There is considerable evidence that the prion protein binds copper. However, there have also been suggestions that prion protein (PrP) binds manganese. We used isothermal titration calorimetry to identify the manganese binding sites in wild-type mouse PrP. The protein showed two manganese binding sites with affinities that would bind manganese at concentrations of 63 and 200 M at pH 5.5. This indicates that PrP binds manganese with affinity similar to other known manganese-binding proteins. Further study indicated that the main manganese binding site is associated with His-95 in the so-called "fifth site" normally associated with copper binding. Additionally, it was shown that occupancy by copper does not prevent manganese binding. Under these conditions, manganese binding resulted in an altered conformation of PrP, displacement of copper, and altered redox chemistry of the metal-protein complex. Cyclic voltammetric measurements suggested a complex redox chemistry involving manganese bound to PrP, whereas copper-bound PrP was able to undergo fully reversible electron cycling. Additionally, manganese binding to PrP converted it to a form able to catalyze aggregation of metal-free PrP. These results further support the notion that manganese binding could cause a conformation change in PrP and trigger changes in the protein similar to those associated with prion disease.
Paramount among issues relating to the transmissible spongiform encephalopathies (also known as prion diseases) is the absence of any effective therapy. This need has been heightened by the substantial European and emerging global problem of bovine spongiform encephalopathy and consequent variant Creutzfeldt-Jakob disease. Stimulated by the recent reports of a potent antiprion effect in cell culture-based clearance assays, we studied the utility of quinacrine in a well-characterized in vivo model of mouse-adapted transmissible spongiform encephalopathy. Our results failed to show any evidence that quinacrine is effective when using the simple but objective measure of survival prolongation.
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