Interaction of calpastatin with calpain: a review

Calpastatin is a multiheaded inhibitor capable of inhibiting more than one calpain molecule. Each inhibitory domain of calpastatin has three subdomains, A, B, and C; A binds to domain IV and C binds to domain VI of the calpains. Crystallographic evidence shows that binding of C to domain VI involves hydrophobic interactions at a site near the first EF-hand in domain VI. Sequence homology suggests that binding of A to calpain domain IV also involves hydrophobic interactions near the EF1-hand of domain IV. Neither subdomain A nor C have inhibitory activity without subdomain B, but both increase the inhibitory activity of B. Subdomain B peptides have no inhibitory activity unless they contain at least 13 amino acids, and inhibitory activity increases with the number of amino acid residues, suggesting that inhibition requires interaction over a large area of the calpain molecule. Although subdomain B inhibition kinetically is competitive in nature, subdomain B does not seem to interact with the active site of the calpains directly, but may bind to domain III of the calpains and act to block access to the active site. It is possible that subdomain B binds to calpain only after it has been activated by Ca2+.     

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+.     

Inhibition of calpain by a synthetic oligopeptide corresponding to an exon of the human calpastatin gene

Calpastatin is a widely distributed endogenous inhibitor protein specifically acting on calpain (Ca2+-dependent cysteine endopeptidase). The inhibitor consists of four inhibitory domains (Domains 1-4) with mutually homologous sequences. NH2-terminal Domain L is non-homologous, and all domains have 120-140 residues each. A human calpastatin genomic DNA clone was isolated using a previously obtained human calpastatin cDNA probe. Sequence analysis has revealed that the clone contains Domain 1 and segments of neighboring domains (Domains L and 2). Each of three highly conserved, restricted regions within Domain 1 was located on separate exons, 1A, 1B, and 1C. Exon 2A, corresponding to the first exon of Domain 2, is homologous to Exon 1A and follows Exon 1D of Domain 1. A 27-residue peptide encoded by Exon 1B, including a 12-residue middle conserved sequence, was chemically synthesized and tested for protease inhibitory activities. The synthetic peptide showed strong inhibition against calpain I (low Ca2+-requiring form), and calpain II (high Ca2+-requiring form), but no inhibition against papain or trypsin. These results indicated that Exon 1B forms a self-sufficient functional subdomain of the calpastatin inhibitory domain.     

Structural determinants of the calpain inhibitory activity of calpastatin peptide B27-WT

Calpastatin is the natural specific inhibitor of calpain. Recent research has linked uncontrolled calpain activation to tissue damage after neuronal and cardiac ischemias, traumatic spine and brain injuries, as well as Alzheimer's disease and cataract formation. An imbalance between the activities of calpain and calpastatin is believed to be responsible for the pathological role of calpain. An important key to understanding calpain regulation by calpastatin is to determine, at the molecular level, how calpastatin interacts with calpain to inhibit its enzymatic activity. A 27-residue peptide (DPMSSTYIEELGKREVTIPPKYRELLA) derived from subdomain 1B of the repetitive domains of calpain, named peptide B27-WT, was previously shown to be a potent inhibitor of mu- and m-calpain. In this report, a combination of beta-alanine scanning mutagenesis and kinetic measurements was used to probe, in a quantitative, systematic, and simultaneous fashion, the relative contribution of the amino acid side chain and backbone functionalities to the overall calpain-inhibitory activity of B27-WT. The study identified two "hot spots," Leu(11)-Gly(12) and Thr(17)-Ile(18)-Pro(19), in B27-WT within which the residues critical for inhibitory function are clustered. Mutation of any one of the key residues in either of the two hot spots resulted in a dramatic loss of inhibitory activity. Furthermore, it was shown that a restricted conformation of the Leu(11)-Gly(12) and Thr(17)-Ile(18)-Pro(19) backbones is required for the peptide inhibitory function. These results suggest a plausible model in which the two hot spots are situated at or near the interface(s) of the calpain-calpastatin complex and act in a concerted fashion to inhibit calpain. The information on the specific contribution of the amide bond and side chain of each key residue to the bioactivity of B27-WT will contribute to a better understanding of the mechanism of calpain inhibition and lead to novel and effective therapies based on the specific inhibition of dysregulated or overactivated calpain.     

Local structural preferences of calpastatin, the intrinsically unstructured protein inhibitor of calpain

Calpain, the calcium-activated intracellular cysteine protease, is under the tight control of its intrinsically unstructured inhibitor, calpastatin. Understanding how potent inhibition by calpastatin can be reconciled with its unstructured nature provides deeper insight into calpain function and a more general understanding of how proteins devoid of a well-defined structure carry out their function. To this end, we performed a full NMR assignment of hCSD1 to characterize it in its solution state. Secondary chemical shift values and NMR relaxation data, R 1, R 2, and hetero-NOE, as well as spectral density function analysis have shown that conserved regions of calpastatin, subdomains A and C, which are responsible for calcium-dependent anchoring of the inhibitor to the enzyme, preferentially sample partially helical backbone conformations of a reduced flexibility. Moreover, the linker regions between subdomains are more flexible with no structural preference. The primary determinant of calpain inhibition, subdomain B, also has a non-fully random conformational preference, resembling a beta-turn structure also ascertained by prior studies of a 27-residue peptide encompassing the inhibitory region. This local structural preference is also confirmed by a deviation in chemical shift values between full-length calpastatin domain 1 and a truncated construct cut in the middle of subdomain B. At the C-terminal end of the molecule, a nascent helical region was found, which in contrast to the overall structural properties of the molecule may indicate a previously unknown functional region. Overall, these observations provide further evidence that supports previous suggestions that intrinsically unstructured proteins use preformed structural elements in efficient partner recognition.     

Interaction between catalytically inactive calpain and calpastatin. Evidence for its occurrence in stimulated cells

Conformational changes in the calpain molecule following interaction with natural ligands can be monitored by the binding of a specific monoclonal antibody directed against the catalytic domain of the protease. None of these conformational states showed catalytic activity and probably represent intermediate forms preceding the active enzyme state. In its native inactive conformation, calpain shows very low affinity for this monoclonal antibody, whereas, on binding to the ligands Ca(2+), substrate or calpastatin, the affinity increases up to 10-fold, with calpastatin being the most effective. This methodology was also used to show that calpain undergoes similar conformational changes in intact cells exposed to stimuli that induce either a rise in intracellular [Ca(2+)] or extensive diffusion of calpastatin into the cytosol without affecting Ca(2+) homeostasis. The fact that the changes in the calpain state are also observed under the latter conditions indicates that calpastatin availability in the cytosol is the triggering event for calpain-calpastatin interaction, which is presumably involved in the control of the extent of calpain activation through translocation to specific sites of action.     

Intracellular regulatory system involving calpain and calpastatin

Seven years have elapsed since the terms calpain and calpastatin were introduced. During these years, significant progress in research has been recorded. Thus, cloning and sequencing of cDNAs for calpains I and II and calpastatin have established amino acid sequences of these molecules. Structure-function relationship of calpastatin has been studied using mutated cDNAs expressed in E. coli. Interleukin 2 receptor-linked expression of calpastatin in HTLV-I-infected T-cells has been reported. Evidence for Ca2+-induced translocation of calpain to the cell membrane, followed by its autolytic activation, has been discussed. A great varieties of proteins such as several kinases, membrane and cytoskeletal proteins, and hormone receptors have been reported to be susceptible to calpains. This paper is to summarize our current knowledge on chemistry and biology of calpain and calpastatin and thereby to speculate the true function of calpains and their regulatory mechanisms.     

Characterization of the calpain/calpastatin system in human hemopoietic cell lines

As previously suggested by PCR analysis [R. DeTullio, R. Stifanese, F. Salamino, S. Pontremoli, E. Melloni, Characterization of a new p94-like calpain form in human lymphocytes, Biochem. J. 375 (2003) 689-696], a p94-like calpain was now established to be present in six different human cells resembling the various peripheral blood cell types. This protease resulted to be the predominant calpain isoforms whereas the conventional mu- and m-calpains are also expressed although at lower or almost undetectable amounts. The p94-like calpain has been identified by a specific mAb and displays unique features such as: Ca2+ requirement for half maximum activity around 30 microM; no autolytic conversion to a low Ca2+ requiring form and lower sensitivity to calpastatin inhibition. Following cell stimulation, the p94-like calpain undergoes inactivation, a process indicating that the protease is activated and participates in the cell responses to stimuli. The involvement of this protease isoform in immunocompetent cell activation is further supported by its partial recruitment on plasma membranes, the site of action of the conventional calpain forms. The amount of calpain translocated to the membranes correlates to the level of calpastatin which has been shown to control this process through the formation of a complex with calpain, which maintains the protease in the cytosol. These results provide new information on the calpain/calpastatin system expressed in immunocompetent cells and on the functional relationship between the p94-like calpain and the biological function of these cells.     

Association of calpain (Ca(2+)-dependent thiol protease) with its endogenous inhibitor calpastatin in myoblasts.

Calpain isozymes (intracellular, Ca(2+)-dependent thiol proteases) are present in the cytoplasm of many cells, along with their endogenous specific inhibitor, calpastatin. Previously, we found that the levels of mu-calpain and m-calpain (activated by microM and mM Ca(2+), respectively) remain about the same during myoblast differentiation and fusion. By contrast, the calpastatin level, which is high during the initial stages of differentiation, diminishes markedly before myoblast fusion, allowing the proteolysis that is required for myotube formation. In the present study, we used immunoprecipitation to investigate the molecular association between calpain and calpastatin in dividing myoblasts and in the initial stages of myoblast differentiation. Immunoprecipitation (IP) was performed in two ways: (1) IP of calpain, using an anti-calpain antibody that recognized both isozymes; and (2) IP of calpastatin (using anti-calpastatin). Calpastatin was co-precipitated when calpain was immunoprecipitated; calpain was co-precipitated when calpastatin was immunoprecipitated. The results indicate that calpastatin is associated with calpain in dividing myoblasts and in myoblasts during the initial stages of differentiation, thereby preventing calpain activation at this stage. Prior studies carried out in vitro have shown a Ca(2+)-dependent interaction of calpain with calpastatin. The results described here suggest that an association between calpain and calpastatin could occur within cells in the presence of physiological Ca(2+)levels. It is proposed that the status of cellular calpain-calpastatin association is modulated by cell constituents, for which some possibilities are suggested.     

Differential compartmentalization of the calpain/calpastatin network with the endoplasmic reticulum and Golgi apparatus

Calpain, a calcium-activated cysteine protease, is involved in modulating a variety of cell activities such as shape change, mobility, and apoptosis. The two ubiquitous isoforms of this protease, calpain I and II, are considered to be cytosolic proteins that can translocate to various sites in the cell. The activity of calpain is modulated by two regulatory proteins, calpastatin, the specific endogenous inhibitor of calpain, and the 28-kDa regulatory subunit. Using velocity gradient centrifugation, the results of this study confirm and greatly expand upon our previous finding that the calpain/calpastatin network is associated with the endoplasmic reticulum and Golgi apparatus in cells. Moreover, confocal microscopy demonstrates that calpain II colocalizes with specific proteins found in these organelles. Additional experiments reveal that hydrophobic rather than electrostatic interactions are responsible for the association of the calpain/calpastatin network with these organelles. Treatment of the organelles with Na2CO3 or deoxycholate reveal that calpain I, 78-kDa calpain II, and the regulatory subunit are "embedded" within the organelle membranes similar to integral membrane proteins. Proteinase K treatment of the organelles shows that calpain I and II, calpastatin, and the regulatory subunit localize to the cytosolic surface of the organelle membranes, and a subset of calpain II and the regulatory subunit are also found within the lumen of these organelles. These results provide a new and novel explanation for how the calpain/calpastatin network is organized in the cell.     

Caspases cleave the amino-terminal calpain inhibitory unit of calpastatin during apoptosis in human Jurkat T cells

We have previously reported the activation of procalpain mu (precursor for low-calcium-requiring calpain) in apoptotic cells using a cleavage-site-directed antibody specific to active calpain [Kikuchi, H. and Imajoh-Ohmi, S. (1995) Cell Death Differ. 2, 195-199]. In this study, calpastatin, the endogenous inhibitor protein for calpain, was cleaved to a 90-kDa polypeptide during apoptosis in human Jurkat T cells. The limited proteolysis of calpastatin preceded the autolytic activation of procalpain. Inhibitors for caspases rescued the cells from apoptosis and simultaneously inhibited the cleavage of calpastatin. The full-length recombinant calpastatin was also cleaved by caspase-3 or caspase-7 at Asp-233 into the same size fragment. Cys-241 was also targeted by these caspases in vitro but not in apoptotic cells. Caspase-digested calpastatin lost its amino-terminal inhibitory unit, and inhibited three moles of calpain per mole. Our findings suggest that caspases trigger the decontrol of calpain activity suppression by degrading calpastatin.     

Changes in contents of calpain and calpastatin in rat liver during growth

Variation of calpain I, calpain II, and calpastatin in rat liver during growth from 0 to 14 weeks was studied by chromatographic fractionation of the liver cytosol and enzyme assays on the eluted fractions. When compared in terms of units per g wet liver, high-Ca2+-requiring calpain II always exceeded low-Ca2+-requiring calpain I in male and female rats. The level of calpain II in neonatal (0 week) rat liver was 1.9-2.9 times higher than that for the adults (7 to 14 weeks). The contents of calpastatin, calpain-specific inhibitor protein, were were always higher than those of calpain II in adult rat liver, but the difference was much less, or sometimes even reversed, in neonatal and young (1 and 2 weeks) animals. In general, the variation was more pronounced in female than in male rats.     

The calpastatin/calpain system in trout Salmo trutta trutta muscle

Many recent reports suggest that the calpastatin/calpain system plays a role in cellular growth and differentiation. Defects of the calpastatin/calpain system have been linked to cellular dysfunctions, apoptosis, myocardial infarct, and dystrophies. The calpastatin/calpain system has also been implicated in post‐mortem tenderization of skeletal muscle through degradation of key myofibrillar and associated proteins, a process of key importance to meat quality. In the present study we investigate the presence and activity of the calpastatin/calpain system in trout muscle samples, collected at 0, 3, 18 and 28 h post‐mortem, by immunohistochemistry method. Calpastatin is a specific endogenous enzyme of cytosol, modulating the ubiquitous calpains. Calpastatin was found in samples obtained in vivo and immediately post‐mortem, but its concentration declined rapidly in samples obtained 3, 18 and 28 h post‐mortem. The ubiquitous m e m‐calpains, which are localized on Z line proteins and activated by intracellular Ca²⁺ increase, showed a rapid decline within 3 h post‐mortem. By contrast p94 calpain, which is specific to skeletal muscle, showed a slow decrease post‐mortem which was independent of intracellular Ca²⁺ increase. Our results suggest that the mechanism of activation and activity of the calpastatin/calpain system in trout is similar to that described in mammals.     

Fragmentation of a 70000-dalton calpastatin molecule upon its complex formation with calpain

Homogenously purified porcine calpain I (Mr 112000), a low-Ca2+-requiring form of Ca2+-dependent cysteine proteinase [EC 3.4.22.17], was coupled to Sepharose 4B gel as an active form. It was used as a ligand to calpastatin (Mr 70000), calpain-specific inhibitor protein, for an affinity chromatography. Only in the presence of Ca2+, calpastatin bound to calpain-Sepharose, but the interaction resulted in rather extensive fragmentation of a calpastatin molecule into several peptides of Mr 14000 to 70000, which still retain inhibitory activities against calpain. Fragmentation was demonstrated both by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and by high-performance liquid chromatography in the presence of 6 M guanidine-HCl.     

Developmental changes of calpain and calpastatin in rabbit brain

A major part of the Ca-activated proteolytic activity in the soluble fraction from rabbit brain could be due to the activity of the neutral thiol-proteases calpain I and II. The activity of calpains exceeded that of the endogenous inhibitor, calpastatin, at all developmental stages studied. The level of calpains increased rapidly from the prenatal stage to reach a peak 10–20 days postnatally. From this period the level of calpains decreased slowly to reach the adult levels. The level of calpastatin increased steadily from the prenatal stage to old age.     

Calcium-induced tripartite binding of intrinsically disordered calpastatin to its cognate enzyme, calpain

The activity of calpain is controlled by the free intracellular calcium level and by the protein's intrinsically disordered endogenous inhibitor, calpastatin, mediated by short conserved segments: subdomains A-C. The exact binding mode of calpastatin to the enzyme has until now been unclear. Our NMR data of the 141 amino acid long inhibitor, with and without calcium and calpain, have revealed structural changes and a tripartite binding mode, in which the disordered inhibitor wraps around, and contacts, the enzyme at three points, facilitated by flexible linkers. This unprecedented binding mode permits a unique combination of specificity, speed and binding strength in regulation.     

A simple one-step procedure for the separation of calpain I, calpain II and calpastatin.

The soluble fraction from rabbit brain was adsorbed on a column of phenyl-Sepharose. By applying a linear gradient with decreasing salt concentration and increasing pH, it was possible to separate calpain I and calpain II from each other and from the endogenous inhibitor calpastatin. Both enzymes were capable of degrading endogenously labelled neuronal proteins, including slowly axonally transported soluble proteins and rapidly transported membrane-bound proteins, as well as casein.     

A region of calpastatin domain L that reprimes cardiac L-type Ca2+ channels

Calpastatin, an endogenous inhibitor of calpain, is composed of domain L and four repetitive homologous domains 1-4. Domains 1-4 inhibit calpain, whereas domain L partially reprimes L-type Ca2+ channels for voltage-gated activation. In the present study, the effects on Ca2+ channel activity of four isoforms and a series of fragments of calpastatin domain L were investigated in guinea-pig ventricular myocytes with the patch-clamp method. With one exception, all the isoforms and fragment peptides that contained amino acid residues 54-64 of domain L reprimed the Ca2+ channels to comparable levels (9-15% of control activity) to those observed previously with a full-length form of calpastatin. These results suggest that the region containing amino acid residues 54-64 (EGKPKEHTEPK) is responsible for the Ca2+ channel repriming function of calpastatin domain L.     

Cystatins as calpain inhibitors: engineered chicken cystatin- and stefin B-kininogen domain 2 hybrids support a cystatin-like mode of interaction with the catalytic subunit of mu-calpain

Within the cystatin superfamily, only kininogen domain 2 (KD2) is able to inhibit mu- and m-calpain. In an attempt to elucidate the structural requirements of cystatins for calpain inhibition, we constructed recombinant hybrids of human stefin B (an intracellular family 1 cystatin) with KD2 and deltaL110 deletion mutants of chicken cystatin-KD2 hybrids. Substitution of the N-terminal contact region of stefin B by the corresponding KD2 sequence resulted in a calpain inhibitor of Ki = 188 nM. Deletion of L110, which forms a beta-bulge in family 1 and 2 cystatins but is lacking in KD2, improved inhibition of mu-calpain 4- to 8-fold. All engineered cystatins were temporary inhibitors of calpain due to slow substrate-like cleavage of a single peptide bond corresponding to Gly9-Ala10 in chicken cystatin. Biomolecular interaction analysis revealed that, unlike calpastatin, the cystatin-type inhibitors do not bind to the calmodulin-like domain of the small subunit of calpain, and their interaction with the mu-calpain heterodimer is completely prevented by a synthetic peptide comprising subdomain B of calpastatin domain 1. Based on these results we propose that (i) cystatin-type calpain inhibitors interact with the active site of the catalytic domain of calpain in a similar cystatin-like mode as with papain and (ii) the potential for calpain inhibition is due to specific subsites within the papain-binding regions of the general cystatin fold.