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Title: Exploring the chaperone function of the mammalian torsinA protein
Author: Adam, Ilectra Stavroula
Awarding Body: University of Kent
Current Institution: University of Kent
Date of Award: 2013
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The molecular chaperone Hspl04, in conjunction with two other chaperones Hsp70 and Hsp40, provides the yeast Saccharomyces cerevisiae with the necessary machinery to ensure that aggregated proteins that form in stressed cells can be remodelled back to a correctly folded and functio nal form. Hspl04 also plays an essential role in non-stressed cells in the propagation of yeast prions such as [PSt1, the prion form of the translation termination factor Sup3S. To date no functional mammalian orthologues of Hspl04 have been described although TorsinA, a 37 kOa ER located protein shows some degree of amino acid identity to Hspl04 predominantly through its conserved AAA+ ATPase domain. Earlier studies in mammalian cells had suggested that TorsinA may share more than sequence similarity with Hspl04 and function as a chaperone although this activity had not been formally demonstrated in vivo. This thesis describes a study of the chaperone function of mammalian TorsinA expressed in yeast. TorsinA was shown to partially rescue the ability of cells lacking Hspl04, to refold-heat denatured luciferase. This putative protein disaggregation function was impaired by mutations in the single ATPase domain of TorsinA and inhibited in cells grown in the presence of guanidine hydrochloride (GdnHCI), an inhibitor of the ATP-dependent function of Hspl04. These findings are consistent with Torsin A acting as an ATP-driven chaperone. However, mutating t he non-canonical Walker A motif of Torsin A nucleotide binding domain into a canonical sequence did not affect the ability of Torsin A to reactivate misfolded luciferase. Several variants of the [PSf1 prion were identified that were destabilised in cells exp ressing TorsinA, and when endogenous Hspl04 levels were elevated. Constitutive expression of Torsin A also protected yeast cells against various forms of stress in absence of Hspl04 demonstrating that TorsinA recapitulates a number of the important chaperone-related properties of Hspl04. A glutamic acid deletion in the (-terminus of Torsin A (associated with a movement disorder called torsion dystonia) reduced the ability of Torsin A to protect cells against stress and to eliminate [PSt] variants but did not affect its ability to reactivate heat-denatured luciferase. Torsin B belongs to the same gene family and shares a high sequence similarity (89.4% similarity) with TOrsin A but the chaperone function of Torsin B had not been established. Consequently its possible chaperone function was examined in yeast but was found not to protect cells against stress or protein aggregation and was not able to eliminate [PSf] but it inh ibited the growth of both yeast and mammalian cells. The ER targeting signal sequence ofTorsin A was recognized in yeast since a significant proportion of Torsin A was detected in the ER. Mutations in Torsin A that would be expected to impair ATP binding and hydrolysis did not affect its localisation although deletion of the N-terminal ER signal sequence resulted in its accumulation in the cytoplasm. 6N Torsin A mutant maintained the protective functions in yeast but exhibited reduced chaperone abilities. Under heat shock conditions Torsin A was translocated to the cytoplasm where it could process substrates, such as luciferase. Identification of a mammalian orthologue of Hspl04 is important for developing therapeutic strategies for protein aggregation-related disorders, such as Parkinson's disease, and improving recombinant protein production. The data in this thesis paint to Torsin A as a potential target.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available