Physicochemical Properties Altered by the Tail Group of Lipid Membranes Influence Huntingtin Aggregation and Lipid Binding.

Huntington's disease is a neurodegenerative disorder caused by an expanded polyglutamine (polyQ) domain within the huntingtin protein (htt) that initiates toxic protein aggregation. Htt directly interacts with membranes, influencing aggregation and spurring membrane abnormalities. These interactions are facilitated by the 17 N-terminal residues (Nt17) that form an amphipathic α-helix implicated in both lipid binding and aggregation. Here, the impact of unsaturation in phospholipid tails on htt-lipid interaction and htt aggregation was determined. There was no correlation between the degree of htt-lipid complexation and the degree of htt aggregation in the presence of each lipid system, indicating that lipid systems with different properties uniquely alter the membrane-mediated aggregation mechanisms. Also, the association between Nt17 and membrane surfaces is determined by complementarity between hydrophobic residues and membrane defects and how easily the peptide can partition into the bilayer. Our results provide critical insights into how membrane physical properties influence downstream htt aggregation.

Macromolecular crowding in solution alters huntingtin interaction and aggregation at interfaces.

Huntington's disease (HD) is a fatal neurodegenerative disease caused by an extended polyglutamine (polyQ) domain within the first exon of the huntingtin protein (htt). PolyQ expansion directly invokes the formation of a heterogenous mixture of toxic htt aggregates, including fibrils and oligomers. While htt is a cytosolic protein, it also associates with numerous membranous surfaces within the cell, leading to altered organelle morphology and dysfunction. Here, the impact of macromolecular crowding on htt aggregation in bulk solution and at solid/liquid or membrane/liquid interfaces was investigated. Dextran, Ficoll, and polyethylene glycol (PEG) were used as crowding agents. In bulk solution, crowding enhanced the heterogeneity of non-fibrillar aggregate species formed in a crowder dependent manner. However, crowding agents interfered with the deposition of htt fibrils on mica, suggesting that a crowded aqueous phase influences the interaction of htt with interfaces. By use of in situ atomic force microcopy (AFM), the aggregation of htt directly at mica and bilayer interfaces was tracked. The predominate aggregates type observed to form at the mica interface was fibrillar, but oligomeric aggregates of various stabilities were also observed. Crowding in the aqueous phase suppressed deposition and formation of htt aggregates on mica. In contrast, the addition of crowders enhanced deposition of htt aggregates onto supported total brain lipid extract (TBLE) bilayers. Different crowding agents led to distinct htt aggregates on supported bilayers with unique morphological impact on bilayer integrity. Collectively, these observations point to the complexity of htt aggregation at interfaces and that crowding in the aqueous phase profoundly influences this process.

Lipid headgroups alter huntingtin aggregation on membranes.

Huntington's Disease is a fatal neurodegenerative disorder caused by expansion of a glutamine repeat region (polyQ) beyond a critical threshold within exon1 of the huntingtin protein (htt). As a consequence of polyQ expansion, htt associates into a variety of aggregate species that are thought to underlie cellular toxicity. Within cells, htt associates with numerous membranous organelles and surfaces that exert influence on the aggregation process. In particular, the first 17 amino acids at the N-terminus of htt (Nt17) serve as a lipid-binding domain that is intrinsically disordered in bulk solution but adopts an amphipathic α-helical structure upon binding membranes. Beyond this, Nt17 is implicated in initiating htt fibrillization. As the interaction between Nt17 and lipid membranes is likely influenced by lipid properties, the impact of lipid headgroups on htt-exon1 aggregation, membrane activity, and the ability to form protein:lipid complexes was determined. Htt-exon1 with a disease-length polyQ domain (46Q) was exposed to lipid vesicles comprised of lipids with either zwitterionic (POPC and POPE) or anionic (POPG and POPS) headgroups. With zwitterionic head groups, large lipid to peptide ratios were required to have a statistically significant impact on htt aggregation. Anionic lipids enhanced htt fibrillization, even at low lipid:protein ratios, and this was accompanied by changes in aggregate morphology. Despite the larger impact of anionic lipids, htt-exon1(46Q) was more membrane active with zwitterionic lipid systems. The ability of Nt17 to form complexes with lipids was also mediated by lipid headgroups as zwitterionic ionic lipids more readily associated with multimeric forms of Nt17 in comparison with anionic lipids. Collectively, these results highlight the complexity of htt/membrane interactions and the resulting impact on the aggregation process.

Phosphomimetic Mutations Impact Huntingtin Aggregation in the Presence of a Variety of Lipid Systems.

Huntington's disease (HD) is a neurodegenerative disorder caused by the abnormal expansion of a polyglutamine (polyQ) tract in the first exon of the htt protein (htt). PolyQ expansion triggers the aggregation of htt into a variety of structures, including oligomers and fibrils. This aggregation is impacted by the first 17 N-terminal amino acids (Nt17) of htt that directly precedes the polyQ domain. Beyond impacting aggregation, Nt17 associates with lipid membranes by forming an amphipathic α-helix. Post-translational modifications within Nt17 are known to modify HD pathology, and in particular, phosphorylation at T3, S13, and/or S16 retards fibrillization and ameliorates the phenotype in HD models. Due to Nt17's propensity to interact with lipid membranes, the impact of introducing phosphomimetic mutations (T3D, S13D, and S16D) into htt-exon1 on aggregation in the presence of a variety of model lipid membranes (total brain lipid extract, 1-palmitoyl-2-oleoyl-glycero-3-phosphatidylcholine, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-1'-rac-glycerol) was investigated. Phosphomimetic mutations altered htt's interaction with and aggregation in the presence of lipids; however, this was dependent on the lipid system.

Lipid Membranes Influence the Ability of Small Molecules To Inhibit Huntingtin Fibrillization.

Several diseases, including Alzheimer's disease, Parkinson's disease, and Huntington's disease (HD), are associated with specific proteins aggregating and depositing within tissues and/or cellular compartments. The aggregation of these proteins is characterized by the formation of extended, ß-sheet rich fibrils, termed amyloid. In addition, a variety of other aggregate species also form, including oligomers and protofibrils. Specifically, HD is caused by the aggregation of the huntingtin (htt) protein that contains an expanded polyglutamine domain. Due to the link between protein aggregation and disease, small molecule aggregation inhibitors have been pursued as potential therapeutic agents. Two such small molecules are epigallocatechin 3-gallate (EGCG) and curcumin, both of which inhibit the fibril formation of several amyloid-forming proteins. However, amyloid formation is a complex process that is strongly influenced by the protein's environment, leading to distinct aggregation pathways. Thus, changes in the protein's environment may alter the effectiveness of aggregation inhibitors. A well-known modulator of amyloid formation is lipid membranes. Here, we investigated if the presence of lipid vesicles altered the ability of EGCG or curcumin to modulate htt aggregation and influence the interaction of htt with lipid membranes. The presence of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine or total brain lipid extract vesicles prevented the curcumin from inhibiting htt fibril formation. In contrast, EGCG's inhibition of htt fibril formation persisted in the presence of lipids. Collectively, these results highlight the complexity of htt aggregation and demonstrate that the presence of lipid membranes is a key modifier of the ability of small molecules to inhibit htt fibril formation.

Proteins Containing Expanded Polyglutamine Tracts and Neurodegenerative Disease.

Several hereditary neurological and neuromuscular diseases are caused by an abnormal expansion of trinucleotide repeats. To date, there have been 10 of these trinucleotide repeat disorders associated with an expansion of the codon CAG encoding glutamine (Q). For these polyglutamine (polyQ) diseases, there is a critical threshold length of the CAG repeat required for disease, and further expansion beyond this threshold is correlated with age of onset and symptom severity. PolyQ expansion in the translated proteins promotes their self-assembly into a variety of oligomeric and fibrillar aggregate species that accumulate into the hallmark proteinaceous inclusion bodies associated with each disease. Here, we review aggregation mechanisms of proteins with expanded polyQ-tracts, structural consequences of expanded polyQ ranging from monomers to fibrillar aggregates, the impact of protein context and post-translational modifications on aggregation, and a potential role for lipid membranes in aggregation. As the pathogenic mechanisms that underlie these disorders are often classified as either a gain of toxic function or loss of normal protein function, some toxic mechanisms associated with mutant polyQ tracts will also be discussed.