Multiple CopperIons Bind to and Promote the Oligomerizationof Huntingtin Protein with Nonpathological Repeat Expansions

Huntington’s disease (HD) is a fatal neurodegenerative disease characterized by the expression of huntingtin protein (htt) that has a polyglutamine (CAG; polyQ) repeat domain consisting of 36 or more glutamines (mhtt). Historically, mhtt is more broadly associated with HD severity, as are elevated metal levels observed in HD patients. The depletion of wild-type (WT) htt (fewer than 36Qs) is also recognized as a contributing factor to HD progression; however, many questions remain about the interactions of biorelevant metals with WT htt and the impact of the interactions on protein aggregation. In the present work, we utilize a combination of biochemical assays and spectroscopic techniques to provide insights into the interaction of copper with an <i>in vitro</i> htt model (N171–17Q). Herein, we use sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and dynamic light scattering to show that the addition of equimolar or higher concentrations of Cu(II) to htt induces time- and temperature-dependent protein oligomerization/aggregation. Additionally, chelation assays, trapped ion mobility spectrometry, and mass spectrometry confirm the (i) rapid reduction of Cu(II) in the presence of N171–17Q htt, (ii) direct binding of multiple copper ions per protein, and (iii) complex Cu:htt speciation profile with a preference for three distinct Cu:htt states. These findings contribute to our molecular level understanding of copper’s role in the depletion and oligomerization/aggregation of WT htt while underscoring the physiological significance of our work, its potential relevance to metal binding in mhtt, and its significance for identifying new avenues for biomarker exploration and therapeutic design strategies.