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

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

Graduation date: 1993

Thesis (M.S.)--West Virginia University, 1999. / Title from document title page. Document formatted into pages; contains vii, 93 p. : ill. (some col.) Includes abstract. Includes bibliographical references (p. 72-80).

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on September 18, 2007) Vita. Includes bibliographical references.

The isolation and purification of calpastatin from ostrich liver is presented, along with its physicochemical and kinetic properties. By using extraction from liver, ion-exchange chromatography on DEAE-Toyopearl, heating to 90 °C for 10 min and rechromatography on Toyopearl Super-Q 650 S, ostrich calpastatin was isolated and purified from ostrich liver. The purified intact calpastatin showed homogeneity on SDS-PAGE (Mr of 105.6 K). Amino acid analysis showed that ostrich calpastatin resembled that of rabbit liver and human erythrocyte calpastatin. An N-terminal sequence could not be obtained because the N-terminus was found to be blocked by an as yet unknown amino acid residue. The Mr values of degradative forms of ostrich liver calpastatin were determined to be 56 K and 90 K. By using PAG-IEF the pI of the intact form was determined to be 5.1. Ostrich liver calpastatin behaved characteristically like other calpastatins during kinetic analysis. Calpastatin inhibited calpain from pH 6 to 9 and was found to be unaffected by temperatures as high as 100 °C. Calpastatin also inhibited calpain activity at Ca2+ concentrations ranging from 1 to 10 mM. The inhibitor was shown to be phosphorylated because after incubation with alkaline phosphatase there was a decrease in inhibitory activity. No inhibitory effects were detected against other proteases such as chymotrypsin and trypsin, with both proteases inactivating calpastatin completely. Ostrich liver calpain was shown to have a pH optimum of 7.5 and a temperature optimum of 30 °C. In terms of its thermodynamic properties it resembled that of other ostrich proteases; DH, DS and DG being 47.07 kJ/mol, -91.1 J/mol/K and 74.237 kJ/mol, respectively. Ostrich liver calpain showed a Km of 0.14 % (w/v). The enzyme was active at both milli- and micro-molar concentrations of Ca2+. Ostrich liver calpastatin showed many physical, chemical and kinetic properties similar to those of other known calpastatins.

Traumatic brain injury (TBI) is a devastating health problem based on its high incidence, economic burden, and lack of effective pharmacological treatment. Individuals who suffer an injury often experience lifelong disability. TBI results in abrupt, initial cell damage leading to delayed neuronal death. The calcium-activated proteases, calpains, are known to contribute to this secondary neurodegenerative cascade. Prolonged activation of calpains results in proteolysis of numerous cellular substrates including cytoskeletal components, membrane receptors, and cytosolic proteins, contributing to cell demise despite coincident expression of calpastatin, the specific inhibitor of calpains. A comprehensive analysis using two separate calpastatin transgenic mouse lines was performed to test the hypothesis that calpastatin overexpression will reduce posttraumatic calpain activity affording neuroprotection and behavioral efficacy. Increased calpastatin expression was achieved using transgenic mice that overexpress the human calpastatin (hCAST) construct under control of a neuron-specific calcium-calmodulin dependent kinase II alpha or a ubiquitous prion protein promoter. Both transgenic lines exhibited enhanced calpastatin expression within the brain, extending into peripheral tissues under the prion protein promoter. hCAST overexpression significantly reduced protease activity confirmed by reductions in acute calpain-mediated substrate proteolysis in the cortex and hippocampus following controlled cortical impact brain injury. Aspects of posttraumatic motor and cognitive behavioral deficits were also lessened in hCAST transgenic mice compared to their wildtype littermates. However, volumetric analyses of neocortical contusion revealed no histological neuroprotection at either acute or long-term time points in either transgenic line. Partial hippocampal neuroprotection observed at a moderate injury severity in neuron-specific calpastatin overexpressing transgenic mice was lost after severe TBI. Greater levels of calpastatin under the prion protein promoter line failed to protect against hippocampal cell loss after severe brain injury. This study underscores the effectiveness of calpastatin overexpression in reducing calpain-mediated proteolysis and behavioral impairment after TBI, supporting the therapeutic potential for calpain inhibition. However, the reduction in proteolysis without accompanied neocortical neuroprotection suggests the involvement of other factors that are critical for neuronal survival after contusion brain injury. Augmenting calpastatin levels may be an effective method for calpain inhibition and may have efficacy in reducing behavioral morbidity after TBI and neurodegenerative disorders.

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Thesis (Ph. D.)--Ohio State University, 2009. / Title from first page of PDF file. Includes bibliographical references (p. 202-241).

Thesis (Ph. D.)--West Virginia University, 2004 / Title from document title page. Document formatted into pages; contains xiii, 119 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.