Calpain 1-titin interactions concentrate calpain 1 in the Z-band edges and in the N2-line region within the skeletal myofibril

Calpain 1, a ubiquitous calcium-dependent intracellular protease, was recently found in a tight association with myofibrils in skeletal muscle tissue [Delgado EF, Geesink GH, Marchello JA, Goll DE & Koohmaraie M (2001) J Anim Sci79, 2097-2107). Our immunofluorescence and immunoelectron microscopy investigations restrain the protease location at the periphery of the Z-band and at the midpoint of the I-band. Furthermore, calpain 1 is found to localize in myofibril fractures, described as proteolysis sites, in postmortem bovine skeletal red muscles, near the calcium deposits located at the N1 and N2 level. This in situ localization of calpain 1 is substantiated by binding assays with two titin regions covering the I-band region: a native fragment of 150 kDa (identified by mass spectrometry) that includes the N-terminal Z8-I5 region and the N1-line region of titin, and an 800 kDa fragment external to the N1 line that bears the PEVK/N2 region. These two titin fragments are shown to tightly bind calpain 1 in the presence of CaCl(2) and E64, a calpain inhibitor. In the absence of E64, they are cleaved by calpain 1. We conclude that titin affords binding sites to calpain 1, which concentrates the protease in the regions restrained by the Z-band edge and the N1-line as well as at the N2-line level, two sarcomeric regions where early postmortem proteolysis is detected.     

Titin-actin interaction in mouse myocardium: passive tension modulation and its regulation by calcium/S100A1

Passive tension in striated muscles derives primarily from the extension of the giant protein titin. However, several studies have suggested that, in cardiac muscle, interactions between titin and actin might also contribute to passive tension. We expressed recombinant fragments representing the subdomains of the extensible region of cardiac N2B titin (tandem-Ig segments, the N2B splice element, and the PEVK domain), and assayed them for binding to F-actin. The PEVK fragment bound F-actin, but no binding was detected for the other fragments. Comparison with a skeletal muscle PEVK fragment revealed that only the cardiac PEVK binds actin at physiological ionic strengths. The significance of PEVK-actin interaction was investigated using in vitro motility and single-myocyte mechanics. As F-actin slid relative to titin in the motility assay, a dynamic interaction between the PEVK domain and F-actin retarded filament sliding. Myocyte results suggest that a similar interaction makes a significant contribution to the passive tension. We also investigated the effect of calcium on PEVK-actin interaction. Although calcium alone had no effect, S100A1, a soluble calcium-binding protein found at high concentrations in the myocardium, inhibited PEVK-actin interaction in a calcium-dependent manner. Gel overlay analysis revealed that S100A1 bound the PEVK region in vitro in a calcium-dependent manner, and S100A1 binding was observed at several sites along titin's extensible region in situ, including the PEVK domain. In vitro motility results indicate that S100A1-PEVK interaction reduces the force that arises as F-actin slides relative to the PEVK domain, and we speculate that S100A1 may provide a mechanism to free the thin filament from titin and reduce titin-based tension before active contraction.     

Domain structure and interactions of the type I and type II modules in the gelatin-binding region of fibronectin. All six modules are independently folded

The gelatin-binding region of fibronectin is isolated easily as a stable and functional 42 kDa fragment containing four type I "finger" modules and two type II "kringle-like" modules arranged in the order I6-II1-II2-I7-I8-I9. This fragment exhibits a single reversible melting transition near 64 degrees C in TBS buffer (0.02 M-Tris buffer containing 0.15 M-NaCl, pH 7.4). The transition is characterized by a calorimetric to van't Hoff enthalpy ratio of 1.6, suggesting a complex domain structure. A 30 kDa fragment with the same NH2 terminus (I6-II1-II2-I7) melts reversibly near 65 degrees C with delta Hcal/delta HvH = 1.3, also consistent with the presence of more than one domain. To elucidate further the domain structure, three non-overlapping subfragments were prepared and characterized with respect to their unfolding induced by heat and guanidinium chloride. The three subfragments, each containing two modules, are designated from amino or carboxyl-terminal location as 13 kDa (I6-II1) 16 kDa (II2-I7) and 21 kDa (I8-I9) according to their apparent Mr in SDS/polyacrylamide gel electrophoresis. All three subfragments exhibited reversible transitions in TBS buffer, behaving in the calorimeter as single co-operative units with delta Hcal/delta HvH close to unity. However, the specific enthalpies and changes in heat capacity associated with the melting of all fragments and subfragments in TBS buffer were low compared to those of most compact globular proteins, suggesting that not all modules are represented. When titrated with guanidinium chloride at 25 degrees C, all fragments exhibited monophasic reversible unfolding transitions detected by changes in fluorescence. Heating in the presence of 6 M-guanidinium chloride revealed three additional transitions not seen in the absence of denaturants. These transitions have been assigned to three of the four type I finger modules (I6, I7 and I9), one of which (I6) was isolated and shown to retain a compact structure as stable as that observed for this module within the parent fragments. Two other modules (II2 and I7) are destabilized when separated from their neighbors. Thus, despite their small size (50 to 60 amino acid residues), all six of the modules in the gelatin-binding region of fibronectin form independently folded domains, three of which (I6, I7 and I9) are unusually stable. Evidence is provided that four of the six modules interact with each other in the parent fragment. This interaction may explain previously noted disruptions in the otherwise uniform strand-like images seen in electron micrographs of fibronectin.     

Altered mechanical properties of titin immunoglobulin domain 27 in the presence of calcium

Titin (connectin) based passive force regulation has been an important physiological mechanism to adjust to varying muscle stretch conditions. Upon stretch, titin behaves as a spring capable of modulating its elastic response in accordance with changes in muscle biochemistry. One such mechanism has been the calcium-dependent stiffening of titin domains that renders the spring inherently more resistant to stretch. This transient titin-calcium interaction may serve a protective function in muscle, which could preclude costly unfolding of select domains when muscles elongate to great lengths. To test this idea, fluorescence spectroscopy was performed revealing a change in the microenvironment of the investigated immunoglobulin domain 27 (I27) of titin with calcium. Additionally, an atomic force microscope was used to evaluate the calcium-dependent regulation of passive force by stretching eight linked titin I27 domains until they unfolded. When stretching in the presence of calcium, the I27 homopolymer chain became stabilized, displaying three novel properties: (1) higher stretching forces were needed to unfold the domains, (2) the stiffness, measured as a persistence length (PL), increased and (3) the peak-to-peak distance between adjacent I27 domains increased. Furthermore, a peak order dependence became apparent for both force and PL, reflecting the importance of characterizing the dynamic unfolding history of a polymer with this approach. Together, this novel titin Ig-calcium interaction may serve to stabilize the I27 domain permitting titin to tune passive force within stretched muscle in a calcium-dependent manner.     

Interaction of 75-106 actin peptide with myosin subfragment-1 and its trypsin modified derivative

To explore the role of a hydrophobic domain of actin in the interaction with a myosin chain we have synthesized a peptide corresponding to residues 75-106 of native actin monomer and studied by fluorescence and ELISA the interaction (13+/-2.6x10(-6) M) with both S-1 and (27 kDa-50 kDa-20 kDa) S-1 trypsin derivative of myosin. The loop corresponding to 96-103 actin residues binds to the S-1 only in the absence of Mg-ATP and under similar conditions but not to the trypsin derivative S-1. Biotinylated C74-K95 and I85-K95 peptide fragments were purified after actin proteolysis with trypsin. The C74-K95 peptide interacted with both S-1 and the S-1 trypsin derivative with an apparent Kd(app) of 6+/-1.2x10(-6) M in the presence or absence of nucleotides. Although peptide fragment I85-K95 binds to S-1 with a Kd(app) of 12+/-2.4x10(-6) M, this fragment did not bind to the trypsin S-1 derivative. We concluded that the actin 85-95 sequence should be a potential binding site to S-1 depending of the conformational state of the intact 70 kDa segment of S-1.     

Myofibrillar tightly bound calcium in skeletal muscle fibers: a possible role of this cation in titin strands aggregation

In muscle cells, part of the calcium is tightly bound to the N1- and N2-line of the sarcomere but its physiological significance was unknown. In the present work we reported the ability of a recombinant titin fragment spanning titin domains Z9 to I1 to tightly bind calcium ions with a K(d) of 0.049+/-0.004 nM. We further showed that calcium induced a spontaneous aggregation of the titin fragment and that the major aggregate is a tetramer. The implication of these findings on the organization of the six titin strands that emanate from the end of the thick filament within the I-band is discussed.     

Expression of human plasma gelsolin in Escherichia coli and dissection of actin binding sites by segmental deletion mutagenesis

Human plasma gelsolin has been expressed in high yield and soluble form in Escherichia coli. The protein has nucleating and severing activities identical to those of plasma gelsolin and is fully calcium sensitive in its interactions with monomeric actin. A number of deletion mutants have been expressed to explore the function of the three actin binding sites. Their design is based on the sixfold segmental repeat in the protein sequence. (These sites are located in segment 1, segments 2-3, and segments 4-6). Two mutants, S1-3 and S4-6, are equivalent to the NH2- and COOH-terminal halves of the molecule obtained by limited proteolysis. S1-3 binds two actin monomers in the presence or absence of calcium, it severs and caps filaments but does not nucleate polymerization. S4-6 binds a single actin monomer but only in calcium. These observations confirm and extend current knowledge on the properties of the two halves of gelsolin. Two novel constructs have also been studied that provide a different pairwise juxtaposition of the three sites. S2-6, which lacks the high affinity site of segment 1 (equivalent to the 14,000-Mr proteolytic fragment) and S1,4-6, which lacks segments 2-3 (the actin filament binding domain previously identified using the 28,000-Mr proteolytic fragment). S2-6 binds two actin monomers in calcium and nucleates polymerization; it associates laterally with filaments in the presence or absence of calcium and has a weak calcium-dependent fragmenting activity. S1,4-6 also binds two actin monomers in calcium and one in EGTA, has weak severing activity but does not nucleate polymerization. A model is presented for the involvement of the three binding sites in the various activities of gelsolin.     

Calcium-activated neutral proteases (calpains) are carbohydrate binding proteins

Calcium-activated neutral proteases (calpain, EC 3.4.22.17) bind to agarose matrices (Bio-Gel A-150m, Sepharose 4B, and Ultrogel AcA 34) with high affinity in the presence of calcium. 6-O-beta-Galactopyranosyl-D-galactose, a disaccharide which closely resembles the repeating unit of the agarose matrices, completely blocks the binding of calpains and can release agarose-bound enzymes in the presence of calcium. At least 1 microM level of free calcium is required for binding. Other calcium binding proteins, including calmodulin, calpastatin, casein, and neurofilament proteins, fail to bind under the same conditions. Both calpain I and calpain II can be readily purified from crude enzyme preparations by agarose chromatography in the presence of calcium and leupeptin. Agarose-bound enzymes are eluted with calcium-free solutions or can be released in the presence of calcium by 1% Triton X-100, but not by 1 M urea or 20% ethylene glycol. Enzymes eluted from agarose are activated, as evidenced by the appearance of faster migrating forms (76 and 78 kDa) of the 80-kDa catalytic subunit of calpain I upon electrophoresis and by the increased sensitivity of calpain II to activation by micromolar levels of calcium. The electrophoretic migration of the 30-kDa regulatory subunit is, however, unaltered in enzyme fractions eluted from an agarose column. When the enzyme subunits are dissociated in 1 M NaSCN, only the 30-kDa subunit binds to the agarose matrix. Furthermore, neither calpain I nor calpain II binds to agarose when their 30-kDa subunit is autocatalyzed to an 18-kDa fragment, indicating that the NH2-terminal of the 30-kDa subunit is important for the binding of calpains to an agarose matrix.     

Cellulosome from Clostridium cellulolyticum: molecular study of the Dockerin/Cohesin interaction.

Clostridium cellulolyticum produces cellulolytic complexes (cellulosomes) made of 10-13 cell wall degrading enzymes tightly bound to a scaffolding protein (CipC) by means of their dockerin domain. It has previously been shown that the receptor domains in CipC are the cohesin domains and that the cohesin/dockerin interaction is calcium-dependent. In the present study, surface plasmon resonance was used to demonstrate that the free cohesin1 from CipC and dockerin from CelA have the same K(D) (2.5 x 10(-)(10) M) as that of the entire CelA and a larger fragment of CipC, the latter of which contains, in addition to cohesin1, a cellulose binding domain and a hydrophilic domain of unknown function. This demonstrates that neither the catalytic domain of CelA nor the noncohesin domains of CipC have any influence on the interaction. Dockerin domains are composed of two conserved segments of 22 residues: removal of the second segment abolishes the affinity for cohesin1, whereas modified dockerins having twice the first segment, twice the second, or both segments but in a reverse order have K(D) values for cohesin1 in the same range as that observed for wild-type dockerin. These data indicate that if two segments are required for the complexation with the cohesin, segments 1 and 2 are similar enough to replace each other. Calcium overlay experiments revealed that the dockerin domain has one calcium binding site per conserved segment. Circular dichroism performed on wild-type and mutant dockerins indicates that this domain is well structured and that removal of calcium only weakly affects the secondary structure, which remains 40-45% helical.     

The neuronal voltage-dependent sodium channel type II IQ motif lowers the calcium affinity of the C-domain of calmodulin

Calmodulin (CaM) is the primary calcium sensor in eukaryotes. Calcium binds cooperatively to pairs of EF-hand motifs in each domain (N and C). This allows CaM to regulate cellular processes via calcium-dependent interactions with a variety of proteins, including ion channels. One neuronal target is NaV1.2, voltage-dependent sodium channel type II, to which CaM binds via an IQ motif within the NaV1.2 C-terminal tail (residues 1901-1938) [Mori, M., et al. (2000) Biochemistry 39, 1316-1323]. Here we report on the use of circular dichroism, fluorescein emission, and fluorescence anisotropy to study the interaction between CaM and NaV1.2 at varying calcium concentrations. At 1 mM MgCl2, both full-length CaM (CaM1-148) and a C-domain fragment (CaM76-148) exhibit tight (nanomolar) calcium-independent binding to the NaV1.2 IQ motif, whereas an N-domain fragment of CaM (CaM1-80) binds weakly, regardless of calcium concentration. Equilibrium calcium titrations of CaM at several concentrations of the NaV1.2 IQ peptide showed that the peptide reduced the calcium affinity of the CaM C-domain sites (III and IV) without affecting the N-domain sites (I and II) significantly. This leads us to propose that the CaM C-domain mediates constitutive binding to the NaV1.2 peptide, but that interaction then distorts calcium-binding sites III and IV, thereby reducing their affinity for calcium. This contrasts with the CaM-binding domains of voltage-dependent Ca2+ channels, kinases, and phosphatases, which increase the calcium binding affinity of the C-domain of CaM.     

Anthracyclines induce calpain-dependent titin proteolysis and necrosis in cardiomyocytes

Titin, the largest myofilament protein, serves as a template for sarcomere assembly and acts as a molecular spring to contribute to diastolic function. Titin is known to be extremely susceptible to calcium-dependent protease degradation in vitro. We hypothesized that titin degradation is an early event in doxorubicin-induced cardiac injury and that titin degradation occurs by activation of the calcium-dependent proteases, the calpains. Treatment of cultured adult rat cardiomyocytes with 1 or 3 micromol/liter doxorubicin for 24 h resulted in degradation of titin in myocyte lysates, which was confirmed by a reduction in immunostaining of an antibody to the spring-like (PEVK) domain of titin at the I-band of the sarcomere. The elastic domain of titin appears to be most susceptible to proteolysis because co-immunostaining with an antibody to titin at the M-line was preserved, suggesting targeted proteolysis of the spring-like domain of titin. Doxorubicin treatment for 1 h resulted in approximately 3-fold increase in calpain activity, which remained elevated at 48 h. Co-treatment with calpain inhibitors resulted in preservation of titin, reduction in myofibrillar disarray, and attenuation of cardiomyocyte necrosis but not apoptosis. Co-treatment with a caspase inhibitor did not prevent the degradation of titin, which precludes caspase-3 as an early mechanism of titin proteolysis. We conclude that calpain activation is an early event after doxorubicin treatment in cardiomyocytes and appears to target the degradation of titin. Proteolysis of the spring-like domain of titin may predispose cardiomyocytes to diastolic dysfunction, myofilament instability, and cell death by necrosis.     

Affinity of S100A1 protein for calcium increases dramatically upon glutathionylation

S100A1 is a typical representative of a group of EF-hand calcium-binding proteins known as the S100 family. The protein is composed of two alpha subunits, each containing two calcium-binding loops (N and C). At physiological pH (7.2) and NaCl concentration (100 mm), we determined the microscopic binding constants of calcium to S100A1 by analysing the Ca(2+)-titration curves of Trp90 fluorescence for both the native protein and its Glu32 --> Gln mutant with an inactive N-loop. Using a chelator method, we also determined the calcium-binding constant for the S100A1 Glu73 --> Gln mutant with an inactive C-loop. The protein binds four calcium ions in a noncooperative way with binding constants of K(1) =4 +/- 2 x 10(3) m(-1) (C-loops) and K(2) approximately 10(2) m(-1) (N-loops). Only when both loops are saturated with calcium does the protein change its global conformation, exposing to the solvent hydrophobic patches, which can be detected by 2-p-toluidinylnaphthalene-6-sulfonic acid - a fluorescent probe of protein-surface hydrophobicity. S-Glutathionylation of the single cysteine residue (85) of the alpha subunits leads to a 10-fold increase in the affinity of the protein C-loops for calcium and an enormous - four orders of magnitude - increase in the calcium-binding constants of its N-loops, owing to a cooperativity effect corresponding to DeltaDeltaG = -6 +/- 1 kcal.mol(-1). A similar effect is observed upon formation of the mixed disulfide with cysteine and 2-mercaptoethanol. The glutathionylated protein binds TRTK-12 peptide in a calcium-dependent manner. S100A1 protein can act, therefore, as a linker between the calcium and redox signalling pathways.     

Integrity of refolded and reoxidized gelatin-binding fragments of fibronectin.

The gelatin-binding region of fibronectin is easily isolated as a stable and functional 42-kDa fragment (42-kDa GBF) containing four type I "finger" modules and two type II "kringle-like" modules arranged in the order I6-II1-II2-I7-I8-I9, where the numbers designate the order of these modules in each of the two polypeptide chains. Each module forms an independently folded domain stabilized by two disulfide bonds. Reduction of disulfides caused large changes in the intrinsic fluorescence and abolished the gelatin-binding activity of 42-kDa GBF and two nonoverlapping gelatin-binding subfragments, 30-kDa GBF (I6-II1-II2-I7) and 21-kDa GBF (I8-I9). However, high yields of active material could be regenerated, without diluting the protein, by dialysis into GdmCl followed by slow overnight removal of GdmCl while maintaining the redox potential with a mixture of oxidized and reduced glutathione. Fluorescence spectroscopic analysis indicated that the tertiary structure and thermodynamic stability of the refolded fragments were similar to those of the originals. The refolded fragments were quantitatively indistinguishable from the originals with respect to their dissociation constants for binding to a fluorescent-labeled collagen fragment. The results suggest that all or most of the cystines, a total of 24 in 42-kDa GBF, are correctly paired in the refolded products and that the tertiary structure was completely recovered. The fact that the 30- and 21-kDa fragments bind with a similar affinity proves the existence of at least two nonoverlapping sites in 42-kDa GBF that recognize gelatin.     

Calpains and cytokines in fibrillating human atria

Atrial fibrillation (AF) is accompanied by intracellular calcium overload. The purpose of this study was to assess the role of calcium-dependent calpains and cytokines during AF. Atrial tissue samples from 32 patients [16 with chronic AF and 16 in sinus rhythm (SR)] undergoing open heart surgery were studied. Atrial expression of calpain I and II, calpastatin, troponin T (TnT), troponin C (TnC), and cytokines [interleukin (IL)-1 beta, IL-2, IL-6, IL-8, IL-10, transforming growth factor (TGF)-beta 1, and tumor necrosis factor-alpha] were determined. Expression of calpain I was increased during AF (461 +/- 201% vs. 100 +/- 34%, P < 0.05). Amounts of calpain II and calpastatin were unchanged. Total calpain enzymatic activity was more than doubled during AF (35.2 +/- 17.7 vs. 12.4 +/- 9.2 units, P < 0.05). In contrast to TnC, TnT levels were reduced in fibrillating atria by 26% (P < 0.05), corresponding to the myofilament disintegration seen by electron microscopy. Small amounts of only IL-2 and TGF-beta 1 mRNA and protein were detected regardless of the underlying cardiac rhythm. In conclusion, atria of patients with permanent AF show evidence of calpain I activation that might contribute to structural remodeling and contractile dysfunction, whereas there is no evidence of activation of tissue cytokines.     

Effect of pressure and calcium on the reversible inhibition of the sarcoplasmic-reticulum calcium-transport enzyme and on its tryptic cleavage pattern

The reversible inhibition of the sarcoplasmic-reticulum calcium-transport enzyme by pressure at room temperature is accompanied by a significant enhancement of the accessibility of the enzyme to tryptic cleavage dependent on the presence of calcium. The calcium-transport enzyme activity was monitored with dinitrophenyl phosphate as substrate. Pressure in the range 0.1-100.0 MPa affects trypsin cleavage of the control substrate N-alpha-benzoyl-L-arginine-4-nitroanilide hydrochloride little in the presence and absence of calcium. In contrast, application of 100.0 MPa to the calcium-transport enzyme at room temperature accelerates subsequent tryptic cleavage at the T2 but not at the T1 cleavage site [C. J. Brandl et al. (1986) Cell 44, 597-607]. Pressure application during tryptic digestion likewise solely affects cleavage at T2 which proceeds slowly in the absence but rapidly in the presence of calcium. At atmospheric pressure in the absence of calcium and at high pressure in the absence and presence of calcium new cleavage sites are exposed giving rise to new subfragments B1-3 in addition to the established peptides A1 and A2. Under pressure and in the presence of calcium, A1 and A2 rapidly disappear indicating the presence of calcium-binding sites in these peptides. In contrast, the B1-3 peptides which are most likely derivates of the B fragment accumulate in the presence and absence of calcium. In contrast to tryptic cleavage at atmospheric pressure, tryptic cleavage of the A as well as the B fragment tends to completion under pressure. In parallel to the disappearance of the A and B fragments calcium-dependent substrate hydrolysis vanishes. Computation of activation volumes for pressure-induced reversible enzyme inhibition and for tryptic cleavage furnished closely related volumes of opposite signs of 20-40 ml/mol and 80-100 ml/mol in the ranges 0.1-40.0 MPa and 40.0-100.0 MPa, respectively. Thus pressure produces reversible changes in the calcium-transport enzyme which activates and modifies tryptic-cleavage patterns at the T2 site of the A segment and at sites in its subfragments in the presence of calcium, i.e. if the enzyme residues in its E1 state. In contrast tryptic cleavage of the B fragment is accelerated by pressure independently of the presence of calcium.     

Localization of the delta-like-1-binding site in human Notch-1 and its modulation by calcium affinity

The Notch signaling pathway plays a key role in a myriad of cellular processes, including cell fate determination. Despite extensive study of the downstream consequences of receptor activation, very little molecular data are available for the initial binding event between the Notch receptor and its ligands. In this study, we have expressed and purified a natively folded wild-type epidermal growth factor-like domain (EGF) 11-14 construct from human Notch-1 and have used flow cytometry and surface plasmon resonance analysis to demonstrate a calcium-dependent interaction with the human ligand Delta-like-1. Site-directed mutagenesis of three of the calcium-binding sites within the Notch-(11-14) fragment indicated that only loss of calcium binding to EGF12, and not EGF11 or EGF13, abrogates ligand binding. Further mapping of the ligand-binding site within this region by limited proteolysis of Notch wild-type and mutant fragments suggested that EGF12 rather than EGF11 contains the major Delta-like-1-binding site. Analysis of an extended fragment EGF-(10-14), where EGF11 is placed in a native context, surprisingly demonstrated a reduction in ligand binding, suggesting that EGF10 modulates binding by limiting access of ligand. This inhibition could be overcome by the introduction of a calcium binding mutation in EGF11, which decouples the EGF-(10-11) module interface. This study therefore demonstrates that long range calcium-dependent structural perturbations can influence the affinity of Notch for its ligand, in the absence of any post-translational modifications.     

Detecting the active conformation of calpain with calpastatin-based reagents

The specific, calcium-dependent, high affinity interaction between calpain and its endogenous inhibitor calpastatin was exploited to selectively detect the calcium-bound, catalytically competent, conformation of calpain in vitro. Modification of calpastatin domain-1 (Val(114)-Ser(270)) or its N-terminal fragment (Val(114)-Pro(202)), at selected unique cysteine residues with maleimide-AlexaFluor546 did not compromise calpastatin function (inhibition of calpain) or its binding with calpain. Ca(2+)-dependent binding between catalytically dead calpain-2 (Cys(105)Ala) fused with eGFP and these fluorigenic calpastatin peptides generates fluorescent resonance energy transfer (FRET). The FRET signal documents proximity of calpain-2, C-terminally linked fluorophore to specific sites within calpastatin when the proteins form a complex. These results provide important insights into the calcium-dependent interaction between calpain and calpastatin and for holo-calpain-2 in solution experimentally validate some key features of their predicted interactions. These data also provide proof of concept that the calpastatin-based reagents may be useful to selectively detect the active conformation of calpain.     

The calpain-calpastatin system in mammalian cells: properties and possible functions.

All mammalian cells contain a calcium-dependent proteolytic system, composed by a proteinase, calpain, and an inhibitor, calpastatin. In some cell types an activator protein has also been identified. Moreover, two calpain isoforms, distinguishable on the basis of a different calcium requirement, can be present in a single cell. Both calpain forms are heterodimers composed of a heavy subunit (80 kDa) that contains the catalytic site and a smaller (regulatory?) subunit (30 kDa). Calpain I expresses full activity at 10-50 microM Ca2+, whereas calpain II requires calcium concentrations in the millimolar range. The removal by autoproteolysis of a fragment from the N-terminus of both calpain subunits generates a proteinase form that can express catalytic activity at concentrations of Ca2+ close to the physiological range. This process is significantly accelerated in the presence of cell membranes or phospholipid vesicles. Calpastatin, the specific inhibitor of calpain, prevents activation and the expression of catalytic activity of calpain. It is in itself a substrate of the proteinase and undergoes a degradation process which correlates with the general mechanism of regulation of the intracellular proteolytic system. The natural calpain activator specifically acts on calpain II isoform, by reducing the Ca2+ required for the autoproteolytic activation process. Based on the general properties of the calpain-calpastatin system and on the substrate specificity, its role in the expression of specific cell functions can be postulated.