Pavanello RCM, Vieira N, Zatz M, Vainzof M. LGMDI-secondary muscle protein alterations in patients with mutations in the fukutin related protein gene. Neuromuscul Disord 2004;14:579-579. abstract.
Haravuori H, Vihola A, Straub V, et al. Secondary calpain3 deficiency in 2q-linked muscular dystrophy: titin is the candidate gene. Neurology 2001;56:869-877.
Garvey SM, Rajan C, Lerner AP, Frankel WN, Cox GA. The muscular dystrophy with myositis (mdm) mouse mutation disrupts a skeletal muscle-specific domain of titin. Genomics 2002;79:146-149.
Hackman P, Richard I, Vihola A, et al. Tibial muscular dystrophy (TMD), 2q31 linked myopathy: sequencing and functional studies of the titin (TTN) gene. J Neurol Sci 2002;199:Suppl 1:S35-S35. abstract.
Hackman P, Vihola A, Haravuori H, et al. Tibial muscular dystrophy is a titinopathy caused by mutations in TTN, the gene encoding the giant skeletal-muscle protein titin. Am J Hum Genet 2002;71:492-500.
Zatz M, Vainzof M, Passos-Bueno MR. Limb-girdle muscular dystrophy: one gene with different phenotypes, one phenotype with different genes. Curr Opin Neurol 2000;13:511-517.
Passos-Bueno MR, Vainzof M, Moreira ES, Zatz M. The seven autosomal recessive limb-girdle muscular dystrophies in the Brazilian population: from LGMD2A to LGMD2G. Am J Med Genet 1999;82:392-398.
Pollitt C, Anderson LV, Pogue R, Davison K, Pyle A, Bushby KM. The phenotype of calpainopathy: diagnosis based on a multidisciplinary approach. Neuromuscul Disord 2001;11:287-296.
Baier LJ, Permana PA, Yang X, et al. A calpain-10 gene polymorphism is associated with reduced muscle mRNA levels and insulin resistance. J Clin Invest 2000;106:R69-R73.
Itani SI, Zhou Q, Pories WJ, MacDonald KG, Dohm GL. Involvement of protein kinase C in human skeletal muscle insulin resistance and obesity. Diabetes 2000;49:1353-1358.
Ortho-Melender M, Klannemark M, Svensson MK, Ridderstrale M, Lidgren CM, Groop L. Variants in the calpain-10 gene predispose to insulin resistance and elevated free fatty acid level. Diabetes 2002;51:2658-2664.
Paul DS, Harmon AW, Winston CP, Patel YM. Calpain facilitates GLUT4 vesicle translocation during insulin-stimulated glucose uptake in adipocytes. Biochem J 2003;376:625-632.
Johnson JD, Han Z, Otani K, et al. RyR2 and calpain-10 delineate a novel apoptosis pathway in pancreatic islets. J Biol Chem 2004;279:24794-24802.
Sato Y, Kuwajima M, Kamiya H, Harashima H. Calpain 10 as a predictive gene for type 2 diabetes: evidence from a novel screening system using white blood cells of Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Biol Pharm Bull 2003;26:1765-1768.
Figueiredo-Pereira ME, Efthimiopoulos S, Tezapsidis N, et al. Distinct secretases, a cysteine protease and a serine protease, generate the C termini of amyloid beta-proteins Abeta1-40 and Abeta1-42, respectively. J Neurochem 1999;72:1417-1422.
Leissring MA, Akbari Y, Fanger CM, Cahalan MD, Mattson MP, LaFerla FM. Capacitative calcium entry deficits and elevated luminal calcium content in mutant presenilin-1 knockin mice. J Cell Biol 2000;149:793-798.
Grynspan F, Griffin WR, Cataldo A, Katayama S, Nixon RA. Active site-directed antibodies identify calpain II as an early-appearing and pervasive component of neurofibrillary pathology in Alzheimer's disease. Brain Res 1997;763:145-158.
Saito K, Elce JS, Hamos JE, Nixon RA. Widespread activation of calcium-activated neutral proteinase (calpain) in the brain in Alzheimer disease: a potential molecular basis for neuronal degeneration. Proc Natl Acad Sci U S A 1993;90:2628-2632.
Kusakawa G, Saito T, Onuki R, Ishiguro K, Kishimoto T, Hisanaga S. Calpain-dependent proteolytic cleavage of the p35 cyclin-dependent kinase 5 activator to p25. J Biol Chem 2000;275:17166-17172.
Selkoe DJ. Alzheimer's disease: genes, proteins, and therapy. Physiol Rev 2001;81:741-766.
Litersky JM, Johnson GV. Phosphorylation by cAMP-dependent protein kinase inhibits the degradation of tau by calpain. J Biol Chem 1992;267:1563-1568.
Reed NA, Castellini MA, Ma H, Shearer TR, Duncan MK. Protein expression patterns for ubiquitous and tissue specific calpains in the developing mouse lens. Exp Eye Res 2003;76:433-443.
Ma H, Fukiage C, Kim YH, et al. Characterization and expression of calpain 10: a novel ubiquitous calpain with nuclear localization. J Biol Chem 2001;276:28525-28531.
Ma H, Fukiage C, Azuma M, Shearer TR. Cloning and expression of mRNA for calpain Lp82 from rat lens: splice variant of p94. Invest Ophthalmol Vis Sci 1998;39:454-461.
Biswas S, Harris F, Dennison S, Singh J, Phoenix DA. Calpains: targets of cataract prevention? Trends Mol Med 2004;10:78-84.
Shearer TR, Ma H, Shih M, Fukiage C, Azuma M. Calpains in the lens of the eye. In: Wang KKW, Yeun P-W, eds. Calpain: pharmacology and toxicology of calcium-dependent protease. Philadelphia: Taylor & Francis, 1999:331-47.
Nakamura Y, Fukiage C, Shih M, et al. Contribution of calpain Lp82-induced proteolysis to experimental cataractogenesis in mice. Invest Ophthalmol Vis Sci 2000;41:1460-1466.
Nakamura M, Yamaguchi M, Sakai O, Inoue J. Exploration of cornea permeable calpain inhibitors as anticataract agents. Bioorg Med Chem 2003;11:1371-1379.
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Pontremoli S, Salamino F, Sparatore B, De Tullio R, Pontremoli R, Melloni E. Characterization of the calpastatin defect in erythrocytes from patients with essential hypertension. Biochem Biophys Res Commun 1988;157:867-874.
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Properties of Calpains
Two members of the calpain superfamily, calpains 1 (μ-calpain) and 2 (m-calpain), have been extensively studied.1 Each of these calpains, which differ in their sensitivity to Ca2+, consists of two different polypeptide subunits. The larger subunit (80 kD) has catalytic activity, whereas the smaller, 30-kD, subunit has a regulatory function (Figure 1).
Figure 1. Schematic Representation of the Domain Structure of the Large and Small Subunits of m-Calpain.
μ-Calpain has a similar heterodimeric structure, but it differs in its sensitivity to Ca2+. Adapted from a diagram provided by Dr. Hiroyuki Sorimachi, Laboratory of Biological Function, University of Tokyo, Tokyo.
The large subunits of μ-calpain and m-calpain are encoded by the CAPN1 and CAPN2 genes, respectively. Calpains 3, 8a, 9, 11, 12, and 13 also have 80-kD and 30-kD subunits. The 80-kD subunit has four domains: domain I is the N-terminal anchoring -helix domain and is important for regulating the activity and dissociation of the subunit2,3,4,5; domain II, a catalytic domain, has two subdomains in the absence of Ca2+6 7; domain III binds Ca2+ and phospholipids8; and domain IV, also called the penta–EF-hand domain (an EF-hand unit consists of two peptide helixes connected by a Ca2+-binding loop), is important for dimer formation.9 The 30-kD regulatory subunit of calpains 1, 2, and 9 consists of two domains: domain V is the N-terminal, glycine-rich, hydrophobic domain and domain VI the penta–EF-hand domain, which is similar to domain IV of the catalytic subunit.6,7 The 80-kD subunit of the other calpains (3, 8a, 11, 12, and 13) do not interact with the 30-kD subunit, although they do have domain IV. Calpains 5, 6, 7, 8b, 10, and 15 are atypical calpains in that some of their domains have been deleted or replaced. They lack domain IV and therefore presumably do not associate with the 30-kD subunit.
There are two classes of calpains: one (comprising calpains 1, 2, 5, 7, 10, 13, and 15) is ubiquitous in cytosol; the other (comprising calpains 3, 6, 8, 9, 11, and 12) occurs only or mainly in certain tissues.10 For example, calpain 8 is stomach-specific, and calpain 3 (CAPN3) is largely specific to skeletal muscle,1,11 although it is also present in cardiac muscle and the liver.12 The amino acid sequence of skeletal-muscle CAPN3 is similar to the sequences of the ubiquitous μ-calpain and m-calpain (which are structural variants that catalyze the same reaction), but it contains three specific insertion sequences.
The mechanisms by which calpains are activated and identify their protein targets are complex and poorly understood. Calpain activity is regulated by a ubiquitous specific inhibitor, calpastatin.13,14 The calpain–calpastatin interaction is important in regulation of the activity of μ-calpain and m-calpain,15 but the nature of this regulation in living cells is not understood. Studies using calpastatin have shown that calpains are clearly involved in some types of apoptosis in specific cell types and in response to certain apoptotic signals.12
An intricate strategy for the regulation of calpain activity seems necessary because calpain is an abundant cytoplasmic protease that can cleave many intracellular signaling and structural proteins. Membrane localization of calpains is an important mechanism for regulating their activity. In early work, it was thought that when a calpain binds to the plasma membrane, it is transformed from an inactive, proenzyme into an active, proteolytic enzyme by autolysis. However, other findings indicate that both μ-calpain and m-calpain are active proteolytic enzymes before autolysis and that interaction with a membrane may bind calpains to their substrates, rather than promote autolysis.16,17,18,19,20,21,22,23
Once activated on the membrane, the calpain presumably diffuses into the cytosol and becomes resistant to the inhibitory action of calpastatin.24 Substrate proteins are digested by the activated calpain on the membrane or in the cytosol.25 According to Gil-Parrado et al.,26 calpain activity is regulated not only by calpastatin but also by differential intracellular localization. In contrast, dissociation of the subunits that constitute a calpain appears to be less critical to its regulation. Ca2+ levels required to initiate autolysis are as high as or even slightly higher than the levels required for proteolytic activity and are much greater than the free Ca2+ levels in living cells.20,27,28 A solution to this paradox appeared when it was discovered that the presence of phospholipids, with phosphatidylinositol, lowered the Ca2+ levels required for autolysis of μ-calpain and m-calpain.29,30 Other studies have shown that autolysis of these two calpains is an intermolecular process,31 rather than an intramolecular process, as previously thought.16
μ-Calpain and m-calpain have diverse functions.11,32 They catalyze the proteolysis of proteins involved in cytoskeletal remodeling, cell-cycle regulation, signal transduction, cell differentiation, apoptosis and necrosis, embryonic development, and vesicular trafficking.3,4,32 For this reason, calpain activity has to be tightly regulated both temporarily and spatially to be effective and limited in scope.
Calpain-Associated Diseases
A number of pathologic conditions have been associated with disturbances of the calpain system. They include type 2 diabetes, cataracts, Duchenne's muscular dystrophy, Parkinson's disease, Alzheimer's disease, rheumatoid arthritis, ischemia, stroke and brain trauma, various platelet syndromes, hypertension, liver dysfunction, and some types of cancer (Table 1). Several oncogenes and tumor-suppressor gene products are substrates for members of the calpain family.48 Mutations in the CAPN3 gene cause LGMD2A, indicating that the protein that it encodes is important for muscle function.
Table 1. Examples of Pathologic Conditions That Have Been Associated with the Calpains.
Limb-Girdle Muscular Dystrophy Type 2A
LGMD2A (Online Mendelian Inheritance in Man number 253600 ) is one of the limb-girdle muscular dystrophies, a heterogeneous group of genetically determined progressive disorders of skeletal muscle. Seventeen genes responsible for various types of limb-girdle muscular dystrophy have been mapped,49,50 and the products of most of them have been identified (Figure 2). LGMD2A, the most prevalent form, accounts for at least 30 percent of all cases.
Figure 2. Schematic Representations of the Proteins Associated with Limb-Girdle Muscular Dystrophies (Top), of the Golgi Complex (Inset), and of the Structure of Muscle Fibers (Bottom).
LGMD2 denotes limb-girdle muscular dystrophy type 2, -DTN -dystrobrevin, FKRP fukutin-related protein, TRIM32 tripartite motif–containing protein 32, and T-cap titin-cap. The variants responsible for different subtypes of limb-girdle muscular dystrophy are shown in red. Adapted from a diagram provided by Peter Hackman and Bjarne Udd, Neurology Department, Vaasa Central Hospital, Vaasa, Finland.
Molecular Mechanisms
LGMD2A is caused by mutations in the CAPN3 (also called p94) gene,51 which encodes CAPN3, the largely skeletal-muscle–specific member of the calpain superfamily. CAPN3, located both in the cytosol and in the nucleus, requires extremely low levels of Ca2+ for activation. In skeletal-muscle cells, CAPN3 binds specifically to certain regions of titin, a giant structural muscle protein; this binding seems to stabilize CAPN3 and thereby prevent its autolysis. Mutations in the CAPN3 gene result in a cascade of events leading to CAPN3 deficiency and, eventually, to muscular dystrophy, but the underlying mechanisms are unknown.12,52 Several studies have suggested that the loss of CAPN3 processing activity, but not its hyperactivation or a defect in its structure, causes LGMD2A.53,54 According to other studies,55 CAPN3 deficiency causes myonuclear apoptosis (i.e., death of muscle-cell nuclei) and profound perturbation of the IB–nuclear factor (nuclear factor of kappa light-chain gene enhancer in B cells inhibitor ) pathway, such that a failure in CAPN3-dependent IB proteolysis results in accumulation of IB in the cytoplasm and nucleus and sequestration of nuclear factor-B in the cytoplasm, both of which culminate in the apoptosis of muscle cells.55
It is unlikely that an influx of extracellular Ca2+ into the sarcoplasm causes muscle fibers to degenerate, because CAPN3 is localized primarily in the myofibril and not on the sarcolemma.13,56 Animal models and findings before the onset of muscular dystrophy in young carriers of CAPN3 mutations suggest a role for CAPN3 in muscle maturation and sarcomere remodeling.57,58,59 In vitro, CAPN3 cleaves filamin C, which is specific to muscle, and regulates its ability to interact with - and -sarcoglycans, two proteins of the dystrophin–glycoprotein complex (dystrophin anchors muscle membranes to actin filaments in myofibrils).60 Since CAPN3 belongs to a large family of Ca2+-dependent proteases, at least seven of which are expressed in muscle, the calpain superfamily could form a proteolytic network in muscle and other cells. In this way, a defect in CAPN3 could distort the calpain system, leading to overactivation of other calpains.14
Clinical Features
Patients with LGMD2A have symmetrical and selective involvement of proximal limb-girdle muscles. They have normal intelligence and no cardiac or facial disturbances. The disease shows wide intrafamilial and interfamilial clinical variability.49,61,62,63,64,65 Findings on examination of muscle-biopsy specimens are consistent with a dystrophic or myopathic process (Figure 3). The serum level of creatine kinase is moderately or markedly increased, particularly in the active phase of the disorder.42,66,67 However, we have seen patients with a normal serum level of creatine kinase or a neurogenic pattern on electromyography,68 suggesting that the spectrum of variability in this calpainopathy might be broader than suspected and that a normal creatine kinase level should not be considered a reason to rule out a possible diagnosis of LGMD2A, even in ambulatory patients.
Figure 3. Histopathologic Features, Results of Muscle-Protein Analysis, and Clinical Characteristics in Limb-Girdle Muscular Dystrophy Type 2A.
Panel A shows the histologic features of normal muscle and muscle from a patient with limb-girdle muscular dystrophy type 2A (LGMD2A) and calpainopathy (hematoxylin–eosin stain). In the latter specimen, there is substantial variation in fiber size, with very large fibers surrounded by small groups of atrophic fibers, connective-tissue infiltration, and splitting. Panel B shows the results of multiplex Western blot analysis of the muscle proteins dystrophin, dysferlin, and calpain 3 from a normal control subject (lane C) and four unrelated patients with LGMD2A (lanes 1 through 4) (performed with monoclonal antibody Calp3C/12A2, which recognizes the 94-kD band of calpain 3). Myosin content served as a control for the amount of muscle protein loaded in the Ponceau-prestained blot. The blot shows almost complete absence of calpain 3 in Patient 1, partial deficiency in Patients 3 and 4, and a normal amount of calpain 3 in Patient 2; there was no clinical correlation with the severity of the phenotype. Image courtesy of Dr. Mariz Vainzof, Human Genome Center, Department of Biology, University of S?o Paulo, S?o Paulo. Panel C shows affected sisters homozygous for a mutation in CAPN3 resulting in the replacement of arginine with glutamine at position 769 of the CAPN3 protein. The intrafamilial variability of LGMD2A is evident: the older sister (left) is still ambulatory at the age of 47 years, whereas the younger sister (right), who is currently 33 years of age, has used a wheelchair since the age of 18.
The age at onset ranges from 2 to 40 years, but the disease usually first appears in the second or third decade of life, with the development of proximal weakness in the lower limbs. Although early-onset cases are usually more severe, cases with a later onset may occasionally involve rapid progression and inability to walk by the third decade. Use of a wheelchair may become necessary during the second through fifth decades, but some patients may remain ambulatory after the sixth decade.49,61,62,64 A more rapid progression in male patients than in female patients has been observed among Brazilian and Italian patients, an intriguing observation for which we have no explanation.62,69
CAPN3 Mutations, CAPN3 Protein Analysis, and Diagnostic Applications
The CAPN3 gene encompasses 24 exons and covers a 50-kb genomic region. It is expressed as a 3.5-kb transcript, which encodes a 94-kD translated protein (Figure 3). More than 130 CAPN3 mutations, with a distribution that varies according to ethnic group, have been identified to date (Leiden Database at http://www.dmd.nl/capn3_home.html)61 (Figure 4). Ten of 13 mutations found in Japanese patients were not found in other populations.63 Others are prevalent in isolated communities (such as the Basque mutation, which is also the most prevalent in Brazil) as a result of a founder effect.70,71 We also observed that 80 percent of the identified Brazilian mutations were concentrated in only six exons (Figure 4). A similar result was recently found in an Italian study.69
Figure 4. Distributions of CAPN3 Gene Mutations Worldwide and in the Brazilian and Italian Populations.
Small circles represent mutations identified only once within the given population and large circles mutations identified more than once, with numbers indicating the number of mutations identified. For example, the Basque mutation (the replacement of AG at positions 2362 and 2363 in exon 22 by TCATCT) was found in 19 of 100 alleles from 50 patients with limb-girdle muscular dystrophy type 2A in Brazil. The horizontal lines represent deletions and the vertical lines insertions. NS denotes N-terminal insertion sequence, and IS insertion sequence.
Western blots of muscle CAPN3 in LGMD2A can show a total or partial deficiency of CAPN3 or, more rarely, no detectable CAPN3 (Figure 3), with no direct correlation between the amount of CAPN3 and the severity of the phenotype. CAPN3 was detectable at very low levels or was undetectable in patients with LGMD2A whose disease ranged from mild to severe,64,72,73 whereas normal or almost normal 94-kD CAPN3 bands were found in 10 to 20 percent of patients with missense mutations in one or both alleles.62,64,72 CAPN3 levels in muscle are normal in some forms of limb-girdle muscular dystrophy, such as sarcoglycanopathy (in which sarcoglycan, part of the dystrophin–glycoprotein complex, is affected) and telethoninopathy (which is a disorder of the Z-disk protein of sarcomeres).73,74 However, a secondary reduction is seen in dysferlinopathy (in which dysferlin, which seems essential for maintaining the structural integrity of the muscle-fiber plasma membrane, is affected), suggesting a possible association between CAPN3 and dysferlin73,75,76 as well as between CAPN3 and muscle proteins related to some other forms of limb-girdle muscular dystrophy, such as LGMD2I (also known as fukutin-related protein limb-girdle dystrophy)77,78 and titinopathy (caused by mutations in the gene encoding titin).79,80,81,82 CAPN3 may influence the role of titin in sarcomere formation through proteolytic cleavage, and some mutations that reduce CAPN3–titin interactions might destabilize and inactivate CAPN3 or remove it from its endogenous substrates.59 In short, a normal amount of CAPN3 on Western blotting may be found in calpainopathies, whereas calpain may be reduced in amount or absent in other forms of LGMD, as a secondary effect. Therefore, for diagnostic purposes, muscle CAPN3 results should always be confirmed by mutation analysis, and screening for mutations should take into account the ethnic origin of affected patients.62,63,69
Genotype–Phenotype Correlation
Despite the marked clinical variability in LGMD2A, missense mutations are usually associated with a milder phenotype than are null mutations.61,64,66,68,72,83,84,85 Studies of patients with compound heterozygosity for missense or null mutations, however, showed that one null mutation is enough to result in a more severe phenotype.62 Null mutations are more often associated with absence of muscle CAPN3, and missense mutations are more often associated with a partial deficiency,61,62,64 although patients carrying the same null mutation may have total or partial CAPN3 deficiency.62 Moreover, the clinical course in patients with apparently normal CAPN3 levels in muscle is not mild, suggesting that although the CAPN3 protein is present, its normal autocatalytic activity is lost. Moreover, the observation that patients with LGMD2A may have an apparently normal amount of calpain suggests that the occurrence of this calpainopathy might be underestimated by protein analysis.
Location of the Second CAPN3 Mutation
In about 10 percent of patients with LGMD2A, only one mutated allele is detectable,61,62,64 suggesting that the second allele has mutations in noncoding regions. We recently identified a family with five affected siblings who carried an in-frame 3-bp deletion in one allele of CAPN3. The clinical course in these patients varied from mild to severe, but all of them had very high serum levels of creatine kinase (50 to 100 times the normal level). Muscle-biopsy specimens from two brothers had normal CAPN3 levels. Linkage analysis ruled out the possibility that the second mutation lay in the CAPN3 gene. Subsequently, we identified a pathogenic mutation in another gene (XK) responsible for their clinical course (unpublished data). We are not aware of other patients carrying one mutation in CAPN3 and a second nonallelic mutation, but the likelihood of finding a patient with two nonallelic or unrelated mutations (double heterozygotes) is at least of the same order of magnitude as the likelihood of finding compound heterozygotes, who are very frequent among patients with LGMD2A.61,62,64
Type 2 Diabetes
Type 2 diabetes affects more than 135 million people worldwide. Determining the genetic risk factors that increase susceptibility to type 2 diabetes will improve our understanding of the mechanism underlying this disorder and perhaps lead to better therapies. The association of the gene encoding calpain 10 (CAPN10), located at 2q37.3, with type 2 diabetes was initially reported by Horikawa and associates.35 Among the known calpains, calpain 10 is atypical, containing a domain III–like structure in place of the usual domain IV (Figure 1). So far, eight splice variants have been identified, among them three with no protease activity. The calpain 10 protein is ubiquitous in adult and fetal human tissues.35
A polymorphism within intron 3 of CAPN10 affects its translation to messenger RNA (mRNA).86 Decreased levels of calpain 10 mRNA were observed in association with the G/G genotype, leading to up-regulation of protein kinase C activity. Since down-regulation of this kinase is important for proper phosphorylation of insulin receptors,87 this polymorphism could cause insulin resistance. Elevated free fatty acid levels are also associated with some variants of CAPN10.88 Calpain 10 may have a role in the actin reorganization that is required for insulin-stimulated translocation of insulin-responsive glucose transporter 4 to the plasma membrane in adipocytes,89 suggesting that there is a link between calpain 10 activity and type 2 diabetes.
More recently, Johnson and associates90 investigated the role of calpain 10 in relation to the type 2 ryanodine receptor (RyR2), which is a Ca2+-release channel on the sarcoplasmic reticulum that may be a central molecule in the control of programmed cell death. They suggested that calpain 10 mediates the death pathway of pancreatic beta cells and that RyR2 plays an essential role in suppressing this pathway. According to these investigators, a novel apoptosis pathway is initiated when Ca2+ flux through RyR2 is blocked. This finding clearly defines a physiologic role for calpain 10 within a specific tissue. Sato and coauthors91 observed that the levels of CAPN10 transcripts in the white cells of rats before and after the onset of type 2 diabetes were significantly lower than those in control animals. They suggest that CAPN10 could be a potential candidate gene for predictive type 2 diabetes tests in humans.
Alzheimer's Disease
The typical brain lesions in Alzheimer's disease are deposits of -amyloid peptides in extracellular amyloid plaques and intracellular neurofibrillar tangles composed of the hyperphosphorylated microtubule-associated tau protein.12 Calpains are not directly related to the production of -amyloid peptides.92 However, Ca2+ and m-calpain levels are elevated,38,93,94 and autolysis of μ-calpain to its 76- and 78-kD forms is enhanced95 in brain tissue from patients with Alzheimer's disease. The regulatory protein p35, which aids in the development of neural tissue, is cleaved by calpains in brain tissue from patients with Alzheimer's disease into a 25-kD form (p25), activating cyclin-dependent kinase 5.96 According to Selkoe,97 p25 is responsible for the hyperphosphorylation of tau in the intracellular neurofibrillary tangles in the brain in Alzheimer's disease. This hyperphosphorylation makes tau highly resistant to degradation by μ-calpain.98 Finally, because the neurofilament proteins are excellent substrates for the calpains, the calpains have an important role in the necrotic neuronal death that accompanies Alzheimer's disease.12
Cataracts
More than 75 percent of cases of cataracts involve elevated levels of Ca2+. The activation of calpains triggered by these high levels of Ca2+ suggests a mechanism for the formation of cataracts. Currently, five calpains have been found in the lens: calpain 1 (μ-calpain), which is expressed only at low levels11; Lp85, which is not yet well characterized99; and the major enzymes — m-calpain,39 calpain 10,100 and Lp82.101 The role of the calpains in the development of cataracts in humans is not as well documented as it is in rats; in animal models of cataractogenesis, m-calpain appears to be the major calpain involved.102 It is the only calpain active in human lenses, and human crystallins are substrates for m-calpain in vitro.
Cataract formation in the lenses of young rats is probably one of the best-documented examples of a relationship between inappropriate calpain activity and tissue lesions.103 A wide variety of insults can cause a large influx of Ca2+ into the lens, with levels of Ca2+ high enough to trigger the proteolytic activity of m-calpains.104 The m-calpain in the lens cleaves the N-terminal region of the lens proteins -crystallin and -crystallin. The truncated crystallin aggregates resist additional proteolysis and form cataracts, which scatter light. The understanding that calpains are involved in some types of human cataracts and their underlying pathogenic mechanisms will be important in the search for calpain inhibitors as anticataract agents, as recently suggested by Nakamura et al.105
Calpain Inhibitors
More than 50 endogenous and exogenous inhibitors of the calpains have been described. They include cellular and extracellular proteins and drugs such as iodoacetate, iodoacetamide, and N-ethylmaleimide, which are inhibitors of cysteine proteases.12,106,107 The intracellular level of calpastatin correlates directly with calpain activation,108 and the affinity of calpastatin for the activated forms of the calpains is greater than its affinity for the proenzyme,109 indicating that both structural and conformational changes due to autolysis favor formation of the enzyme–inhibitor complex. The inhibitory efficiency of calpastatin and its level are important in the prevention of calpain-mediated lesions. Phosphorylation of calpastatin may alter its efficiency and specificity in isolated cells and skeletal muscle.110,111,112 This potential inhibitor has possible therapeutic applications in diseases involving the calpains.12,113
Intramuscular administration of the synthetic calpain inhibitor leupeptin to dystrophic mdx mice apparently prevents decreases in muscle-fiber diameter, suggesting that this protease inhibitor could prevent the loss of muscle mass in dystrophic mice.114 In rats, leupeptin, calpain inhibitor 1, and caspase 3 inhibitors reduce infarct size and postischemic apoptosis in hearts without modifying contractile recovery.115 Other studies suggest that treatment with leupeptin may rescue motor neurons from cell death and improve muscle function after nerve injury.116,117 The possibility that calpain inhibitors can restore cognition and synaptic transmission in transgenic models of Alzheimer's disease is being tested.113
Further elucidation of the roles of members of the calpain superfamily and of potential calpain inhibitors will be important for delineating the various approaches to be used in the treatment of diseases related to these proteins.
Supported by the Funda??o de Amparo à Pesquisa do Estado de S?o Paulo–Centro de Estudos do Genoma Humano and Conselho Nacional de Desenvolvimento Científico e Tecnológico.
We are indebted to Dr. Maria Rita Passos-Bueno, Dr. Mariz Vainzof, Dr. Eloisa S. Moreira, Dr. Rita C.M. Pavanello, Dr. Ivo Pavanello, Dr. Viviane Abreu N.C. Dantas, Marta Cánovas, Antonia Cerqueira, and Constancia Urbani for many years of invaluable collaboration.
Source Information
From the Human Genome Research Center, Departamento de Biologia, Instituto de Biociências; Universidade de S?o Paulo, S?o Paulo.
Address reprint requests to Dr. Zatz at the Departamento de Biologia, Instituto de Biociências, Universidade de S?o Paulo, Rua do Mat?o 277, Cidade Universitária, CEP 05508-900, S?o Paulo, Brazil, or at mayazatz@usp.br.
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