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Please use this identifier to cite or link to this item: http://hdl.handle.net/1974/1489

Title: CALPAIN: TRANSITIONING FROM THE USE OF THE PROTEASE CORE TO THE FULL-LENGTH ENZYME FOR THE DEVELOPMENT OF SPECIFIC SUBSTRATES AND INHIBITORS
Authors: KELLY, JACQUELINE

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Keywords: Calpain
Calpastatin
Issue Date: 2008
Series/Report no.: Canadian theses
Abstract: 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.
Description: Thesis (Master, Biochemistry) -- Queen's University, 2008-09-26 15:49:17.317
URI: http://hdl.handle.net/1974/1489
Appears in Collections:Biochemistry Graduate Theses
Queen's Theses & Dissertations

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