PhD Defense: Zara Akram

Clinically identified missense mutations in the Wilson Disease gene interfere with in vivo folding of ATP7B Copper ATPase

Supervisor: Professor Per Amstrup Pedersen

Evaluation committee:
Associate Professor Henriette Pilegaard, Biologisk Institut (chair)
Professor Steven Karlish, Weizmann Institute of Science, Israel
Professor Dan Klærke, Faculty of Life Science, University of Copenhagen

Wilson disease (WND) is an autosomal recessive disorder of copper excretion caused by mutations in the ATP7B gene encoding a copper P_1B-type ATPase. To investigate whether compromised in vivo folding of ATP7B is part of the molecular mechanism behind WND, we selected 10 clinically identified WND associated amino acid substitutions located in the N domain, the P domain or transmembrane domain 6 (TM6) and tested their effect on protein production, in vivo protein folding and protein function. Furthermore, we explored whether endogenous metabolites could act as chemical chaperones and correct the protein folding abnormalities caused by the investigated amino acid substitutions and improve ATP7B activity. Yeast complementation assays performed at 15ºC, 20ºC, 25ºC, 30ºC or 35ºC identified and number of variants to be temperature-sensitive with respect to delivery of copper to the yeast ferroxidase, Fet3p. Live cell bioimaging showed that nine out of ten investigated disease causing ATP7B variants could be localized to the Golgi compartment. In contrast to wild type monitoring the Unfolded Protein Response revealed that production of all disease causing ATP7B variants, except one, induced the unfolded protein response in a temperature dependent way. In contrast to temperature independent accumulation of wild type ATP7B, western blotting demonstrated temperature dependent accumulation for all variants except three. Supplementation of endogenous small molecules to the growth medium partially rescued activity of some the clinically identified missense mutations.