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dc.contributor.authorTondfekr, Reza
dc.date.accessioned2021-01-04T01:15:25Z
dc.date.available2021-01-04T01:15:25Z
dc.date.issued2020
dc.identifier.urihttp://hdl.handle.net/11343/258506
dc.description© 2020 Reza Tondfekr
dc.description.abstractCopper is a vital but potentially toxic element for all living organisms. Therefore, it is necessary to maintain a balance between a deficiency and an excess level of copper. Cu-ATPase proteins are selective for Cu(I) and are crucial for removal of excess copper from cellular cytosols and for supply of essential copper to the required enzymes. Using the energy derived from ATP hydrolysis, the ATPase ATP7A (Menkes' protein, MNK) plays a key role in maturation of copper enzymes by inserting the essential metal cofactor into the nascent apo-forms. It also maintains copper homeostasis by controlling copper levels in cells. Mutations in the ATP7A gene appear to lead to neurodegenerative diseases such as Menkes' disease (MD), Occipital Horn Syndrome (OHS), X-linked Distal Hereditary Motor Neuropathy (dHMNx), and Brachial Amyotrophic Diplegia (BAD). In the past decade, in vivo studies of the human copper pumps ATP7A/ATP7B have improved the understanding of their cellular functions. However, the investigation of the Cu-ATPase pump at the molecular level has fallen behind due to a number of challenges. In general, molecular characterization of transmembrane proteins requires large amounts of highly purified samples plus the challenge of producing a Cu pump in unmodified native form. It has also proven difficult to express and isolate the Cu pumps. This study details methods to address those challenges. This work also aims to undertake molecular characterisation of ATP7A and selected mutants using a 1H-NMR approach as a sensitive probe of ATPase activity. Asp1044 is the primary phosphorylation site that is essential for ATP7A function and the variant D1044E served as a negative control for the functional assay. The two variants P1386S and M1311V are two mutations identified in patients with dHMNx and BAD diseases, respectively. A baculovirus expression system was used to express His-tagged wild-type protein ATP7Awt and the variants in fully functional form. When dissolved in the detergent n-dodecyl-Beta-D-maltopyranoside (DDM) or embedded in Sf21 microsomes, the expressed proteins were each active in acyl-phosphorylation using the BODIPY FL ATP-Gamma-S assay system. Rates varied with Cu(I) availability, ATP concentration, enzyme concentration and temperature, and other conditions were optimized. The effects of competition for Cu(I) by glutathione (GSH), bathocuproine disulfonate anion (BCS), the metal binding site 1 from the E. coli analogue EcCopA (MBS1), metal binding site 6 of human Wilson disease protein (WLN6) and antioxidant protein 1 (ATOX1) were investigated. The ATPase activity assays evaluated by 1H-NMR demonstrated that: ATPase activity is dependent upon Cu(I) availability to the enzyme; the ATP7Awt protein is most active in the presence of its native metallo-chaperone ATOX1. The M1113V mutation does not affect ATPase activity significantly. Consequently, the problem of this mutation in BAD is likely related to the trafficking of Cu(I) rather than to its enzyme activity. The P1386S mutation associated with dHMNx, however, exhibited a reduced ATPase activity.
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dc.subjectCopper transport, ATP7A protein, ATPase activity, P1386S and M1311V mutations
dc.titleMolecular characterization of the ATP7A protein and selected mutants
dc.typePhD thesis
melbourne.affiliation.departmentSchool of Chemistry
melbourne.affiliation.facultyScience
melbourne.thesis.supervisornameAnthony Wedd
melbourne.contributor.authorTondfekr, Reza
melbourne.thesis.supervisorothernameZhiguang Xiao
melbourne.tes.fieldofresearch1340201 Bioinorganic chemistry
melbourne.accessrightsOpen Access


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