polyQ Length Research Articles

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.

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OBJECTIVES/SPECIFIC AIMS: Polyglutamine (polyQ) neurodegenerative diseases, associated with the unstable expansion of polyQ tracts, are devastating diseases for which no treatments exist. Moreover, most drug discovery attempts have been hindered by the lack of understanding on the relevant pathogenic mechanisms. Here, using previously reported 3D protein predicted structures of ataxin-2 and ataxin-3, we analyze the effect of polyQ enlargement on hydrogen bonding and water accessibility patterns as a possible mechanism for pathogenesis thought enhanced protein aggregation. METHODS/STUDY POPULATION: Using the I-TASSER predicted structures of ataxin-2 and ataxin-3 with different numbers of glutamine repeats representing polyQ lengths characteristic of both normal and pathological tracts (Journal of Biomolecular Structure and Dynamics, 2016: 1–16), we identified hydrogen bonds (HBs, UCSF Chimera FindHBond module) and calculated solvent-accessible surface areas (SASA, DSSP program) for the polyQ tracts available in the 3D structures. RESULTS/ANTICIPATED RESULTS: The identified HBs were analyzed as the function of the number of glutamines in the polyQ tracts and characterized as those intra-polyQ and exter-polyQ, respectively. The SASA of the polyQ region was also studied as the function of the polyQ tract length. DISCUSSION/SIGNIFICANCE OF IMPACT: The results obtained here indicate that polyQ regions increasingly prefer self-interactions, which consistently can lead to more compact polyQ structures. The results strongly support the notion that the expansion of the polyQ region can be an intrinsic force leading to self-aggregation of polyglutamine proteins and suggest that the modulation of solvent-polyQ interactions could be a possible therapeutic strategy for polyQ diseases.

Molecular dynamics analysis of the aggregation propensity of polyglutamine segments.

Protein misfolding and aggregation is a pathogenic feature shared among at least ten polyglutamine (polyQ) neurodegenerative diseases. While solvent-solution interaction is a key factor driving protein folding and aggregation, the solvation properties of expanded polyQ tracts are not well understood. By using GPU-enabled all-atom molecular dynamics simulations of polyQ monomers in an explicit solvent environment, this study shows that solvent-polyQ interaction propensity decreases as the lengths of polyQ tract increases. This study finds a predominance in long-distance interactions between residues far apart in polyQ sequences with longer polyQ segments, that leads to significant conformational differences. This study also indicates that large loops, comprised of parallel β-structures, appear in long polyQ tracts and present new aggregation building blocks with aggregation driven by long-distance intra-polyQ interactions. Finally, consistent with previous observations using coarse-grain simulations, this study demonstrates that there is a gain in the aggregation propensity with increased polyQ length, and that this gain is correlated with decreasing ability of solvent-polyQ interaction. These results suggest the modulation of solvent-polyQ interactions as a possible therapeutic strategy for treating polyQ diseases.

N-Terminal Fragments of Huntingtin Longer than Residue 170 form Visible Aggregates Independently to Polyglutamine Expansion.

A hallmark of Huntington's disease is the progressive aggregation of full length and N-terminal fragments of polyglutamine (polyQ)-expanded Huntingtin (Htt) into intracellular inclusions. The production of N-terminal fragments appears important for enabling pathology and aggregation; and hence the direct expression of a variety of N-terminal fragments are commonly used to model HD in animal and cellular models. It remains unclear how the length of the N-terminal fragments relates to polyQ - mediated aggregation. We investigated the fundamental intracellular aggregation process of eight different-length N-terminal fragments of Htt in both short (25Q) and long polyQ (97Q). N-terminal fragments were fused to fluorescent proteins and transiently expressed in mammalian cell culture models. These included the classic exon 1 fragment (90 amino acids) and longer forms of 105, 117, 171, 513, 536, 552, and 586 amino acids based on wild-type Htt (of 23Q) sequence length nomenclature. N-terminal fragments of less than 171 amino acids only formed inclusions in polyQ-expanded form. By contrast the longer fragments formed inclusions irrespective of Q-length, with Q-length playing a negligible role in extent of aggregation. The inclusions could be classified into 3 distinct morphological categories. One type (Type A) was universally associated with polyQ expansions whereas the other two types (Types B and C) formed independently of polyQ length expansion. PolyQ-expansion was only required for fragments of less than 171 amino acids to aggregate. Longer fragments aggregated predominately through a non-polyQ mechanism, involving at least one, and probably more distinct clustering mechanisms.

Lattice Models for Modeling Sequence-Specific Phase Behavior of Low Complexity Domains

Low complexity domains (LCDs), enriched in charged, polar, and aromatic residues are drivers of aggregation and phase separation in a variety of proteins including those that drive the formation of RNA-protein granules. Polyglutamine (polyQ) tracts are a distinct archetype of LCDs. Mutational expansion of polyQ tracts in specific proteins is associated with onset and progression of at least nine different neurodegenerative disorders including Huntington's disease. The latter is associated with an expanded polyQ tract in the protein huntingtin. Recent experiments have uncovered complex thermodynamic and kinetic phase diagrams for N-terminal fragments of huntingtin. These phase diagrams are modulated by polyQ length, sequence contexts, concentration, and solution conditions. The details of the molecular and supramolecular interactions that give rise to emergent phase behavior of polyQ containing at distinct length scales remains poorly understood. Computations play an important role in predicting the phase behavior of LCDs. These calculations require simulations encompassing several hundreds of thousands of protein molecules spanning several orders of magnitude in protein concentrations. Such simulations are beyond the scope of all atom models. Since phase behavior results from the synergistic interactions amongst small numbers of collective coordinates, simulations designed to uncover sequence-encoded phase behavior can be pursued using either systematic coarse-graining or phenomenological models. Recent work has demonstrated the utility of both approaches. However, these models lack the requisite molecular detail to enable a complete understanding of sequence-specific contributions that determine similarities and differences in sequence-encoded phase behavior of LCDs. Here we present a lattice model that is tailored for modeling sequence-specific phase behaviors of LCDs. The model serves as an intermediate description between ultra coarse-graining and all-atom extremes. In the lattice model, a single bead on a 3-dimensional lattice site describes each residue. Each bead has multiple faces and this helps us capture the directionality of backbone hydrogen bonding and sidechain-specific interactions. Monte Carlo simulations based on these lattice models help us uncover the interplay amongst different sequence-encoded energy scales and phase behaviors of LCDs with polyQ tracts. Specifically, we focus on explaining the role of spherical mesophases as mediators of fibril formation in polyQ containing proteins. We also show that the model can be deployed for understanding the interplay amongst liquids, fibrils, and gels for other LCDs.

No effect of AR polyG polymorphism on spinal and bulbar muscular atrophy phenotype.

Disease severity varies considerably among patients with Spinal and Bulbar Muscular Atrophy (SBMA). Our aim was to investigate the role of androgen receptor (AR) polymorphic repeats in SBMA phenotype. We analyzed the length of AR polyQ and polyG tracts in 159 SBMA patients. No relationship between polyG size or polyG/polyQ haplotypes and clinical phenotype was found. An independent negative correlation between polyQ-length and onset of weakness was confirmed (P<0.001). The negative results of our study prompt to continue the search for potential disease modifiers in SBMA outside the AR gene.

Loss of C9 ORF 72 impairs autophagy and synergizes with polyQ Ataxin‐2 to induce motor neuron dysfunction and cell death

An intronic expansion of GGGGCC repeats within the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). Ataxin-2 with intermediate length of polyglutamine expansions (Ataxin-2 Q30x) is a genetic modifier of the disease. Here, we found that C9ORF72 forms a complex with the WDR41 and SMCR8 proteins to act as a GDP/GTP exchange factor for RAB8a and RAB39b and to thereby control autophagic flux. Depletion of C9orf72 in neurons partly impairs autophagy and leads to accumulation of aggregates of TDP-43 and P62 proteins, which are histopathological hallmarks of ALS-FTD SMCR8 is phosphorylated by TBK1 and depletion of TBK1 can be rescued by phosphomimetic mutants of SMCR8 or by constitutively active RAB39b, suggesting that TBK1, SMCR8, C9ORF72, and RAB39b belong to a common pathway regulating autophagy. While depletion of C9ORF72 only has a partial deleterious effect on neuron survival, it synergizes with Ataxin-2 Q30x toxicity to induce motor neuron dysfunction and neuronal cell death. These results indicate that partial loss of function of C9ORF72 is not deleterious by itself but synergizes with Ataxin-2 toxicity, suggesting a double-hit pathological mechanism in ALS-FTD.

Conformational Shifts in Huntingtin Exon 1 Monomer are Dependent on Temperature and Polyglutamine Length

Expansion of the polyglutamine (polyQ) region in the first exon of huntingtin (htt) is the root of Huntington's disease (HD). Misfolding and aggregation have long been observed to a greater extent in polyQ-expanded htt, but studies have also uncovered many implications of polyQ expansion in alterations to the dynamics and intermolecular interactions of monomeric htt. Here we find that htt exon 1 (HDx1), which is composed of the polyQ tract and flanking regions and in expanded form is sufficient to cause HD-like symptoms in transgenic mice, has a greater proclivity for adopting α-helical structure in the presence of a polyQ expansion. Our circular dichroism (CD) studies show that cooler temperature causes a gain in α-helical structure in HDx1 of a variety of polyQ lengths but a greater gain at disease-level polyQ length. We performed continuous wave electron paramagnetic resonance (EPR) with site-directed spin labeling (SDSL) and found the gain in structure to occur in the HDx1 N-terminus and the N-terminal region of the polyQ tract. Performing pulsed EPR on double spin-labeled mutants of HDx1 confirmed the α-helical structure adopted by the residues in those regions. Our findings fall in step with the previously proposed ‘rusty hinge’ hypothesis, which suggests that the normal, but not expanded, polyQ region serves as a flexible hinge that enables intra- and intermolecular interactions of htt. Stiffening of the hinge by polyQ expansion may lead to gains and losses of physiological functions for the protein. Our next step is to investigate the effect of phosphorylation to the N-terminus. To this end, we have already generated a number of phosphorylation-mimicking HDx1 mutants.Support: HDF, CHDI

The role of androgen receptor gene variants on SBMA phenotype

Background: Spinal and bulbar muscular atrophy (SBMA), also known as Kennedy’s disease (KD), is a lower motor neuron disorder, caused by expansion over 36 of a polymorphic CAG repeat sequence encoding a polyglutamine tract (polyQ), in the androgen steroid hormone receptor (AR) gene on chromosome X. PolyQ tract length negatively correlates with age at onset, without affecting disease progression. Objective: To assess the role of AR variants on disease phenotype. Patients and methods: A cohort of 159 SBMA patients were genotyped for AR variants and a genotype-phenotype correlation study was performed. Results: Besides the pathogenetic polyQ tract, the only AR variant to result polymorphic (minor allele frequency > 5%) was a polyglycine tract (polyG) encoded by GGN spanning amino-acids 451–473. We confirmed an inverse correlation between the polyQ length and the age at onset of muscle weakness (r = -0.49; p < 0.0001, Fig. 1). No correlation between GGN length and milestones of disease progression was found. The number of CAG repeats could explain approximately 25% variance of age at onset of muscle weakness and GGN length was not significantly correlated (p = 0.28) in a multiple regression analysis. Conclusions: AR polyG tract is not a genetic modifier of SBMA phenotype.

Computational investigation of molecular mechanism and neuropathological implications in Huntington disease.

Huntington's disorder (HD), caused by mutations of the IT-15 gene, is an autosomal genetic disease that causes the breakdown of the nerve cells in the brain. The IT-15 gene encodes the huntingtin (Htt) protein. Htt, along with its interacting partners, are involved in maintaining proper communication among neurons. Our work is based on the interaction behavior between Htt (in three polyglutamine (polyQ) states that is Htt 0Q, 17Q and 36Q) and SH3GL3 interacting protein by using computational methods. We used the HADDOCK docking platform to find out the extent of interaction between Htt polyQ models and SH3GL3. The Htt36Q (mutated) showed higher interaction than Htt17Q (native) with SH3GL3. Molecular dynamics simulation was performed to uncover the structural fluctuations of polyQ models and their complexes. RMSD, Rg, SASA, and total interaction energy graph showed significant results, where as mutant Htt showed higher fluctuations and flexibility than native Htt. The increase in the length of polyQ was found to affect the stability, flexibility, and compactness of the protein and its complex. Our research provided a propitious approach to understand the consequence of polyglutamination in Htt and its relation with HD.

The emerging role of the first 17 amino acids of huntingtin in Huntington's disease.

Huntington's disease (HD) is caused by a polyglutamine (polyQ) domain that is expanded beyond a critical threshold near the N-terminus of the huntingtin (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 the polyQ domain, such as the first 17 amino acids on the N terminus (Nt17), influence aggregation, aggregate stability, influence other important biochemical properties of the protein and ultimately its role in pathogenesis. Here, we review the impact of Nt17 on htt aggregation mechanisms and kinetics, structural properties of Nt17 in both monomeric and aggregate forms, the potential role of posttranslational modifications (PTMs) that occur in Nt17 in HD, and the function of Nt17 as a membrane targeting domain.

Molecular Interaction between the Chaperone Hsc70 and the N-terminal Flank of Huntingtin Exon 1 Modulates Aggregation

The aggregation of polyglutamine (polyQ)-containing proteins is at the origin of nine neurodegenerative diseases. Molecular chaperones prevent the aggregation of polyQ-containing proteins. The exact mechanism by which they interact with polyQ-containing, aggregation-prone proteins and interfere with their assembly is unknown. Here we dissect the mechanism of interaction between a huntingtin exon 1 fragment of increasing polyQ lengths (HttEx1Qn), the aggregation of which is tightly associated with Huntington's disease, and molecular chaperone Hsc70. We show that Hsc70, together with its Hsp40 co-chaperones, inhibits HttEx1Qn aggregation and modifies the structural, seeding, and infectious properties of the resulting fibrils in a polyQ-independent manner. We demonstrate that Hsc70 binds the 17-residue-long N-terminal flank of HttEx1Qn, and we map Hsc70-HttEx1Qn surface interfaces at the residue level. Finally, we show that this interaction competes with homotypic interactions between the N termini of different HttEx1Qn molecules that trigger the aggregation process. Our results lay the foundations of future therapeutic strategies targeting huntingtin aggregation in Huntington disease.

Influence of the protein context on the polyglutamine length-dependent elongation of amyloid fibrils

Polyglutamine (polyQ) diseases, including Huntington's disease, are neurodegenerative disorders associated with the abnormal expansion of a polyQ tract within nine proteins. The polyQ expansion is thought to be a major determinant in the development of neurotoxicity, triggering protein aggregation into amyloid fibrils, although non-polyQ regions play a modulating role. In this work, we investigate the relative importance of the polyQ length, its location within a host protein, and the conformational state of the latter in the amyloid fibril elongation. Model polyQ proteins made of the β-lactamase BlaP containing up to 79Q inserted at two different positions, and quartz crystal microbalance and atomic force microscopy were used for this purpose. We demonstrate that, independently of the polyQ tract location and the conformational state of the host protein, the relative elongation rate of fibrils increases linearly with the polyQ length. The slope of the linear fit is similar for both sets of chimeras (i.e., the elongation rate increases by ~1.9% for each additional glutamine), and is also similar to that previously observed for polyQ peptides. The elongation rate is, however, strongly influenced by the location of the polyQ tract within BlaP and the conformational state of BlaP. Moreover, comparison of our results with those previously reported for aggregation in solution indicates that these two parameters also modulate the ability of BlaP-polyQ chimeras to form the aggregation nucleus. Altogether our results suggest that non-polyQ regions are valuable targets in order to interfere with the process of amyloid fibril formation associated with polyQ diseases.

Depletion of p62 reduces nuclear inclusions and paradoxically ameliorates disease phenotypes in Huntington's model mice.

Huntington's disease (HD) is a dominantly inherited genetic disease caused by mutant huntingtin (htt) protein with expanded polyglutamine (polyQ) tracts. A neuropathological hallmark of HD is the presence of neuronal inclusions of mutant htt. p62 is an important regulatory protein in selective autophagy, a process by which aggregated proteins are degraded, and it is associated with several neurodegenerative disorders including HD. Here, we investigated the effect of p62 depletion in three HD model mice: R6/2, HD190QG and HD120QG mice. We found that loss of p62 in these models led to longer life spans and reduced nuclear inclusions, although cytoplasmic inclusions increased with polyQ length. In mouse embryonic fibroblasts (MEFs) with or without p62, mutant htt with a nuclear localization signal (NLS) showed no difference in nuclear inclusion between the two MEF types. In the case of mutant htt without NLS, however, p62 depletion increased cytoplasmic inclusions. Furthermore, to examine the effect of impaired autophagy in HD model mice, we crossed R6/2 mice with Atg5 conditional knockout mice. These mice also showed decreased nuclear inclusions and increased cytoplasmic inclusions, similar to HD mice lacking p62. These data suggest that the genetic ablation of p62 in HD model mice enhances cytoplasmic inclusion formation by interrupting autophagic clearance of polyQ inclusions. This reduces polyQ nuclear influx and paradoxically ameliorates disease phenotypes by decreasing toxic nuclear inclusions.

Architecture of Polyglutamine-containing Fibrils from Time-resolved Fluorescence Decay

The disease risk and age of onset of Huntington disease (HD) and nine other repeat disorders strongly depend on the expansion of CAG repeats encoding consecutive polyglutamines (polyQ) in the corresponding disease protein. PolyQ length-dependent misfolding and aggregation are the hallmarks of CAG pathologies. Despite intense effort, the overall structure of these aggregates remains poorly understood. Here, we used sensitive time-dependent fluorescent decay measurements to assess the architecture of mature fibrils of huntingtin (Htt) exon 1 implicated in HD pathology. Varying the position of the fluorescent labels in the Htt monomer with expanded 51Q (Htt51Q) and using structural models of putative fibril structures, we generated distance distributions between donors and acceptors covering all possible distances between the monomers or monomer dimensions within the polyQ amyloid fibril. Using Monte Carlo simulations, we systematically scanned all possible monomer conformations that fit the experimentally measured decay times. Monomers with four-stranded 51Q stretches organized into five-layered β-sheets with alternating N termini of the monomers perpendicular to the fibril axis gave the best fit to our data. Alternatively, the core structure of the polyQ fibrils might also be a zipper layer with antiparallel four-stranded stretches as this structure showed the next best fit. All other remaining arrangements are clearly excluded by the data. Furthermore, the assessed dimensions of the polyQ stretch of each monomer provide structural evidence for the observed polyQ length threshold in HD pathology. Our approach can be used to validate the effect of pharmacological substances that inhibit or alter amyloid growth and structure.

Role of Intrinsic Helicity Within N-Terminal Flanking Sequences on Huntingtin Aggregation Mechanisms

Huntington's disease (HD) is associated with CAG repeats within exon 1 that lead to polyglutamine (polyQ) expansions in the protein huntingtin (htt). Aberrant splicing of htt transcripts generates N-terminal fragments in a polyQ length dependent manner that span exon 1 of htt. These N-terminal fragments can be trafficked into the nucleus and form neuronal nuclear inclusions. Within exon 1 of htt the polyQ region is flanked by a N-terminal 17-residue amphipathic stretch (N17) and a C-terminal proline-rich stretch. Modulation of these flanking sequences has profound effects on HD progression. Specifically, post-translational modifications and single / double point mutations within N17 can modulate the intrinsic helicity of N17, as well as aggregation in vitro and HD phenotypes in animal models. However, the role of helicity within N17 on the modulation of polyQ aggregation remains unresolved. Here, we focus on understanding the synergy between the intrinsic helicity within N17 and polyQ length on htt aggregation mechanisms. Through the use of atomistic Monte Carlo simulations utilizing the ABSINTH implicit solvation model, we examine the monomeric properties of N17 permutants with varying degrees of intrinsic helicity as a function of polyQ length. Results suggest that the degree of coupling between N17 constructs and polyQ is dependent on the intrinsic helicity within N17 constructs. A comparative analysis of N17 constructs in cis with a range of polyQ lengths will be combined with biophysical experiments to understand how monomeric properties of N17 modulate the solubility and mechanism of polyQ aggregation.

Polyglutamine Flanking Regions in Huntingtin Highlight Key Structural Intermediates Relevant for Molecular Chaperone Interaction and Huntington's Disease Pathogenesis

Huntington's Disease (HD) is a neurodegenerative disorder caused by an expansion in the polyglutamine (polyQ) tract of the Huntingtin (Htt) protein. The clinical hallmark of this mutation is the accumulation of amyloid Htt aggregates in neurons. While Htt aggregation is highly correlated with the length of this polyQ tract both in vitro and in vivo, recent studies suggest that the regions flanking the polyQ tract can influence Htt aggregation and toxicity independent of polyQ length. In addition, it is well established that two polyQ flanking regions in exon1 of Htt, the N-terminal first 17 amino acids (N17) and a proline-rich region (PolyPro), influence Htt aggregation propensity. Here, we show that the N17 and PolyPro regions of Htt dramatically influence the rate of the Htt aggregation pathway. We also show that mutations within these polyQ-flanking regions alter Htt toxicity in a brain slice model. The influence of these flanking regions on the heterogeneous distribution of the aggregate species may account for these differences in toxicity. This theory is increasingly relevant in light of recent thought that Htt toxicity may derive from a species other than the amyloid fibril itself. Finally, previous work has shown that the N17 region of Htt interacts with the molecular chaperonin TRiC, and this interaction exerts a protective effect against both Htt aggregation and toxicity. An understanding how these flanking regions influence Htt aggregation will inform how TRiC works through the N17 element. Together, this information can provide the basis to design HD therapeutics that exploit these flanking regions to suppress HD pathogenesis.

Linear and extended: a common polyglutamine conformation recognized by the three antibodies MW1, 1C2 and 3B5H10

A long-standing pathomechanistic model proposes that the polyglutamine (polyQ)-length-dependent toxicity threshold observed in all polyQ diseases is triggered by a conformational change within the monomer that occurs only above a certain polyQ length. If true, this yet undefined and elusive mutant-specific toxic conformation would constitute a direct therapeutic target. Three anti-polyQ antibodies-MW1, 1C2 and 3B5H10-have been extensively used to probe the conformation of polyQ. The crystal structure of the MW1 epitope reveals a linear, non-pathogenic polyQ. In contrast, although the detailed structure of its epitope is unknown, the 3B5H10 antibody is widely advertised and used as a conformational antibody that recognizes the toxic conformation of expanded polyQ. We solved the crystal structure of the 1C2 antigen-binding domain (1C2-Fab) and performed a direct comparison between the 1C2, MW1 and 3B5H10 structures. The MW1 and 1C2 antibodies have similar sequences and structures, consistent with their binding to short polyQ and their polyQ length-discrimination properties. Unexpectedly, the 3B5H10 antibody also shares striking features with MW1 and 1C2, which prompted us to revisit its binding properties. We show that the 3B5H10 epitope is actually a short, non-pathogenic polyQ. All three antibodies MW1, 1C2 and 3B5H10 interact similarly with polyQ of various lengths, and bind small polyQ epitopes in similar linear and extended conformations. Together with studies published during the recent years, our work argues against the hypothesis that a mutant-specific conformation in monomeric polyQ molecules is the toxic entity responsible for polyQ diseases.

Modulation of Polyglutamine Conformations and Associations by C-Terminal Proline Rich Regions from Exon 1 of Huntingtin

Huntington's disease (HD) is associated with CAG repeats within exon 1 that lead to polyglutamine (polyQ) expansions in the protein huntingtin (htt). Proteolysis of htt leads to products that can be trafficked into the nucleus and form neuronal intranuclear inclusions. The presence / absence of flanking sequences namely, the N-terminal 17-residue amphipathic stretch (Nt17h-htt) and the C-terminal 38-residue proline-rich stretch (Ct38h-htt) in products of proteolysis have a profound effect on the HD phenotype in animal models (where h-htt denotes human-htt). The biophysical basis for the effects of these flanking sequences remains unresolved. Here, we focus on the influence of different proline-rich C-terminal sequences on the conformations of and intermolecular associations through the polyQ tract. The flanking sequences include Ct38h-htt and Ct31m-htt (the mouse 31-residue C-terminal proline-rich region).We used atomistic simulations based on the ABSINTH implicit solvation model and underlying forcefield paradigm to quantify the nature of the coupling between C-terminal flanking sequences and polyQ tracts for different polyQ lengths. There are clear differences between the intrinsic and context dependent properties of Ct31m-htt and Ct38h-htt. Detailed analyses of the monomer conformations and intermolecular associations of polyQ in cis with different synthetic and naturally occurring C-terminal proline-rich stretches identify how flanking sequences modulate aggregation mechanisms. These results might be important for recent studies in animal models that show different toxicity profiles between transgenic animals expressing different C-terminal proline-rich sequences.This work was supported by grant 5R01NS056114 from the National Institutes of Health.

Sedimentation Velocity Analysis of Polyglutamine Assembly in C. elegans using a Fluorescence Detection System

The presence of different size protein aggregates is a common feature of neurodegenerative diseases, and which types of aggregates (whether oligomeric or fibrillar) are toxic are still not well defined. using expression of various polyQ expression constructs in C. elegans, we apply analytical ultracentrifugation methods with fluorescence detection capability to determine size distribution of protein aggregation. We find that an increase in intermediate size range pools (80 S-120 S) is a direct result of an increase in polyQ length. Strains of worms expressing polyQ0-YFP, polyQ24-YFP, polyQ35-YFP, polyQ40-YFP, and polyQ40 (no YFP) in muscle wall were synchronized and grown at 20° C and harvested as one-day old adults. PolyQ40 without the fluorescent YFP fusion was labeled using an extrinsic dye, DCVJ, that emits fluorescence when bound to punctates. Quantitative analysis of samples prepared with minimal fractionation for polyQ40-YFP shows evidence of polydisperse aggregates with size range from 30-150 S similar in size to the oligomeric states observed in situ in a mouse neuroblastoma cell line. These intermediates were not observed in polyQ0-YFP control constructs. Our data show that the presence of a polydisperse oligomeric pool, and we suggest that this heterogeneity is associated with the cellular context since such polydispersity is not observed in in vitro model systems.