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Study of calcineurin interaction with inhibitor-1 and natural products. Towards novel calcineurin inhibitors Open Access
- Other title
- Type of item
- Degree grantor
University of Alberta
- Author or creator
- Supervisor and department
Holmes, Charles (Biochemistry)
- Examining committee member and department
Baksh, Shairaz (Pediatrics)
Moorhead, Greg (Biochemistry)
Holmes, Charles (Biochemistry)
Michalak, Marek (Biochemistry)
Fliegel, Larry (Biochemistry)
Department of Biochemistry
- Date accepted
- Graduation date
Doctor of Philosophy
- Degree level
Calcineurin (CaN) is an eukaryotic Ser/Thr protein phosphatase that plays an important role in lymphocyte activation. Inhibition of CaN leads to immune system suppression, a necessary procedure in organ transplantation. However, use of CaN inhibitors results in toxic side effects. The search for more specific immunosuppressants is therefore an important and ongoing endeavor. The focus of this thesis was to investigate methods that could lead to the discovery of novel CaN inhibitors.
CaN is closely related to protein phosphatase-1 (PP-1) but both phosphatases retain distinct substrates, regulatory proteins, and inhibitors. Two such inhibitors, okadaic acid (OA) and microcystin-LR (MCLR), potently inhibit PP-1 but are markedly less effective against CaN. Mutagenesis of CaN was undertaken to generate a form of CaN more sensitive to OA and MCLR. The optimal construct was a Y159I:F160Y:L312C:Y315L quadruple point mutant that showed 600-fold and 37-fold increased sensitivity to MCLR and OA, respectively. These studies provide the basis for chemical engineering to generate analogs of OA and MCLR that are CaN specific.
Multiple proteins use the PXIXIT amino acid sequence to interact with CaN, most notably the nuclear factor of activated T-cells (NFAT). NFAT dephosphorylation by CaN is necessary for immune system activation. The second chapter of results demonstrates that inhibitor-1 (I-1), a CaN substrate, contains the PXIXIT motif used for CaN binding. Disruption of the PXIXIT motif of I-1 reduced its dephosphorylation by CaN. I-1 therefore is a suitable substrate for identification of novel immunosuppressants that could block the interaction between CaN and the PXIXIT motif of NFAT proteins.
A new method of bioassay-guided isolation of novel CaN inhibitors from extracts of marine organisms was established in the final chapter of results. Monitoring the inhibition of CaN activity using the I-1 substrate during extract purification led to identification of three compounds: halisulfate-7, hipposulfate C, and a novel one, irregularsulfate. These compounds were identified as equipotent µM inhibitors of CaN and PP-1. The method of bioassay-guided isolation of CaN inhibitors is applicable to searching for potential drugs that could block access to the active site of CaN, or to the PXIXIT motif binding groove.
- Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
- Citation for previous publication
Carr, G., Raszek, M., Van Soest, R., Matainaho, T., Shopik, M., Holmes, C. F. and Andersen, R. J. (2007). Protein phosphatase inhibitors isolated from Spongia irregularis collected in Papua New Guinea. J Nat Prod 70 (11): 1812-5
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