Copper transfer studies between the N-terminal copper binding domains one and four of human Wilson protein.

Human Wilson protein functions in the secretory pathway to insert copper ultimately into the multicopper oxidase ceruloplasmin and also plays a role in the excretion of excess copper to the bile. This copper-transporting P-type ATPase possesses six N-terminal cytosolic copper-binding domains contained within an approximately 72 amino acid consensus motif and the first four of these domains, denoted WLN1-4, are implicated in copper acquisition from the metallochaperone HAH1, whereas the domains closest to the membrane portion of the enzyme, WLN5-6, are essential for copper transport across the membrane. In order to test our hypothesis that copper transfer occurs between domains in the N-terminus of Wilson protein, we expressed and purified to homogeneity copper-binding domains 1, 3, 4, 5-6, and 6, denoted by WLN1, WLN3, WLN4, WLN5-6, and WLN6, respectively. Since we determined WLN1 and WLN4 to have the highest and lowest isoelectric points (6.77 and 3.85, respectively) and thus are readily separated via ion exchange chromatography, we developed a copper transfer assay between these domains. We anaerobically incubated either Cu(I)-WLN1 with apo-WLN4 or apo-WLN1 with Cu(I)-WLN4, then separated these domains and quantified the amount of copper that migrates from one domain to another by ICP-MS. Regardless of whether we start with Cu(I)-WLN1 or Cu(I)-WLN4 as the initial copper donor, we demonstrate facile copper transfer between WLN1 and WLN4, thereby demonstrating the feasibility of copper transfer between these domains in vivo.

Gait and cognition: Mapping the global and discrete relationships in ageing and neurodegenerative disease.

Recent research highlights the association of gait and cognition in older adults but a stronger understanding is needed to discern coincident pathophysiology, patterns of change, examine underlying mechanisms and aid diagnosis. This structured review mapped associations and predictors of gait and cognition in older adults with and without cognitive impairment, and Parkinson's disease. Fifty papers out of an initial yield of 22,128 were reviewed and a model of gait guided analysis and interpretation. Associations were dominated by the pace domain of gait; the most frequently studied domain. In older adults pace was identified as a predictor for cognitive decline. Where comprehensive measurement of gait was conducted, more specific pathological patterns of association were evident highlighting the importance of this approach. This review confirmed a robust association between gait and cognition and argues for a selective, comprehensive measurement approach. Results suggest gait may be a surrogate marker of cognitive impairment and cognitive decline. Understanding the specific nature of this relationship is essential for refinement of diagnostics and development of novel therapies.

Structure of human Wilson protein domains 5 and 6 and their interplay with domain 4 and the copper chaperone HAH1 in copper uptake.

Human Wilson protein is a copper-transporting ATPase located in the secretory pathway possessing six N-terminal metal-binding domains. Here we focus on the function of the metal-binding domains closest to the vesicular portion of the copper pump, i.e., domain 4 (WLN4), and a construct of domains 5 and 6 (WLN5-6). For comparison purposes, some experiments were also performed with domain 2 (WLN2). The solution structure of apoWLN5-6 consists of two ferredoxin folds connected by a short linker, and (15)N relaxation rate measurements show that it behaves as a unit in solution. An NMR titration of apoWLN5-6 with the metallochaperone Cu(I)HAH1 reveals no complex formation and no copper exchange between the two proteins, whereas titration of Cu(I)HAH1 with WLN4 shows the formation of an adduct that is in fast exchange on the NMR time scale with the isolated protein species as confirmed by (15)N relaxation data. A similar interaction is also observed between Cu(I)HAH1 and WLN2; however, the relative amount of the adduct in the protein mixture is lower. An NMR titration of apoWLN5-6 with Cu(I)WLN4 shows copper transfer, first to WLN6 then to WLN5, without the formation of an adduct. Therefore, we suggest that WLN4 and WLN2 are two acceptors of Cu(I) from HAH1, which then somehow route copper to WLN5-6, before the ATP-driven transport of copper across the vesicular membrane.