Oxidation of Huntingtin Modulates its Aggregation and Interaction with Membranes

Abstract

Huntington's disease is an autosomal dominant disease caused by the expansion of a polyglutamine (polyQ) domain within the first exon of the huntingtin (htt) protein. PolyQ expansion promotes htt aggregation into oligomers, b-sheet rich fibrils, and other amorphous species. Htt aggregation is mediated by the first 17 N-terminal amino acids (N17) that directly precede the polyQ domain. In addition, N17 is a membrane binding domain. Both the ability of N17 to enhance htt aggregation and bind lipid membranes are attributed to its ability to form an amphipathic α-helix. N17 also contains several sites for potential posttranslational modifications (PTMs), including oxidation of two methionine residues. Here, the impact of oxidation of methionine residues within N17 on htt aggregation in the presence or absence of lipid membranes comprised of total brain lipid extract (TBLE) was investigated. Two experimental systems were used, a synthetic N-terminal peptide fragment of htt (N17-Q35-P10) and full-length htt-exon1 with 46 repeat glutamines expressed as a GST-fusion protein. For the second system, cleavage of the GST moiety initiates aggregation. To oxidize the methionine residues in N17, the experimental htt systems were treated with hydrogen peroxide. Based on ThT assays, hydrogen peroxide treatment reduced N17-Q35-P10 fibril formation in a dose-dependent manner. Fibrillization of full-length htt-exon1 appeared inhibited with similar hydrogen peroxide treatments as assessed by ex situ atomic force microscopy (AFM) assays. That is, increasing peroxide treatments led to the emergence of shorter and unbranched fibrils while promoting oligomerization. The addition of lipid vesicles generally reduced fibrilization of both experimental systems with and without hydrogen peroxide treatment. However, similar to the trend observed in the absence of lipid, increasing oxidant concentration favored the formation of oligomers. In addition, the aggregation of unoxidized and oxidized htt-exon1 was directly tracked on supported TBLE lipid bilayers using in situ AFM. Both oxidized and unoxidized lipid bilayers were investigated. In general, oxidation of either htt-exon1 alone or both htt and TBLE resulted in reduced membrane damage and disruption. Unoxidized htt deposited quickly and aggressively on TBLE bilayers, forming lumpy amorphous aggregates. With oxidized lipid membranes, htt-exon1 deposited less aggressively on the bilayer, forming a mix of amorphous and smaller grainy oligomeric aggregates over a longer period. In contrast, when both htt-exon1 and TBLE were oxidized, the aggregates formed were mostly grainy oligomeric aggregates. Collectively, these observations suggest that oxidation plays an important role in both the aggregation of htt and its ability to bind lipid membranes, which may ultimately be related to the location of the methionine residues within the amphipathic α-helix of N17.