Huntingtin Exon Research Articles

Structural insights into the aggregation mechanism of huntingtin exon 1 protein fragment with different polyQ-lengths.

Huntington disease is a neurodegenerative disorder caused by the expansion of polyglutamine (polyQ) at the N-terminal of the huntingtin exon 1 protein. The detailed structure and the mechanism behind this aggregation remain unclear and it is assumed that the polyQ undergoes a conformational transition to the β-sheet structure when it aggregates. Investigating the misfolding of polyQ facilitates the determination of the molecular mechanism of aggregation and can potentially help in developing a novel approach to inhibit polyQ aggregation. Moreover, the flanking sequences of the polyQ region play a vital role in structural changes and the aggregation mechanism. We performed all-atom molecular dynamics simulations to gain structural insights into the aggregation mechanism using eight different models with glutamine repeat lengths Q27 , Q27 P11 , Q34 , Q35 , Q36 , Q40 , Q50 , and Q50 P11 . In the models without flanking polyPs, we noticed that the transformation of a random coil to β-sheet occurs when the number of Q increases. We also found that the flanking polyPs prevent aggregation by decreasing the probability of forming a β-sheet structure. When polyQ length increases, the 17 N-terminal flanking residues are more likely to adopt a β-sheet conformation from α-helix and coil. From our simulations, we suggest that at least 34 glutamines are required for initiating aggregation and 40 residues length is critical for the aggregation of huntingtin exon 1 protein for disease onset. This study provides structural insights into misfolding and the role of flanking sequences in huntingtin aggregation which will further help in developing therapeutic strategies for Huntington's disease.

The folding equilibrium of huntingtin exon 1 monomer depends on its polyglutamine tract

Expansion of the polyglutamine (polyQ) tract in exon 1 of the huntingtin protein (Httex1) leads to Huntington's disease resulting in fatal neurodegeneration. However, it remains poorly understood how polyQ expansions alter protein structure and cause toxicity. Using CD, EPR, and NMR spectroscopy, we found here that monomeric Httex1 consists of two co-existing structural states whose ratio is determined by polyQ tract length. We observed that short Q-lengths favor a largely random-coil state, whereas long Q-lengths increase the proportion of a predominantly α-helical state. We also note that by following a mobility gradient, Httex1 α-helical conformation is restricted to the N-terminal N17 region and to the N-terminal portion of the adjoining polyQ tract. Structuring in both regions was interdependent and likely stabilized by tertiary contacts. Although little helicity was present in N17 alone, each Gln residue in Httex1 enhanced helix stability by 0.03-0.05 kcal/mol, causing a pronounced preference for the α-helical state at pathological Q-lengths. The Q-length-dependent structuring and rigidification could be mimicked in proteins with shorter Q-lengths by a decrease in temperature, indicating that lower temperatures similarly stabilize N17 and polyQ intramolecular contacts. The more rigid α-helical state of Httex1 with an expanded polyQ tract is expected to alter interactions with cellular proteins and modulate the toxic Httex1 misfolding process. We propose that the polyQ-dependent shift in the structural equilibrium may enable future therapeutic strategies that specifically target Httex1 with toxic Q-lengths.

FAN1 modifies Huntington's disease progression by stabilizing the expanded HTT CAG repeat.

Huntington’s disease (HD) is an inherited neurodegenerative disease caused by an expanded CAG repeat in the huntingtin (HTT) gene. CAG repeat length explains around half of the variation in age at onset (AAO) but genetic variation elsewhere in the genome accounts for a significant proportion of the remainder. Genome-wide association studies have identified a bidirectional signal on chromosome 15, likely underlain by FANCD2- and FANCI-associated nuclease 1 (FAN1), a nuclease involved in DNA interstrand cross link repair. Here we show that increased FAN1 expression is significantly associated with delayed AAO and slower progression of HD, suggesting FAN1 is protective in the context of an expanded HTT CAG repeat. FAN1 overexpression in human cells reduces CAG repeat expansion in exogenously expressed mutant HTT exon 1, and in patient-derived stem cells and differentiated medium spiny neurons, FAN1 knockdown increases CAG repeat expansion. The stabilizing effects are FAN1 concentration and CAG repeat length-dependent. We show that FAN1 binds to the expanded HTT CAG repeat DNA and its nuclease activity is not required for protection against CAG repeat expansion. These data shed new mechanistic insights into how the genetic modifiers of HD act to alter disease progression and show that FAN1 affects somatic expansion of the CAG repeat through a nuclease-independent mechanism. This provides new avenues for therapeutic interventions in HD and potentially other triplet repeat disorders.

Dynamics of the Proline-Rich C-Terminus of Huntingtin Exon-1 Fibrils.

Intrinsically disordered protein domains not only are found in soluble proteins but also can be part of large protein complexes or protein aggregates. For example, several amyloid fibrils have intrinsically disordered domains framing a rigid β-sheet-rich core. These disordered domains can often be observed using solution NMR methods in combination with modest magic angle spinning and without perdeuteration. But how can these regions be detected using solution NMR methods when they are part of a fibril that is not tumbling isotropically in solution? Here we addressed this question by investigating the dynamic C-terminus of huntingtin exon-1 (HTTex1) fibrils that are important in Huntington's disease. We assigned the most dynamic regions of the C-terminus of three HTTex1 variants. On the basis of this assignment, we measured site-specific secondary chemical shifts, peak intensities, and R1, R'2, and R1ρ 15N relaxation rates. In addition, we determined the residual 1H-15N dipolar couplings of this region. Our results show that the dipolar couplings are averaged to a very high degree, resulting in an order parameter that is essentially zero. Together, our data show that the C-terminus of HTTex1 is intrinsically disordered and undergoes motions in the high picosecond to low nanosecond range.

Mutational analysis implicates the amyloid fibril as the toxic entity in Huntington's disease

In Huntington disease (HD), an expanded polyglutamine (polyQ > 37) sequence within huntingtin (htt) exon1 leads to enhanced disease risk. It has proved difficult, however, to determine whether the toxic form generated by polyQ expansion is a misfolded or avid-binding monomer, an α-helix-rich oligomer, or a β-sheet-rich amyloid fibril. Here we describe an engineered htt exon1 analog featuring a short polyQ sequence that nonetheless quickly forms amyloid fibrils and causes HD-like toxicity in rat neurons and Drosophila. Additional modifications within the polyQ segment produce htt exon1 analogs that populate only spherical oligomers and are non-toxic in cells and flies. Furthermore, in mixture with expanded-polyQ htt exon1, the latter analogs in vitro suppress amyloid formation and promote oligomer formation, and in vivo rescue neurons and flies expressing mhtt exon1 from dysfunction and death. Thus, in our experiments, while htt exon1 toxicity tracks with aggregation propensity, it does so in spite of the toxic construct's possessing polyQ tracts well below those normally considered to be disease-associated. That is, aggregation propensity proves to be a more accurate surrogate for toxicity than is polyQ repeat length itself, strongly supporting a major toxic role for htt exon1 aggregation in HD. In addition, the results suggest that the aggregates that are most toxic in these model systems are amyloid-related. These engineered analogs are novel tools for mapping properties of polyQ self-assembly intermediates and products that should similarly be useful in the analysis of other expanded polyQ diseases. Small molecules with similar amyloid inhibitory properties might be developed into effective therapeutic agents.

Decorrelating Kinetic and Relaxation Parameters in Exchange Saturation Transfer NMR: A Case Study of N-Terminal Huntingtin Peptides Binding to Unilamellar Lipid Vesicles.

Dark state exchange saturation transfer (DEST) and lifetime line-broadening (Δ R2, the difference in the measured transverse relaxation rates for the observable species in the presence and absence of exchange with a species characterized by very large intrinsic transverse relaxation rates) have proven to be powerful NMR tools for studying exchange phenomena between a NMR visible species and a high-molecular weight, "dark", NMR invisible state. However, in the exchange regime, where the transverse spin relaxation rates in the bound state ( R2bound) are smaller than the strength of the DEST saturation radio frequency field, typically corresponding to systems below ∼6 MDa, the combination of DEST and Δ R2 data, while sufficient to define the apparent association rate constant, cannot unambiguously determine the population of the bound state pB and R2bound values independently. We show that the latter exchange and relaxation parameters can be decorrelated by the measurement of the maximal value of the contribution of the fast-relaxing magnetization component to the total NMR signal, Cfastmax, an observable that is directly proportional to pB. When integrated into the analysis of DEST/Δ R2 data, Cfastmax provides an indispensable source of information for quantitative studies of exchange involving high-molecular-weight dark states. We demonstrate the utility of this approach by investigating the binding kinetics of two huntingtin exon-1-derived peptides to small unilamellar lipid vesicles (SUV), ∼ 31 nm in diameter and 4.3 MDa in molecular weight. The interaction of the N-terminal amphiphilic domain of huntingtin exon-1 with membrane surfaces promotes polyglutamine-mediated aggregation and, as such, is thought to play a role in the etiology of Huntington's disease, an autosomal dominant fatal neurodegenerative condition. The first peptide comprises the 16-residue N-terminal amphiphilic domain (httNT) alone, while the second contains an additional seven residue polyglutamine tract at the C-terminus (httNTQ7). At a peptide-to-lipid molar ratio of 1:4, the population of peptide bound to the SUV surface is substantial, ∼ 7-8%, while exchange between the free and SUV-bound peptide is slow on the relaxation time-scale ( kex ∼ 200 s-1). The last two C-terminal residues of httNT and the last 9 of httNTQ7 remain flexible in the SUV-bound form due to transient detachment from the lipid surface that occurs on a time-scale several-fold faster than binding.

N-Acyldopamine induces aggresome formation without proteasome inhibition and enhances protein aggregation via p62/SQSTM1 expression

Accumulation of ubiquitinated protein aggregates is a common pathology associated with a number of neurodegenerative diseases and selective autophagy plays a critical role in their elimination. Although aging-related decreases in protein degradation properties may enhance protein aggregation, it remains unclear whether proteasome dysfunction is indispensable for ubiquitinated-protein aggregation in neurodegenerative diseases. Here, we show that N-oleoyl-dopamine and N-arachidonyl-dopamine, which are endogenous brain substances and belong to the N-acyldopamine (AcylDA) family, generate cellular inclusions through aggresome formation without proteasome inhibition. Although AcylDA itself does not inhibit proteasome activity in vitro, it activates the rearrangement of vimentin distribution to form a vimentin cage surrounding aggresomes and sequesters ubiquitinated proteins in aggresomes. The gene transcription of p62/SQSTM1 was significantly increased by AcylDAs, whereas the transcription of other ubiquitin-dependent autophagy receptors was unaffected. Genetic depletion of p62 resulted in the loss of ubiquitinated-protein sequestration in aggresomes, indicating that p62 is a critical component of aggresomes. Furthermore, AcylDAs accelerate the aggregation of mutant huntingtin exon 1 proteins. These results suggest that aggresome formation does not require proteasome dysfunction and AcylDA-induced aggresome formation may participate in forming cytoplasmic protein inclusions.

Interaction of Huntingtin Exon-1 Peptides with Lipid-Based Micellar Nanoparticles Probed by Solution NMR and Q-Band Pulsed EPR.

Lipid-based micellar nanoparticles promote aggregation of huntingtin exon-1 peptides. Here we characterize the interaction of two such peptides, httNTQ 7 and httNTQ 10 comprising the N-terminal amphiphilic domain of huntingtin followed by 7 and 10 glutamine repeats, respectively, with 8 nm lipid micelles using NMR chemical exchange saturation transfer (CEST), circular dichroism and pulsed Q-band EPR. Exchange between free and micelle-bound httNTQ n peptides occurs on the millisecond time scale with a KD ∼ 0.5-1 mM. Upon binding micelles, residues 1-15 adopt a helical conformation. Oxidation of Met7 to a sulfoxide reduces the binding affinity for micelles ∼3-4-fold and increases the length of the helix by a further two residues. A structure of the bound monomer unit is calculated from the backbone chemical shifts of the micelle-bound state obtained from CEST. Pulsed Q-band EPR shows that a monomer-dimer equilibrium exists on the surface of the micelles and that the two helices of the dimer adopt a parallel orientation, thereby bringing two disordered polyQ tails into close proximity which may promote aggregation upon dissociation from the micelle surface.

A Liquid to Solid Phase Transition Underlying Pathological Huntingtin Exon1 Aggregation

SummaryHuntington’s disease is caused by an abnormally long polyglutamine tract in the huntingtin protein. This leads to the generation and deposition of N-terminal exon1 fragments of the protein in intracellular aggregates. We combined electron tomography and quantitative fluorescence microscopy to analyze the structural and material properties of huntingtin exon1 assemblies in mammalian cells, in yeast, and in vitro. We found that huntingtin exon1 proteins can form reversible liquid-like assemblies, a process driven by huntingtin’s polyQ tract and proline-rich region. In cells and in vitro, the liquid-like assemblies converted to solid-like assemblies with a fibrillar structure. Intracellular phase transitions of polyglutamine proteins could play a role in initiating irreversible pathological aggregation.

Does N-terminal huntingtin function as a 'holdase' for inhibiting cellular protein aggregation?

Proteolytic cleavage of huntingtin gives rise to N-terminal fragments. While the role of truncated mutant huntingtin is described in Huntington's disease (HD) pathogenesis, the function of N-terminal wild-type protein is less studied. The yeast model of HD is generated by the presence of FLAG tag and absence of polyproline tract as flanking sequences of the elongated polyglutamine stretch. We show that the same sequence derived from wild-type huntingtin exon1 is able to inhibit the aggregation of proteins in vitro and in yeast cells. It is able to stabilize client proteins as varied as luciferase, α-synuclein, and p53 in a soluble but non-native state. This is somewhat similar to the 'holdase' function of small heat shock proteins and 'nonchaperone proteins' which are able to stabilize partially unfolded client proteins in a nonspecific manner, slowing down their aggregation. Mutagenesis studies show this property to be localized at the N17 domain preceding the polyglutamine tract. Distortion of this ordered segment, either by deletion of this segment or mutation of a single residue (L4A), leads to decreased stability and increased aggregation of client proteins. It is interesting to note that the helical conformation of the N17 domain is also essential for aggregation of the N-terminal mutant protein. Our results provide evidence for a novel function for the amphipathic helix derived from exon1 of wild-type huntingtin.

Tadpole-like Conformations of Huntingtin Exon 1 Are Characterized by Conformational Heterogeneity that Persists regardless of Polyglutamine Length

Soluble huntingtin exon 1 (Httex1) with expanded polyglutamine (polyQ) engenders neurotoxicity in Huntington's disease. To uncover the physical basis of this toxicity, we performed structural studies of soluble Httex1 for wild-type and mutant polyQ lengths. Nuclear magnetic resonance experiments show evidence for conformational rigidity across the polyQ region. In contrast, hydrogen–deuterium exchange shows absence of backbone amide protection, suggesting negligible persistence of hydrogen bonds. The seemingly conflicting results are explained by all-atom simulations, which show that Httex1 adopts tadpole-like structures with a globular head encompassing the N-terminal amphipathic and polyQ regions and the tail encompassing the C-terminal proline-rich region. The surface area of the globular domain increases monotonically with polyQ length. This stimulates sharp increases in gain-of-function interactions in cells for expanded polyQ, and one of these interactions is with the stress-granule protein Fus. Our results highlight plausible connections between Httex1 structure and routes to neurotoxicity.

Self-assembly of Mutant Huntingtin Exon-1 Fragments into Large Complex Fibrillar Structures Involves Nucleated Branching

Huntingtin (HTT) fragments with extended polyglutamine tracts self-assemble into amyloid-like fibrillar aggregates. Elucidating the fibril formation mechanism is critical for understanding Huntington's disease pathology and for developing novel therapeutic strategies. Here, we performed systematic experimental and theoretical studies to examine the self-assembly of an aggregation-prone N-terminal HTT exon-1 fragment with 49 glutamines (Ex1Q49). Using high-resolution imaging techniques such as electron microscopy and atomic force microscopy, we show that Ex1Q49 fragments in cell-free assays spontaneously convert into large, highly complex bundles of amyloid fibrils with multiple ends and fibril branching points. Furthermore, we present experimental evidence that two nucleation mechanisms control spontaneous Ex1Q49 fibrillogenesis: (1) a relatively slow primary fibril-independent nucleation process, which involves the spontaneous formation of aggregation-competent fibrillary structures, and (2) a fast secondary fibril-dependent nucleation process, which involves nucleated branching and promotes the rapid assembly of highly complex fibril bundles with multiple ends. The proposed aggregation mechanism is supported by studies with the small molecule O4, which perturbs early events in the aggregation cascade and delays Ex1Q49 fibril assembly, comprehensive mathematical and computational modeling studies, and seeding experiments with small, preformed fibrillar Ex1Q49 aggregates that promote the assembly of amyloid fibrils. Together, our results suggest that nucleated branching in vitro plays a critical role in the formation of complex fibrillar HTT exon-1 aggregates with multiple ends.

Molecular and structural architecture of polyQ aggregates in yeast

Huntington's disease is caused by the expansion of a polyglutamine (polyQ) tract in the N-terminal exon of huntingtin (HttEx1), but the cellular mechanisms leading to neurodegeneration remain poorly understood. Here we present in situ structural studies by cryo-electron tomography of an established yeast model system of polyQ toxicity. We find that expression of polyQ-expanded HttEx1 results in the formation of unstructured inclusion bodies and in some cases fibrillar aggregates. This contrasts with recent findings in mammalian cells, where polyQ inclusions were exclusively fibrillar. In yeast, polyQ toxicity correlates with alterations in mitochondrial and lipid droplet morphology, which do not arise from physical interactions with inclusions or fibrils. Quantitative proteomic analysis shows that polyQ aggregates sequester numerous cellular proteins and cause a major change in proteome composition, most significantly in proteins related to energy metabolism. Thus, our data point to a multifaceted toxic gain-of-function of polyQ aggregates, driven by sequestration of endogenous proteins and mitochondrial and lipid droplet dysfunction.

A General Strategy to Access Structural Information at Atomic Resolution in Polyglutamine Homorepeats.

Homorepeat (HR) proteins are involved in key biological processes and multiple pathologies, however their high‐resolution characterization has been impaired due to their homotypic nature. To overcome this problem, we have developed a strategy to isotopically label individual glutamines within HRs by combining nonsense suppression and cell‐free expression. Our method has enabled the NMR investigation of huntingtin exon1 with a 16‐residue polyglutamine (poly‐Q) tract, and the results indicate the presence of an N‐terminal α‐helix at near neutral pH that vanishes towards the end of the HR. The generality of the strategy was demonstrated by introducing a labeled glutamine into a pathological version of huntingtin with 46 glutamines. This methodology paves the way to decipher the structural and dynamic perturbations induced by HR extensions in poly‐Q‐related diseases. Our approach can be extended to other amino acids to investigate biological processes involving proteins containing low‐complexity regions (LCRs).

A General Strategy to Access Structural Information at Atomic Resolution in Polyglutamine Homorepeats

AbstractHomorepeat (HR) proteins are involved in key biological processes and multiple pathologies, however their high‐resolution characterization has been impaired due to their homotypic nature. To overcome this problem, we have developed a strategy to isotopically label individual glutamines within HRs by combining nonsense suppression and cell‐free expression. Our method has enabled the NMR investigation of huntingtin exon1 with a 16‐residue polyglutamine (poly‐Q) tract, and the results indicate the presence of an N‐terminal α‐helix at near neutral pH that vanishes towards the end of the HR. The generality of the strategy was demonstrated by introducing a labeled glutamine into a pathological version of huntingtin with 46 glutamines. This methodology paves the way to decipher the structural and dynamic perturbations induced by HR extensions in poly‐Q‐related diseases. Our approach can be extended to other amino acids to investigate biological processes involving proteins containing low‐complexity regions (LCRs).

Huntington Mice Demonstrate Diminished Pain Response in Inflammatory Pain Model.

Huntington disease (HD) affects the nervous system and leads to mental and motor dysfunction. Previous studies have shown that HD is caused by the exon 1 region of the huntingtin (HTT) gene having expanded CAG trinucleotide repeats. However, few studies have focused on the relationship between HD and pain. The purpose of this study is to investigate the relationship between HD and pain response. We used clinical similar transgenic HD mice carrying a mutant HTT exon 1 containing 84 CAG trinucleotide repeats to evaluate the relationship between HD and pain. Inflammatory pain models were induced by either formalin or complete Freund adjuvant injection over the hind paw. Spinal cord, dorsal root ganglion, and paw skin tissues were harvested at the end of the behavioral inflammatory pain studies. Immunofluorescence assay, Western blotting, and enzyme-linked immunosorbent assay were used to identify changes in cells and cytokines. Our data demonstrate that preonset HD mice exhibited less pain behavior than wild-type (WT) mice in both young (n = 11 [WT], 13 [HD]) and aged (n = 8 [WT], 9 [HD]) mice. Western blotting and immunohistological examination of lumbar spinal cord tissue and dorsal root ganglion indicate less activation of glial cells and astrocytes in young HD mice (n = 6-7) compared to that in WT mice (n = 6-7). The production levels of tumor necrosis factor-α, interleukin-1β, and substance P were also lower in young HD mice (n = 6-7). Our data demonstrate less pain behavior and pain-related cytokine response at the spinal cord level for HD mice compared to those for WT mice. Further studies are needed for determining the mechanism as to how mutant HTT leads to altered pain behavior and pain-related cytokine response.

Cell-to-cell Transmission of Polyglutamine Aggregates in C. elegans.

Huntington disease (HD) is an inherited neurodegenerative disorder characterized by motor and cognitive dysfunction caused by expansion of polyglutamine (polyQ) repeat in exon 1 of huntingtin (HTT). In patients, the number of glutamine residues in polyQ tracts are over 35, and it is correlated with age of onset, severity, and disease progression. Expansion of polyQ increases the propensity for HTT protein aggregation, process known to be implicated in neurodegeneration. These pathological aggregates can be transmitted from neuron to another neuron, and this process may explain the pathological spreading of polyQ aggregates. Here, we developed an in vivo model for studying transmission of polyQ aggregates in a highly quantitative manner in real time. HTT exon 1 with expanded polyQ was fused with either N-terminal or C-terminal fragments of Venus fluorescence protein and expressed in pharyngeal muscles and associated neurons, respectively, of C. elegans. Transmission of polyQ proteins was detected using bimolecular fluorescence complementation (BiFC). Mutant polyQ (Q97) was transmitted much more efficiently than wild type polyQ (Q25) and forms numerous inclusion bodies as well. The transmission of Q97 was gradually increased with aging of animal. The animals with polyQ transmission exhibited degenerative phenotypes, such as nerve degeneration, impaired pharyngeal pumping behavior, and reduced life span. The C. elegans model presented here would be a useful in vivo model system for the study of polyQ aggregate propagation and might be applied to the screening of genetic and chemical modifiers of the propagation.

Phosphorylation of huntingtin at residue T3 is decreased in Huntington’s disease and modulates mutant huntingtin protein conformation

Posttranslational modifications can have profound effects on the biological and biophysical properties of proteins associated with misfolding and aggregation. However, their detection and quantification in clinical samples and an understanding of the mechanisms underlying the pathological properties of misfolding- and aggregation-prone proteins remain a challenge for diagnostics and therapeutics development. We have applied an ultrasensitive immunoassay platform to develop and validate a quantitative assay for detecting a posttranslational modification (phosphorylation at residue T3) of a protein associated with polyglutamine repeat expansion, namely Huntingtin, and characterized its presence in a variety of preclinical and clinical samples. We find that T3 phosphorylation is greatly reduced in samples from Huntington's disease models and in Huntington's disease patients, and we provide evidence that bona-fide T3 phosphorylation alters Huntingtin exon 1 protein conformation and aggregation properties. These findings have significant implications for both mechanisms of disease pathogenesis and the development of therapeutics and diagnostics for Huntington's disease.

Spatiotemporal Proteomic Profiling of Huntington’s Disease Inclusions Reveals Widespread Loss of Protein Function

SummaryAggregation of polyglutamine-expanded huntingtin exon 1 (HttEx1) in Huntington’s disease (HD) proceeds from soluble oligomers to late-stage inclusions. The nature of the aggregates and how they lead to neuronal dysfunction is not well understood. We employed mass spectrometry (MS)-based quantitative proteomics to dissect spatiotemporal mechanisms of neurodegeneration using the R6/2 mouse model of HD. Extensive remodeling of the soluble brain proteome correlated with insoluble aggregate formation during disease progression. In-depth and quantitative characterization of the aggregates uncovered an unprecedented complexity of several hundred proteins. Sequestration to aggregates depended on protein expression levels and sequence features such as low-complexity regions or coiled-coil domains. In a cell-based HD model, overexpression of a subset of the sequestered proteins in most cases rescued viability and reduced aggregate size. Our spatiotemporally resolved proteome resource of HD progression indicates that widespread loss of cellular protein function contributes to aggregate-mediated toxicity.

Monomeric Huntingtin Exon 1 Has Similar Overall Structural Features for Wild-Type and Pathological Polyglutamine Lengths.

Huntington’s disease is caused by expansion of a polyglutamine (polyQ) domain within exon 1 of the huntingtin gene (Httex1). The prevailing hypothesis is that the monomeric Httex1 protein undergoes sharp conformational changes as the polyQ length exceeds a threshold of 36–37 residues. Here, we test this hypothesis by combining novel semi-synthesis strategies with state-of-the-art single-molecule Förster resonance energy transfer measurements on biologically relevant, monomeric Httex1 proteins of five different polyQ lengths. Our results, integrated with atomistic simulations, negate the hypothesis of a sharp, polyQ length-dependent change in the structure of monomeric Httex1. Instead, they support a continuous global compaction with increasing polyQ length that derives from increased prominence of the globular polyQ domain. Importantly, we show that monomeric Httex1 adopts tadpole-like architectures for polyQ lengths below and above the pathological threshold. Our results suggest that higher order homotypic and/or heterotypic interactions within distinct sub-populations of neurons, which are inevitable at finite cellular concentrations, are likely to be the main source of sharp polyQ length dependencies of HD.