Investigation of Early Complex Formation of Huntingtin Protein With and Without Lipids

Abstract

Huntington’s disease (HD) is a fatal neurodegenerative disease caused by the expansion of the polyglutamine (polyQ) domain of the huntingtin protein (htt). The expansion of the polyQ domain beyond a threshold of approximately 35 repeats triggers complex toxic aggregation mechanisms and results in altered interactions between htt and lipid membranes. Many factors modulate these processes. One such modulator includes sequences flanking the polyQ domain, most notably the first 17 amino acids at the N-terminus of the protein (Nt17), and environmental factors including the presence of membranous structures. Nt17 has the propensity to form an amphipathic a-helix in the presence of binding partners, which can facilitate aggregation and lipid binding. These processes can be further modulated by overall membrane composition and physiochemical properties. Considering the influence of membrane composition and the known dysregulation of cholesterol homeostasis in HD, cholesterol may be a crucial membrane component that can modulate early htt interactions through influencing membrane properties such as permeability, fluidity, and overall organization. Early interactions may also be modulated through altered electrostatics, hydrophobicity, and hydrogen bonding introduced through mutations to residues within the Nt17 domain. A mechanistic understanding of such modulating factors and their impacts on early htt interactions can provide crucial insights into the toxic mechanism of HD. Early htt interactions were further explored in the studies presented here. Thioflavin T (ThT) aggregation assays, atomic force microscopy (AFM), polydiacetylene (PDA) lipid binding assays, and mass spectrometry (MS) were used to identify changes to aggregation, aggregate morphologies, htt/lipid binding, and htt/lipid complexation respectively. When htt was exposed to lipid systems composed of pure POPC, DOPC, and POPG distinctly different htt interactions with increasing amounts of exogenously added cholesterol were observed. Increasing cholesterol content increased aggregation for the DOPC systems, but reduced aggregation for the POPC and POPG systems. Htt/lipid binding decreased with increasing cholesterol for the POPC systems, while binding increased for the DOPC and POPG systems. Differences in htt/lipid complexation were also observed for each pure lipid system with increasing cholesterol content. When htt was incubated with Nt17 peptides with mutations that altered residue charges aggregation was significantly reduced, though the extent was dependent on the type of modification. Incorporation of Nt17 peptides into oligomeric structures resulted in minimal changes to oligomer/lipid interactions, though monomeric peptide/lipid complexation was altered with the introduction of modifications.