Valery F. Thompson

Role of the calpain system in muscle growth

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 myofibril 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.

INTERACTION OF CALPASTATIN WITH CALPAIN: A REVIEW

Abstract Calpastatin is a multiheaded inhibitor capable of inhibiting more than one calpain molecule. Each inhibitory domain of calpastatin has three subdomains, A, B, and C; A binds to domain IV and C binds to domain VI of the calpains. Crystallographic evidence shows that binding of C to domain VI involves hydrophobic interactions at a site near the first EF-hand in domain VI. Sequence homology suggests that binding of A to calpain domain IV also involves hydrophobic interactions near the EF1- hand of domain IV. Neither subdomain A nor C have inhibitory activity without subdomain B, but both increase the inhibitory activity of B. Subdomain B peptides have no inhibitory activity unless they contain at least 13 amino acids, and inhibitory activity increases with the number of amino acid residues, suggesting that inhibition requires interaction over a large area of the calpain molecule. Although subdomain B inhibition kinetically is competitive in nature, subdomain B does not seem to interact with the active site of the calpains directly, but may bind to domain III of the calpains and act to block access to the active site. It is possible that subdomain B binds to calpain only after it has been activated by Ca[2+].

Comparison of the autolyzed and unautolyzed forms of μ- and m-calpain from bovine skeletal muscle

Bovine skeletal muscle mu- and m-calpain autolyze when incubated with Ca2+. During the first 30 to 300 s, autolysis: (1) has little effect on the specific proteolytic activity of either mu- or m-calpain when assayed at 5 mM Ca2+; and (2) produces two new proteolytically active forms of calpain in addition to the original mu- and m-calpain. The four proteolytically active forms of calpain are: (1) autolyzed mu-calpain, having polypeptide subunits of 76 and 18 kDa and requiring 0.60 microM Ca2+ for half-maximal activity; (2) mu-calpain with 80- and 28-kDa subunits and requiring 7.1 microM Ca2+ for half-maximal activity; (3) autolyzed m-calpain with 78- and 18-kDa subunits and requiring 180 microM Ca2+ for half-maximal activity; and (4) m-calpain with 80- and 28-kDa subunits and requiring 1000 microM Ca2+ for half-maximal activity. All four forms of the calpains have similar pH optima (7.4 to 7.6) and almost identical circular dichroism spectra in the far ultraviolet (all four have little secondary structure with 26-30% alpha-helix and less than 10% beta-sheet structure). Autolyzed mu- and unautolyzed mu-calpain are fully activated proteolytically by Mn2+ with activity starting at 125 microM Mn2+. Autolyzed m-calpain is also activated by Mn2+ up to 80% of the maximum proteolytic activity obtained with Ca2+; Mn2+ activation begins at 320 microM Mn2+. Unautolyzed m-calpain has only 6 to 8% as much activity in the presence of Mn2+ as it does in the presence of Ca2+. Autolysis increases the axial ratios of the calpains from 3.5 to 4.6 for mu-calpain and from 3.7 to 5.0 for m-calpain (assuming 20% hydration). The estimated length of the calpain molecules increases by 13% upon autolysis from 73 to 84 A for mu-calpain and from 76 to 90 A for m-calpain (assuming 20% hydration). The autolyzed calpains elute after their unautolyzed counterparts off a DEAE-ion exchange column. Because autolyzed forms of the calpains are not found in DEAE elution profiles of cell extracts, bovine skeletal muscle cells must contain very little (less than 5% of total calpain) or none of the autolyzed form of the calpains.

The calpain system and skeletal muscle growth

The first protein of a group of proteins now identified as belonging to the calpain system was purified in 1976. The calpain system presently is known to be constituted of three well-characterized proteins; several lesser studied proteins that have been isolated from invertebrates; and 10 mRNAs, two each in Drosophila and C. elegans and six in vertebrates, that encode proteins, which, based on sequence homology, belong to the calpain family. The three well-characterized proteins in the calpain family include two Ca2+-dependent proteolytic enzymes, µ-calpain and m-calpain, and a protein, calpastatin, that has no known activity other than to inhibit the two calpains. A substantial amount of experimental evidence accumulated during the past 25 yr has shown that the calpain system has an important role both in rate of skeletal muscle growth and in rate and extent of postmortem tenderization. Calpastatin seems to be the variable component of the calpain system, and skeletal muscle calpastatin activity is highl...

The Bovine Calpastatin Gene Promoter and a New N-terminal Region of the Protein Are Targets for cAMP-dependent Protein Kinase Activity

To investigate the regulation of calpastatin gene expression, we isolated bovine heart calpastatin cDNAs and 5′-regions of the calpastatin gene. Analysis of 5′-cDNA sequence identified a new translation initiation site that is in frame and 204 nucleotides upstream of the previously designated start site. Conceptual translation from this upstream AUG produces a protein containing 68 additional N-terminal amino acids. This “XL” region contains three potential PKA phosphorylation sites but shares no homology with other regions of calpastatin or with any known protein. Immunoblot studies demonstrated that heart and liver contain a calpastatin protein of 145 kDa on SDS-polyacrylamide gel electrophoresis that comigrates with full-length bacterially expressed calpastatin and calpastatin produced by coupled in vitro transcription-translation from the upstream AUG. An antibody raised against the XL region recognized the 145-kDa band, demonstrating that the upstream AUG is utilized and that the 145-kDa band represents full-length calpastatin in vivo. Transient transfection assays demonstrated that sequence within 272 nucleotides upstream of transcription initiation of the calpastatin gene is sufficient to direct moderate level transcription. Promoter sequences further upstream act to inhibit or stimulate transcriptional activity. Exposure of transfected cells to dibutyryl cAMP resulted in a 7–20-fold increase in promoter activity for constructs containing at least 272 nucleotides of upstream promoter sequence. Deletion analysis indicates that at least one cAMP-responsive element resides within 102 nucleotides of transcription initiation.

Effect of substrate on Ca2+-concentration required for activity of the Ca2+-dependent proteinases, μ- and m-calpain

The Ca2+ concentrations required for half-maximal activity of mu- and m-calpain purified from bovine skeletal muscle were tested using four different protein substrates and three different synthetic peptide substrates. Hammersten casein, the commonly used substrate for measuring mu- and m-calpain activity, required 2.5 microM Ca2+ for half-maximal activity of mu-calpain and 290 microM Ca2+ for half-maximal activity of m-calpain. When Hammersten casein was dialyzed against 8 M urea and 10 mM EDTA to remove all endogenous Ca2+, it required 1.9 and 290 microM Ca2+ for half-maximal activity of mu- and m-calpain, respectively. Rabbit skeletal muscle myofibrils and rabbit skeletal muscle troponin required 65 microM and 24 microM Ca2+ for half-maximal activity of mu-calpain and 380 microM and 580 microM Ca2+ for half-maximal activity of m-calpain, respectively. The three synthetic substrates tested, Suc-Leu-Tyr-MCA, Boc-Leu-Thr-Arg-MCA, and Suc-Leu-Leu-Val-Tyr-MCA, required 1.6 microM to 3.7 microM Ca2+ for half-maximal activity of mu-calpain and 200 to 560 microM Ca2+ for half-maximal activity of m-calpain.

Properties of easily releasable myofilaments: are they the first step in myofibrillar protein turnover?

Myofibrillar proteins must be removed from the myofibril before they can be turned over metabolically in functioning muscle cells. It is uncertain how this removal is accomplished without disruption of the contractile function of the myofibril. It has been proposed that the calpains could remove the outer layer of filaments from myofibrils as a first step in myofibrillar protein turnover. Several studies have found that myofilaments can be removed from myofibrils by trituration in the presence of ATP. These easily releasable myofilaments (ERMs) were proposed to be intermediates in myofibrillar protein turnover. It was unclear, however, whether the ERMs were an identifiable entity in muscle or whether additional trituration would remove more myofilaments until the myofibril was gone and whether calpains could release ERMs from intact myofibrils. The present study shows that few ERMs could be obtained from the residue after the first removal of ERMs, and the yield of ERMs from well-washed myofibrils was reduced, probably because some ERMs had been removed by the washing process. Mild calpain treatment of myofibrils released filaments that had a polypeptide composition and were ultrastructurally similar to ERMs. The yield of calpain-released ERMs was two- to threefold greater than the normal yield. Hence, ERMs are an identifiable entity in myofibrils, and calpain releases filaments that are similar to ERMs. The role of ERMs in myofibrillar protein turnover is unclear, because only filaments on the surface of the myofibril would turn over, and changes in myofibrillar protein isoforms during development could not occur via the ERM mechanism.

Autolysis of µ- and m-Calpain from Bovine Skeletal Muscle

Abstract The rate of autolysis of and mcalpain from bovine skeletal muscle was measured by using densitometry of SDS polyacrylamide gels and determining the rate of disappearance of the 28 and 80 kDa subunits of the native, unautolyzed calpain molecules. Rate of autolysis of both the 28 and 80 kDa subunits of calpain decreased when calpain concentration decreased and when ?casein, a good substrate for the calpains, was present. Hence, autolysis of both calpain subunits is an intermolecular process at pH 7.5, 0 or 25.0 C, and low ionic strength. The 78 kDa subunit formed in the first step of autolysis of mcalpain was not resolved from the 80 kDa subunit of the native, unautolyzed mcalpain by our densitometer, so autolysis of mcalpain was measured by determining rate of disappearance of the 28 kDa subunit and the 78/80 kDa complex. At Ca2+ concentrations of 1000 mM or higher, neither the mcalpain concentration nor the presence of bcasein affected the rate of autolysis of mcalpain. Hence, mcalpain autolysis is intramolecular at Ca[2+] concentrations of 1000 M or higher and pH 7.5. At Ca[2+] concentrations of 350 M or less, the rate of mcalpain autolysis decreased with decreasing mcalpain concentration and in the presence of bcasein. Thus, mcalpain autolysis is an intermolecular process at Ca[2+] concentrations of 350 M or less. If calpain autolysis is an intermolecular process, autolysis of a membranebound calpain would require selective participation of a second, cytosolic calpain, making it an inefficient process. By incubating the calpains at Ca[2+] concentrations below those required for halfmaximal activity, it is possible to show that unautolyzed calpains degrade a ?casein substrate, proving that unautolyzed calpains are active proteases.

Intrinsically disordered RGG/RG domains mediate degenerate specificity in RNA binding

Abstract RGG/RG domains are the second most common RNA binding domain in the human genome, yet their RNA-binding properties remain poorly understood. Here, we report a detailed analysis of the RNA binding characteristics of intrinsically disordered RGG/RG domains from Fused in Sarcoma (FUS), FMRP and hnRNPU. For FUS, previous studies defined RNA binding as mediated by its well-folded domains; however, we show that RGG/RG domains are the primary mediators of binding. RGG/RG domains coupled to adjacent folded domains can achieve affinities approaching that of full-length FUS. Analysis of RGG/RG domains from FUS, FMRP and hnRNPU against a spectrum of contrasting RNAs reveals that each display degenerate binding specificity, while still displaying different degrees of preference for RNA.

Effect of two dietary concentrate levels on tenderness, calpain and calpastatin activities, and carcass merit in Waguli and Brahman steers

The objective of this study was to compare carcass characteristics of a newly introduced breed, the Waguli (Wagyu x Tuli), with the carcass characteristics of the Brahman breed. Brahman cattle are used extensively in the Southwest of the United States because of their tolerance to adverse environmental conditions. However, Brahman carcasses are discounted according to the height of their humps because of meat tenderness issues. The Waguli was developed in an attempt to obtain a breed that retained the heat tolerance of the Brahman but had meat quality attributes similar to the Wagyu. Twenty-four animals were used. Six steers from each breed were fed a 94% concentrate diet and 6 steers from each breed were fed an 86% concentrate diet. Eight steers, 2 from each group, were harvested after 128 d, after 142 d, and after 156 d on feed. Waguli steers had larger LM, greater backfat thickness, greater marbling scores, and greater quality grades than the Brahman steers (P < 0.05). The Japanese Wagyu breed is well known for its highly marbled and tender meat, and these traits are also present in the Waguli. The Waguli had significantly lower Warner-Bratzler shear force values than the Brahman steers after 7 and 10 d of postmortem aging (P < 0.05); this difference decreased after 14 d postmortem (P = 0.2), when tenderness of the slower aging Brahman had increased to acceptable levels. Toughness of the Brahman has been associated with high levels of calpastatin in Brahman muscle, and the Waguli LM had significantly less calpastatin activity (P = 0.02) at 0 h postmortem than the Brahman LM. At 0-h postmortem, the total LM calpain activity did not differ between the Brahman and Waguli (P = 0.57). Neither diet nor days on feed had any significant effect on the 0-h postmortem calpain or at 0-h postmortem calpastatin activity, nor an effect on Warner-Bratzler shear-force values. In conclusion, LM muscle from the Waguli steers had a high degree of marbling, lower shear force values, and low calpastatin activity, all of which are related to more tender meat.