Polyglutamine Induced Misfolding of Huntingtin Exon1 is Modulated by the Flanking Sequences

Polyglutamine (polyQ) expansion in exon1 (XN1) of the huntingtin protein is linked to Huntington's disease. When the number of glutamines exceeds a threshold of approximately 36–40 repeats, XN1 can readily form amyloid aggregates similar to those associated with disease. Many experiments suggest that misfolding of monomeric XN1 plays an important role in the length-dependent aggregation. Elucidating the misfolding of a XN1 monomer can help determine the molecular mechanism of XN1 aggregation and potentially help develop strategies to inhibit XN1 aggregation. The flanking sequences surrounding the polyQ region can play a critical role in determining the structural rearrangement and aggregation mechanism of XN1. Few experiments have studied XN1 in its entirety, with all flanking regions. To obtain structural insights into the misfolding of XN1 toward amyloid aggregation, we perform molecular dynamics simulations on monomeric XN1 with full flanking regions, a variant missing the polyproline regions, which are hypothesized to prevent aggregation, and an isolated polyQ peptide (Qn). For each of these three constructs, we study glutamine repeat lengths of 23, 36, 40 and 47. We find that polyQ peptides have a positive correlation between their probability to form a β-rich misfolded state and their expansion length. We also find that the flanking regions of XN1 affect its probability to^x_page_count=28 form a β-rich state compared to the isolated polyQ. Particularly, the polyproline regions form polyproline type II helices and decrease the probability of the polyQ region to form a β-rich state. Additionally, by lengthening polyQ, the first N-terminal 17 residues are more likely to adopt a β-sheet conformation rather than an α-helix conformation. Therefore, our molecular dynamics study provides a structural insight of XN1 misfolding and elucidates the possible role of the flanking sequences in XN1 aggregation

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

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

F-Actin Binding Regions on the Androgen Receptor and Huntingtin Increase Aggregation and Alter Aggregate Characteristics

Protein aggregation is associated with neurodegeneration. Polyglutamine expansion diseases such as spinobulbar muscular atrophy and Huntington disease feature proteins that are destabilized by an expanded polyglutamine tract in their N-termini. It has previously been reported that intracellular aggregation of these target proteins, the androgen receptor (AR) and huntingtin (Htt), is modulated by actin-regulatory pathways. Sequences that flank the polyglutamine tract of AR and Htt might influence protein aggregation and toxicity through protein-protein interactions, but this has not been studied in detail. Here we have evaluated an N-terminal 127 amino acid fragment of AR and Htt exon 1. The first 50 amino acids of ARN127 and the first 14 amino acids of Htt exon 1 mediate binding to filamentous actin in vitro. Deletion of these actin-binding regions renders the polyglutamine-expanded forms of ARN127 and Htt exon 1 less aggregation-prone, and increases the SDS-solubility of aggregates that do form. These regions thus appear to alter the aggregation frequency and type of polyglutamine-induced aggregation. These findings highlight the importance of flanking sequences in determining the propensity of unstable proteins to misfold

Lipid binding properties of huntingtin as a novel therapeutic target

As protein aggregation is the defining hallmark of all amyloid diseases, a common therapeutic strategy is to develop molecules that inhibit aggregation. However, this approach has yielded limited success. Many amyloid proteins directly interact with lipid membranes. These interactions promote distinct aggregation pathways and often result in membrane damage leading to toxicity. As a result, directly targeting the ability of amyloids to bind lipid membranes represents a novel therapeutic strategy. As a proof of principle, the interaction between lipid membranes and mutant huntingtin protein (htt) aggregates was used to test this strategy. Mutant htt containing an expanded polygulatmine (polyQ) domain causes Huntington’s disease (HD). Using a colorimetric lipid binding assay over 1200 compounds were screened for their ability to block htt/lipid binding. The screen was set up to only identify compounds that directly interacted with htt, not the lipid membrane. Three compounds were identified having the ability to inhibit htt/lipid interaction, Ro-90-7501 (Ro), benzamil hydrochloride (ben) and ruthenium red. As these compounds directly interact with htt, ThT and AFM assays were performed to assess their impact on aggregation. Ro and ben did not inhibit fibril formation; however, oligomer precursors were significantly smaller when exposed to Benzamil. Molecular dynamic simulations (MD) revealed that the two compounds have unique mechanisms of interaction with htt aggregates. Unlike Ro and ben, ruthenium red altered htt aggregation and inhibit fibrilization. Having established that the compound prevented htt from binding membranes, a C. elegans model of HD was used to determine if this strategy could alleviate phenotype. Despite have a minimal impact on punctate formation, all three compounds reduced a thrashing deficit in animals caused by mutant htt expression, suggesting that this strategy reduces htt toxicity

Fibril polymorphism affects immobilized non-amyloid flanking domains of huntingtin exon1 rather than its polyglutamine core

Polyglutamine expansion in the huntingtin protein is the primary genetic cause of Huntington's disease (HD). Fragments coinciding with mutant huntingtin exon1 aggregate in vivo and induce HD-like pathology in mouse models. The resulting aggregates can have different structures that affect their biochemical behaviour and cytotoxic activity. Here we report our studies of the structure and functional characteristics of multiple mutant htt exon1 fibrils by complementary techniques, including infrared and solid-state NMR spectroscopies. Magic-angle-spinning NMR reveals that fibrillar exon1 has a partly mobile α-helix in its aggregation-accelerating N terminus, and semi-rigid polyproline II helices in the proline-rich flanking domain (PRD). The polyglutamine-proximal portions of these domains are immobilized and clustered, limiting access to aggregation-modulating antibodies. The polymorphic fibrils differ in their flanking domains rather than the polyglutamine amyloid structure. They are effective at seeding polyglutamine aggregation and exhibit cytotoxic effects when applied to neuronal cells

Physicochemical modulation of huntingtin aggregation on lipid membranes: Implications for Huntington\u27s disease

Huntington disease is a genetic, neurodegenerative disease caused by an expanded polyglutamine (polyQ) in the first exon of the huntingtin (htt) protein, facilitating its aggregation, leading to the formation of a diverse population of potentially toxic aggregate species, such as oligomers, fibrils, and annular aggregates. Htt interacts with a variety of membranous structures within the cell, and the first seventeen amino acids (Nt17) of htt directly flanking the polyQ domain is an amphipathic alpha-helix (AH) lipid-binding domain. AHs are also known to detect membrane curvature. Nt17 also promotes diverse aggregate species of htt and undergoes a number of posttranslational modifications that can modulate htt\u27s toxicity, subcellular localization, and trafficking of vesicles. To get in-depth mechanistic insights of huntingtin aggregation, both chemical and physical modulators of the aggregation process were explored. Specifically, the importance of htt acetylation and the role of membrane curvature on htt aggregation were investigated using atomic force microscopy (AFM), which has become a robust technique to obtain physical insights into the formation of toxic protein aggregates associated with amyloid diseases on lipid membranes. Acetylation of htt exon 1, and synthetic truncated htt exon1 mimicking peptide (Nt 17Q35P10KK) was achieved using a selective covalent label sulfo-N-hydroxysuccinimide (NHSA) in molar ratios of 1x, 2x, and 3x NHSA per peptide. Htt acetylation was found to decrease fibril formation in solution and promoted the formation of larger globular aggregates. Acetylation strongly altered htt\u27s ability to bind lipid membranes. However, one of the several limitations associated with using these current flat, supported bilayers as model surfaces is the absence of membrane curvature, which can heavily influence the interaction of proteins at lipid interfaces. Using an AFM force reconstruction technique, silicon substrate, and silica nanobeads, model lipid bilayer system was developed and validated in which the underlying solid support is comprised of flat and curved regions to induce regions of curvature in the bilayer. This model bilayer system was exposed to Nt17Q35P10KK peptide, and this peptide preferentially bound and accumulated to curved membranes, consistent with the ability of AHs to sense membrane curvature

Cholesterol Modulates Huntingtin Binding to and Aggregation on Lipid Membranes

Huntington disease is an autosomal dominant neurodegenerative disorder. The abnormally long CAG-repeats in the huntingtin gene that encode an expanded polyglutamine stretch which promotes self-assemble of huntingtin into different aggregation species that are disease related. Huntingtin intimately interacts with a variety of lipid membranes. Lipid composition is altered in HD, especially cholesterol content. Here we investigate how cholesterol content modulates the interaction between huntingtin and lipid membranes. TBLE/PDA binding assay is performed to test the binding affinity of huntingtin with lipid bilayers containing different amount of cholesterol. As the cholesterol content increases, the extent of huntingtin binding to lipid bilayers decreases. Also, atomic force microscopy (AFM) is used to directly monitor the formation of aggregates on supported lipid bilayers containing exogenously added cholesterol. Morphological and mechanical changes in the bilayers exposed to huntingtin are observed by the presence of cholesterol. On pure TBLE, globular aggregates are formed and grainy in appearance. Most of the bilayers are disrupted. In contrast, lipid bilayers enriched in different amount of cholesterol facilitate the formation of plateau-like aggregates with a smooth appearance. With the increase of cholesterol content in lipid bilayers, the percentage of the surface disrupted by the protein aggregation decreases. In sum, the presence and amount of cholesterol in lipid bilayers modulates the huntingtin binding and aggregation on lipid membranes

Structure of a single-chain Fv bound to the 17 N-terminal residues of huntingtin provides insights into pathogenic amyloid formation and suppression.

Huntington's disease is triggered by misfolding of fragments of mutant forms of the huntingtin protein (mHTT) with aberrant polyglutamine expansions. The C4 single-chain Fv antibody (scFv) binds to the first 17 residues of huntingtin [HTT(1-17)] and generates substantial protection against multiple phenotypic pathologies in situ and in vivo. We show in this paper that C4 scFv inhibits amyloid formation by exon1 fragments of huntingtin in vitro and elucidate the structural basis for this inhibition and protection by determining the crystal structure of the complex of C4 scFv and HTT(1-17). The peptide binds with residues 3-11 forming an amphipathic helix that makes contact with the antibody fragment in such a way that the hydrophobic face of this helix is shielded from the solvent. Residues 12-17 of the peptide are in an extended conformation and interact with the same region of another C4 scFv:HTT(1-17) complex in the asymmetric unit, resulting in a β-sheet interface within a dimeric C4 scFv:HTT(1-17) complex. The nature of this scFv-peptide complex was further explored in solution by high-resolution NMR and physicochemical analysis of species in solution. The results provide insights into the manner in which C4 scFv inhibits the aggregation of HTT, and hence into its therapeutic potential, and suggests a structural basis for the initial interactions that underlie the formation of disease-associated amyloid fibrils by HTT.E.D.G. and C.M.D. are grateful for support by the Medical Research Council (G1002272). We also thank the Hereditary Disease Foundation (A.M.). D.Y.C. is supported by the Crystallographic X-ray Facility at the Department of Biochemistry, University of Cambridge. We would like to acknowledge Dr. Katherine Stott at the Biophysics Facility at the Department of Biochemistry, University of Cambridge, for her help with the ultracentrifugation experiments and Prof. Weiss and Dr. Desplancq at the Ecole Supérieure de Biotechnologie de Strasbourg for the kind gift of the gankyrin-specific scFv, scFvR19 as a control for our in vitro aggregation experiments.This is the final published version. It first appeared at http://www.sciencedirect.com/science/article/pii/S002228361500217X#

Factors Influencing Huntingtin Aggregation at Surfaces: Implications for Huntington’s Disease

Huntington’s Disease (HD) is a genetic, neurodegenerative disease characterized by an abnormal polyglutamine (polyQ) expansion in the first exon of the huntingtin protein (htt). The polyQ domain facilitates aggregation and initiates the formation of a diverse collection of aggregate species, including fibrils, oligomers and annular aggregates. The first 17 amino acids of htt (Nt17) directly flank the polyQ domain and is a key factor in htt’s association to membranous structures. In addition to Nt17 being an amphipathic αhelix, it also promotes aggregation through self-association and contains numerous posttranslational modifications (PTMs) that can modulate toxicity and subcellular localization. For in depth understanding of these mechanisms, particularly in the presence of lipid membrane surfaces, the PTM phosphorylation and macromolecular crowders found in subcellular environments were explored. Through the application of phosphomimetic mutations of htt to a variety of lipid systems, lipid-specific impacts of electrostatic interactions involved in htt/lipid interactions were elucidated. Cytosolic conditions mimicked through the addition of macromolecular crowders and htt were evaluated at both solid/liquid and membrane/liquid interfaces, with each crowder having a distinct effect on htt aggregation. The results presented here aid in the understanding of the multi-faceted nature of htt aggregation in the presence of cellular and subcellular surfaces

Conformational studies of pathogenic expanded polyglutamine protein deposits from Huntington’s disease

Huntington’s disease, like other neurodegenerative diseases, continues to lack an effective cure. Current treatments that address early symptoms ultimately fail Huntington’s disease patients and their families, with the disease typically being fatal within 10–15 years from onset. Huntington’s disease is an inherited disorder with motor and mental impairment, and is associated with the genetic expansion of a CAG codon repeat encoding a polyglutamine-segment-containing protein called huntingtin. These Huntington’s disease mutations cause misfolding and aggregation of fragments of the mutant huntingtin protein, thereby likely contributing to disease toxicity through a combination of gain-of-toxic-function for the misfolded aggregates and a loss of function from sequestration of huntingtin and other proteins. As with other amyloid diseases, the mutant protein forms non-native fibrillar structures, which in Huntington’s disease are found within patients’ neurons. The intracellular deposits are associated with dysregulation of vital processes, and inter-neuronal transport of aggregates may contribute to disease progression. However, a molecular understanding of these aggregates and their detrimental effects has been frustrated by insufficient structural data on the misfolded protein state. In this review, we examine recent developments in the structural biology of polyglutamine-expanded huntingtin fragments, and especially the contributions enabled by advances in solid-state nuclear magnetic resonance spectroscopy. We summarize and discuss our current structural understanding of the huntingtin deposits and how this information furthers our understanding of the misfolding mechanism and disease toxicity mechanisms