Huntington's Disease (HD) is a neurodegenerative disorder that is defined by the accumulation of nanoscale aggregates comprised of the huntingtin (htt) protein. Aggregation is directly caused by an expanded polyglutamine (polyQ) domain in htt, leading to a diverse population of aggregate species, such as oligomers, fibrils, and annular aggregates. Furthermore, the length of this polyQ domain is directly related to onset and severity of disease. The first 17 N-terminal amino acids of htt have been shown to further modulate aggregation. Additionally, these 17 amino acids appear to have lipid binding properties as htt interacts with a variety of membrane-containing structures present in cells, such as organelles, and interactions with these membrane surfaces may further modulate htt aggregation. To investigate the interaction between htt exon1 and lipid bilayers, in situ atomic force microscopy (AFM) was used to directly monitor the aggregation of htt exon1 constructs with varying Q-lengths (35Q, 46Q, 51Q, and myc-53Q) on supported lipid membranes comprised of total brain lipid extract. The exon1 fragments accumulated on the lipid membranes, causing disruption of the membrane, in a polyQ dependent manner. Furthermore, the addition of an N-terminal myc-tag to the htt exon1 fragments impeded the interaction of htt with the bilayer.

Huntington's disease (HD) is a genetic neurodegenerative disorder caused by an expanded polyglutamine (polyQ) domain near the N-terminus of the huntingtin (htt) protein. Expanded polyQ leads to htt aggregation. The first 17 amino acids (Nt(17)) in htt comprise a lipid-binding domain that undergoes a number of posttranslational modifications that can modulate htt toxicity and subcellular localization. As there are three lysines within Nt(17), we evaluated the impact of lysine acetylation on htt aggregation in solution and on model lipid bilayers. Acetylation of htt-exon1(51Q) and synthetic truncated htt-exon 1 mimicking peptides (Nt(17)-Q35-P10-KK) was achieved using a selective covalent label, sulfo-N-hydroxysuccinimide (NHSA). With this treatment, all three lysine residues (K6, K9, and K15) in Nt(17) were significantly acetylated. N-terminal htt acetylation retarded fibril formation in solution and promoted the formation of larger globular aggregates. Acetylated htt also bound lipid membranes and disrupted the lipid bilayer morphology less aggressively compared with the wild-type. Computational studies provided mechanistic insights into how acetylation alters the interaction of Nt(17) with lipid membranes. Our results highlight that N-terminal acetylation influences the aggregation of htt and its interaction with lipid bilayers.

Huntington’s disease (HD) is caused by a polyglutamine (polyQ) domain that is expanded beyond a critical threshold near the N-terminus of the huningtin (htt) protein, directly leading to htt aggregation. While full-length htt is a large (on the order of ~350 kDa) protein, it is proteolyzed into a variety of N-terminal fragments that accumulate in oligomers, fibrils, and larger aggregates. It is clear that polyQ length is a key determinant of htt aggregation and toxicity. However, the flanking sequences around polyQ domain, such as the first seventeen amino acids on the N terminus (Nt17), influence aggregation, aggregate stability, other important biochemical properties of the protein, and ultimately it role in pathogenesis. Here, we review the impact of Nt17 on both htt aggregation mechanisms and kinetics, structural properties of Nt17 in both monomeric and aggregate forms, the potential role of post translational modifications (PTMs) that occur in Nt17 in HD, and Nt17’s function as a membrane targeting domain.

Huntington’s disease (HD) is an inherited neurodegenerative disease caused by abnormally long CAG-repeats in the huntingtin gene that encode an expanded polyglutamine (polyQ) domain near the N-terminus of the huntingtin (htt) protein. Expanded polyQ domains are directly correlated to disease-related htt aggregation. Htt is found highly associated with a variety of cellular and subcellular membranes that are predominantly comprised of lipids. Since cholesterol homeostasis is altered in HD, we investigated how varying cholesterol content modifies the interactions between htt and lipid membranes. A combination of Langmuir trough monolayer techniques, vesicle permeability and binding assays, and in situ atomic force microscopy were used to directly monitor the interaction of a model, synthetic htt peptide and a full-length htt-exon1 recombinant protein with model membranes comprised of total brain lipid extract (TBLE) and varying amounts of exogenously added cholesterol. As the cholesterol content of the membrane increased, the extent of htt insertion decreased. Vesicles containing extra cholesterol were resistant to htt-induced permeabilization. Morphological and mechanical changes in the bilayer associated with exposure to htt were also drastically altered by the presence of cholesterol. Disrupted regions of pure TBLE bilayers were grainy in appearance and associated with a large number of globular aggregates. In contrast, morphological changes induced by htt in bilayers enriched in cholesterol were plateau-like with a smooth appearance. Collectively, these observations suggest that the presence and amount of cholesterol in lipid membranes play a critical role in htt binding and aggregation on lipid membranes.

Huntington disease (HD) is a genetic neurodegenerative disease caused by an expanded polyglutamine (polyQ) domain in the first exon of the huntingtin (Htt) protein, facilitating its aggregation. Htt interacts with a variety of membraneous structures within the cell, and the first 17 amino acids (Nt17) of Htt directly flanking the polyQ domain comprise an amphiphathic α-helix (AH) lipid-binding domain. AHs are also known to detect membrane curvature. To determine if Htt exon 1 preferentially binds curved membranes, in situ atomic force microscopy (AFM) studies were performed. Supported lipid bilayers are commonly used as model membranes for AFM studies of protein aggregation. However, these supported bilayers usually lack curvature. By forming a bilayer on top of silica nanobeads (50 ± 10 nm) deposited on a silicon substrate, model supported lipid bilayers with flat and curved regions were developed for AFM studies. The presence of the bilayer over the beads was validated by continual imaging of the formation of the bilayer, height measurements, and spatially resolved mechanical measurements of the resulting bilayer using scanning probe acceleration microscopy. Interpretation of this data was facilitated by numerical simulations of the entire imaging process. The curved supported bilayers associated with the beads were found to be more compliant than flat supported bilayers, consistent with the altered packing density of lipids caused by the induced curvature. This model bilayer system was exposed to a synthetic truncated Htt exon 1 peptide (Nt17Q35P10KK), and this peptide preferentially accumulated on curved membranes, consistent with the ability of AHs to sense membrane curvature.