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• W1983477773 abstract "The inhibitory domains of calpastatin contain three highly conserved regions, A, B, and C, of which A and C bind calpain in a strictly Ca2+-dependent manner but have no inhibitory activity whereas region B inhibits calpain on its own. We synthesized the 19-mer oligopeptides corresponding to regions A and C of human calpastatin domain I and tested their effect on human erythrocyte μ-calpain and rat m-calpain. The two peptides significantly activate both calpains: the Ca2+ concentration required for half-maximal activity is lowered from 4.3 to 2.4 μm for μ-calpain and from 250 to 140 μm for m-calpain. The EC50concentration of the peptides is 7.5 μm for μ-calpain and 25 μm for m-calpain. It is noteworthy that at low Ca2+ concentrations (1–2 μm for μ-calpain and 70–110 μm for m-calpain) both enzymes are activated about 10-fold by the peptides. Based on these findings, it is suggested that calpastatin fragments may have a role in calpain activationin vivo. Furthermore, these activators open new avenues to cell biological studies of calpain function and eventually may alleviate pathological states caused by calpain malfunction. The inhibitory domains of calpastatin contain three highly conserved regions, A, B, and C, of which A and C bind calpain in a strictly Ca2+-dependent manner but have no inhibitory activity whereas region B inhibits calpain on its own. We synthesized the 19-mer oligopeptides corresponding to regions A and C of human calpastatin domain I and tested their effect on human erythrocyte μ-calpain and rat m-calpain. The two peptides significantly activate both calpains: the Ca2+ concentration required for half-maximal activity is lowered from 4.3 to 2.4 μm for μ-calpain and from 250 to 140 μm for m-calpain. The EC50concentration of the peptides is 7.5 μm for μ-calpain and 25 μm for m-calpain. It is noteworthy that at low Ca2+ concentrations (1–2 μm for μ-calpain and 70–110 μm for m-calpain) both enzymes are activated about 10-fold by the peptides. Based on these findings, it is suggested that calpastatin fragments may have a role in calpain activationin vivo. Furthermore, these activators open new avenues to cell biological studies of calpain function and eventually may alleviate pathological states caused by calpain malfunction. t-butoxycarbonyl N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin Calpains represent a superfamily of proteases related by their homology in a papain-like protease domain (1Sorimachi H. Ishiura S. Suzuki K. Biochem. J. 1997; 328: 721-732Crossref PubMed Scopus (620) Google Scholar, 2Sorimachi H. Suzuki K. J. Biochem. (Tokyo). 2001; 129: 653-664Crossref PubMed Scopus (246) Google Scholar). Several members of this family, such as the best studied typical forms, μ- and m-calpain, are thought to be activated by a Ca2+ signal; their activation then leads to the limited proteolytic modification of a variety of substrate proteins. Recent x-ray crystallographic studies of rat (3Hosfield C.M. Elce J.S. Davies P.L. Jia Z. EMBO J. 1999; 18: 6880-6889Crossref PubMed Scopus (290) Google Scholar) and human (4Strobl S. Fernandez-Catalan C. Braun M. Huber R. Masumoto H. Nakagawa K. Irie A. Sorimachi H. Bourenkow G. Bartunik H. Suzuki K. Bode W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 588-592Crossref PubMed Scopus (314) Google Scholar) m-calpain have revealed the structural background of the Ca2+ dependence of calpain activity. Without Ca2+ the structure of the active site is distorted; large scale conformational changes have to occur for the assembly of a catalytically competent active site. Although mechanistically clear, key aspects of the activation process in vivo are still enigmatic as calpains require non-physiologically high Ca2+concentrations in vitro. μ- and m-Calpains reach half-maximal activation in the high μm to mmfree Ca2+ concentration range (cf. Ref. 5Baki A. Tompa P. Alexa A. Molnár O. Friedrich P. Biochem. J. 1996; 318: 897-901Crossref PubMed Scopus (99) Google Scholar), hardly attainable under normal cellular conditions. Several suggestions have been put forward on how to close this gap. First, calpain undergoes autolytic activation with a concomitant sensitization to Ca2+ (5Baki A. Tompa P. Alexa A. Molnár O. Friedrich P. Biochem. J. 1996; 318: 897-901Crossref PubMed Scopus (99) Google Scholar, 6Suzuki K. Tsuji S. Ishiura S. Kimura Y. Kubota S. Imahori K. J. Biochem. (Tokyo). 1981; 90: 1787-1793Crossref PubMed Scopus (97) Google Scholar); this, of course, does not alleviate the initial high Ca2+ requirement. Second, phospholipids increase calcium sensitivity of calpain (7Saido T.C. Shibata M. Takenawa T. Murofushi H. Suzuki K. J. Biol. Chem. 1992; 267: 24585-24590Abstract Full Text PDF PubMed Google Scholar, 8Tompa P. Emori Y. Sorimachi H. Suzuki K. Friedrich P. Biochem. Biophys. Res. Commun. 2001; 280: 1333-1339Crossref PubMed Scopus (137) Google Scholar), and activation probably involves the translocation of the enzyme to the plasma membrane (9Michetti M. Salamino F. Tedesco I. Averna M. Minafra R. Melloni E. Pontremoli S. FEBS Lett. 1996; 392: 11-15Crossref PubMed Scopus (65) Google Scholar, 10Suzuki K. Sorimachi H. FEBS Lett. 1998; 433: 1-4Crossref PubMed Scopus (140) Google Scholar). Third, calpain might also be sensitized to Ca2+ by a specific activator protein that affects membrane localization and autolysis of the enzyme (11Melloni E. Michetti M. Salamino F. Sparatore B. Pontremoli S. Biochem. Biophys. Res. Commun. 1998; 249: 583-588Crossref PubMed Scopus (26) Google Scholar). Nuclear DNA, shown to promote calpain effect on transcription factors and other nuclear proteins, may be another positive effector (12Mellgren R.L. Song K. Mericle M.T. J. Biol. Chem. 1993; 268: 653-657Abstract Full Text PDF PubMed Google Scholar). The above factors have been characterized to different extents, and there is no consensus with respect to their contribution. In this paper we raise the possibility of an additional, rather unexpected mode of calpain sensitization to Ca2+ by fragments of its endogenous inhibitor, calpastatin. Typical calpastatin molecules have five structural domains; the function of the N-terminal domain is not clear, the other four homologous domains are all capable of inhibiting a calpain molecule (13Maki M. Takano E. Mori H. Sato A. Murachi T. Hatanaka M. FEBS Lett. 1987; 223: 174-180Crossref PubMed Scopus (94) Google Scholar, 14Emori Y. Kawasaki H. Imajoh S. Minami Y. Suzuki K. J. Biol. Chem. 1988; 263: 2364-2370Abstract Full Text PDF PubMed Google Scholar). The presence of Ca2+ is mandatory for calpain binding and inhibition by calpastatin (15Imajoh S. Suzuki K. FEBS Lett. 1985; 187: 47-50Crossref PubMed Scopus (23) Google Scholar, 16Nishimura T. Goll D.E. J. Biol. Chem. 1991; 266: 11842-11850Abstract Full Text PDF PubMed Google Scholar, 17Yang H.Q. Ma H. Takano E. Hatanaka M. Maki M. J. Biol. Chem. 1994; 269: 18977-18984Abstract Full Text PDF PubMed Google Scholar), and calpastatin binding invariably occurs at a Ca2+ concentration significantly lower than that needed for enzyme activity (18Kapprell H.P. Goll D.E. J. Biol. Chem. 1989; 264: 17888-17896Abstract Full Text PDF PubMed Google Scholar). Each inhibitory domain contains three short conserved stretches of about 20 amino acids, termed subdomains A, B, and C (cf. Fig. 1). In functional studies it was shown that subdomain B is responsible for inhibition (19Maki M. Bagci H. Hamaguchi K. Ueda M. Murachi T. Hatanaka M. J. Biol. Chem. 1989; 264: 18866-18869Abstract Full Text PDF PubMed Google Scholar, 20Ma H. Yang H.Q. Takano E. Lee W.J. Hatanaka M. Maki M. J. Biochem. (Tokyo). 1993; 113: 591-599Crossref PubMed Scopus (29) Google Scholar, 21Takano E. Ma H. Yang H.Q. Maki M. Hatanaka M. FEBS Lett. 1995; 362: 93-97Crossref PubMed Scopus (73) Google Scholar), whereas subdomains A and C potentiate this inhibitory effect by binding to the calmodulin-like domains of the large and small subunit, respectively, in a strictly Ca2+-dependent manner (17Yang H.Q. Ma H. Takano E. Hatanaka M. Maki M. J. Biol. Chem. 1994; 269: 18977-18984Abstract Full Text PDF PubMed Google Scholar, 20Ma H. Yang H.Q. Takano E. Lee W.J. Hatanaka M. Maki M. J. Biochem. (Tokyo). 1993; 113: 591-599Crossref PubMed Scopus (29) Google Scholar, 21Takano E. Ma H. Yang H.Q. Maki M. Hatanaka M. FEBS Lett. 1995; 362: 93-97Crossref PubMed Scopus (73) Google Scholar, 22Ma H. Yang H.Q. Takano E. Hatanaka M. Maki M. J. Biol. Chem. 1994; 269: 24430-24436Abstract Full Text PDF PubMed Google Scholar). Subdomains A and C have no inhibitory effect on calpain (20Ma H. Yang H.Q. Takano E. Lee W.J. Hatanaka M. Maki M. J. Biochem. (Tokyo). 1993; 113: 591-599Crossref PubMed Scopus (29) Google Scholar, 21Takano E. Ma H. Yang H.Q. Maki M. Hatanaka M. FEBS Lett. 1995; 362: 93-97Crossref PubMed Scopus (73) Google Scholar, 22Ma H. Yang H.Q. Takano E. Hatanaka M. Maki M. J. Biol. Chem. 1994; 269: 24430-24436Abstract Full Text PDF PubMed Google Scholar, 23Maki M. Takano E. Osawa T. Ooi T. Murachi T. Hatanaka M. J. Biol. Chem. 1988; 263: 10254-10261Abstract Full Text PDF PubMed Google Scholar). These observations allow for a very intriguing possibility, theactivation of calpain by calpastatin fragments. Namely, as binding of calpastatin and its subdomains to calpain is facilitated by Ca2+, thermodynamic necessity demands that the reverse be also true: calpastatin should facilitate Ca2+ binding to calpain. In other words, calpastatin or its subdomains must lower the Ca2+ demand of calpain. This is clearly seen with whole calpastatin, which binds at a Ca2+ concentration significantly lower than that needed for calpain activity (18Kapprell H.P. Goll D.E. J. Biol. Chem. 1989; 264: 17888-17896Abstract Full Text PDF PubMed Google Scholar). In essence, calpastatin shifts the conformational equilibrium of calpain toward the active form; activation with whole calpastatin, of course, is not seen because of blocking of the active site by subdomain B. Subdomains A and C, on the other hand, lack this inhibitory potential. In earlier experiments they did not inhibit calpain at high Ca2+ concentrations (20Ma H. Yang H.Q. Takano E. Lee W.J. Hatanaka M. Maki M. J. Biochem. (Tokyo). 1993; 113: 591-599Crossref PubMed Scopus (29) Google Scholar, 21Takano E. Ma H. Yang H.Q. Maki M. Hatanaka M. FEBS Lett. 1995; 362: 93-97Crossref PubMed Scopus (73) Google Scholar, 22Ma H. Yang H.Q. Takano E. Hatanaka M. Maki M. J. Biol. Chem. 1994; 269: 24430-24436Abstract Full Text PDF PubMed Google Scholar, 23Maki M. Takano E. Osawa T. Ooi T. Murachi T. Hatanaka M. J. Biol. Chem. 1988; 263: 10254-10261Abstract Full Text PDF PubMed Google Scholar). Our results here show that they in fact potentiate Ca2+ binding and thus activate the enzyme at subsaturating Ca2+ levels. Human erythrocyte μ-calpain was purchased fromCalbiochem (catalog no. 208713). Rat m-calpain composed of an 80-kDa large subunit and a 21-kDa truncated small subunit was expressed in Escherichia coli and purified via the C-terminal His tag attached to its large subunit as given in Ref. 24Elce J.S. Hegadorn C. Gauthier S. Vince J.W. Davies P.L. Protein Eng. 1995; 8: 843-848Crossref PubMed Scopus (65) Google Scholar. The enzymes were dialyzed against three changes of 10 mmHEPES, 150 mm NaCl, 1 mm EDTA, 2 mmbenzamidine, 0.2 mm phenylmethylsulfonyl fluoride, and 15 mm β-mercaptoethanol (calpain buffer) and stored at 4 °C until use. On the day of use a small aliquot was taken out and reactivated with 15 mm β-mercaptoethanol for 1 h prior to the experiments. The acetylated oligopeptide amides used in the present study correspond to subdomains A and C of human calpastatin inhibitory domain I (cf. Fig. 1 and Ref. 21Takano E. Ma H. Yang H.Q. Maki M. Hatanaka M. FEBS Lett. 1995; 362: 93-97Crossref PubMed Scopus (73) Google Scholar) and have the sequence as follows. A, SGKSGMDAALDDLIDTLGG; C, SKPIGPDDAIDALSSDFTS. The peptides were synthesized on 4-methylbenzhydrylamine resin by solid phase method using the Boc1technique. The side protection groups were as follows: Boc-Asp(cHex), Boc-Lys(2-Cl-Z), Boc-Ser(Bzl), Boc-Thr(Bzl). All Boc derivatives were obtained from Reanal, Budapest, Hungary. The coupling was carried out by using 2 eq of protected amino acid derivative and 1.9 eq ofO-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate (dissolved in 1-methyl-2-pyrrolidone or inN,N-dimethylformamide), in the presence ofN,N-diisopropylethylamine for 30 min. In case of positive ninhydrin assay the coupling was repeated until no free amino groups were detected. The Boc group was cleaved by 100% trifluoroacetic acid for 1 × 1 min. After the cleavage the resin was washed with 5%N,N-diisopropylethylamine in dichloromethane and with dichloromethane. The N-terminal aminoacetyl group was introduced by reacting the resin-bound peptide with 10 eq of 4-nitrophenyl acetate. The peptides were cleaved from the resin by liquid HF (20 ml) at −5 °C, in the presence of 2 ml of anisole and 100 mg of dithiothreitol for 1.5 h. The peptides were purified by gel filtration in 30% acetic acid followed by reverse phase-high performance liquid chromatography on a Vydac 5-μm C18 column (250 × 4.6 mm) (Hesperia, CA) using 0.045% trifluoroacetic acid in water as eluent A, 0.036% trifluoroacetic acid in acetonitrile as eluent B, and linear gradient 30–55% B in 60 min and 20–52% B in 30 min for peptide A or 8.5–30% B in 100 min and 5–40% B in 30 min for peptide C. The homogeneity of peptide A was >98% and of peptide C was >92%. The peptides were checked by amino acid analysis using a Beckman model 6300 amino acid analyzer, and mass spectra were recorded on a PerkinElmer Sciex API2000 triple quadrupole instrument (Toronto, Canada) equipped with TurboIonspray source. Both peptides were dissolved in calpain buffer at 5 mg/ml concentration and stored in aliquots at −20 °C until use. Calpain activity was measured in a Jasco FP 777 spectrofluorometer at excitation and emission wavelengths of 380 and 460 nm in a 3 × 3 mm quartz cuvette. The reaction mixture in 50 μl of calpain buffer contained 1 mm LY-AMC as substrate at various peptide and Ca2+ concentrations as indicated. The actual free Ca2+ concentration was calculated from the total concentrations by using the stability constant logK app = 7.815 for Ca2+-EDTA. The reaction was started by the rapid mixing of the enzyme at a final concentration of 0.1 μm. The fluorescent trace recorded was transferred to a PC and evaluated by the MicroCal Origin data analysis software for determining the initial slope of fluorescence change; it was checked that this slope was proportional to enzyme amount/activity under the given conditions. The fluorescent substrate LY-AMC (catalog no. S 1153) and all other chemicals were purchased from Sigma. The buffer was prepared with ion-exchanged distilled water. To see if oligopeptides A and C have an effect on μ-calpain activity, an intermediate Ca2+ concentration was used at which the enzyme activity could be conveniently measured but was significantly lower than that at saturating Ca2+concentrations (13% at 2.4 μm free Ca2+). At this Ca2+ level the enzyme was titrated with increasing concentrations of a mixture of peptides A + C. Fig.2 shows that the oligopeptides increase calpain activity about 5-fold with an EC50 concentration around 7.5 μm. The concentration dependence of activation has a slight sigmoidal shape, which hints at a cooperative effect between the two peptides. Subdomains A and C, when applied separately, also activate the enzyme (Fig. 2). Under the conditions used both increase the activity about 2–3-fold, with a half-maximal effect around 15 μm concentration. Activation of calpain at subsaturating Ca2+ concentration might result from the expected sensitization to Ca2+, from a net increase in maximal activity without affecting Ca2+sensitivity, or both. To distinguish between these alternatives, the Ca2+ dependence of μ-calpain in the absence of peptides and in the presence of peptides at saturating concentration (40 μm each) was measured (Fig.3 A). Activation is clearly due to a significant increase in Ca2+ sensitivity of the enzyme, the concentration of half-maximal activity shifts from 4.3 to 2.4 μm upon the addition of the peptide mixture. The maximal activity at saturating Ca2+ concentrations, on the other hand, is unchanged within experimental error. With respect to the physiological activation of the enzyme, it is potentially very significant that an activation of about an order of magnitude is seen at low Ca2+ concentrations (Fig. 3 B). A similar set of experiments was carried out with m-calpain,i.e. with rat 80/21. Subdomains A and C exert a qualitatively similar activating effect: at 140 μm free Ca2+, where enzyme activity is 10% of that at saturating Ca2+ concentrations, the peptides cause an about 5-fold activation of the enzyme (Fig. 4). Half-maximal peptide concentration is somewhat higher (25 μm), and the course of activation has a more distinct sigmoidal shape than with μ-calpain. The peptides themselves activate the enzyme about 2–3-fold with a half-effective concentration of 55 μm. The activation again can be accounted for by an increase in the Ca2+ sensitivity of the enzyme: the half-maximal Ca2+ concentration shifts from 250 to 140 μm at saturating (75 μm) concentrations of the peptides with no effect on maximal activity (Fig.5 A). At low Ca2+concentrations the activation approaches one order of magnitude (Fig.5 B).Figure 5Ca2+ dependence of rat m-calpain activation by calpastatin subdomains A and C. A, the activity of m-calpain was measured by LY-AMC at the free Ca2+ concentrations indicated in the absence (▵) or presence (▴) of 75 μm peptides A and C. The initial rate of substrate consumption in the percentage of the activity measured at saturating (3 mm) Ca2+concentration is given. Free Ca2+ concentration needed for half-maximal activation shifted from 250 to 140 μm by the peptides. B, the extent of activation exerted by the peptides at various Ca2+ concentrations is calculated as given in Fig. 3 B for μ-calpain.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The results presented unequivocally demonstrate that peptides corresponding to subdomains A and C of human calpastatin domain I markedly sensitize both μ- and m-calpain to Ca2+. At saturating peptide concentrations the peptides lower the Ca2+ concentration needed for half-maximal activity from 4.3 to 2.4 μm for μ-calpain and from 250 to 140 μm for m-calpain. Perhaps more significantly, at low Ca2+ concentrations approaching the physiological range both μ- and m-calpain are activated by an order of magnitude. The activation shows a sigmoidal concentration dependence, which indicates cooperativity between binding of the two peptides. In structural terms this implies that the conformational change of the two subunits is functionally linked and occurs in a concerted manner. The peptide concentration needed for half-maximal effect is somewhat lower for μ-calpain (7.5 μm) than m-calpain (25 μm), which might be due to the different sensitivity of isoforms, but may also reflect species difference as the peptides are derived from human calpastatin. Either way, this difference indicates that the development of isoform-specific activator peptides is conceivable. Overall, our observations have three important implications. The first implication relates to the issue of the activation of calpains under physiological Ca2+ concentrations. As outlined in the introduction, several factors may be involved in lowering the Ca2+ demand of calpains. Although this issue is not settled yet, it seems that phospholipids (7Saido T.C. Shibata M. Takenawa T. Murofushi H. Suzuki K. J. Biol. Chem. 1992; 267: 24585-24590Abstract Full Text PDF PubMed Google Scholar, 8Tompa P. Emori Y. Sorimachi H. Suzuki K. Friedrich P. Biochem. Biophys. Res. Commun. 2001; 280: 1333-1339Crossref PubMed Scopus (137) Google Scholar), nuclear DNA (12Mellgren R.L. Song K. Mericle M.T. J. Biol. Chem. 1993; 268: 653-657Abstract Full Text PDF PubMed Google Scholar), a specific activator protein (11Melloni E. Michetti M. Salamino F. Sparatore B. Pontremoli S. Biochem. Biophys. Res. Commun. 1998; 249: 583-588Crossref PubMed Scopus (26) Google Scholar) and autolytic processing of calpain itself (5Baki A. Tompa P. Alexa A. Molnár O. Friedrich P. Biochem. J. 1996; 318: 897-901Crossref PubMed Scopus (99) Google Scholar, 6Suzuki K. Tsuji S. Ishiura S. Kimura Y. Kubota S. Imahori K. J. Biochem. (Tokyo). 1981; 90: 1787-1793Crossref PubMed Scopus (97) Google Scholar) may all contribute to shifting the Ca2+ need of calpain toward the physiological range. Our findings, perhaps paradoxically, bring calpastatin, the specific calpain inhibitor, into this picture. In principle, it is possible that calpastatin is fragmented in such a way in vivo that degrades its inhibitory subdomain (B) but leaves activator subdomains A and C more or less intact. Under such circumstances activation might override inhibition and calpastatin fragments stimulate calpain. Calpastatin is very sensitive to proteolysis in vitro (25Takano E. Maki M. Mori H. Hatanaka M. Marti T. Titani K. Kannagi R. Ooi T. Murachi T. Biochemistry. 1988; 27: 1964-1972Crossref PubMed Scopus (146) Google Scholar) and is degraded by caspases (26Kato M. Nonaka T. Maki M. Kikuchi H. Imajoh-Ohmi S. J. Biochem. (Tokyo). 2000; 127: 297-305Crossref PubMed Scopus (68) Google Scholar) and calpain itself (27De Tullio R. Averna M. Salamino F. Pontremoli S. Melloni E. FEBS Lett. 2000; 475: 17-21Crossref PubMed Scopus (31) Google Scholar) in vivo. A significant difference in the sequence of calpastatin subdomains A, B, and C suggests that the action of a protease with a trypsin-like specificity may result in such a fragmentation: subdomain B has several Lys and Arg residues making this part of calpastatin sensitive whereas subdomains A and C have only a single N-terminal Lys residue offering limited access to proteolysis. The other condition for this mechanism to operate in vivo is that the intracellular concentration of calpastatin be high enough for the peptides to reach a level sufficient for activation. Quantitative considerations argue that in some tissues this condition is met. The physiological concentration of calpastatin varies in different tissues from about 0.035 to 4.4 μm (28Otsuka Y. Goll D.E. J. Biol. Chem. 1987; 262: 5839-5851Abstract Full Text PDF PubMed Google Scholar, 29Thompson V.F. Goll D.E. Elce J.S. Calpain Methods and Protocols. 144. Humana Press, Totowa, NJ2000: 3-16Google Scholar); as calpastatin consists of four homologous inhibitory domains (cf. Fig. 1), each capable of inhibiting one calpain molecule in a Ca2+-dependent manner (13Maki M. Takano E. Mori H. Sato A. Murachi T. Hatanaka M. FEBS Lett. 1987; 223: 174-180Crossref PubMed Scopus (94) Google Scholar, 14Emori Y. Kawasaki H. Imajoh S. Minami Y. Suzuki K. J. Biol. Chem. 1988; 263: 2364-2370Abstract Full Text PDF PubMed Google Scholar), the concentration of inhibitory domains, and the potential concentration of activator peptides, is four times higher, i.e. is in the range of 0.14 to 17.6 μm. Thus, in certain tissues specific fragmentation of calpastatin may produce the activator peptides in concentrations commensurable with the range used in our studies. Furthermore, two other considerations support this conclusion in a wider range. First, it seems that calpastatin concentrations at certain locations within the cell is markedly higher than the average values just mentioned. In an unstimulated cell calpastatin localizes in an aggregated state close to the cell nucleus (30Tullio R.D. Passalacqua M. Averna M. Salamino F. Melloni E. Pontremoli S. Biochem. J. 1999; 343: 467-472Crossref PubMed Scopus (75) Google Scholar), at local concentrations which significantly exceed the values calculated for homogeneous distribution. Second, macromolecular crowding caused by the extreme protein concentration in the cytoplasm favors interactions to such an extent that reaction rates and equilibrium constants of interactions may be orders of magnitude higher in vivo than under test tube conditions (31Ellis R.J. Trends Biochem. Sci. 2001; 26: 597-604Abstract Full Text Full Text PDF PubMed Scopus (1731) Google Scholar). Because of this mechanism, fragments corresponding to the same amount of calpastatin subdomains A and C may give a much higher effective concentration in the cell than in dilute solution in the test tube and activate calpain at concentrations significantly lower than the values reported in this work. The second general implication of our work is related to previous efforts at characterizing the physiological function of calpains. It is known from knockout experiments that calpains play an essential role as deletion of the ubiquitous small subunit is lethal in mice (32Arthur J.S. Elce J.S. Hegadorn C. Williams K. Greer P.A. Mol. Cell. Biol. 2000; 20: 4474-4481Crossref PubMed Scopus (295) Google Scholar). The role of various calpain forms has been extensively studied, almost invariably by the application of calpain inhibitors. From such studies we know that calpain(s) play a regulatory role in basic cellular processes such as the cell cycle (33Mellgren R.L. Biochem. Biophys. Res. Commun. 1997; 236: 555-558Crossref PubMed Scopus (42) Google Scholar), cell motility (34Glading A. Chang P. Lauffenburger D.A. Wells A. J. Biol. Chem. 2000; 275: 2390-2398Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar), cell spreading (35Croce K. Flaumenhaft R. Rivers M. Furie B. Furie B.C. Herman I.M. Potter D.A. J. Biol. Chem. 1999; 274: 36321-36327Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), apoptosis (36Blomgren K. Zhu C. Wang X. Karlsson J.O. Leverin A.L. Bahr B.A. Mallard C. Hagberg H. J. Biol. Chem. 2001; 276: 10191-10198Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar), and many more. The inhibitors, however, lack strict specificity. They may react with the proteosome, lysosomal proteinases, or even non-proteolytic enzymes (37Wang K.K. Yuen P.W. Trends Pharmacol. Sci. 1994; 15: 412-419Abstract Full Text PDF PubMed Scopus (272) Google Scholar, 38Donkor I.O. Curr. Med. Chem. 2000; 7: 1171-1188Crossref PubMed Scopus (140) Google Scholar). These findings have prompted the development of strictly specific calpain inhibitors (39Wang K.K. Nath R. Posner A. Raser K.J. Buroker-Kilgore M. Hajimohammadreza I. Probert Jr., A.W. Marcoux F.W. Ye Q. Takano E. Hatanaka M. Maki M. Caner H. Collins J.L. Fergus A. Lee K.S. Lunney E.A. Hays S.J. Yuen P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6687-6692Crossref PubMed Scopus (255) Google Scholar) or the use of a cell-permeable variety of calpastatin subdomain B for cell biological studies. In general, the results with calpain inhibitors cannot always be unequivocally associated with the action of calpain. Thus, it is desirable to develop activators for calpain based on our observations with subdomains A and C. As these peptides are highly charged, both subdomains have to be fused with “carrier” peptides that make other substances membrane-permeable. The development of such constructs, which will probably complement the use of calpain inhibitors in further analysis of calpain function, is under way. 2P. Tompa and P. Friedrich (December 10, 2001) Hungarian Patent P0105289. The third implication worth considering is related to the pathological role of calpain. Overactivation of calpain seems to be important in a range of disorders (37Wang K.K. Yuen P.W. Trends Pharmacol. Sci. 1994; 15: 412-419Abstract Full Text PDF PubMed Scopus (272) Google Scholar, 40Wang K.K. Yuen P.W. Adv. Pharmacol. 1997; 37: 117-152Crossref PubMed Scopus (94) Google Scholar); more recent findings, however, point to the link of diminished activity and the development of pathological states. Positional cloning in patients with limb girdle muscular dystrophy 2A linked the gene of calpain 3 (p94) with this disease (41Richard I. Broux O. Allamand V. Fougerousse F. Chiannilkulchai N. Bourg N. Brenguier L. Devaud C. Pasturaud P. Roudaut C. et al.Cell. 1995; 81: 27-40Abstract Full Text PDF PubMed Scopus (863) Google Scholar). Later studies revealed that disease is associated with mutations in this gene, which invariably leads to a partial or complete loss of enzyme activity (42Ono Y. Shimada H. Sorimachi H. Richard I. Saido T.C. Beckmann J.S. Ishiura S. Suzuki K. J. Biol. Chem. 1998; 273: 17073-17078Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 43Horikawa Y. Oda N. Cox N.J. Li X. Orho-Melander M. Hara M. Hinokio Y. Lindner T.H. Mashima H. Schwarz P.E. del Bosque-Plata L. Oda Y. Yoshiuchi I. Colilla S. Polonsky K.S. Wei S. Concannon P. Iwasaki N. Schulze J. Baier L.J. Bogardus C. Groop L. Boerwinkle E. Hanis C.L. Bell G.I. Nat. Genet. 2000; 26: 163-175Crossref PubMed Scopus (1252) Google Scholar). In a similar approach, diabetes mellitus type 2, which accounts for about 90% of the incidence of diabetes, was linked to the gene of calpain 10 (43); a single-nucleotide polymorphism within one intron of the gene is associated with a significantly reduced mRNA level (44Baier L.J. Permana P.A. Yang X. Pratley R.E. Hanson R.L. Shen G.Q. Mott D. Knowler W.C. Cox N.J. Horikawa Y. Oda N. Bell G.I. Bogardus C. J. Clin. Invest. 2000; 106: R69-R73Crossref PubMed Scopus (261) Google Scholar). Thus, the enzyme in this case is fully active, but it is expressed at a lower level. The two diseases, and possibly others, have in common a diminution in calpain activity; apparently, drugs that could boost activity might be beneficial. We thank Prof. J. S. Elce (Queen's University, Kingston, Ontario, Canada) for kindly providing the expression construct of rat m-calpain (80/21)." @default.
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• W1983477773 date "2002-03-01" @default.
• W1983477773 modified "2023-09-28" @default.
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