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Title: Characterisation of tropomyosin heterodimers carrying single cardiomyopathy mutations
Author: Janco, Miroslav
Awarding Body: University of Kent
Current Institution: University of Kent
Date of Award: 2013
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It is known that different point mutations in α-tropomyosin (Tm) can cause either hypertrophic (HCM) or dilated (DCM) cardiomyopathy. Both of these serious pathologies have a distinct phenotype with unknown mechanisms of development. Biochemical in vitro studies provide valuable information for exploring downstream consequences of cardiomyopathy mutations in sarcomeric proteins leading to cardiac remodelling and consequent heart failure. Tm is a linear a-helical coil-coiled dimer involved in calcium dependent regulation of muscle contraction. Prior to this work, the effects of mutations in Tm on regulation of muscle contraction have been made exclusively with Tm mutant homodimers. However, individuals with a heterozygous background may express mutant and WT proteins in a 1 : 1 ratio which can assembly into a mixture of αα, αα* and α*α* Tm dimers. We found that presence of mutation has little effect on dimer formation between the mutant and the WT monomers, therefore theoretical ratio of the Tm dimers in vivo may be 1 : 2 : 1, respectively. This assumption would make the heterodimer predominant. The properties of in vitro assembled Tm heterodimers carrying HCM (WT-D175N and WT-E180G) and DCM (WT-E40K, WT-E54K, and WT-D230N) causing mutations were examined including thermal stability, flexibility, actin affinity, calcium regulation of 51 biding, and calcium regulation of myofibril force. We showed that various properties of the heterodimers can be similar to those of the wild-type (thermal stability of reduced WTD175N; actin affinity of WT-E40K, and WT-D175N; ΔpCa of WT-E40K, WT-E54K, WT-E180G, WT-D230N), similar of those to the mutant homodimer (ΔpCa of WT-D175N, flexibility of WT-D175N and WT-E180G), intermediate between the two (actin affinity of WT-E180G), or different from both (thermal stability of reduced WT-E40K, WT-E54K, WT-E180G, and WTD230N; actin affinity ofWT-E40, WT-E54K, and WT-D230N; ΔpCa of D175N). The results demonstrate that the properties of Tm heterodimers cannot be predicted from the interpolation of the WT and mutant homodimer data. The distinct properties of heterodimers establish that it will be important to define if the pathogenic agent is a homodimer, a heterodimer or both for each known cardiomyopathy mutation in Tm.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: QP517 Biochemistry