Calpains are intracellular enzymes that merge cysteine protease and calcium sensing activities together in one molecule. They respond to Ca2+ signals and modify the activity of their targets by selective proteolysis. Calpains are involved in normal cellular process like cell migration and apoptosis. The over-activation of calpain due to disturbances in Ca2+ homeostasis or inactivation due to mutations, contribute to diseases like ischemic injury and muscular dystrophy.
The classical calpains 1 and 2 are heterodimeric enzymes containing a large (80 kDa) subunit and a small subunit (28 kDa). Dimerization occurs through the 5th EF-hand of penta-EF-hand (PEF) domains present in both large and small subunits. In this study, I have used structural genomics approaches to explore the PEF and C2-like regulatory domains of some of the other 12 human calpain isoforms. I have shown that recombinant PEF domain of skeletal muscle-specific calpain 3 exists as a stable homodimer when produced alone. Modelling studies suggest that there would be no barriers for dimerization of the full-length enzyme through the PEF domains which would place the protease cores at opposite ends of the dimer. Co-expression studies using small subunit were performed with PEF domains of calpains 1, 3, 8, 9, 11, 12 and 13. A differential tagging system was devised to differentiate heterodimers from homodimers. The PEF domains of calpains 1, 3, 9 and 13 co-expressed with the small subunit, while the others failed to express. The PEF domains of calpains 1 and 9 formed heterodimers. Conversely, the PEF domain of calpain 3 formed a homodimer and that of calpain 13 predominantly formed a homodimer with a small amount of heterodimer. Homodimerization of calpains implies they are less-likely to be inhibited by the endogenous calpain inhibitor, calpastatin.
C2-like regulatory domains of calpains 5-13 were also studied. The structure of the distal C2-like domain of calpain 7 was solved. It is markedly different from canonical C2 domains and may not bind Ca2+. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2009-02-11 12:30:29.18
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OKQ.1974/5343 |
| Date | 03 December 2009 |
| Creators | RAVULAPALLI, RAVIKIRAN |
| Contributors | Queen's University (Kingston, Ont.). Theses (Queen's University (Kingston, Ont.)) |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Format | 4908594 bytes, application/pdf |
| Rights | This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner. |
| Relation | Canadian theses |
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