Dissertation (Ph.D.)--University of Toledo, 2007. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Title from title page of PDF document. Bibliography: p. 117-147.

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Solving the problem of inconsistent meat tenderness is a top priority of the meat industry. This requires a greater understanding of the processes that affect meat tenderness and the adoption of such information by the meat industry. It is essential that we understand the mechanism of meat tenderisation of which, the calpain protease system is believed to play a central role. This thesis focuses on three aspects; characterisation of calpain activity, the effect of porcine μ-calpain on myofibril degradation and the effect of μ-calpain on specific proteins desmin and troponin-T. To study the effect of calpain activity, fluorogenic assays were used to determine: μ-calpain concentration for optimal peptide cleavage; calcium requirements and the effect of chelating substances on the activity of μ-calpain. In addition, the affinity of μ-calpain for substrates CalS-I and CalS-III were assessed. The effect of μ-calpain on myofibril degradation was evaluated through the use of myofibrillar fragmentation index and density marker beads. Myofibrils were digested at three different temperatures for varying time periods. Conflicting results were displayed and it was concluded that these methods are not accurate, thus further research should be conducted to ensure inconsistencies are eliminated. Specific proteins desmin and troponin-T have previously been shown to exhibit degradation in the presence of calcium and μ-calpain. SDS-polyacrylamide electrophoresis, western blotting and densitometry measurements were utilized to investigate this effect. It was concluded that μ-calpain plays a significant role in the post mortem proteolysis of myofibrillar protein. This thesis provides information and strives to give a better understanding of the proteolytic changes that occur within muscle. Understanding how these mechanisms affect meat on a cellular level, can help to control the influence they inflict on meat quality.

Calpains are a family of calcium-dependent cysteine proteases involved in intracellular signaling. They participate in many normal cellular processes such as cell motility and apoptosis but when over-activated they contribute to diseases ranging from ischemic injury to neurodegenerative disorders. The major calpain isoforms µ- and m- are large heterodimeric enzymes that are subject to autoproteolysis and aggregation when activated by Ca2+. To avoid these complications the protease core (domains I and II) has been used to screen inhibitors and design substrates. Using the protease core of µ-calpain, I showed that the superior calpain substrate, PLFMER, is cut at the intended scissile bond between F and M. Alanine substitutions at each position optimized the sequence to PLFAAR, which has a 2.3-fold higher turnover rate. The set of substrates derived from this study provided a tool for profiling the activity of calpain isoforms. One disadvantage of the protease core is that it is less active than the whole enzyme. This was even more apparent with the protease cores of the tissue-specific calpains 3, 8, 9 and 15 such that it prevented their use in substrate and inhibitor screening. The recently solved crystal structure of calcium-bound full-length m-calpain has revealed additional sites for the interaction of substrates and inhibitors in the unprimed side of the catalytic cleft provided by domain III. To sample these sites, it is necessary to prevent the full-length calpain from aggregating and precipitating upon calcium binding. I have developed a method here that uses portions of calpastatin (CAST), the natural endogenous inhibitor and stabilizer of calpain, to keep the enzyme soluble. By artificially connecting those portions of calpastatin that bind to calpain domains IV and VI, it is possible to stabilize the enzyme without blocking its active site. Of the three constructs made, 1C-2A, 2C-3A, and 3C-4A, the 3C-4A peptide was shown to completely inhibit aggregation of m-calpain at a 1:1 molar ratio, as monitored by turbidity. This mechanism of stabilization will permit the use of full-length calpains for the development of specific substrates and inhibitors. / Thesis (Master, Biochemistry) -- Queen's University, 2008-09-26 15:49:17.317

Thesis (M. Sc.)--University of Canterbury, 2007. / Typescript (photocopy). Includes bibliographical references. Also available via the World Wide Web.

This thesis summarises the progress made in the design and synthesis of conformationally constrained β-strand peptidomimetic compounds using ring closing metathesis methodology under microwave irridation conditions. The best macrocycle were elaborated into an inhibitor for a specific protease target. Calpain was used as an example of protease targeting cataract disease. Chapter One introduces proteases in general centring on the general context of protease inhibitor design. The significant of the β-strand 'bioactive' conformation is discussed in details in particular the exploitation of conformationally constrained to potential lock the 'bioactive' conformation. Chapter Two illustrates in silico methods used to design a series of β-strand macrocycle 2.1-2.7. The analysis of these is performed using molecular modelling software Schrodinger suite (2005). A brief discussion of ring closing metathesis methodology is also included. Chapter Three describes the synthesis of the precursor required for RCM reactions (tripeptides dienes). Various types of allylated amino acid side chains were synthesised. The tripeptides were obtained using standard peptide coupling methodology utilising reagents such as HATU, EDC and HOAT. Chapter Four describes the application of ring closing metathesis for the synthesis of β-strand macrocycles. The development of a new reaction conditions to optimise the ring closing metathesis reaction is discussed. In particular the effect of the use of a Lewis acid (chlorodicyclohexylborane) additive in RCM reactions is investigated. Chapter Five discusses the mechanism of cataract formation, cataract treatment and the potential development of calpain inhibitors. One of the macrocycles synthesised in chapter 4 is elaborated into a calpain inhibitor. The in-vitro assay result of this is presented and this compound is currently undergoing in vivo evaluation.

Recombinant human calpain-5 was expressed in insect cells using a baculovirus system. The expressed calpain-5 was purified by both traditional chromatography and by affinity-column chromatography. Both methods yielded active protease. Calpain-5 displayed very limited hydrophobicity. This indicated that calpain-5 is not a membrane binding protein. Calpain-5 had pI of 8.3. The recombinant calpain-5 also exhibited calcium-dependent proteolytic activity. The calculated calcium requirement for half-maximal activity was 9.6 mM when incubated at 37��C and 26.5 mM when incubated at 30��C. Compared to traditional calpains, which require less than 1 mM calcium for half-maximal activity, calpain-5 exhibited weaker proteolytic activity. This is an unusual observation because calpain-5 lacks the typical calcium-binding domain of the calpains and implied that other calcium-binding region of the protein account for calcium-binding and sensitivity. Our results also showed that calpain-5 was different from traditional calpains because its activity was higher at 37��C compared to 30��C and remained active at 37��C for more than 2 hours. This differs from traditional calpains which display better proteolytic activity at lower temperatures and become inactive within 30 minutes of incubation in 37��C. Calpain-specific inhibitors, calpastatin and E64, did not inhibit calpain-5. Only one calcium-binding inhibitor, PD150606, inhibited calpain-5 proteolytic activity. These results confirmed that calpain's calcium-binding domain is important in calpastatin binding and calpain-5 possesses other calcium-binding regions. Calpain-5 was able to degrade spectrin, a ubiquitous cytoskeletal protein. This indicates that calpain-5 might have a role in cell remodeling. Finally, calpain-5 has the ability to degrade itself. It is not clear if this is the result of inter- or intra-molecular proteolysis and whether this leads to activation of the protein or is, instead, the first step in its degradation. Calpain-5 is expressed at highest concentrations in testis, brain, liver and gastrointestinal tract. It is not clear why these tissues require a unique calpain. Calpain-5 may provide these tissues with an additional calcium-dependent proteolytic activity which is not regulated by calpastatin and which could participate in cytoskeletal protein turnover. / Graduation date: 2004

The first goal of this study was to understand the role of calpains in skeletal muscle protein degradation in cultured cells. We have developed a genetic approach to inhibit endogenous calpain activity through over-expressing dominant negative m-calpain (DN), antisense m-calpain (AS) and calpastatin inhibitory domain (CID). We observed that, under conditions of accelerated degradation (serum withdrawal), inhibition of m-calpain through DN-m-calpain over-expression caused a 30% inhibition of total protein degradation whereas CID over-expression reduced degradation by 63%. These constructs did not significantly affect degradation in the presence of serum. These data indicate that calpains participate in the accelerated degradation associated with serum withdrawal. Inhibition of calpain also stabilized nebulin, a major structural protein of the sarcomere. These observations indicate that calpains play significant roles in muscle protein turnover. Finally, over-expression of antisense m-calpain caused a transient reduction in m-calpain concentration after which normal m-calpain concentration was quickly re-established. These observations indicate that m-calpain is a short half-life protein in muscle cells. The second goal of this study is to investigate the role of calpain in the mediation of PARP protein level in differentiating myoblasts. Poly(ADP-ribosyl)ation, catalyzed by PARP, is involved in various physiological events, such as DNA excision repair, DNA recombination, DNA replication, cell differentiation, cell growth and transformation, and apoptosis. A protease participating in PARP turnover could be a significant regulator to the events which PARP is involved. A relationship between apoptosis and myofibrillar protein degradation via a common protease might suggest the basis for muscle wasting and atrophy which characterize in many muscle diseases. We established a genetic approach to inhibit endogenous calpain activity through over-expressing calpastatin inhibitory domain (CID). We observed that (1) inhibition of calpain activity increased PARP concentration when post-confluent myoblasts were cultured with 2% HS medium, an inducer of differentiation and (2) inhibition of calpain activity prevented PARP degradation induced by A23187 and etoposide in differentiating myoblasts. These data demonstrate that calpain is involved in regulation of PARP in cultured cells. / Graduation date: 1998

Objectives of this study were to examine effects of age, castration and gestation on actvities of calpains and calpastatin in skeletal muscle. Also, the regulation of calpains and calpastatin at the molecular level was investigated. Two experiments were designed. Experiment 1 was designed to evaluate effects of age and castration on calpains and calpastatin in sheep skeletal muscle. Experiment 2 was designed to examine the effects of age and gestation on calpains, calpastatin and their molecular regulation in rabbit skeletal muscle. In Experiment 1, ten newborn male lambs, six weaned wethers (92 days), six weaned rams (92 days), six market weight wethers (89 days post-weaning) and six market weight rams (89 days post-weaning) were slaughtered and gluteobiceps femoris was taken for assay of calpain I, calpain II and calpastatin. Body weights were not different between wethers and rams in weaned animals; however, post-weaning, body weight gain of rams was greater (p < .05) than that of wethers. Ram gluteobiceps femoris weight at market weight was greater than that of wethers (p < .05). Activities of muscle calpain I, calpain II and calpastatin in newborn lambs were higher than those of weaned and market weight lambs. The age-dependent reduction of calpain and calpastatin activities was complete at weaning and may be associated with or cause age-dependent attenuation of muscle protein degradation. Although muscle weights were greater in rams compared to wethers, no differences in activities of muscle calpains and calpastatin were detected between these two groups at weaning and at market weight. Hence, castration does not influence lamb muscle growth by changing muscle total calpain or calpastatin activities. In Experiment 2, biceps femoris muscle was collected from newborn, weaned (1 month), market weight (2 months), non-gestational adults (5 months) and gestational (20 day) adult rabbits and for assay of calpains, calpastatin activities, muscle NI-- methylhistidine (NMH) and mRNA encoding calpains and calpastatin. Muscle protein content (mg/g tissue) increased as animals aged indicating proliferation of myofibrillar protein. Total RNA content and ribosomal capacity (mg/g protein) declined (p < .05) as the rabbits aged; however, no differences in protein content and ribosomal capacity were detected in muscles taken from gestational versus non-gestational adult rabbits. Concentration of muscle NMH decreased as animal aged; however, no differences were detected in 20-day gestational muscle versus non-gestational muscle. Activities of calpains and calpastatin declined (p < .05) as animal aged. Most of the loss of activity occurred before 4 weeks of age (weaning). No differences in calpain and calpastatin activities were detected in gestational versus non-gestational animals. Reductions in activities of calpains and calpastatin were associated with reduced RNA concentration (ribosomal capacity) and reduced concentrations of muscle calpains and calpastatin mRNA (expressed as densitometry units per g tissue protein). The reduction of mRNA appeared to be associated with an overall loss of RNA from skeletal muscle because mRNA/g protein was greatly reduced whereas mRNA/total RNA for both calpains and calpastatin did not change greatly; however, mRNA/total RNA in adult rabbits compared to newborn, weaned and market weight animal declined significantly. In addition, from the Northern blot assay, it was determinated that newborn and weaned rabbits only expressed an intermediate length calpastatin mRNA isoform (II). However, the longest calpastatin mRNA isoform (I) was expressed in market weight and adult rabbits. The reason why muscle cells express different forms of calpastatin mRNA at different ages remains unknown. In pregnant rabbits, neither calpains nor calpastatin changed significantly. Also, muscle NMH concentration did not change. However, at the mRNA level, the steadystate concentration of mRNA encoding calpain II increased and calpastatin band II mRNA decreased in pregnant rabbits. This implies that in late catabolic stage of gestation, increased calpain II activity and decreased calpastatin activity may facilitate proteolysis. / Graduation date: 1991

There have been several reports that cataract development results from unregulated Ca2+ mediated degradation of lens crystallins. The calpain isoform m-calpain, a cysteine protease, is known to be a major player in cataract formation in rodent lenses and recent evidence indicates that over-activation by Ca2+ causes cataractogenesis in other mammals. Molecular modelling studies of seventeen analogues of compound SJA6017 (our lead compound) in a calpain model are compared to measured IC50 values against ovine calpain. The studies validated the potential of the ‘model’, method and defined activity criteria that could be used as a tool to select molecules to synthesize as potential calpain inhibitors. Using this screening methodology and two virtual libraries of potential inhibitory molecules led to the synthesis of several inhibitors including macrocyclic 811. In vitro sheep eye lens culture experiments showed that macrocycle 811 possessed the characteristics to slow cataractogenesis.