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The role of calmodulin in the regulation of calcium signalling proteins in health and disease

Calver, Brian 2018. The role of calmodulin in the regulation of calcium signalling proteins in health and disease. PhD Thesis, Cardiff University.
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Calcium ions (Ca2+) are major secondary signalling messengers, controlling many biological processes. Signalling relies on the activity of multiple proteins to bind, sequester, transport, release and respond to Ca2+. Phospholipase C (PLC) catalyses the production of inositol 1,4,5-trisphosphate (IP3) which stimulates the intracellular release of Ca2+ via IP3 receptor (IP3R). Another primary Ca2+ release channel is the ryanodine receptor (RyR). In response to changes in Ca2+ concentration the Ca2+ sensitive protein calmodulin (CaM) binds to and releases multiple proteins, including PLC and RyR ,altering function and regulating activity. The sperm-specific PLCζ isoform stimulates oocyte activation upon fertilisation by triggering, via IP3, subsequent Ca2+ release events to produce the oscillations in the Ca2+ concentration required for embryogenesis. Dysfunctional PLCζ causes male infertility and subfertility. The structure of PLCζ and the full mechanism of action are unknown. Recently, a novel inhibitory interaction between PLCζ and CaM was observed. The cardiac-specific RyR2 isoform releases Ca2+ in cardiac myocytes during the heartbeat. Dysfunctional RyR2 activity causes life-threatening arrhythmias. CaM binding to RyR2 inhibits Ca2+ release, and dysfunctional CaM binding is arrhythmogenic. Mutations in CaM and the CaM-binding sites of RyR2 cosegregate with arrhythmias. This thesis develops the tools for subsequent investigation of the structure of PLCζ, interaction between PLCζ and CaM, altered characteristics of interaction with RyR2 by arrhythmogenic CaM mutations. Varying fusion partners and expressed amino acid coordinates improved the yield and solubility of recombinant PLCζ. Recombinant CaM protein recapitulated established parameters of CaM and Ca2+ dependent interactions between RyR2 and CaM. Arrhythmia patient mutations of CaM perturbed these divergently without altering protein secondary structure. However, the mutations altered the Ca2+ binding affinity and thermal stability of CaM. Ca2+ dependent binding between CaM and PLCζ occurred between the C-lobe of CaM with contribution from the N-lobe and no Ca2+-free binding was observed.

Item Type: Thesis (PhD)
Status: Unpublished
Schools: Medicine
Date of First Compliant Deposit: 10 July 2019
Last Modified: 10 Jul 2020 02:05

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