Autolysis of mu- and m-calpain from bovine skeletal muscle

The rate of autolysis of mu- and m-calpain from bovine skeletal muscle was measured by using densitometry of SDS polyacrylamide gels and determining the rate of disappearance of the 28 and 80 kDa subunits of the native, unautolyzed calpain molecules. Rate of autolysis of both the 28 and 80 kDa subunits of mu-calpain decreased when mu-calpain concentration decreased and when beta-casein, a good substrate for the calpains, was present. Hence, autolysis of both mu-calpain subunits is an intermolecular process at pH 7.5, 0 or 25.0 degrees C, and low ionic strength. The 78 kDa subunit formed in the first step of autolysis of m-calpain was not resolved from the 80 kDa subunit of the native, unautolyzed m-calpain by our densitometer, so autolysis of m-calpain was measured by determining rate of disappearance of the 28 kDa subunit and the 78/80 kDa complex. At Ca2+ concentrations of 1000 microM or higher, neither the m-calpain concentration nor the presence of beta-casein affected the rate of autolysis of m-calpain. Hence, m-calpain autolysis is intramolecular at Ca2+ concentrations of 1000 microM or higher and pH 7.5. At Ca2+ concentrations of 350 microM or less, the rate of m-calpain autolysis decreased with decreasing m-calpain concentration and in the presence of beta-casein. Thus, m-calpain autolysis is an intermolecular process at Ca2+ concentrations of 350 microM or less. If calpain autolysis is an intermolecular process, autolysis of a membrane-bound calpain would require selective participation of a second, cytosolic calpain, making it an inefficient process. By incubating the calpains at Ca2+ concentrations below those required for half-maximal activity, it is possible to show that unautolyzed calpains degrade a beta-casein substrate, proving that unautolyzed calpains are active proteases.     

Efficient expression and purification of recombinant human m-calpain using an Escherichia coli expression system at low temperature

Calpain belongs to the superfamily of Ca(2+)-regulated cysteine proteases, which are indispensable to the regulation of various cellular functions. Of the 15 mammalian calpain isoforms, μ- and m-calpains are the best characterized. Both μ- and m-calpain are ubiquitously expressed and exist as heterodimers, containing a distinct 80-kDa catalytic subunit (CAPN1 and CAPN2, respectively) and the common, 30-kDa regulatory subunit (CAPNS1). To date, various expression systems have been developed for producing recombinant calpains for use in structural and physiological studies, however Escherichia coli systems have proven incompatible with large-scale preparation of calpain, with the exception of rat m-calpain. Here, we have established a highly efficient method to purify active recombinant human m-calpain using an E. coli expression system at low temperature (22°C). This was achieved by co-expressing CAPN2 with a C-terminal histidine-tag, and CAPNS1, lacking the first Gly-repeated region at the N-terminal. After three sequential passes through a chromatographic column, ~5 mg of human m-calpain was homogenously purified from 1 l of E. coli culture. Proteins were stable for several months. This is the first report of efficient, large-scale purification of recombinant human m-calpain using an E. coli expression system.     

Calpain as a novel regulator of autophagosome formation

Ubiquitously expressed micro- and m-calpain proteases consist of 80-kDa catalytic subunits encoded by the Capn1 and Capn2 genes, respectively, and a common 28-kDa regulatory subunit encoded by the calpain small 1 (Capns1) gene. The micro- and m-calpain proteases have been implicated in both pro- or anti-apoptotic functions. We have found that Capns1 depletion is coupled to increased sensitivity to apoptosis triggered by a number of autophagy-inducing stimuli in mammalian cells. Therefore we investigated the involvement of calpains in autophagy using MEFs derived from Capns1 knockout mice and Capns1 depleted human cells as model systems. We found that autophagy is impaired in Capns1-deficient cells by immunostaining of the endogenous autophagosome marker LC3 and electron microscopy experiments. Accordingly, the enhancement of lysosomal activity and long-lived proteins degradation, normally occurring upon starvation, are also reduced. In Capns1-depleted cells ectopic LC3 accumulates in early endosome-like vesicles that might represent a salvage pathway for protein degradation when autophagy is defective.     

Binding of calpain fragments to calpastatin

Their cDNA-derived amino acid sequences predict that the 80-kDa subunits of the micromolar and millimolar Ca(2+)-requiring forms of the Ca(2+)-dependent proteinase (mu- and m-calpain, respectively) each consist of four domains and that the 28-kDa subunit common to both mu- and m-calpain consists of two domains. The calpains were allowed to autolyze to completion, and the autolysis products were separated and were characterized by using gel permeation chromatography, calpastatin affinity chromatography, and sequence analysis. Three major fragments were obtained after autolysis of either calpain. The largest fragment (34 kDa for mu-calpain, 35 kDa for m-calpain) contains all of domain II, the catalytic domain, plus part of domain I of the 80-kDa subunit of mu- or m-calpain. This fragment does not bind to calpastatin, a competitive inhibitor of the calpains, and has no proteolytic activity in either the absence or presence of Ca2+. The second major fragment (21 kDa for mu-calpain and 22 kDa for m-calpain) contains domain IV, the calmodulin-like domain, plus approximately 50 amino acids from domain III of the 80-kDa subunit of mu- or m-calpain. The third major fragment (18 kDa) contains domain VI, the calmodulin-like domain of the 28-kDa subunit. The second and third major fragments bind to a calpastatin affinity column in the presence of Ca2+ and are eluted with EDTA. The second and third fragments are noncovalently bound, so the 80- and 28-kDa subunits of the intact calpain molecules are noncovalently bound at domains IV and VI. After separation in 1 M NaSCN, the 28-kDa subunit binds completely to calpastatin, approximately 30-40% of the 80-kDa subunit of mu-calpain binds to calpastatin, and the 80-kDa subunit of m-calpain does not bind to calpastatin in the presence of 1 mM Ca2+.     

Digestion of mu- and m-calpain by trypsin and chymotrypsin

Proteolytic digestion by trypsin and chymotrypsin was used to probe conformation and domain structure of the mu- and m-calpain molecules in the presence and the absence of Ca(2+). Both calpains have a compact structure in the absence of Ca(2+); incubation with either protease for 120 min results in only three or four major fragments. A 24-kDa fragment was produced by removal of the Gly-rich area in domain V of the 28-kDa subunit. The other fragments were from the 80-kDa subunit. Except for trypsin digestion of m-calpain, the region between amino acids 245 and 265 (human sequence) was very susceptible to cleavage by both proteases in the absence of Ca(2+); this region is in domain II (IIb of the crystallographic structure). Although no proteolytically active fragments could be isolated from either tryptic or chymotryptic digests, the calpain molecule can remain assembled in a proteolytically active complex even after the 80-kDa subunit has been completely degraded. The results suggest that interaction among different regions of the entire calpain molecule is required for its full proteolytic activity. In the presence of 1 mM Ca(2+), both calpains are degraded to fragments less than 40-kDa in less than 5 min. The C-terminal ends of both subunits, from amino acids 503 to 506 to the end of the 80-kDa subunit and from amino acids 85 to 88 to the end of the 28-kDa subunit, were resistant to degradation by either protease in the presence or in the absence of Ca(2+). Hence, this part of the calpain molecule is in a compact structure that does not change significantly in the presence of Ca(2+).     

Ubiquitous calpains promote caspase-12 and JNK activation during endoplasmic reticulum stress-induced apoptosis

Ubiquitously expressed mu- and m-calpain proteases are implicated in development and apoptosis. They consist of 80-kDa catalytic subunits encoded by the capn1 and capn2 genes, respectively, and a common 28-kDa regulatory subunit encoded by the capn4 gene. The regulatory subunit is required to maintain the stability and activity of mu- and m-calpains. Accordingly, genetic disruption of capn4 in the mouse eliminated both ubiquitous calpain activities. In embryonic fibroblasts derived from these mice, calpain deficiency correlated with resistance to endoplasmic reticulum (ER) stress-induced apoptosis, and this was directly related to a calpain requirement for activation of both caspase-12 and the ASK1-JNK cascade. This study provides compelling genetic evidence for calpain's role in caspase-12 activation at the ER, and reveals a novel role for the ubiquitous calpains in ER-stress induced apoptosis and JNK activation.     

Sequence comparison among muscle-specific calpain, p94, and calpain subunits.

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.     

Dissociation of m-calpain subunits occurs after autolysis of the N-terminus of the catalytic subunit, and is not required for activation.

Calpain is a heterodimeric, intracellular Ca(2+)-dependent, "bio-modulator" that alters the properties of substrates through site-specific proteolysis. It has been proposed that calpains are activated by autolysis of the N-terminus of the large subunit and/or its dissociation into the subunits. It is, however, unclear whether the dissociation into subunits is required for the expression of protease activity and/or for in vivo function. Recently, the crystal structure of m-calpain in the absence of Ca(2+) has been resolved. The 3D structure clearly shows that the N-terminus of the m-calpain large subunit (mCL) makes contact with the 30K subunit, suggesting that autolysis of the N-terminus of mCL changes the interaction of both subunits. To examine the relationship between autolysis, dissociation, and activation, we made and analysed a series of N-terminal mutants of mCL that mimic the autolysed forms or have substituted amino acid residue(s) interacting with 30K. As a result, the mutant m-calpains, which are incapable of autolysis, did not dissociate into subunits, whereas those lacking the N-terminal 19 residues (Delta 19), but not those lacking only nine residues (Delta 9), dissociated into subunits even in the absence of Ca(2+). Moreover, both Delta 9 and Delta 19 mutants showed an equivalent reduced Ca(2+) requirement for protease activity. These results indicate that autolysis is necessary for the dissociation of the m-calpain subunits, and that the dissociation occurs after, but is not necessary for, activation.     

Dissociation and aggregation of calpain in the presence of calcium

Calpain is a heterodimeric Ca(2+)-dependent cysteine protease consisting of a large (80 kDa) catalytic subunit and a small (28 kDa) regulatory subunit. The effects of Ca(2+) on the enzyme include activation, aggregation, and autolysis. They may also include subunit dissociation, which has been the subject of some debate. Using the inactive C105S-80k/21k form of calpain to eliminate autolysis, we have studied its disassociation and aggregation in the presence of Ca(2+) and the inhibition of its aggregation by means of crystallization, light scattering, and sedimentation. Aggregation, as assessed by light scattering, depended on the ionic strength and pH of the buffer, on the Ca(2+) concentration, and on the presence or absence of calpastatin. At low ionic strength, calpain aggregated rapidly in the presence of Ca(2+), but this was fully reversible by EDTA. With Ca(2+) in 0.2 m NaCl, no aggregation was visible but ultracentrifugation showed that a mixture of soluble high molecular weight complexes was present. Calpastatin prevented aggregation, leading instead to the formation of a calpastatin-calpain complex. Crystallization in the presence of Ca(2+) gave rise to crystals mixed with an amorphous precipitate. The crystals contained only the small subunit, thereby demonstrating subunit dissociation, and the precipitate was highly enriched in the large subunit. Reversible dissociation in the presence of Ca(2+) was also unequivocally demonstrated by the exchange of slightly different small subunits between mu-calpain and m-calpain. We conclude that subunit dissociation is a dynamic process and is not complete in most buffer conditions unless driven by factors such as crystal formation or autolysis of active enzymes. Exposure of the hydrophobic dimerization surface following subunit dissociation may be the main factor responsible for Ca(2+)-induced aggregation of calpain. It is likely that dissociation serves as an early step in calpain activation by releasing the constraints upon protease domain I.     

m-Calpain activation in vitro does not require autolysis or subunit dissociation

Calpains are Ca(2+)-dependent, intracellular cysteine proteases involved in many physiological functions. How calpains are activated in the cell is unknown because the average intracellular concentration of Ca(2+) is orders of magnitude lower than that needed for half-maximal activation of the enzyme in vitro. Two of the proposed mechanisms by which calpains can overcome this Ca(2+) concentration differential are autoproteolysis (autolysis) and subunit dissociation, both of which could release constraints on the core by breaking the link between the anchor helix and the small subunit to allow the active site to form. By measuring the rate of autolysis at different sites in calpain, we show that while the anchor helix is one of the first targets to be cut, this occurs in the same time-frame as several potentially inactivating cleavages in Domain III. Thus autolytic activation would overlap with inactivation. We also show that the small subunit does not dissociate from the large subunit, but is proteolyzed to a 40-45k heterodimer of Domains IV and VI. It is likely that this autolysis-generated heterodimer has previously been misidentified as the small subunit homodimer produced by subunit dissociation. We propose a model for m-calpain activation that does not involve either autolysis or subunit dissociation.     

Effects of autolysis on properties of mu- and m-calpain

Although the biochemical changes that occur during autolysis of mu- and m-calpain are well characterized, there have been few studies on properties of the autolyzed calpain molecules themselves. The present study shows that both autolyzed mu- and m-calpain lose 50-55% of their proteolytic activity within 5 min during incubation at pH 7.5 in 300 mM or higher salt and at a slower rate in 100 mM salt. This loss of activity is not reversed by dialysis for 18 h against a low-ionic-strength buffer at pH 7.5. Proteolytic activity of the unautolyzed calpains is not affected by incubation for 45 min at ionic strengths up to 1000 mM. Size-exclusion chromatography shows that ionic strengths of 100 mM or above cause dissociation of the two subunits of autolyzed calpains and that the dissociated large subunits (76- or 78-kDa) aggregate to form dimers and trimers, which are proteolytically inactive. Hence, instability of autolyzed calpains is due to aggregation of dissociated heavy chains. Autolysis removes the N-terminal 19 (m-calpain) or 27 (mu-calpain) amino acids from the large subunit and approximately 90 amino acids from the N-terminus of the small subunit. These regions form contacts between the two subunits in unautolyzed calpains, and their removal leaves only contacts between domain IV in the large subunit and domain VI in the small subunit. Although many of these contacts are hydrophobic in nature, ionic-strength-induced dissociation of the two subunits in the autolyzed calpains indicates that salt bridges have an important, possibly indirect, role in the domain IV/domain VI interaction.     

Conditional disruption of ubiquitous calpains in the mouse

Ubiquitous mu- and m-calpain proteases are implicated in development and apoptosis. They are heterodimers consisting of 80-kDa catalytic subunits encoded by capn1 and capn2, respectively, and a common 28-kDa regulatory subunit encoded by capn4. The regulatory subunit is required to maintain stability and activity of mu- and m-calpains; thus, genetic disruption of capn4 was predicted to eliminate both calpain activities. Germline disruption of capn4 caused embryonic lethality, hampering the use of those mouse models to explore physiological calpain functions. Here we describe a loxP/cre conditional capn4 targeted mouse model that enables tissue-specific and temporal deletion of calpain activity. Disruption of the floxed capn4 gene using a ubiquitous cytomegalovirus promoter driven Cre recombinase transgene led to midgestation embryonic lethality. Fibroblasts from these embryos lacked detectable regulatory subunit expression, had reduced levels of the mu- and m-calpain catalytic subunits, and had no detectable mu- and m-calpain activities. These defects were corrected with a capn4-encoding lentivirus.     

Fetuin A stabilizes m-calpain and facilitates plasma membrane repair

Yeast two-hybrid experiments identified alpha(2)-Heremans-Schmid glycoprotein (human fetuin A) as a binding partner for calpain domain III (DIII). The tandem DIIIs of calpain-10 interacted under the most selective culture conditions, but DIIIs of m-calpain, calpain-3, and calpain-5 also interacted under less stringent selection. DIIIs of mu-calpain, calpain-6, and the tandem DIII-like domains of the Dictyostelium Cpl protein did not interact with alpha(2)-Heremans-Schmid glycoprotein in the yeast two-hybrid system. Bovine fetuin A stabilized proteolytic activity of purified m-calpain incubated in the presence of mm calcium chloride and prevented calcium-dependent m-calpain aggregation. Consistent with the yeast two-hybrid studies, fetuin A neither stabilized mu-calpain nor prevented its aggregation. Confocal immunofluorescence microscopy of scratch-damaged L6 myotubes demonstrated accumulation of m-calpain at the wound site in association with the membrane repair protein, dysferlin. m-Calpain also co-localized with fluorescein-labeled fetuin A at the wound site. The effect of fetuin A on calpain-mediated plasma membrane resealing was investigated using fibroblasts from Capns1(-/-) and Capns1(+/+) mouse embryos. Capns1 encodes the small noncatalytic subunit that is required for the proteolytic function of m- and mu-calpains. Thus, Capns1(-/-) fibroblasts do not express these calpains in active form. Fetuin A increased resealing of scrape-damaged wild-type fibroblasts but not Capns1(-/-) fibroblasts. These studies identify fetuin A as a potential extracellular regulator of m-calpain at nascent sites of plasma membrane wounding.     

Purification and characterization of calpain and calpastatin from rainbow trout, Oncorhynchus mykiss

Although the calpain system has been studied extensively in mammalian animals, much less is known about the properties of μ-calpain, m-calpain, and calpastatin in lower vertebrates such as fish. These three proteins were isolated and partly characterized from rainbow trout, Oncorhynchus mykiss, muscle. Trout m-calpain contains an 80-kDa large subunit, but the  26-kDa small subunit from trout m-calpain is smaller than the 28-kDa small subunit from mammalian calpains. Trout μ-calpain and calpastatin were only partly purified; identity of trout μ-calpain was confirmed by labeling with antibodies to bovine skeletal muscle μ-calpain, and identity of trout calpastatin was confirmed by specific inhibition of bovine skeletal muscle μ- and m-calpain. Trout μ-calpain requires 4.4 ± 2.8 μM and trout m-calpain requires 585 ± 51 μM Ca²⁺ for half-maximal activity, similar to the Ca²⁺ requirements of μ- and m-calpain from mammalian tissues. Sequencing tryptic peptides indicated that the amino acid sequence of trout calpastatin shares little homology with the amino acid sequences of mammalian calpastatins. Screening a rainbow trout cDNA library identified three cDNAs encoding for the large subunit of a putative m-calpain. The amino acid sequence predicted by trout m-calpain cDNA was 65% identical to the human 80-kDa m-calpain sequence. Gene duplication and polyploidy occur in fish, and the amino acid sequence of the trout m-calpain 80-kDa subunit identified in this study was 83% identical to the sequence of a trout m-calpain 80-kDa subunit described earlier. This is the first report of two isoforms of m-calpain in a single species.     

Disruption of the murine calpain small subunit gene, Capn4: calpain is essential for embryonic development but not for cell growth and division

Calpains are a family of Ca(2+)-dependent intracellular cysteine proteases, including the ubiquitously expressed micro- and m-calpains. Both mu- and m-calpains are heterodimers, consisting of a distinct large 80-kDa catalytic subunit, encoded by the genes Capn1 and Capn2, and a common small 28-kDa regulatory subunit (Capn4). The physiological roles and possible functional distinctions of mu- and m-calpains remain unclear, but suggested functions include participation in cell division and migration, integrin-mediated signal transduction, apoptosis, and regulation of cellular control proteins such as cyclin D1 and p53. Homozygous disruption of murine Capn4 eliminated both mu- and m-calpain activities, but this did not affect survival and proliferation of cultured embryonic stem cells or embryonic fibroblasts, or the early stages of organogenesis. However, mutant embryos died at midgestation and displayed defects in the cardiovascular system, hemorrhaging, and accumulation of erythroid progenitors.     

Tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induces phosphorylation of mu- and m-calpain in association with increased secretion, cell migration, and invasion

Mounting evidence indicates that cigarette smoking not only promotes tumorigenesis but also may increase the spread of cancer cells in the body. However, the intracellular mechanism(s) by which cigarette smoking promotes metastasis of human lung cancer remains enigmatic. Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an important component in cigarette smoke and is formed by nitrosation of nicotine. mu- and m-calpain (calpain I and calpain II) are major members of the calpain family, which are ubiquitously expressed in both small cell lung cancer and non-small cell lung cancer cells. Our findings indicated that NNK potently induces phosphorylation of both mu- and m-calpain in association with their activation and increased migration as well as invasion of lung cancer cells. Treatment of cells with PD98059 blocked phosphorylation of m- and mu-calpain and resulted in suppression of NNK-induced cell migration and invasion. p44 MAPK/extracellular signal-regulated kinase 1 (ERK1) and p42 MAPK/ERK2 were activated by NNK, co-localized with mu- and m-calpain in cytoplasm, and directly phosphorylated mu- and m-calpain in vitro. These findings suggest a role for the ERK1/2 kinases as NNK-activated physiological calpain kinases. Specific knock-down of mu- and/or m-calpain expression by RNA interference blocked NNK-stimulated migration and invasion, suggesting that mu- and m-calpain may act as required targets in a NNK-induced metastatic signaling pathway. Furthermore, NNK promotes secretion of active mu- and m-calpain from lung cancer cells through vesicles, which may have the potential to cleave substrates in the extracellular matrix. Thus, NNK-induced cell migration and invasion may occur, at least in part, through a novel mechanism involving phosphorylation of calpains that leads to their activation and secretion, which may contribute to metastasis and/or progression of lung cancer.     

Selenoprotein K is a novel target of m-calpain, and cleavage is regulated by Toll-like receptor-induced calpastatin in macrophages

Calpains are proteolytic enzymes that modulate cellular function through cleavage of targets, thereby modifying their actions. An important role is emerging for calpains in regulating inflammation and immune responses, although specific mechanisms by which this occurs have not been clearly defined. In this study, we identify a novel target of calpain, selenoprotein K (SelK), which is an endoplasmic reticulum transmembrane protein important for Ca(2+) flux in immune cells. Calpain-mediated cleavage of SelK was detected in myeloid cells (macrophages, neutrophils, and dendritic cells) but not in lymphoid cells (B and T cells). Both m- and μ-calpain were capable of cleaving immunoprecipitated SelK, but m-calpain was the predominant isoform expressed in mouse immune cells. Consistent with these results, specific inhibitors were used to show that only m-calpain cleaved SelK in macrophages. The cleavage site in SelK was identified between Arg(81) and Gly(82) and the resulting truncated SelK was shown to lack selenocysteine, the amino acid that defines selenoproteins. Resting macrophages predominantly expressed cleaved SelK and, when activated through different Toll-like receptors (TLRs), SelK cleavage was inhibited. We found that decreased calpain cleavage was due to TLR-induced up-regulation of the endogenous inhibitor, calpastatin. TLR-induced calpastatin expression not only inhibited SelK cleavage, but cleavage of another calpain target, talin. Moreover, the expression of the calpain isoforms and calpastatin in macrophages were different from T and B cells. Overall, our findings identify SelK as a novel calpain target and reveal dynamic changes in the calpain/calpastatin system during TLR-induced activation of macrophages.     

小鼠canstatin C端片段基因的克隆及其在大肠杆菌中的表达

为了构建小鼠canstatinC端片段的原核表达载体并在大肠杆菌中表达。以小鼠肝脏组织总RNA为模板,通过RT-PCR扩增小鼠canstatinC端片段(mCan-C)基因,克隆到pMD18-T载体中并进行序列分析。将mCan-C基因定向克隆于原核表达载体pET30a(+)中,构建表达载体pET／mCan-C,转化大肠杆菌BL21(DE3),IPTG诱导表达。结果表明,小鼠canstatinC端片段的cDNA长度为399bp,含有1个终止密码,编码132个氨基酸,与已知的人canstatinC端片段氨基酸的同源性为61％。IPTG诱导mCan-C在大肠杆菌E．coliBL21中表达,表达量约占菌体总蛋白量的28％,重组蛋白主要以包涵体形式存在。首次克隆了小鼠canstatinC端片段的cDNA,IPTG诱导mCan-C在大肠杆菌E．coliBL21中高效表达。小鼠canstatinC端片段的cDNA序列已收入GenBank,接受号为:AY502947。     

Activation of calpain by Ca2+: roles of the large subunit N-terminal and domain III-IV linker peptides

The calpains are a family of cysteine proteases with closely related amino acid sequences, but a wide range of Ca(2+) requirements (K(d)). For m-calpain, K(d) is approximately 325microM, for mu-calpain it is approximately 50microM, and for calpain 3 it is not strictly known but may be approximately 0.1microM. On the basis of previous structure determination of m-calpain we postulated that two regions of the calpain large subunits, the N-terminal peptide (residues 1-20) and a domain III-IV linker peptide (residues 514-530 in m-calpain) were important in defining K(d). The mutations Lys10Thr in the N-terminal peptide, and Glu517Pro in the domain linker peptide, reduced K(d) of m-calpain by 30% and 42%, respectively, revealing that these two regions are functionally important. The increased Ca(2+)-sensitivity of these mutants demonstrate that the Lys10-Asp148 salt link and the short beta-sheet interaction involving Glu517 are factors contributing to the high K(d) of m-calpain. Though these two regions are physically remote from the active site and Ca(2+)-binding site, they play significant roles in regulating the response of calpain to Ca(2+). Differences in these interactions in mu-calpain and in calpain 3 are also consistent with their progressively lower K(d) values.     

Acyl-CoA-binding protein is a potent m-calpain activator

Acyl-CoA-binding protein, a 20-kDa homodimer that exerts many physiological functions, promotes activation of the classic calpain forms, most markedly that of the m-isozyme. This protein factor was purified from rat skeletal muscle and was also expressed in Escherichia coli. Both native and recombinant acyl-CoA-binding proteins show the same molecular properties and an identical capacity to decrease the [Ca(2+)] required for m-calpain activity. The binding of long-chain acyl-CoAs to acyl-CoA-binding protein does not modify the activating effect on calpains. Acyl-CoA-binding protein seems to be involved in the m-calpain regulation process, whereas the previously identified UK114 activator is a specific modulator of micro-calpain. Acyl-CoA-binding protein is proposed as a new component of the Ca(2+)-dependent proteolytic system. A comparative analysis among levels of classic calpains and their activator proteins is also reported.