Huntingtin Exon Research Articles

A Closed-Form Expression for Analysis of Dark State Exchange Saturation Transfer (DEST) NMR Experiments.

Closed-form expressions for the analysis of Dark state Exchange Saturation Transfer (DEST) NMR experiments, a powerful experimental tool for characterizing exchange processes involving the interaction of NMR visible species with very high molecular weight partners, is presented. Essentially identical exchange and relaxation parameters are derived from the analytical and numerical best fits of the DEST profiles obtained for a protein construct derived from huntingtin exon-1, comprising the N-terminal amphiphilic sequence followed by a seven-residue glutamine repeat, httNTQ7, in the presence of small (SUV) and large (LUV) unilamellar lipid vesicles. The use of analytical expressions significantly speeds up the fitting of experimental DEST profiles to a two-state exchange model and simplifies the analysis of the DEST effects.

M6A modification of mutant huntingtin RNA promotes the biogenesis of pathogenic huntingtin transcripts

In Huntington’s disease (HD), aberrant processing of huntingtin (HTT) mRNA produces HTT1a transcripts that encode the pathogenic HTT exon 1 protein. The mechanisms behind HTT1a production are not fully understood. Considering the role of m6A in RNA processing and splicing, we investigated its involvement in HTT1a generation. Here, we show that m6A methylation is increased before the cryptic poly(A) sites (IpA1 and IpA2) within the huntingtin RNA in the striatum of Hdh+/Q111 mice and human HD samples. We further assessed m6A’s role in mutant Htt mRNA processing by pharmacological inhibition and knockdown of METTL3, as well as targeted demethylation of Htt intron 1 using a dCas13-ALKBH5 system in HD mouse cells. Our data reveal that Htt1a transcript levels are regulated by both METTL3 and the methylation status of Htt intron 1. They also show that m6A methylation in intron 1 depends on expanded CAG repeats. Our findings highlight a potential role for m6A in aberrant splicing of Htt mRNA.

Integrative determination of the atomic structure of mutant huntingtin exon 1 fibrils implicated in Huntington's disease.

Neurodegeneration in Huntington's disease (HD) is accompanied by the aggregation of fragments of the mutant huntingtin protein, a biomarker of disease progression. A particular pathogenic role has been attributed to the aggregation-prone huntingtin exon 1 (HTTex1), generated by aberrant splicing or proteolysis, and containing the expanded polyglutamine (polyQ) segment. Unlike amyloid fibrils from Parkinson's and Alzheimer's diseases, the atomic-level structure of HTTex1 fibrils has remained unknown, limiting diagnostic and treatment efforts. We present and analyze the structure of fibrils formed by polyQ peptides and polyQ-expanded HTTex1 in vitro. Atomic-resolution perspectives are enabled by an integrative analysis and unrestrained all-atom molecular dynamics (MD) simulations incorporating experimental data from electron microscopy (EM), solid-state NMR, and other techniques. Alongside the use of prior data, we report new magic angle spinning NMR studies of glutamine residues of the polyQ fibril core and surface, distinguished via hydrogen-deuterium exchange (HDX). Our study provides a new understanding of the structure of the core as well as surface of aggregated HTTex1, including the fuzzy coat and polyQ-water interface. The obtained data are discussed in context of their implications for understanding the detection of such aggregates (diagnostics) as well as known biological properties of the fibrils.

A Peptide Strategy for Inhibiting Different Protein Aggregation Pathways.

Protein aggregation correlates with many human diseases. Protein aggregates differ in structure and shape. Strategies to develop effective aggregation inhibitors that reach the clinic failed so far. Here, we developed a family of peptides targeting early aggregation stages for both amorphous and fibrillar aggregates of proteins unrelated in sequence and structure. They act on dynamic precursors before mechanistic differentiation takes place. Using peptide arrays, we first identified peptides inhibiting the amorphous aggregation of a molten globular, aggregation-prone mutant of the Axin tumor suppressor. Optimization revealed that the peptides activity did not depend on their sequences but rather on their molecular determinants: a composition of 20-30 % flexible, 30-40 % aliphatic and 20-30 % aromatic residues, a hydrophobicity/hydrophilicity ratio close to 1, and an even distribution of residues of different nature throughout the sequence. The peptides also suppressed fibrillation of Tau, a disordered protein that forms amyloids in Alzheimer's disease, and slowed down that of Huntingtin Exon1, an amyloidogenic protein in Huntington's disease, both entirely unrelated to Axin. Our compounds thus target early stages of different aggregation mechanisms, inhibiting both amorphous and amyloid aggregation. Such cross-mechanistic, multi-targeting aggregation inhibitors may be lead compounds for developing drug candidates against various protein aggregation diseases.

TDP43 and huntingtin Exon-1 undergo a conformationally specific interaction that strongly alters the fibril formation of both proteins

Protein aggregation is a common feature of many neurodegenerative diseases. In Huntington's disease, mutant huntingtin is the primary aggregating protein, but the aggregation of other proteins, such as TDP43, is likely to further contribute to toxicity. Moreover, mutant huntingtin is also a risk factor for TDP pathology in ALS. Despite this co-pathology of huntingtin and TDP43, it remains unknown whether these amyloidogenic proteins directly interact with each other. Using a combination of biophysical methods, we show that the aggregation-prone regions of both proteins, huntingtin exon-1 (Httex1) and the TDP43 low complexity domain (TDP43-LCD), interact in a conformationally specific manner. This interaction significantly slows Httex1 aggregation, while it accelerates TDP43-LCD aggregation. A key intermediate responsible for both effects is a complex formed by liquid TDP43-LCD condensates and Httex1 fibrils. This complex shields seeding competent surfaces of Httex1 fibrils from Httex1 monomers, which are excluded from the condensates. In contrast, TDP43-LCD condensates undergo an accelerated liquid-to-solid transition upon exposure to Httex1 fibrils. Cellular studies show co-aggregation of untagged Httex1 with TDP43. This interaction causes mislocalization of TDP43, which has been linked to TDP43 toxicity. The protection from Httex1 aggregation in lieu of TDP43-LCD aggregation is interesting, as it mirrors what has been found in disease models, namely that TDP43 can protect from huntingtin toxicity, while mutant huntingtin can promote TDP43 pathology. These results suggest that direct protein interaction could, at least in part, be responsible for the linked pathologies of both proteins.

Binding structures of SERF1a with NT17-polyQ peptides of huntingtin exon 1 revealed by SEC-SWAXS, NMR and molecular simulation.

The aberrant fibrillization of huntingtin exon 1 (Httex1) characterized by an expanded polyglutamine (polyQ) tract is a defining feature of Huntington's disease, a neurodegenerative disorder. Recent investigations underscore the involvement of a small EDRK-rich factor 1a (SERF1a) in promoting Httex1 fibrillization through interactions with its N terminus. By establishing an integrated approach with size-exclusion-column-based small- and wide-angle X-ray scattering (SEC-SWAXS), NMR, and molecular simulations using Rosetta, the analysis here reveals a tight binding of two NT17 fragments of Httex1 (comprising the initial 17 amino acids at the N terminus) to the N-terminal region of SERF1a. In contrast, examination of the complex structure of SERF1a with a coiled NT17-polyQ peptide (33 amino acids in total) indicates sparse contacts of the NT17 and polyQ segments with the N-terminal side of SERF1a. Furthermore, the integrated SEC-SWAXS and molecular-simulation analysis suggests that the coiled NT17 segment can transform into a helical conformation when associated with a polyQ segment exhibiting high helical content. Intriguingly, NT17-polyQ peptides with enhanced secondary structures display diminished interactions with SERF1a. This insight into the conformation-dependent binding of NT17 provides clues to a catalytic association mechanism underlying SERF1a's facilitation of Httext1 fibrillization.

Insulin-Degrading Enzyme Efficiently Degrades polyQ Peptides but not Expanded polyQ Huntingtin Fragments.

Huntington's disease is an inheritable autosomal dominant disorder caused by an expanded CAG trinucleotide repeat within the Huntingtin gene, leading to a polyglutamine (polyQ) expansion in the mutant protein. A potential therapeutic approach for delaying or preventing the onset of the disease involves enhancing the degradation of the aggregation-prone polyQ-expanded N-terminal mutant huntingtin (mHTT) exon1 fragment. A few proteases and peptidases have been identified that are able to cleave polyQ fragments with low efficiency. This study aims to identify a potent polyQ-degrading endopeptidase. Here we used quenched polyQ peptides to identify a polyQ-degrading endopeptidase. Next we investigated its role on HTT turnover, using purified polyQ-expanded HTT fragments and striatal cells expressing mHTT exon1 peptides. We identified insulin-degrading enzyme (IDE) as a novel endopeptidase for degrading polyQ peptides. IDE was, however, ineffective in reducing purified polyQ-expanded HTT fragments. Similarly, in striatal cells expressing mHTT exon1 peptides, IDE did not enhance mHTT turnover. This study shows that despite IDE's efficiency in degrading polyQ peptides, it does not contribute to the direct degradation of polyQ-expanded mHTT fragments.

Deconvoluting Monomer- and Dimer-Specific Distance Distributions between Spin Labels in a Monomer/Dimer Mixture Using T1-Edited DEER EPR Spectroscopy.

Double electron-electron resonance (DEER) EPR is a powerful tool in structural biology, providing distances between pairs of spin labels. When the sample consists of a mixture of oligomeric species (e.g., monomer and dimer), the question arises as to how to assign the peaks in the DEER-derived probability distance distribution to the individual species. Here, we propose incorporating an EPR longitudinal electron relaxation (T1) inversion recovery experiment within a DEER pulse sequence to resolve this problem. The apparent T1 between dipolar coupled electron spins measured from the inversion recovery time (τinv) dependence of the peak intensities in the T1-edited DEER-derived probability P(r) distance distribution will be affected by the number of nitroxide labels attached to the biomolecule of interest, for example, two for a monomer and four for a dimer. We show that global fitting of all the T1-edited DEER echo curves, recorded over a range of τinv values, permits the deconvolution of distances between spin labels originating from monomeric (longer T1) and dimeric (shorter T1) species. This is especially useful when the trapping of spin labels in different conformational states during freezing gives rise to complex P(r) distance distributions. The utility of this approach is demonstrated for two systems, the β1 adrenergic receptor and a construct of the huntingtin exon-1 protein fused to the immunoglobulin domain of protein G, both of which exist in a monomer-dimer equilibrium.

Effects of Macromolecular Cosolutes on the Kinetics of Huntingtin Aggregation Monitored by NMR Spectroscopy.

The effects of two macromolecular cosolutes, specifically the polysaccharide dextran-20 and the protein lysozyme, on the aggregation kinetics of a pathogenic huntingtin exon-1 protein (hhtex1) with a 35 polyglutamine repeat, httex1Q35, are described. A unified kinetic model that establishes a direct connection between reversible tetramerization occurring on the microsecond time scale and irreversible fibril formation on a time scale of hours/days forms the basis for quantitative analysis of httex1Q35 aggregation, monitored by measuring cross-peak intensities in a series of 2D 1H-15N NMR correlation spectra acquired during the course of aggregation. The primary effects of the two cosolutes are associated with shifts in the prenucleation tetramerization equilibrium resulting in substantial changes in concentration of "preformed" httex1Q35 tetramers. Similar effects of the two cosolutes on the tetramerization equilibrium observed for a shorter, nonaggregating huntingtin variant with a 7-glutamine repeat, httex1Q7, lend confidence to the conclusions drawn from the fits to the httex1Q35 aggregation kinetics.

A Targetable Self-association Surface of the Huntingtin exon1 Helical Tetramer Required for Assembly of Amyloid Pre-nucleation Oligomers

Polyglutamine (polyQ) sequences undergo repeat-length dependent formation of disease-associated, amyloid-like cross-β core structures with kinetics and aggregate morphologies often influenced by the flanking sequences. In Huntington’s disease (HD), the httNT segment on the polyQ’s N-terminal flank enhances aggregation rates by changing amyloid nucleation from a classical homogeneous mechanism to a two-step process requiring an ɑ-helix-rich oligomeric intermediate. A folded, helix-rich httNT tetrameric structure suggested to be this critical intermediate was recently reported. Here we employ single alanine replacements along the httNT sequence to assess this proposed structure and refine the mechanistic model. We find that Ala replacement of hydrophobic residues within simple httNT peptides greatly suppresses helicity, supporting the tetramer model. These same helix-disruptive replacements in the httNT segment of an exon-1 analog greatly reduce aggregation kinetics, suggesting that an ɑ-helix rich multimer – either the tetramer or a larger multimer – plays an on-pathway role in nucleation. Surprisingly, several other Ala replacements actually enhance helicity and/or amyloid aggregation. The spatial localization of these residues on the tetramer surface suggests a self-association interface responsible for formation of the octomers and higher-order multimers most likely required for polyQ amyloid nucleation. Multimer docking of the tetramer, using the protein–protein docking algorithm ClusPro, predicts this symmetric surface to be a viable tetramer dimerization interface. Intriguingly, octomer formation brings the emerging polyQ chains into closer proximity at this tetramer-tetramer interface. Further supporting the potential importance of tetramer super-assembly, computational docking with a known exon-1 aggregation inhibitor predicts ligand contacts with residues at this interface.

Nucleation of Huntingtin Aggregation Proceeds via Conformational Conversion of Pre-Formed, Sparsely-Populated Tetramers.

Pathogenic huntingtin exon-1 protein (httex1), characterized by an expanded polyglutamine tract located between the N-terminal amphiphilic region and a C-terminal polyproline-rich domain, forms fibrils that accumulate in neuronal inclusion bodies, and is associated with a fatal, autosomal dominant neurodegenerative condition known as Huntington's disease. Here a complete kinetic model is described for aggregation/fibril formation of a httex1 construct with a 35-residue polyglutamine repeat, httex1Q35. Using exchange NMR spectroscopy, it is previously shown that the reversible formation of a sparsely-populated tetramer of the N-terminal amphiphilic domain of httex1Q35, comprising a D2 symmetric four-helix bundle, occurs on the microsecond time-scale and is a prerequisite for subsequent nucleation and fibril formation on a time scale that is many orders of magnitude slower (hours). Here a unified kinetic model of httex1Q35 aggregation is developed in which fast, reversible tetramerization is directly linked to slow irreversible fibril formation via conversion of pre-equilibrated tetrameric species to "active", chain elongation-capable nuclei by conformational re-arrangement with a finite, monomer-independent rate. The unified model permits global quantitative analysis of reversible tetramerization and irreversible fibril formation from a time series of 1H-15N correlation spectra recorded during the course of httex1Q35 aggregation.

Huntingtin HTT1a is generated in a CAG repeat-length-dependent manner in human tissues

BackgroundThe disease-causing mutation in Huntington disease (HD) is a CAG trinucleotide expansion in the huntingtin (HTT) gene. The mutated CAG tract results in the production of a small RNA, HTT1a, coding for only exon 1 of HTT. HTT1a is generated by a block in the splicing reaction of HTT exon 1 to exon 2 followed by cleavage in intron 1 and polyadenylation. Translation of HTT1a leads to the expression of the highly toxic HTT exon 1 protein fragment. We have previously shown that the levels of HTT1a expression in mouse models of HD is dependent on the CAG repeat length. However, these data are lacking for human tissues.MethodsTo answer this question, we developed highly sensitive digital PCR assays to determine HTT1a levels in human samples. These assays allow the absolute quantification of transcript numbers and thus also facilitate the comparison of HTT1a levels between tissues, cell types and across different studies. Furthermore, we measured CAG repeat sizes for every sample used in the study. Finally, we analysed our data with ANOVA and linear modelling to determine the correlation of HTT1a expression levels with CAG repeat sizes.ResultsIn summary, we show that HTT1a is indeed expressed in a CAG repeat-length-dependent manner in human post mortem brain tissues as well as in several peripheral cell types. In particular, PBMCs show a statistically significant positive correlation of HTT1a expression with CAG repeat length, and elevated HTT1a expression levels even in the adult-onset CAG repeat range.ConclusionsOur results show that HTT1a expression occurs throughout a wide range of tissues and likely with all CAG lengths. Our data from peripheral sample sources demonstrate that HTT1a is indeed generated throughout the body in a CAG repeat-length-dependent manner. Therefore, the levels of HTT1a might be a sensitive marker of disease state and/or progression and should be monitored over time, especially in clinical trials targeting HTT expression.

Dynamic interdomain interactions in oligomeric and higher-order assemblies can modulate the intrinsic structural features of huntingtin exon 1

Dynamic interdomain interactions in oligomeric and higher-order assemblies can modulate the intrinsic structural features of huntingtin exon 1

Fluorescent protein tagging promotes phase separation and alters the aggregation pathway of huntingtin exon-1

Fluorescent protein tags are convenient tools for tracking the aggregation states of amyloidogenic or phase separating proteins, but the effect of the tags is often not well understood. Here, we investigated the impact of a C-terminal red fluorescent protein (RFP) tag on the phase separation of huntingtin exon-1 (Httex1), an N-terminal portion of the huntingtin protein that aggregates in Huntington's disease. We found that the RFP-tagged Httex1 rapidly formed micron-sized, phase separated states in the presence of a crowding agent. The formed structures had a rounded appearance and were highly dynamic according to electron paramagnetic resonance and fluorescence recovery after photobleaching, suggesting that the phase separated state was largely liquid in nature. Remarkably, the untagged protein did not undergo any detectable liquid condensate formation under the same conditions. In addition to strongly promoting liquid-liquid phase separation, the RFP tag also facilitated fibril formation, as the tag-dependent liquid condensates rapidly underwent a liquid-to-solid transition. The rate of fibril formation under these conditions was significantly faster than that of the untagged protein. When expressed in cells, the RFP-tagged Httex1 formed larger aggregates with different antibody staining patterns compared to untagged Httex1. Collectively, these data reveal that the addition of a fluorescent protein tag significantly impacts liquid and solid phase separations of Httex1 invitro and leads to altered aggregation in cells. Considering that the tagged Httex1 is commonly used to study the mechanisms of Httex1 misfolding and toxicity, our findings highlight the importance to validate the conclusions with untagged protein.

Interaction between huntingtin exon 1 and HEAT repeat structure probed by chimeric model proteins.

Huntington disease (HD) is associated with aggregation of huntingtin (HTT) protein containing over 35 continuous Q residues within the N-terminal exon 1 encoded region. The C-terminal of the HTT protein consists mainly of HEAT repeat structure which serves as a scaffold for multiple cellular activities. Structural and biochemical analysis of the intact HTT protein has been hampered by its huge size (~300 kDa) and most in vitro studies to date have focused on the properties of the exon 1 region. To explore the interaction between HTT exon 1 and the HEAT repeat structure, we constructed chimeric proteins containing the N-terminal HTT exon 1 region and the HEAT repeat protein PR65/A. The results indicate that HTT exon 1 slightly destabilizes the downstream HEAT repeat structure and endows the HEAT repeat structure with more conformational flexibility. Wild-type and pathological lengths of polyQ did not show differences in the interaction between HTT exon 1 and the HEAT repeats. With the C-terminal fusion of PR65/A, HTT exon 1 containing pathological lengths of polyQ could still form amyloid fibrils, but the higher-order architecture of fibrils and kinetics of fibril formation were affected by the C-terminal fusion of HEAT repeats. This indicates that interaction between HTT exon 1 and HEAT repeat structure is compatible with both normal function of HTT protein and the pathogenesis of HD, and this study provides a potential model for further exploration.

Limitations of Dual-Single Guide RNA CRISPR Strategies for the Treatment of Central Nervous System Genetic Disorders.

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by a toxic gain-of-function CAG expansion in the first exon of the huntingtin (HTT) gene. The monogenic nature of HD makes mutant HTT (mHTT) inactivation a promising therapeutic strategy. Single nucleotide polymorphisms frequently associated with CAG expansion have been explored to selectively inactivate mHTT allele using the CRISPR/Cas9 system. One of such allele-selective approaches consists of excising a region flanking the first exon of mHTT by inducing simultaneous double-strand breaks at upstream and downstream positions of the mHTT exon 1. The removal of the first exon of mHTT deletes the CAG expansion and important transcription regulatory sites, leading to mHTT inactivation. However, the frequency of deletion events is yet to be quantified either in vitro or in vivo. Here, we developed accurate quantitative digital polymerase chain reaction-based assays to assess HTT exon 1 deletion in vitro and in fully humanized HU97/18 mice. Our results demonstrate that dual-single guide RNA (sgRNA) strategies are efficient and that 67% of HTT editing events are leading to exon 1 deletion in HEK293T cells. In contrast, these sgRNA actively cleaved HTT in HU97/18 mice, but most editing events do not lead to exon 1 deletion (10% exon 1 deletion). We also showed that the in vivo editing pattern is not affected by CAG expansion but may potentially be due to the presence of multiple copies of wildtype (wt)/mHTT genes HU97/18 mice as well as the slow kinetics of AAV-mediated CRISPR/Cas9 delivery.

The yeast guanine nucleotide exchange factor Sec7 is a bottleneck in spatial protein quality control and detoxifies neurological disease proteins

ER-to-Golgi trafficking partakes in the sorting of misfolded cytoplasmic proteins to reduce their cytological toxicity. We show here that yeast Sec7, a protein involved in proliferation of the Golgi, is part of this pathway and participates in an Hsp70-dependent formation of insoluble protein deposits (IPOD). Sec7 associates with the disaggregase Hsp104 during a mild heat shock and increases the rate of Hsp104 diffusion in an Hsp70-dependent manner when overproduced. Sec7 overproduction increased formation of IPODs from smaller aggregates and mitigated the toxicity of Huntingtin exon-1 upon heat stress while Sec7 depletion increased sensitivity to aẞ42 of the Alzheimer’s disease and α-synuclein of the Parkinson’s disease, suggesting a role of Sec7 in mitigating proteotoxicity.

Site-Specific Introduction of Alanines for the Nuclear Magnetic Resonance Investigation of Low-Complexity Regions and Large Biomolecular Assemblies.

Nuclear magnetic resonance (NMR) studies of large biomolecular machines and highly repetitive proteins remain challenging due to the difficulty of assigning frequencies to individual nuclei. Here, we present an efficient strategy to address this challenge by engineering a Pyrococcus horikoshii tRNA/alanyl-tRNA synthetase pair that enables the incorporation of up to three isotopically labeled alanine residues in a site-specific manner using in vitro protein expression. The general applicability of this approach for NMR assignment has been demonstrated by introducing isotopically labeled alanines into four distinct proteins: huntingtin exon-1, HMA8 ATPase, the 300 kDa molecular chaperone ClpP, and the alanine-rich Phox2B transcription factor. For large protein assemblies, our labeling approach enabled unambiguous assignments while avoiding potential artifacts induced by site-specific mutations. When applied to Phox2B, which contains two poly-alanine tracts of nine and twenty alanines, we observed that the helical stability is strongly dependent on the homorepeat length. The capacity to selectively introduce alanines with distinct labeling patterns is a powerful tool to probe structure and dynamics of challenging biomolecular systems.

Targeting of biomolecular condensates to the autophagy pathway.

Biomolecular condensates are membraneless compartments formed by liquid-liquid phase separation. They can phase transit into gel-like and solid states. The amount and state of biomolecular condensates must be tightly regulated to maintain normal cellular function. Autophagy targets biomolecular condensates to the lysosome for degradation or other purposes, which we term biocondensophagy. In biocondensophagy, autophagy receptors recognize biomolecular condensates and target them to the autophagosome, the vesicle carrier of autophagy. Multiple types of autophagy receptors have been identified and they are specifically involved in targeting biomolecular condensates with different phase transition states. The receptors also organize the phase transition of biomolecular condensate to facilitate biocondensophagy. Here, we briefly discuss the latest discoveries regarding how biomolecular condensates are recognized by autophagy receptors.

Multi-site-specific isotopic labeling accelerates high-resolution structural investigations of pathogenic huntingtin exon-1

Huntington's disease neurodegeneration occurs when the number of consecutive glutamines in the huntingtin exon-1 (HTTExon1) exceeds a pathological threshold of 35. The sequence homogeneity of HTTExon1 reduces the signal dispersion in NMR spectra, hampering its structural characterization. By simultaneously introducing three isotopically labeled glutamines in a site-specific manner in multiple concatenated samples, 18 glutamines of a pathogenic HTTExon1 with 36 glutamines were unambiguously assigned. Chemical shift analyses indicate the α-helical persistence in the homorepeat and the absence of an emerging toxic conformation around the pathological threshold. Using the same type of samples, the recognition mechanism of Hsc70 molecular chaperone has been investigated, indicating that it binds to the N17 region of HTTExon1, inducing the partial unfolding of the poly-Q. The proposed strategy facilitates high-resolution structural and functional studies in low-complexity regions.