Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.548023
Title: The molecular basis of the interactions of copper metallochaperones
Author: Aggarwal, Shilpa
Awarding Body: Newcastle University
Current Institution: University of Newcastle upon Tyne
Date of Award: 2011
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Abstract:
Copper metallochaperones help prevent mis-metallation of proteins and ensure copper incorporation in specific Cu(I)-binding proteins by ligand-exchange reactions. The human copper metallochaperone (hCCS) for superoxide dismutase-1 (hSOD1) can interact via domain 1 with the carboxyl-terminal domain (CTD) of human β-secretase-1 (hBACE1). hBACE1 initiates the production of amyloid-β peptide (Aβ), the major constituent of senile plaques in Alzheimer’s disease (AD). One of the aims of this thesis was to identify the residues involved in mediating the interaction between hCCS and hBACE1 CTD and to investigate the role of copper in mediating this interaction using a yeast two-hybrid system. The data show that the Cys residues in the CXXC motifs of hCCS and hBACE1 CTD are essential for this interaction. The interaction is optimised by residues located close to the Cu(I)-binding sites. The hCCS/hBACE1 CTD interaction decreases in copper-deficient conditions. hBACE1 CTD was also shown to interact with the copper metallochaperone HAH1 involving the Cys residues in the CXXC motif of hBACE1 CTD. A model has been proposed based on the physiological implications of these data with respect to AD pathology. The interaction of hCCS with hSOD1 was also investigated using the yeast two-hybrid system. The mutation of the Cu(I)-binding residues in hCCS do not have a significant effect on the hSOD1/hCCS interaction. Interestingly, the hSOD1/hCCS interaction increases in copper-deficient conditions which can also have implications for AD pathology, as discussed in the proposed model. The yeast two-hybrid system was also used to study the interaction of the copper metallochaperone Atx1 from Synechocystis PCC 6803 (ScAtx1) with the metal-binding domains (MBDs) of the Cu(I)-transporting ATPases, CtaA (CtaAN) and PacS (PacSN). The data show that the residues on loop 5 of the ferredoxin-like fold of ScAtx1 (His61), CtaAN (Phe87) and PacSN (Tyr65) are important for mediating proteinprotein interactions. Swapping the loop 5 residue disrupted complex formation of ScAtx1 with CtaAN and PacSN. The mutation of ScAtx1 His61 to a charged residue or to a neutral Ala also weakens the interactions with the MBDs, demonstrating that His61 is the best choice of amino acid in this position to enable ScAtx1 to interact optimally with both of its target proteins. The possibility that ScAtx1 may form two structurally different complexes (side-to-side or head-to-head) with CtaAN and PacSN was also investigated. The data indicate that both CtaAN and PacSN can likely form a side-toside complex although the possibility that they can also form a head-to-head complex can not be excluded.
Supervisor: Not available Sponsor: BBSRC
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.548023  DOI: Not available
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