FIGURE 1.

Copper promotes oligomerization of cell-free N171 huntingtin. N171-17Q huntingtin was expressed and purified from bacteria (see “Experimental Procedures”). Aliquots containing 2 μm protein were incubated for 1 h at 37 °C under conditions outlined below and then evaluated by Western blot analysis. A, purified N171 huntingtin spontaneously forms reduction-sensitive dimers. Copper incubation (5 μm) promotes formation of additional dimer and higher molecular mass oligomers. B–D, all samples were analyzed by nonreducing SDS-PAGE. B, copper dose-dependently promotes N171 oligomerization. As little as 50 nm added copper (II) promotes N171 oligomerization. C, hydrogen peroxide (2 μm) promotes N171-17Q oligomerization, but the effect is blocked by metal chelation. D, copper-selective chelators inhibit hydrogen peroxide (4 μm)-promoted oligomerization. E, iron and manganese do not promote N171 oligomerization. F, copper co-purifies with N171 huntingtin. N171-17Q huntingtin was expressed in bacteria and then purified using a DEAE column (see “Experimental Procedures”). Protein was quantified using BSA as standards. Metals were quantified by inductively coupled plasma-mass spectrometry. There is an average of 1 copper per 3 N171 huntingtin protein molecules. Manganese is present but in significantly smaller amounts. n = 3; beta-ME, β-mercaptoethanol; BC, bathocuproine.