Role of the calpain system in muscle growth

Biochimie (1992) 74, 225-237

225

© Soci6t6 franqaise de biochimie et biologic mol6culaire / Elsevier. Paris

Role of the caipain system in muscle growth DE Goll, VF Thompson,

RG Taylor, JA Christiansen

Muscle Biology Group. University of Arizona. Tucson, AZ 85721, USA

(Received 12 April 199t; accepted 18 January 1992)

Summary - - Muscle protein degradation has an important role in rate of muscle growth. It has been difficult to develop procedures for measuring rate of muscle protein degradation in living animals, and most studies have used in vitro systems and muscle strips to determine rate of protein degradation. The relationship between results obtained by using muscle strips and rate of muscle protein turnover in living animals is unclear because these strips are in negative nitrogen balance and often develop hypoxic cores. Also, rate of protein degradation is usually estimated by release of labeled amino acids, which reflects an average rate of degradation of all cellular proteins and does not distinguish between rates of degradation of different groups of proteins such as the sarcoplasmic and the myofibrillar proteins in muscle. A number of studies have suggested that the calpain system initiates turnover of myofibrillar proteins, which are the major group of proteins in striated muscle, by making specific cleavages that release thick and thin filaments from the surface of the myofibrill and large polypeptide fragments from some of the other myofibrillar proteins. The calpains do not degrade myofibrillar proteins to small peptides or to amino acids, and they cause no bulk degradation of sarcoplasmic proteins. Hence, the calpains are not directly responsible for release of amino acids during muscle protein turnover. Activity of the calpains in living cells is regulated by calpastatin and Ca2+. but the nature of this regulation is still unclear. calpain / muscle protein degradation / muscle growth Introduction Three factors ultimately determine the rate and extent of muscle growth: i) the number of muscle cells; ii) the rate of muscle protein synthesis; and iii) the rate of muscle protein degradation. Although a large number o f treatments or conditions such as hormone administration, nutritional status, environmental management, trauma, etc may affect rate of muscle growth, the influence of these treatments or conditions is mediated through their effects on one or more of these three basic factors. For example, genetic selection m a y alter the number of cells in a muscle and thereby increase rate of muscle growth. Increasing either the total or the quality of an animal's nutrient intake may enhance rate of protein synthesis and hence increase rate of muscle growth. A number of hormones affect rate of muscle growth by altering either rate of protein synthesis or rate of protein Abbreviations: ta-calpain, the micromolar Ca2÷-requiring Ca2÷-

dependent proteinsae; m-calpain, the millimolar Ca2÷-requiring Ca2+-dependent proteinase; EDTA, ethylene-diaminetetraacetare; MCE, 2-mercaptoethanol; SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis; "Iris. 2-amino-2hydroxymethyl-1,3-propanediol.

degradation or both these factors [1-6]. Because of the complex array of interactions among the various hormones and the physiological status of the animal, it has been difficult to specify the exact nature that any given hormone has on rate of muscle protein synthesis or degradation [I-3]. In general, for animals in the fed state, insulin is one of the most important hormones involved in muscle protein metabolism [3]. The presence of insulin generally accelerates rate of total muscle protein synthesis and lowers rate of muscle protein degradation [1, 3, 4]. The effects of insulin on muscle protein breakdown seem to involve an alteration in lysosomal function [4, 7, 81, and insulin does not affect rate of myofibrillar protein degradation [4]. Insulin-like growth factor I affects muscle protein synthesis and degradation in a manner similar to that of insulin; rate of protein breakdown in rat soleus or extensor digitorum longus muscles is decreased by 20-30%, principally through suppression of lysosomal degradation [4]. The glucocorticoid hormones have a variety of effects on rates of muscle protein synthesis and degradation. In general, the responses of muscle protein synthesis and degradation to glucocorticoid levels depend on insulin levels. In fed animals (high insulin), glucocorticoid administration decreases protein syn-

226 thesis but has little effect on muscle protein degradation [3, 4]. The synthesis of myofibrillar proteins seems to be primarily affected [4, 9]. Large doses of glucocorticoids increase rate of muscle protein degradation, at least for a few days after administration, and again, degradation of the myofibrillar proteins as measured by the release of 3-methylhistidine is primarily affected [3, 4, 10, ll]. In fasted animals (low insulin levels), glucocorticoids generally increase the rate of muscle protein degradation [3, 4]. Thyroid hormones also affect rates of muscle protein synthesis and degradation [1-4]. In hypophysectomized or thyroidectomized rats, physiological doses of thyroid hormones stimulate muscle protein synthesis and cause a small increase in muscle protein degradation [3, 41. Pharmacological doses of thyroid hormones, however, greatly increase the rate of muscle protein degradation and elicit a small increase in rate of muscle protein synthesis [3, 4]. Most studies on the effects of hormones on rates of muscle protein synthesis and degradation have been done by using in vin'o assays on isolated muscle strips. These assays use release of tyrosine or phenylalanine to estimate rates of muscle protein degradation and the incorporation of radiolabeled amino acids into muscle proteins to estimate rates of muscle protein synthesis. The rate of protein degradation greatly exceeds the rate of protein synthesis in these isolated strips, and it is difficult to maintain these strips longer than several hours. Consequently, although measurements of muscle protein degradation and synthesis can be made rapidly and reproducibly by using these h~ vitro systems and although the isolated muscles respond to various hormones and other treatments that affect protein turnover in vivo, many of these in vitro experiments are 'replacement' experiments in which protein turnover rates are restored toward their in vivo values. It is unclear how accurately such measurements reflect the in vivo situation [2]. In addition, even small, thin muscle strips rapidly develop hypoxic cores in vitro, and the presence of these hypoxic cores affects the rates of protein synthesis and degradation measured on the whole strip [2]. Because of these difficulties, caution is necessary when extrapolating results obtained from in vitro experiments on muscle protein synthesis and degradation to in vivo systems. In addition to the difficulties caused by the differences between isolated muscle strips and muscle tissue in vivo, most studies of muscle protein degradation in isolated strips have used the release of free amino acids to estimate ra~te of muscle protein deqradation [ 1-5]. Free amino acids represent the end point of protein degradation, and an unknown number of proteolytic events exist between the intact, native protein and its complete degradation to individual amino acids. Indeed, it would seem advantageous to cells if

regulation of protein degradation occurred at the first step in the series of events eventually leading to the release of free amino acids. It is unclear whether the rate of release of free amino acids would always be directly related to the rate o f this first degradation step. Hence, measurements o f tyrosine or phenylalanine release from muscle strips in vitro include an unknown number of processes and may reflect rate of a change in protein conformation, rate of a proteolytic cleavage, or even rate of release of free amino acids from a muscle cell. The number of these intermediate steps is likely to be greatest in turnover of those proteins assembled into subcellular structures such as organelles or, in striated muscles, myofibrils. Because lysosomal enzymes are largely inactive at the pH values in cell cytosol [1 l l, most lysosomal degradation takes place in secondary lysosomes. Myofibrils are so large that they could not be assimilated by lysosomes. Therefore, striated muscle myofibrils would have to be disassembled to individual filaments and possibly to filament fragments before the lysosomal cathepsins could have a role in their turnover. Furthermore, it seems unlikely that large proteolytic complexes such as macropain (the multicatalytic protease or the proteosome) could effectively degrade intact myofibrils before they were disassembled into filaments. Consequently, the initial step in the metabolic turnover of myofibrillar proteins, which constitute 50-60% of total protein in mature skeletal muscle cells, is probably disassembly of myofibrils into filaments. This initial step may or may not be the ratelimiting step in myofibrillar protein turnover. Although 3-methylhistidine released from muscle strips likely originates from the actin or myosin in these strips, this 3-methylhistidine is the product of the last step in myofibrillar protein degradation. Therefore, it is hazardous to attempt to relate rate of 3-methylhistidine to the activity of any single proteolytic system. A number of studies have suggested that disassembly of striated muscle myofibrils into filaments is the first step in the metabolic turnover of myofibrillar proteins and that the calpain system is involved in this disassembly. These studies are summarized in the following sections.

Effect of the calpains on skeletal muscle myofibriis in vitro The calpains were discovered because Z-disks in muscle strips incubated in a Ca-'+-containing solution disappeared with no other ultrastructurally detectable change [ 12, 13]. Subsequent studies showed that incubation of muscle strips or myofibrils with purified Itor m-calpain in the presence of Ca2+ caused complete removal of Z-disks in striated muscle and a loss of

227 periodicity along thin filaments without producing any ultrastructurally detectable change in the thick or thin filaments themselves [14-19]. Recent studies [20] have shown that the first structural component removed from myofibrils by purified IX- or m-calpain seems to be the increased density observed at a line perpendicular to the myofibril axis on either side of the Z-disk [20]. This line has been named the N2 line and has been reported to be composed of nebulin or titin or both. The micrographs in figure 1 confirm that the calpains have no effect on the structure of thick or thin filaments even after they have completely removed Z-disks (fig 1D). As reported earlier [20], the first ultrastructurally detectable effects of the calpains are removal of the N2 line and the appearance of small gaps in the continuous density of the Z-disk (arrow, fig IB). Longer periods of incubation with the calpains cause a continued, gradual loss in Z-disk density (fig IC). It is unclear whether the open gaps seen in the Z-disk regions of myofibrils after longer periods of incubation with the eaipains (arrow, fig 1D) are due to continued degradation of proteins in the Z-disk region or whether this gap occurs because the thin filaments withdraw into the A-band after their connection to the Z-disk has been severed. SDS-PAGE of myofibrils after incubation with the calpains shows that the calpains have a very limited and specific effect on myofibrlllar proteins (fig 2). The lanes on the right sides of the two gels in figure 2 were loaded lightly to facilitate detection of any degradation of myosin and actin. These lanes clearly show that neither IX- nor m-calpain causes any detectable degradation of myosin or actin in myofbrils. Our earlier studies showed that m-calpain did not degrade purified, undenatured myosin or actin I15], and a recent study has demonstrated that, even after prolonged incubation with purified m-calpain, the Nand C-termini of undenatured actin and tx-actinin polypeptides remain unchanged [20]. Although several studies have reported that the calpains cleave myosin, actin, and ¢t-actinin, it is unclear whether these earlier studies used native, undenatured proteins. The calpains rapidly cleave denatured myosin, actin, and 0t-actinin (Wolfe and Goll, unpublished results). The gels shown in figure 2 are gels of myofibril pellets after centrifugation to remove any proteins or peptides that have been released by the calpains (see legend to fig 2) and are similar to our earlier SDS-gels of myofbrils after incubation with IX- or m-calpain [20]. The 100-kDa tx-actinin polypeptide (ct-actinin is a major protein in the Z-disk [21-231) is released from myofbrils during the first 10 min of incubation with Ix- or m-calpain (see also [20]) and is replaced by a group of three polypeptides, two of which are slightly larger than 100 kDa. The origin of these three polypeptides is not known; they are not from the myosin

heavy chain because the myosin heavy chain is not degraded by the calpains. These three polypeptides m a t emanate from filamin, nebulin, or titin, all of which are degraded by the calpains. A very large polypeptide fragment migrating above the myosin heavy chain at an approximate Mr of 300 000 appears after 5 (m-calpain) or 10 (ix-calpain) min; this polypeptide fragment probably originates from titin because titin is the only protein in myofibrils that is present in sufficient quantities to produce such a prominent fragment and that is larger than 300 kDa. As has been reported by many investigators [24], the 37-kDa troponin T polypeptide is rapidly degraded by both ~tand m-calpain. The experiments shown in figure 2 used equal amounts of It- and m-calpain and indicate that m-calpain cleaves myofibrillar proteins more rapidly than Ix-calpain at the Ca2+ concentrations used in these experiments. We have previously reported that m-calpain cleaves a casein substrate more rapidly than Ix-calpain [25], and it seems likely that m-calpain has an intrinsically higher turnover number than Ix-calpain. The polypeptide fragments produced by m-calpain cleavage of myofibrils are similar to those produced by Ix-calpain, however, and suggest that Itand m-calpain have similar, if not identical, subsite specificities. The results shown in figures 1 and 2 demonstrate that the calpains cause a very limited and specific degr[ldation of myofibrillar proteins and that they do not cleave these proteins to free amino acids. Because the calpains do not cleave undenatured actin or myosin and because these two proteins are the principal sources of 3-methylhistidine released during muscle protein turnover, release of 3-methylhistidine cannot be used to indicate whether the calpain system has a role in myofibrillar protein turnover. In addition to their limited and specific effects on myofibrillar proteins, the calpains also cause no bulk degradation of the sarcoplasmic proteins in skeletal muscle (table I). The sarcoplasmic protein fraction tested in table I was prepared by adjusting the pH of a low ionic strength extract of skeletal muscle to 4.9 and centrifuging to remove all endogenous calpain activity in this extract. The pH of the supematant was raised to 7.5, its protein content adjusted, and then used as a protein substrate in the standard assay for calpain activity. Although this extract still contains the endogenous calpain inhibitor, calpastatin, its calpastatin content was not nigh enough to prevent calpain degradation of a cascin substrate when the casein and the sarcoplasmic protein extract were mixed together (table I). Consequently, although the calpains rapidly cleave a few specific sarcoplasmic proteins such as phospherylase kinase and a variety of other kinases and phosphatases [27], they do not cause general degradation of sarcoplasmic proteins to fragments

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229 Fig 1. Electron micrographs showing the effects of ~t- or m-calpain on bovine skeletal muscle. Glyerinated bovine skeletal muscle strips were rinsed with phosphate-buffered saline, and one strip each was then immersed in one of three different solutions: 1) 50 llg ~t-calpain/ml, 100 mM KCi, 50 mM Tris-HCl, pH 7.5, 1.0 mM CaCI2, 0.1% MCE; 2) 50 lig m-calpain/ml in the same solution described for I.tcalpain; and 3) a control solution similar to that described for li-calpain but containing 1.0 mM EDTA instead of ! m M CaCI: and no calpain. After incubation for 1.5 h at 25°C, the strips were transferred to a second set of incubation tubes containing the same solution but having fresh calpain (the calpains autolyze and lose their activity after approximately 1.5 h at 25°C). The strips were incubated in the second solution for 1.5 h at 25°C and were then removed, washed with phosphate-buffered saline, and processed for electron microscopy. A. Micrograph of a strip incubated in a control solution containing EDTA and no calpain; Z-disks are prominent and faint N, lines (arrow) are evident. B. Micrograph of a strip incubated with m-calpain and Ca2+; the N_, lines are gone and gaps or breaks in the continuity of the Z-disk (arrow) can be seen; this section was taken from the interior of the strip, and the calpain had just reached this area after 3 h of incubation. C. Micrograph of a strip incubated with g-calpain and Ca"+; density of the Z-disks is gone but filaments still remain in the Z-disk area; this section was taken from a distance intermediate between the surface and the interior of the strip. D. Micrograph of a strip incubated with ~t-calpain and Ca'-+; the Z-disks are completely gone in this section that was taken from the surface of the strip and only an open gap remains where the Zdisks once were. The bar in A represents 1 lam; the bars in B, C, and D represent 0.25 ~tm. soluble in 2.5% trichloroacetic acid. These results emphasize that, if the calpains are involved in muscle protein turnover, their action is directed exclusively at the myofibrillar or cytoskeletal proteins where their effects result in disassembly of the myofibril and in release of large polypeptide fragments and not in liberation of free amino acids.

Possible mechanism for initiation of myofibrillar protein turnover The arguments summarized in the two preceding sections suggest that myofibrillar protein turnover is initiated by disassembly of intact myofibrils to filaments or polypeptide fragments that are subsequently degraded to free amino acids. The schematic diagram in figure 3 summarizes how myofibrillar protein turnover may occur. A portion of a longitudinal section of a myofibril containing four thick filaments and five pairs of thin filaments per sarcomere is shown at the top of the diagram. Myofibrils in mammalian skeletal muscle are cylindrical structures between 1 and 3 ~tm in diameter and extending from one end of the muscle

cell to the other (-- 1--40 mm). Consequently, a myofibril is much larger than lysosomes or ihe multicatalytic protease (macropain) complex, which is approximately 11 nm high and 16 nm in diameter [28]. If the outer layer of filaments in a myofibril were released, the remaining myofibril would have a smaller diameter (two thick filaments and four pairs of thin filaments in fig 3) but would remain functionally intact (although its strength would eventually be diminished). The released filaments could either reassociate with its parent or another myofibril (arrows in both directions) or could be degraded to free amino acids by one or more cytosolic proteases or by lysosomal cathepsins. The intact, released filaments would still be large compared with muscle lysosomes (mammalian skeletal muscle thin filaments are 1000 nm long and 4 - 6 nm in diameter, and mammalian skeletal muscle thick filaments are 1500 nm long and 1416 nm in diameter), and it seems unlikely that the released filaments could be taken up by lysosomes unless they were at least partly degraded by some cytosolic protease. It has recently been shown that skeletal muscle cells contain the multicatalytic protease or macropain system [29, 30], and this proteolytic system would be a good candidate for degradation of filaments released from myofibrils to amino acids. Release of filaments from the surface of myofibrils as shown in figure 3 would require that the Z-disk, which anchors the thin filaments to the myofibril, and titin and nebulin, which anchor both the thin filaments and the thick filaments to the myofibril [31], be cleaved. Dissociation of thin filaments to actin monomers and of thick filaments to myosin monomers would be facilitated if troponin and tropomyosin (thin filaments) and C-protein (thick filaments) were also degradated (fig 3). Dissociation of the thick and thin filaments into monomers would likely enhance their susceptibility to cytosolic proteases or to being taken up by lysosomes. Consequently, a protease that degrades Z-disks, titin, nebulin, tropomyosin, troponin, and C-protein but that leaves the myosin and actin molecules intact (thereby allowing the possibility for these major myofibrillar proteins to reassemble into functional structures and conserve metabolic energy) would be the ideal enzyme for initiating disassembly of myofibrils.

Evidence indicating that the calpain system initiates turnover of myofibrillar protein A number of lines of evidence (summarized in fig 4) indicate that the calpain system has a role in initiating myofibrillar disassembly. As shown earlier in figures 1 and 2, the calpains selectively remove Z-disks and

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Fig 2. SDS-PAGE of rabbit skeletal muscle myofibrils after incubation with g- or m-calpain. Conditions: 2.0 mg of rabbit skeletal muwle myofibrils/ml in 71 mM KCI, 50 mM Tris-HCI, pH 7.5, 0.1% MCE were incubated with 2.0 mM CaCI2 and 0.04 mg ~t- or m-calpain/ml (I calpain: 50 myofibrils, w/w) at 25°C for the times indicated. The digestion was stopped by adding EDTA to a final concentration of 10 mM; the EDTA was added before the Ca2+ for the zero-time samples. After incubation, the myofibrils were centrifuged at 25°C and 1200 g (max) for 10 min to remove those proteins and peptide fragments that had been released by the calpains (t~-actinin released from myofibrils by the calpains rebinds if the myofibrils are incubated or centrifuged at 0-2 °C). The minigels had 10 l.tg protein (the left sides, lanes 1-4 for the I.t-calpain and m-calpain gels) or 2.5 ktg protein (the right sides, lanes 5-8 for the g-calpain and m-calpain gels) loaded on each lane.

Fig 3. Schematic diagram showing how the calpains could initiate turnover of myofibrillar proteins. The angled lines on the thick filaments represent bands of C-protein that occur at regular intervals along the length of the thick filament. The crossbridges and C-protein bands on the thick filaments are not drawn to scale and represent only a few of the total cross-bridges and C-protein bands on a thick filament. The lines on the surface of the thin filaments represent troponin molecules, which are distributed at 38.5-nm intervals along the surface of the thin filament (again, not all the troponin molecules that actually exist on thin filaments are shown). Both It- and m-caipain degrade Z-disks, troponin T, and C-protein, so the thick and thin filaments released by the calpains no longer have C-protein or troponin. After incubation with the calpains, the resulting myofibril is narrower by two thick filaments and two thin filaments but is otherwise unchanged. The thick and thin filaments released by the calpain may either reassociate with the myofibril or be degraded by cytosolic proteases, possibly the multicatalytic protease (macropain).

231 !. Comparison of the rates of hydrolysis of casein and rabbit skeletal muscle sarcoplasmic and myofibrillar protein fractions by m-calpaina

Table

Protein substrate

surface of myofibrils, and the calpains are unique among the known proteases in that they do not cleave actin and myosin. No other proteolytic enzyme has yet been detected that has these unique properties, tha't is present inside muscle cells, and that is active at the physiological conditions of pH and ionic strength that exist inside living striated muscle cells. Consequently, if, as the available evidence indicates, myofibrillar proteins must be disassembled from myofibrils before they can be turned over metabolically, the calpains are the only known proteolytic enzymes that could perform this task. Quantitative immunoelectron localization studies indicate that both It- and m-calpain are located exclusively inside skeletal muscle cells; that they tend to be associated with subcellular structures such as mitochondria or myofibrils rather than free in the cell cytoplasm, and that their relative concentrations are two times greater at the Z-disk - the structure they remove - than elsewhere on tee myofibril [32]. The immunogold miclographs in figure 5 also indicate that the calpains are more prevalent at the Z-disk than else-

0D278 units solubilized mg m-calpain

Casein Myofibrillar protein Sarcoplasmic protein Sarcoplasmic protein + casein

81.94 __+ 4.90 (6) 45.02 + 1.51 (8)

1.33 + 0.44 (8) 73.80 _+ 5.65 (4)

aAssay conditions: 100 mM KCI, 100 mM Tris-acetate, pH 7.5, 10 mM MCE, 1 mM NAN,, 5.0 mg of the indicated protein/ml except for those assays having casein and sareoplasmic protein where 2.5 mg casein/ml and 2.5 mg sarcoplasmic protein/ml were used, 25°C for 30 min. The data were taken from Tan et al [26].

degrade titin, nebulin, C-protein, tropomyosin, and troponin (see also [15]) without cleaving myosin and actin. These cleavages are exactly the cleavages needed for a protease to remove filaments from the

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232 where on the myofibril. Immunogold particles in figure 5 seem more numerous along the edges of the myofibrils (which would be on the surface of the cylindrical myofibrils in living cells) than they are in the interior of the thick and thin filament lattice. Evidently, the Z-disk lattice is large enough to permit penetration of the calpain molecules to the center of the myofibril, but the lattice in the remainder of the myofibril (which contains titin and nebulin filaments in addition to thick and thin filaments) does not permit entry of calpain molecules. Consequently, immunolocalization studies show that the calpains are located in Z-disks and on the ~urface of the remainder of the myofibril and are positionecl perfectly to initiate myofibrillar disassembly as depicted in figure 3. Several studies have shown that striated muscle cells contain approximately 5-10% of their total myofibrillar protein in the |brm of easily releasable myofilaments [34, 35]. These easily releasable myofilaments are removed from the remainder of the myofibril by gentle agitation in the presence of ATP, suggesting that they are loosely associated with the surface of myofibrils. Pulse-labeling studies show that

specific activities of the myosin heavy chain in easily releasable filaments are 3-6 times higher than in the myosin heavy chain in the remainder of the myofibril [35]. These properties indicate that easily releasable flaments are intermediates in the turnover of myofibrillar proteins [34, 35]. Incubation of muscles with leupeptin, a protease inhibitor that inhibits the calpains (and several other proteases), or passive stretch, a treatment that reduces the rate of muscle protein turnover, decreases the amount of the easily releasable filaments [35]. Incubation of muscles with a calcium ionophore, A23187, which would activate the calpains, or fasting the animals or treating them with corticosterone, both of which would increase the rate of myofibriilar protein turnover, increases the size of the easily releasable filament pool. The increase due to fasting could be reduced by treatment of the animals with E-64, a cysteine protease inhibitor that inhibits the calpains. Treatment of isolated myofibrils with a crude calpain preparation releases filaments from the myofibrils that are identical to the easily releasable filaments (they lack ¢t-actinin and highmolecular weight proteins). Studies done 20 years ago

A The available evidence indicates that myofibrils grow and turnover by adding and removing filaments from their surfaces; approximately 5-10% of total myofibriilar protein is in the |brrn of easily released myofilaments that are rapidly labeled and seem to be intermediates in metabolic turnover of myofibrils (Dahlmann et al [34]; van der Westhuyzen et al [35]; and growing myofibrils add radiolabeled amino acids to their surfaces (Morkin et al [361). 1 The unique specificity of calpain proteolysis would result in release of filaments from myofibrils: the proportion of easily released myofibrilr~increases in the presence of excess Ca -'+ and decreases in the presence of compounds that inhibit the calpains. B One of the most consistent structural features of rapidly atrophying muscle is degradation of Z-disks and various Z-disk aherations such as 'streaming'. 1 The calpains are unique among the known proteases in their specific degradation of Z-disks. C SDS-PAGE of myofibrils isolated from rapidly atrophying muscle shows that many of the myofibrillar proteins including myosin and actin are intact in these myofibrils. ! The calpains are unique among the known pmtea~es in that they do not degrade undenatured myosin and actin. D Immunolocalization studies indicate that the calpains are located inside muscle cells on myofibrils with their highest concentration at the Z-disk, the structure they degrade. 1 The calpains are optimally active at intracellular pH and ionic strength (but not intraceilular Ca2+ concentration). E The calpains do not degrade myosin or actin and do not cause bulk degradation of sarcoplasmic proteins. I The calpains do not degrade muscle proteins to amino acids. F Studies that measure muscle protein degradation by the release of free amino acids agree that elevated Ca2~-concentrations increase the rate of unuscle protein degradation (to free amino acids), but do not agree as to whether calpain inhibitors affect this Ca2÷-induced increase in rate of muscle protein degradation. I These discrepancies may arise because the calpains are involved in only myofibrillar protein turnover and in only the first step of myofibrillar protein turnover: other proteases, some of them possibly activated by Ca2÷, are required to degrade the released myofilaments to amino acids,

Fig 4. Summary of the evidence suggesting that the calpain system has a role in muscle protein turnover by initiating myofibrillar disassembly but does not degrade myofibrillar or sarcoplasmic proteins to amino acids.

233

Properties and regulation of the calpain system

Fig 5. Electron micrographs showing immunogold localization of the calpains in bovine skeletal muscle. Samples were prepared foe immunoelectron localization by using the procedures described by Yoshimura et al [33]. Sections were incubated with a monoclonai antibody that reacts with the 80-kDa subunits of both It- and m-calpain followed by incubation with protein A conjugated to lO-nm gold particles. The calpains are located principally at the Z-disks (arrow) and on the fibers themselves. Bar represents 0.5 Itm.

indicated that newly synthesized myofibrillar proteins (containing radiolabeled amino acids) were added to the surface of existing myofibrils in skeletal muscle and did not assemble to form new myofibrils de n o v o [36]. Together, these findings strongly suggest that metabolic turnover of myofibrillar proteins involves at least several different processes and that it is initiated by disassembly of myofilaments from the surface of myofibrils as shown in figure 3. Consequently, the mechanism for metabolic turnover of myofibrillar proteins differs substantially from that for sarcoplasmic or cytosolic proteins, which do not require disassembly before being degraded to amino acids, and regulation of myofibrillar protein turnover is likely to differ substantially from regulation of cytosolic protein turnover. A numbe:- of studies have indicated that myofibri!!ar and non.myofibrillar proteins are degraded by different pathway~ [2, 4]°

Because the calpain system likely has an important role in initiating metabolic turnover of myofibrillar proteins, understanding the properties of the calpain system and how its activity is regulated in living cells is important in understanding the regulation of myofibrillar protein turnover. A great deal of information on the properties of the calpain system has been obtained since m-calpain was first purified in 1976 [13-16]. Some general properties of the calpain system are summarized in figure 6. The cDNAs for I.tcalpain, m-calpain, and calpastatin from several different species have been cloned and sequenced (see [37, 38] for reviews), and a cDNA for a third calpain found only in skeletal muscle tissue has been sequenced [39]. SDS-PAGE of the purified la- and mcalpains had shown earlier that these molecules each contained two polypeptide chains, one of 80 kDa and one of 28 kDa [14, 18, 191. The cDNA-derived sequences demonstrated that the 28-kDa subunit of ~t- and m-calpain was identical and that the 80-kDa subunits of the two calpains were highly related with 50% sequence homology [37]. The cDNA-derived sequences also showed that the C-terminal domain of the 80-kDa subunits of la- and m-calpain had an amino acid sequence homologous to calmodulin with four sequences similar to the E-F hand Ca2+-binding sequences. The amino acid sequence of the C-terminal domain of the 28-kDa subunit common to It- and mcalpain also is homologous to calmodulin and contains four sets of sequences analogous to the E-F hand Ca2÷-binding sequences. Consequently, the amino acid sequences of the la- and m-calpain molecules predict eight potential Ca2+-binding sites on each molecule. The number of Ca2+ atoms actually bound by the calpains has not yet been established although it is probably five or more. Some of the physical properties of the calpain molecules are summarized in table II. Calpain activity in living cells is almost certainly regulated by Ca2+ and by calpastatin, the protein inhibitor specific for the calpains. The nature of this regulation, however, is still unclear. The Ca2÷ concentration required for calpastatin to bind to and inactivate the calpains is approximately the same (~tcalpain) or significantly less (m-caipain) than the Ca2+ concentration required for proteolytic activity of the calpains themselves [41]. Consequently, if the calpains and calpastatin are located in the same places inside skeletal muscle cells, as immunolocalization studies show they are [32], rising Ca 2÷ concentrations would induce calpastatin binding before activating proteolytic activity of the calpains. Recent results in our laboratory indicate that, in the absence of Ca 2÷, the active, catalytic site in the calpain molecules is blocked

234

A It has been detected in all vertebrate cells that have been examined for its presence and in Drosophila but has not been found in plants. B It consists of at least four proteins. 1 It-Calpain - proteinase requiring 5-70 gM Ca2÷ for activity. 2 m-Calpain - proteinase requmng 100--2000 ItM Ca2+ for activity. 3 A third calpain - identified only in muscle cells; sequence homology to It- and m-calpain- a n-calpain or high- Ca2+-cal pain. 4 Calpastatin - an inhibitor specific for the calpains. C All three proteinases require Ca2÷ for activity, and Ca2+ is required for calpastatin to bind to the proteinases. D The physiological function of the system remains unclear; it has been shown to degrade three classes of substrate proteins in

vitro. 1 Cytoskeletal proteins 2 Kinases and phosphatases 3 Hormone receptors a In each instance, calpain degradation seems specific and leaves large peptide fragments, sometimes with altered physiological properties. Fig 6. Summary of some of the general properties of the Ca2+-dependent proteinase (calpain) system. and is unavailable to small (sterically) inhibitors such as E-64 [42]. Addition o f Ca 2+ concentrations high enough to induce proteolytic activity unblocks these active sites and makes them available to E-64 (and presumably also to protein substrates). Hence, Ca 2÷ concentrations high enough to induce proteolytic ac-

tivity evidently cause a significant conformational change in the calpain molecule, making its active site available to substrate. We recently found that Ca2+dependent binding o f calpastatin occurs at two places on the calpain molecule; one on the calmodulin-like domain o f the 28-kDa subunit and one on the cal-

Table !I. Comparison of some properties of the autolyzed and unautolyzed forms of It- and m-calpain from bovine skeletal muscle "~.

Propert3'

Autoly',ed

Autolyzed

It-calpain

It-calpain

m-calpain

m-calpain

Stokes radius b (A)

41.0 + !.0

39.4 + 1.0

41.5 + !.0

39.8 4- 1.0

Molecular dimensions c

18 × 84

21 x 73

18 x 90

20 × 76

% ¢t-helixa

29.9 + 1.5

29.8 + 1.6

27.2 + 1.6

25.8 + 2.6

% 13-sheeta

5.0 + 2.8

2.7 + 1.7

7.6 4. 3.3

11.3 + 5.4

(20% hydration) (A)

[Ca2+l for half-

0,60

7.1

180

1000

7.6

7.5

7.4

7.6

-maximal activity (itM) pH optimum

aData were taken from Edmunds et ai [40]: bobtained from gel permeation chromatography; ccalculated from Stokes' radius and molecular weight obtained from the amino acid composition; aobtained from far ultraviolet circular dichroism spectra.

235

II

INACTIVE

III

Co 2+

ft.,"

II

i ""

III

I

•

I

INACTIVE

? I / c . ,+ I I

It

/ I

NI

ACTIVE

~

Ill

V

INACTIVE

Fig 7. Schematic diagram showing interaction of calpain with Ca2+ and calpastatin. The calpain molecules are shown with an axial ratio of 3.5-3.7 as suggested by hydrodynamic measurements. The active site (the SH group) is shown as being sterically blocked by the N-terminal regions of the 28- and 80-kDa subunits. Roman numerals l-IV represent the four domains of the 80-kDa subunit and Roman numerals V and VI represent the two domains of the 28-kDa subunit. Only a small part of the elongated calpastatin molecule (dashed line) is included because one calpastatin molecule can inhibit four calpain molecules.

modulin-like domain of the 80-kDa subunit of the molecule [43]. Calpastatin is a competitive inhibitor of the calpains [44, 45], so it must also bind to a third site, the active site, on the calpains. Because the Ca2+ concentration required for calpastatin to bind to the calpains is lower than that required for proteolytic activity (ie for the conformational change that opens the active site), calpastatin initially probably binds just to the two calmodulin-like domains at the Ctermini of the two polypeptide chains of the inactive calpain molecule (upper half of fig 7). Rising Ca2+ concentrations would then induce the conformational change that opens the active site, and the calpastatin molecule already bound to the calmodulin-like domains would immediately bind to the active site and inactivate the enzyme (lower right molecule in fig 7). If calpastatin was not located in the same place

in the cell as the calpain molecule, rising Ca2* concentrations could induce the conformational change and open the active site on the calpain molecule creating an active protease before it bound calpastatin (lower left hand molecule in fig 7). If calpastatin relocated to the same place as the calpain, it would then bind to the calpain and inactivate it (the Ca 2÷ concentrations would already be high enough to induce calpastatin binding, so no additional Ca e+ would be required lower half of fig 7). No evidence yet exists, however, to indicate that calpastatin undergoes such relocations in living cells. The Ca2~- concentrations required to induce proteolytic activity (or the conformational change in the caipain molecule) are much higher than the free Ca2* concentrations that normally exist inside living cells (2-20 ~tM free Ca 2÷ or 200-700 ~tM free Ca 2÷

236 requiled for half-maximal activity o f IX- and m - c a l pain, respectively, c o m p a r e d with 0.2-0.8 laM free Ca -'+ inside cells). Consequently, cells must contain some m e c h a n i s m to lower the C a 2+ concentration required for proteolytic activity o f the calpains. T h e nature o f this m e c h a n i s m is unknown, but it too probably requires Ca 2+. This m e c h a n i s m would also nullify the inhibition by calpastatin because the Ca2+ concentration required for calpastatin to bind to the calpains is also higher than the free C a 2÷ concentrations inside cells. Hence, this ' a c t i v a t o r ' , if it exists, would have a crucial role in regulating calpain activity and therefore, in regulating the rate o f calpaininduced myofibrillar protein turnover. Obviously, a great deal remains to be learned about the function o f the calpain system and how its activity is regulated in living cells, and additional studies are needed in this area.

10 11

12 13

14

15

Acknowledgments

We thank Janet Christner for her excellent work in preparing this manuscript in the very short time allowed her. The research reported in this paper that originated from the authors' laboratory was supported by USDA Competitive Grant 87-CRCR-I2283, by the Muscular Dystrophy Association, by the National Livestock and Meat Board, and by the Arizona Agriculture Experiment Station. Project 28, a contributing project to USDA Regional Project NC- 131. References

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