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Stable Polyglutamine Dimers Can Contain β-Hairpins with Interdigitated Side Chains—But Not α-Helices, β-Nanotubes, β-Pseudohelices, or Steric Zippers
Abstract
A common thread connecting nine fatal neurodegenerative protein aggregation diseases is an abnormally expanded polyglutamine tract found in the respective proteins. Although the structure of this tract in the large mature aggregates is increasingly well described, its structure in the small early aggregates remains largely unknown. As experimental evidence suggests that the most toxic species along the aggregation pathway are the small early ones, developing strategies to alleviate disease pathology calls for understanding the structure of polyglutamine peptides in the early stages of aggregation. Here, we present a criterion, grounded in available experimental data, that allows for using kinetic stability of dimers to assess whether a given polyglutamine conformer can be on the aggregation path. We then demonstrate that this criterion can be assessed using present-day molecular dynamics simulations. We find that although the α-helical conformer of polyglutamine is very stable, dimers of α-helices lack the kinetic stability necessary to support further oligomerization. Dimers of steric zipper, β-nanotube, and β-pseudohelix conformers are also too short-lived to initiate aggregation. The β-hairpin-containing conformers, instead, invariably form very stable dimers when their side chains are interdigitated. Combining these findings with the implications of recent solid-state NMR data on mature fibrils, we propose a possible pathway for the initial stages of polyglutamine aggregation, in which β-hairpin-containing conformers act as templates for fibril formation.
... Once a minimal nucleus is formed, the elongation process is thermodynamically favorable and spontaneous. 99,101 For polyglutamine, K n* increases with longer repeat lengths, leading to ever faster aggregation of proteins with longer polyglutamine repeats. 96 Thus, Htt fragments with very long glutamine repeats nucleate more quickly than those with short repeats. ...
... The rate-limiting step for long polyglutamine manifests instead as a monomeric event. 100,101 Various lines of evidence support the idea that monomeric nucleation reflects the formation of a b-hairpin within the expanded polyglutamine monomer. Structural studies detect b-hairpin structures in the endproduct of aggregated polyglutamine and Q44-HttEx1. ...
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.
Impact statement
Many incurable neurodegenerative disorders are associated with, and potentially caused by, the amyloidogenic misfolding and aggregation of proteins. Usually, complex genetic and behavioral factors dictate disease risk and age of onset. Due to its principally mono-genic origin, which strongly predicts the age-of-onset by the extent of CAG repeat expansion, Huntington’s disease (HD) presents a unique opportunity to dissect the underlying disease-causing processes in molecular detail. Yet, until recently, the mutant huntingtin protein with its expanded polyglutamine domain has resisted structural study at the atomic level. We present here a review of recent developments in HD structural biology, facilitated by breakthrough data from solid-state NMR spectroscopy, electron microscopy, and complementary methods. The misfolded structures of the fibrillar proteins inform our mechanistic understanding of the disease-causing molecular processes in HD, other CAG repeat expansion disorders, and, more generally, protein deposition disease.
... In addition, studies of polyglutamine monomers have been carried on but only few works focus on systematic studies by varying the number of the glutamine residues [14,15]. A recent theoretical study with polyQ segments of 40 residues has shown that the polyQ stretch may populate the α-helical conformation as a monomer, while the elongation into fibrils would probably be feasible only after a conformational conversion [16]. Nagai et al. [17] reported the study by circular dichroism of a polyQ stretch of 62 residues (Q62) that has been kept in solution. ...
... In this work the results of more than 1500 ns time of simulations of five different polyQ chains such as Q7, Q11, Q23, Q37 and Q45 are presented. PolyQ chains have been modeled in α-helix conformation as starting conformation due to the available data in the literature about the existence of α-helices, polymorphism and the recent X-ray crystallographic results [11][12][13][16][17][18][19]. Detailed analyses along the generated MD trajectories were performed, and results of different properties such as the structures, RMSD, radius of gyration, clustering, interaction of these peptides with water (hydrogen bonds), torsional angles and analysis of secondary structures are presented. ...
Polyglutamine peptides with an abnormal repeat length are the causative agent of at least nine genetic diseases, known as polyglutamine diseases, which include Huntigton’s disease (HD) and some ataxias, in between the others. In the case of HD the disease appears when the polyQ segment has approximately 36 glutamines (Q). It is known that conformational changes occur and that polyQ aggregation is modulated by the number of Q present in the chain. In the present study, the conformations of monomeric polyQ chains of different lengths (from 7 Q to 45 Q) are systematically investigated by performing all-atom molecular dynamics simulations in explicit water at 310 K. All simulations are performed without any structure constraints. For each system trajectories of 300 ns are generated. Analysis of structures, radius of gyration, root-mean-squared deviation, hydrogen bond analysis, clustering, dihedral angles and analysis of the secondary structures of all studied polyQ repeats are presented. This study provides information about the conformational changes of the considered monomeric peptides with increasing the number of Q. The obtained results are compared with available data. This work confirms that a structural change from the polyQ segment containing 24 Q to the one containing 37 residues occurs.
... There are some studies suggesting that it is the N-terminal helix which self-associates and brings the polyQ stretch together [48,58]. This leads to backbone-backbone and side chain-side chain interaction [59], rearranging into the β sheet which can be observed in the final aggregates. Similar to IAPP, it is proposed that N-and C-terminus are important for the formation of higher ordered aggregate structure [60,61,62]. ...
... The challenge to study polyQ peptides is the low solubility of the polyQ chain. Regardless of the structure of polyQ stretch in monomer, the final aggregates have the β sheets of the polyQ stretch [59,91]. Earlier studies performed on htt exon1 show polyQ repeat dependence of formation of aggregates [78]. ...
The huntingtin protein forms amyloid aggregates in the brain of the patient suffering from Huntinton's disorder. The first exon of huntingtin protein consists of polyglutamine stretch, the extention of which (>36) is responsible for the aggregation. It is not yet clear what kind of conformational changes are involved in the early aggregation event. There are many studies are performed on the synthetic polyglutamine peptides, however, not much research has been conducted on recombinant huntingtin exon1. The study performed here investigates the conformational changes associated with polyglutamine extention in recombinant htt exon1. The conformations and the aggregation propensity of huntingtin exon1 are also studied in the presence of macromolecular crowders and osmolytes to have insights into the effects of the environment mimicking that of the cell. Additionally, the effects of special class of chemical modulators known as molecular tweezers are studied on huntingtin exon1.
... In our model, and consistent with early proposals by Wetzel and colleagues (Kar et al., 2011), the termini of the hairpin fold back toward the beta turn. Unlike other models for the polyQ nucleus (Chen and Wolynes, 2017;Chen et al., 2016;Miettinen et al., 2014; T. T. M. Phan and Schmit, 2020), the AHA monomer contains an embryo of exquisitely ordered Q zipper structure necessary for the entropic bottleneck. We note that the AHA conformer is one of a small number of configurations for a minimal tertiary motif known as a beta-arcade, which was previously proposed to serve as an amyloid nucleus based on its favorable energetics and characteristic occurrence in amyloid fibrils (Kajava et al., 2010). ...
A long-standing goal of the study of amyloids has been to characterize the physical nature of the rate-determining nucleating event. However, the transience and rarity of that event within the heterogeneous ensemble of states populated by amyloid-forming proteins make it inaccessible to classical biochemistry, structural biology, and computational approaches. Here, we address these limitations by measuring the dependence of amyloid formation on concentration and conformational templates in living cells, whose volumes are sufficiently small to resolve independent nucleation events. We characterized over one hundred rationally designed sequence variants of polyglutamine (polyQ), a polypeptide that precipitates Huntingtons and other amyloid-associated neurodegenerative diseases when its length exceeds a characteristic threshold. We deduce that amyloid formation by polyQ begins with a steric zipper embryo of approximately twelve interdigitated glutamine side chains within an individual polypeptide molecule. Formation of the embryo was limited to polypeptides longer than the pathogenic threshold, and involved neither phase separation nor oligomerization. We found that different amyloid propensities of polyQ sequence variants can be rationalized by steric zipper ordering orthogonally to the axis of polymerization, and validated this intuition using all-atom molecular dynamics simulations. The unique ability of the polyQ sequence to fold in this fashion not only allowed for polyQ amyloid to nucleate from low concentrations; it also stalled amyloid growth with a concomitant accumulation of partially-ordered oligomers. By illuminating the structural mechanism of polyQ amyloid formation in cells, our findings reveal a potential molecular etiology for polyQ diseases, and may provide a roadmap for the design of new therapies.
Investigating protein–protein interactions is crucial for understanding cellular biological processes because proteins often function within molecular complexes rather than in isolation. While experimental and computational methods have provided valuable insights into these interactions, they often overlook a critical factor: the crowded cellular environment. This environment significantly impacts protein behavior, including structural stability, diffusion, and ultimately the nature of binding. In this review, we discuss theoretical and computational approaches that allow the modeling of biological systems to guide and complement experiments and can thus significantly advance the investigation, and possibly the predictions, of protein–protein interactions in the crowded environment of cell cytoplasm. We explore topics such as statistical mechanics for lattice simulations, hydrodynamic interactions, diffusion processes in high-viscosity environments, and several methods based on molecular dynamics simulations. By synergistically leveraging methods from biophysics and computational biology, we review the state of the art of computational methods to study the impact of molecular crowding on protein–protein interactions and discuss its potential revolutionizing effects on the characterization of the human interactome.
A family of neurodegenerative diseases, including Huntington’s disease (HD) and spinocerebellar ataxias, are associated with an abnormal polyglutamine (polyQ) expansion in mutant proteins that become prone to form amyloid-like aggregates. Prior studies have suggested a key role for β-hairpin formation as a driver of nucleation and aggregation, but direct experimental studies have been challenging. Toward such research, we set out to enable spatiotemporal control over β-hairpin formation by the introduction of a photosensitive β-turn mimic in the polypeptide backbone, consisting of a newly designed azobenzene derivative. The reported derivative overcomes the limitations of prior approaches associated with poor photochemical properties and imperfect structural compatibility with the desired β-turn structure. A new azobenzene-based β-turn mimic was designed, synthesized, and found to display improved photochemical properties, both prior and after incorporation into the backbone of a polyQ polypeptide. The two isomers of the azobenzene-polyQ peptide showed different aggregate structures of the polyQ peptide fibrils, as demonstrated by electron microscopy and solid-state NMR (ssNMR). Notably, only peptides in which the β-turn structure was stabilized (azobenzene in the cis configuration) closely reproduced the spectral fingerprints of toxic, β-hairpin-containing fibrils formed by mutant huntingtin protein fragments implicated in HD. These approaches and findings will enable better deciphering of the roles of β-hairpin structures in protein aggregation processes in HD and other amyloid-related neurodegenerative diseases.
The accumulation of aggregated protein is a typical hallmark of many human neurodegenerative disorders, including polyglutamine-related diseases such as chorea Huntington. Misfolding of the amyloidogenic proteins gives rise to self-assembled complexes and fibres. The huntingtin protein is characterised by a segment of consecutive glutamines which, when exceeding ~ 37 residues, results in the occurrence of the disease. Furthermore, it has also been demonstrated that the 17-residue amino-terminal domain of the protein (htt17), located upstream of this polyglutamine tract, strongly correlates with aggregate formation and pathology. Here, we demonstrate that membrane interactions strongly accelerate the oligomerisation and β-amyloid fibril formation of htt17-polyglutamine segments. By using a combination of biophysical approaches, the kinetics of fibre formation is investigated and found to be strongly dependent on the presence of lipids, the length of the polyQ expansion, and the polypeptide-to-lipid ratio. Finally, the implications for therapeutic approaches are discussed.
We address polyglutamine‐14 in aqueous solution with specific chromophores and a solubility chain by means of a multiscale simulation approach, combining atomistic molecular dynamics simulations and coarse‐grained Monte‐Carlo conformational sampling. Despite the intrinsically disordered nature of the amyloidogenic polyglutamine, we observe transient characteristic structural motifs which exhibit a specific hydrogen bonding pattern. We illustrate the relationship between structure pattern and the distance distribution of a pair of chromophores attached to the peptide termini, in light of specific influence of a short solubility tail and the chromophores themselves on the conformational ensemble.
The size of the critical nucleus acting as intermediate in the amyloid formation of a model peptide is calculated. The theoretical approach is based on experimentally determined amyloid formation rates and gives new insights into the amyloid formation pathway.
The disease risk and age of onset of Huntingtons 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.
A systematic analysis is performed on the effectiveness of removing degrees of freedom from hydrogen atoms and/or increasing hydrogen masses to increase the efficiency of molecular dynamics simulations of hydrogen-rich systems such as proteins in water. In proteins, high-frequency bond-angle vibrations involving hydrogen atoms limit the time step to 3 fs, which is already a factor of 1.5 beyond the commonly used time step of 2 fs. Removing these degrees of freedom from the system by constructing hydrogen atoms as dummy atoms, allows the time step to be increased to 7 fs, a factor of 3.5 compared with 2 fs. Additionally, a gain in simulation stability can be achieved by increasing the masses of hydrogen atoms with remaining degrees of freedom from 1 to 4 u. Increasing hydrogen mass without removing the high-frequency degrees of freedom allows the time step to be increased only to 4 fs, a factor of two, compared with 2 fs. The net gain in efficiency of sampling configurational space may be up to 15% lower than expected from the increase in time step due to the increase in viscosity and decrease in diffusion constant. In principle, introducing dummy atoms and increasing hydrogen mass do not influence thermodynamical properties of the system and dynamical properties are shown to be influenced only to a moderate degree. Comparing the maximum time step attainable with these methods (7 fs) to the time step of 2 fs that is routinely used in simulation, and taking into account the increase in viscosity and decrease in diffusion constant, we can say that a net gain in simulation efficiency of a factor of 3 to 3.5 can be achieved. (C) 1999 John Wiley & Sons, Inc.
We have compared molecular dynamics (MD) simulations of a β-hairpin forming peptide derived from the protein Nrf2 with 10 biomolecular force fields using trajectories of at least 1 μs. The total simulation time was 37.2 μs. Previous studies have shown that different force fields, water models, simulation methods, and parameters can affect simulation outcomes. The MD simulations were done in explicit solvent with a 16-mer Nrf2 β-hairpin forming peptide using Amber ff99SB-ILDN, Amber ff99SB*-ILDN, Amber ff99SB, Amber ff99SB*, Amber ff03, Amber ff03*, GROMOS96 43a1p, GROMOS96 53a6, CHARMM27, and OPLS-AA/L force fields. The effects of charge-groups, terminal capping, and phosphorylation on the peptide folding were also examined. Despite using identical starting structures and simulation parameters, we observed clear differences among the various force fields and even between replicates using the same force field. Our simulations show that the uncapped peptide folds into a native-like β-hairpin structure at 310 K when Amber ff99SB-ILDN, Amber ff99SB*-ILDN, Amber ff99SB, Amber ff99SB*, Amber ff03, Amber ff03*, GROMOS96 43a1p, or GROMOS96 53a6 were used. The CHARMM27 simulations were able to form native hairpins in some of the elevated temperature simulations, while the OPLS-AA/L simulations did not yield native hairpin structures at any temperatures tested. Simulations that used charge-groups or peptide capping groups were not largely different from their uncapped counterparts with single atom charge-groups. On the other hand, phosphorylation of the threonine residue located at the β-turn significantly affected the hairpin formation. To our knowledge, this is the first study comparing such a large set of force fields with respect to β-hairpin folding. Such a comprehensive comparison will offer useful guidance to others conducting similar types of simulations.
The previously developed particle mesh Ewald method is reformulated in terms of efficient B‐spline interpolation of the structure factors. This reformulation allows a natural extension of the method to potentials of the form 1/r p with p≥1. Furthermore, efficient calculation of the virial tensor follows. Use of B‐splines in place of Lagrange interpolation leads to analytic gradients as well as a significant improvement in the accuracy. We demonstrate that arbitrary accuracy can be achieved, independent of system size N, at a cost that scales as N log(N). For biomolecular systems with many thousands of atoms this method permits the use of Ewald summation at a computational cost comparable to that of a simple truncation method of 10 Å or less.
Classical Monte Carlo simulations have been carried out for liquid water in the NPT ensemble at 25 °C and 1 atm using six of the simpler intermolecular potential functions for the water dimer: Bernal–Fowler (BF), SPC, ST2, TIPS2, TIP3P, and TIP4P. Comparisons are made with experimental thermodynamic and structural data including the recent neutron diffraction results of Thiessen and Narten. The computed densities and potential energies are in reasonable accord with experiment except for the original BF model, which yields an 18% overestimate of the density and poor structural results. The TIPS2 and TIP4P potentials yield oxygen–oxygen partial structure functions in good agreement with the neutron diffraction results. The accord with the experimental OH and HH partial structure functions is poorer; however, the computed results for these functions are similar for all the potential functions. Consequently, the discrepancy may be due to the correction terms needed in processing the neutron data or to an effect uniformly neglected in the computations. Comparisons are also made for self‐diffusion coefficients obtained from molecular dynamics simulations. Overall, the SPC, ST2, TIPS2, and TIP4P models give reasonable structural and thermodynamic descriptions of liquid water and they should be useful in simulations of aqueous solutions. The simplicity of the SPC, TIPS2, and TIP4P functions is also attractive from a computational standpoint.
For molecular dynamics simulations of hydrated proteins a simple yet reliable model for the intermolecular potential for water is required. Such a model must be an effective pair potential valid for liquid densities that takes average many-body interactions into account. We have developed a three-point charge model (on hydrogen and oxygen positions) with a Lennard-Jones 6–12 potential on the oxygen positions only. Parameters for the model were determined from 12 molecular dynamics runs covering the two-dimensional parameter space of charge and oxygen repulsion. Both potential energy and pressure were required to coincide with experimental values. The model has very satisfactory properties, is easily incorporated into protein-water potentials, and requires only 0.25 sec computertime per dynamics step (for 216 molecules) on a CRAY-1 computer.
Molecular dynamics simulations provide a vehicle for capturing the structures, motions, and interactions of biological macromolecules in full atomic detail. The accuracy of such simulations, however, is critically dependent on the force field--the mathematical model used to approximate the atomic-level forces acting on the simulated molecular system. Here we present a systematic and extensive evaluation of eight different protein force fields based on comparisons of experimental data with molecular dynamics simulations that reach a previously inaccessible timescale. First, through extensive comparisons with experimental NMR data, we examined the force fields' abilities to describe the structure and fluctuations of folded proteins. Second, we quantified potential biases towards different secondary structure types by comparing experimental and simulation data for small peptides that preferentially populate either helical or sheet-like structures. Third, we tested the force fields' abilities to fold two small proteins--one α-helical, the other with β-sheet structure. The results suggest that force fields have improved over time, and that the most recent versions, while not perfect, provide an accurate description of many structural and dynamical properties of proteins.
Abnormal polyglutamine (polyQ) tracts are the only common feature in nine proteins that each cause a dominant neurodegenerative disorder. In Huntington's disease, tracts longer than 36 glutamines in the protein huntingtin (htt) cause degeneration. In situ, monoclonal antibody 3B5H10 binds to different htt fragments in neurons in proportion to their toxicity. Here, we determined the structure of 3B5H10 Fab to 1.9 Å resolution by X-ray crystallography. Modeling demonstrates that the paratope forms a groove suitable for binding two β-rich polyQ strands. Using small-angle X-ray scattering, we confirmed that the polyQ epitope recognized by 3B5H10 is a compact two-stranded hairpin within monomeric htt and is abundant in htt fragments unbound to antibody. Thus, disease-associated polyQ stretches preferentially adopt compact conformations. Since 3B5H10 binding predicts degeneration, this compact polyQ structure may be neurotoxic.
Preferential accumulation of mutant proteins in the nucleus has been suggested to be the molecular culprit that confers cellular toxicity in the neurodegenerative disorders caused by polyglutamine (polyQ) expansion. Here, we use dynamic imaging approaches, orthogonal cross-seeding, and composition analysis to examine the dynamics and structure of nuclear and cytoplasmic inclusions of atrophin-1, implicated in dentatorubropallidoluysian atrophy, a polyQ-based disease with complex clinical features. Our results reveal a large heterogeneity in the dynamics of the nuclear inclusions compared with the compact and immobile cytoplasmic aggregates. At least two types of inclusions of expanded atrophin-1 with different mobility of the molecular species and ability to exchange with the surrounding monomer pool coexist in the nucleus. Intriguingly, the enrichment of nuclear inclusions with slow dynamics parallels changes in the aggregate core architecture that are dominated by the polyQ stretch. We propose that the observed complexity in the dynamics of the nuclear inclusions provides a molecular explanation for the enhanced cellular toxicity of the nuclear aggregates in polyQ-based neurodegeneration.
Polyglutamine (polyQ) stretches exceeding a threshold length confer a toxic function to proteins that contain them and cause at least nine neurological disorders. The basis for this toxicity threshold is unclear. Although polyQ expansions render proteins prone to aggregate into inclusion bodies, this may be a neuronal coping response to more toxic forms of polyQ. The exact structure of these more toxic forms is unknown. Here we show that the monoclonal antibody 3B5H10 recognizes a species of polyQ protein in situ that strongly predicts neuronal death. The epitope selectively appears among some of the many low-molecular-weight conformational states assumed by expanded polyQ and disappears in higher-molecular-weight aggregated forms, such as inclusion bodies. These results suggest that protein monomers and possibly small oligomers containing expanded polyQ stretches can adopt a conformation that is recognized by 3B5H10 and is toxic or closely related to a toxic species.
Because polyglutamine (polyQ) aggregate formation has been implicated as playing an important role in expanded CAG repeat diseases, it is important to understand the biophysics underlying the initiation of aggregation. Previously, we showed that relatively long polyQ peptides aggregate by nucleated growth polymerization and a monomeric critical nucleus. We show here that over a short range of repeat lengths, from Q(23) to Q(26), the size of the critical nucleus for aggregation increases from monomeric to dimeric to tetrameric. This variation in nucleus size suggests a common duplex antiparallel β-sheet framework for the nucleus, and it further supports the feasibility of an organized monomeric aggregation nucleus for longer polyQ repeat peptides. The data also suggest that a change in the size of aggregation nuclei may have a role in the pathogenicity of polyQ expansion in this series of familial neurodegenerative diseases.
Huntington disease results from an expanded polyglutamine region in the N terminus of the huntingtin protein. HD pathology
is characterized by neuronal degeneration and protein inclusions containing N-terminal fragments of mutant huntingtin. Structural
information is minimal, though it is believed that mutant huntingtin polyglutamine adopts β structure upon conversion to a
toxic form. To this end, we designed mammalian cell expression constructs encoding compact β variants of Htt exon 1 N-terminal
fragment and tested their ability to aggregate and induce toxicity in cultured neuronal cells. In parallel, we performed molecular
dynamics simulations, which indicate that constructs with expanded polyglutamine β-strands are stabilized by main-chain hydrogen
bonding. Finally, we found a correlation between the reactivity to 3B5H10, an expanded polyglutamine antibody that recognizes
a compact β rich hairpin structure, and the ability to induce cell toxicity. These data are consistent with an important role
for a compact β structure in mutant huntingtin-induced cell toxicity.
An expanded polyglutamine (polyQ) stretch in the protein huntingtin (htt) induces self-aggregation into inclusion bodies (IBs) and causes Huntington's disease (HD). Defining precise relationships between early observable variables and neuronal death at the molecular and cellular levels should improve our understanding of HD pathogenesis. Here, we used an automated microscope that tracks thousands of neurons individually over their entire lifetime to quantify interconnected relationships between early variables, such as htt levels, polyQ length, and IB formation, and neuronal death in a primary striatal model of HD. The resulting model revealed that mutant htt increases the risk of death by tonically interfering with homeostatic coping mechanisms rather than producing accumulated damage to the neuron, htt toxicity is saturable, the rate-limiting steps for inclusion body formation and death can be traced to different conformational changes in monomeric htt, and IB formation reduces the impact of the starting levels of htt of a neuron on its risk of death. Finally, the model that emerges from our quantitative measurements places critical limits on the potential mechanisms by which mutant htt might induce neurodegeneration, which should help direct future research.
Expansion of CAG trinucleotide repeat within the coding region of several genes results in the production of proteins with expanded polyglutamine (PolyQ) stretch. The expression of these pathogenic proteins leads to PolyQ diseases, such as Huntington's disease or several types of spinocerebellar ataxias. This family of neurodegenerative disorders is characterized by constant progression of the symptoms and molecularly, by the accumulation of mutant proteins inside neurons causing their dysfunction and eventually death. So far, no effective therapy actually preventing the physical and/or mental decline has been developed. Experimental therapeutic strategies either target the levels or processing of mutant proteins in an attempt to prevent cellular deterioration, or they are aimed at the downstream pathologic effects to reverse or ameliorate the caused damages. Certain pathomechanistic aspects of PolyQ disorders are discussed here. Relevance of disease models and recent knowledge of therapeutic possibilities is reviewed and updated.
Expansion of poiygiutamine (poSyQ) tracts m proteins results m protein aggregation and is associated with cei death m at feast arte rseyrodegenerative diseases. Disease age of onset as eorreiated with the poSyQ insert Sengih above a critica vaiue of 35-40 gSutamines. The aggregation kinetics of isoiated poSyQ peptides in vitro aSso shows a simiiar edticaS-Sength dependence. WhiSe recersi experimenta work has provided corssiderabie insights irsto pofyQ aggregation, the molecular mecharilsm of aggregation is not weH understood. Here, using computer simiaiationis of isoaated poiyQ peptides, we show that a mechanism of aggregation is the conformatiorsai transition in a singie poiyQ peptide chairs from random coif to a parafSeS β-heix. This transition occurs selectively in peptides Songer than 37 gfytamines. fn the β-heHces observed in simyiations, aH residues adopt p-strand backbone dihedra angles, and the poypeptke chain coais around a centrai heiica axis with 18.5 ±2 residues per tym. We aiso fmd that mytant poiyQ peptides with prourse-gSycisie inserts show formation of antaparaHe β-hairpins in their ground state, ira agreement with experiments. The Sower stability of mutant β-heuces expiains their iower aggregation rates compared to wiid type. Our resuits provide a moiecuiar mechanism for poiyQ-medlated aggregation.
Expansion of trinucleotide repeat sequences is the mutational mechanism in at least 16 neurological disorders, including fragile X type A syndrome, myotonic dystrophy, spinobulbar muscular atrophy, Huntington disease, and several others. The discovery of trinucleotide repeat expansions provides a biological explanation for anticipation,the increase in disease severity and decrease in age of onset from one generation to the next, observed in all of these disorders. This chapter provides an overview of trinucleotide repeat disorders and discusses some of the findings in pathogenesis studies.
An N·log(N) method for evaluating electrostatic energies and forces of large periodic systems is presented. The method is based on interpolation of the reciprocal space Ewald sums and evaluation of the resulting convolutions using fast Fourier transforms. Timings and accuracies are presented for three large crystalline ionic systems. The Journal of Chemical Physics is copyrighted by The American Institute of Physics.
CHARMM27 is a widespread and popular force field for biomolecular simulation, and several recent algorithms such as implicit solvent models have been developed specifically for it. We have here implemented the CHARMM force field and all necessary extended functional forms in the GROMACS molecular simulation package, to make CHARMM-specific features available and to test them in combination with techniques for extended time steps, to make all major force fields available for comparison studies in GROMACS, and to test various solvent model optimizations, in particular the effect of Lennard-Jones interactions on hydrogens. The implementation has full support both for CHARMM-specific features such as multiple potentials over the same dihedral angle and the grid-based energy correction map on the , ψ protein backbone dihedrals, as well as all GROMACS features such as virtual hydrogen interaction sites that enable 5 fs time steps. The medium-to-long time effects of both the correction maps and virtual sites have been tested by performing a series of 100 ns simulations using different models for water representation, including comparisons between CHARMM and traditional TIP3P. Including the correction maps improves sampling of near native-state conformations in our systems, and to some extent it is even able to refine distorted protein conformations. Finally, we show that this accuracy is largely maintained with a new implicit solvent implementation that works with virtual interaction sites, which enables performance in excess of 250 ns/day for a 900-atom protein on a quad-core desktop computer.
Molecular simulation is an extremely useful, but computationally very expensive tool for studies of chemical and biomolecular systems. Here, we present a new implementation of our molecular simulation toolkit GROMACS which now both achieves extremely high performance on single processors from algorithmic optimizations and hand-coded routines and simultaneously scales very well on parallel machines. The code encompasses a minimal-communication domain decomposition algorithm, full dynamic load balancing, a state-of-the-art parallel constraint solver, and efficient virtual site algorithms that allow removal of hydrogen atom degrees of freedom to enable integration time steps up to 5 fs for atomistic simulations also in parallel. To improve the scaling properties of the common particle mesh Ewald electrostatics algorithms, we have in addition used a Multiple-Program, Multiple-Data approach, with separate node domains responsible for direct and reciprocal space interactions. Not only does this combination of algorithms enable extremely long simulations of large systems but also it provides that simulation performance on quite modest numbers of standard cluster nodes.
By removing the fastest degrees of freedom, constraints allow for an increase of the time step in molecular simulations. In the last decade parallel simulations have become commonplace. However, up till now efficient parallel constraint algorithms have not been used with domain decomposition. In this paper the parallel linear constraint solver (P-LINCS) is presented, which allows the constraining of all bonds in macromolecules. Additionally the energy conservation properties of (P-)LINCS are assessed in view of improvements in the accuracy of uncoupled angle constraints and integration in single precision.
Several neurodegenerative diseases are associated with the polyglutamine (polyQ) repeat disorder in which a segment of consecutive glutamines in the native protein is produced with too many glutamines. Huntington’s disease, for example, is related to the misfolding of the Huntingtin protein which occurs when the polyQ segment has more than approximately 36 glutamines. Experimentally, it is known that the polyQ segment alone aggregates into β-rich conformations such as amyloid fibrils. Its aggregation is modulated by the number of glutamine residues as well as by the surrounding amino acid sequences such as the 17-amino-acid N-terminal fragment of Huntingtin which increases the aggregation rate. Little structural information is available, however, regarding the first steps of aggregation and the atomistic mechanisms of oligomerization are yet to be described. Following previous coarse-grained replica-exchange molecular dynamics simulations that show the spontaneous formation of a nanotube consisting of two intertwined antiparallel strands (Laghaei, R.; Mousseau, N. J. Chem. Phys.2010, 132, 165102), we study this configuration and some extensions of it using all-atom explicit solvent MD simulations. We compare two different lengths for the polyQ segment, 40 and 30 glutamines, and we investigate the impact of the Huntingtin N-terminal residues (httNT). Our results show that the dimeric nanotubes can provide a building block for the formation of longer nanotubes (hexamers and octamers). These longer nanotubes are characterized by large β-sheet propensities and a small solvent exposure of the main-chain atoms. Moreover, the oligomerization between two nanotubes occurs through the formation of protein/protein H-bonds and can result in an elongation of the water-filled core. Our results also show that the httNT enhances the structural stability of the β-rich seeds, suggesting a new mechanism by which it can increase the aggregation rate of the amyloidogenic polyQ sequence.
Polyglutamine (polyQ) diseases comprise a group of dominantly inherited pathology caused by an expansion of an unstable polyQ stretch which is presumed to form β-sheets. Similar to other amyloid pathologies, polyQ amyloidogenesis occurs via a nucleated polymerization mechanism, and proceeds through energetically unfavorable nucleus whose existence and structure are difficult to detect. Here, we use atomistic molecular dynamics simulations in explicit solvent to assess the conformation of the polyQ stretch in the nucleus that initiates polyQ fibrillization. Comparison of the kinetic stability of various structures of polyQ peptide with a Q-length in the pathological range (Q(40)) revealed that steric zipper or nanotube-like structures (β-nanotube or β-pseudohelix) are not kinetically stable enough to serve as a template to initiate polyQ fibrillization as opposed to β-hairpin-based (β-sheet and β-sheetstack) or α-helical conformations. The selection of different structures of the polyQ stretch in the aggregation-initiating event may provide an alternative explanation for polyQ aggregate polymorphism.
An analytical algorithm, called SETTLE, for resetting the positions and velocities to satisfy the holonomic constraints on the rigid water model is presented. This method is still based on the Cartesian coordinate system and can be used in place of SHAKE and RATTLE. We implemented this algorithm in the SPASMS package of molecular mechanics and dynamics. Several series of molecular dynamics simulations were carried out to examine the performance of the new algorithm in comparison with the original RATTLE method. It was found that SETTLE is of higher accuracy and is faster than RATTLE with reasonable tolerances by three to nine times on a scalar machine. Furthermore, the performance improvement ranged from factors of 26 to 98 on a vector machine since the method presented is not iterative. © 1992 by John Wiley & Sons, Inc.
Several neurodegenerative diseases have been found to be strongly associated with proteins containing a polyglutamine stretch which is greatly expanded from approximately 20 glutamines in normal individuals to more than 40 in affected individuals. A conformational change in the expanded polyglutamine stretch has been suggested to form the molecular basis for disease onset. Model peptides containing polyglutamine tend to aggregate and become insoluble. We have synthesized readily water-soluble monomeric peptides by flanking polyglutamine stretches with sequences rich in alanine and lysine. Circular dichroism measurements show that polyglutamine stretches of length 9 or 17 adopt a random coil configuration in aqueous solution. We think that in the disease-associated peptides for normal individuals the stretches of ∼20 glutamines are in a random coil conformation, whereas in affected individuals the polyglutamine stretch may be in some other conformation. Our method to design soluble monomeric peptides containing extended polyglutamine stretches may be generally useful in studying other highly aggregating peptides.
VMD is a molecular graphics program designed for the display and analysis of molecular assemblies, in particular biopolymers such as proteins and nucleic acids. VMD can simultaneously display any number of structures using a wide variety of rendering styles and coloring methods. Molecules are displayed as one or more "representations," in which each representation embodies a particular rendering method and coloring scheme for a selected subset of atoms. The atoms displayed in each representation are chosen using an extensive atom selection syntax, which includes Boolean operators and regular expressions. VMD provides a complete graphical user interface for program control, as well as a text interface using the Tcl embeddable parser to allow for complex scripts with variable substitution, control loops, and function calls. Full session logging is supported, which produces a VMD command script for later playback. High-resolution raster images of displayed molecules may be produced by generating input scripts for use by a number of photorealistic image-rendering applications. VMD has also been expressly designed with the ability to animate molecular dynamics (MD) simulation trajectories, imported either from files or from a direct connection to a running MD simulation. VMD is the visualization component of MDScope, a set of tools for interactive problem solving in structural biology, which also includes the parallel MD program NAMD, and the MDCOMM software used to connect the visualization and simulation programs. VMD is written in C++, using an object-oriented design; the program, including source code and extensive documentation, is freely available via anonymous ftp and through the World Wide Web.
A new Lagrangian formulation is introduced. It can be used to make molecular dynamics (MD) calculations on systems under the most general, externally applied, conditions of stress. In this formulation the MD cell shape and size can change according to dynamical equations given by this Lagrangian. This new MD technique is well suited to the study of structural transformations in solids under external stress and at finite temperature. As an example of the use of this technique we show how a single crystal of Ni behaves under uniform uniaxial compressive and tensile loads. This work confirms some of the results of static (i.e., zero temperature) calculations reported in the literature. We also show that some results regarding the stress‐strain relation obtained by static calculations are invalid at finite temperature. We find that, under compressive loading, our model of Ni shows a bifurcation in its stress‐strain relation; this bifurcation provides a link in configuration space between cubic and hexagonal close packing. It is suggested that such a transformation could perhaps be observed experimentally under extreme conditions of shock.
Polyglutamine (polyGln) aggregates are neuropathological markers of expanded CAG repeat disorders, and may also play a critical role in the development of these diseases. We have established a highly sensitive, fast, reproducible, and specific assay capable of monitoring aggregate-dependent deposition of polyglutamine peptides. This assay allows detailed studies on various aspects of aggregation kinetics, and also makes possible the detection and quantitation of low levels of “extension-competent” aggregates. In the simplest form of this assay, polyGln aggregates are made from chemically synthesized peptides and immobilized onto microplate wells. These wells are incubated for different times with low concentrations of a soluble biotinylated polyGln peptide. Europium-streptavidin complexation of the immobilized biotin, followed by time-resolved fluorescence detection of the deposited europium, allows us to calculate the rate (fmol/h) of incorporation of polyGln peptides into polyGln aggregates. This assay will make possible basic studies on the assembly mechanism of polyGln aggregates and on critical features of the reaction, such as polyGln length dependence. The assay also will be a valuable tool for screening and characterizing anti-aggregation inhibitors. It will also be useful for detection and quantitation of aggregation-competent polyGln aggregates in biological materials, which may prove to be of critical importance in understanding the disease mechanism.
In expanded CAG repeat diseases such as Huntington’s disease, proteins containing polyglutamine (poly(Gln)) sequences with repeat lengths of about 37 residues or more are associated with development of both disease symptoms and neuronal intranuclear inclusions (NIIs). Disease physiology in animal and cellular models does not always correlate with NII formation, however, and the mechanism by which aggregate formation might lead to cytotoxicity is unknown. To help evaluate various possible mechanisms, we determined the biophysical properties of a series of simple poly(Gln) peptides. The circular dichroism spectra of poly(Gln) peptides with repeat lengths of five, 15, 28 and 44 residues are all nearly identical and are consistent with a high degree of random coil structure, suggesting that the length-dependence of disease is not related to a conformational change in the monomeric states of expanded poly(Gln) sequences. In contrast, there is a dramatic increase in both the kinetics and the thermodynamic favorability of the spontaneous formation of ordered, amyloid-like aggregates for poly(Gln) peptides with repeat lengths of greater than 37 residues. At the same time, poly(Gln) peptides with repeat lengths in the 15–20 residue range, despite their poor abilities to support spontaneous, self-nucleated aggregation, are capable of efficiently adding to an already-formed aggregate. We also find that morphologically small, finely divided aggregates are much more efficient at recruiting poly(Gln) peptides than are large aggregates, suggesting a possible explanation for why disease pathology does not always correlate with the observable NII burden. Together, these data are consistent with a model for disease pathology in which critical cellular proteins possessing poly(Gln) sequences of modest length become inactivated when they are recruited into aggregates of an expanded poly(Gln) protein.
Polyglutamine (polyQ) sequences of unknown normal function are present in a significant number of proteins, and their repeat expansion is associated with a number of genetic neurodegenerative diseases. PolyQ solution structure and properties are important not only because of the normal and abnormal biology associated with these sequences but also because they represent an interesting case of a biologically relevant homopolymer. As the common thread in expanded polyQ repeat diseases, it is important to understand the structure and properties of simple polyQ sequences. At the same time, experience has shown that sequences attached to polyQ, whether in artificial constructs or in disease proteins, can influence structure and properties. The two major contenders for the molecular source of the neurotoxicity implicit in polyQ expansion within disease proteins are a populated toxic conformation in the monomer ensemble and a toxic aggregated species. This review summarizes experimental and computational studies on the solution structure and aggregation properties of both simple and complex polyQ sequences, and their repeat-length dependence. As a representative of complex polyQ proteins, the behavior of huntingtin N-terminal fragments, such as exon-1, receives special attention.
In genetic disorders associated with premature neuronal death, symptoms may not appear for years or decades. This delay in clinical onset is often assumed to reflect the occurrence of age-dependent cumulative damage. For example, it has been suggested that oxidative stress disrupts metabolism in neurological degenerative disorders by the cumulative damage of essential macromolecules. A prediction of the cumulative damage hypothesis is that the probability of cell death will increase over time. Here we show in contrast that the kinetics of neuronal death in 12 models of photoreceptor degeneration, hippocampal neurons undergoing excitotoxic cell death, a mouse model of cerebellar degeneration and Parkinson's and Huntington's diseases are all exponential and better explained by mathematical models in which the risk of cell death remains constant or decreases exponentially with age. These kinetics argue against the cumulative damage hypothesis; instead, the time of death of any neuron is random. Our findings are most simply accommodated by a 'one-hit' biochemical model in which mutation imposes a mutant steady state on the neuron and a single event randomly initiates cell death. This model appears to be common to many forms of neurodegeneration and has implications for therapeutic strategies.
Over the past 4 years the GROMOS96 force field has been successfully used in biomolecular simulations, for example in peptide folding studies and detailed protein investigations, but no applications to lipid systems have been published yet. Here we provide a detailed investigation of aliphatic liquid systems. For liquids of larger aliphatic chains, n-heptane and longer, the standard GROMOS96 parameter sets 43A1 and 43A2 yield a too low pressure at the experimental density. Therefore, a reparametrization of the GROMOS96 force field regarding aliphatic carbons was initiated. The new force field parameter set 45A3 shows considerable improvements for n-alkanes, cyclo-, iso-, and neoalkanes and other branched aliphatics. Liquid densities and heat of vaporization are reproduced for almost all of these molecules. Excellent agreement is found with experiment for the free energy of hydration for alkanes. The GROMOS96 45A3 parameter set should, therefore, be suitable for application to lipid aggregates such as membranes and micelles, for mixed systems of aliphatics with or without water, for polymers, and other apolar systems that may interact with different biomolecules. © 2001 John Wiley & Sons, Inc. J Comput Chem 22: 1205–1218, 2001
Protein aggregation via polyglutamine stretches occurs in a number of severe neurodegenerative diseases such as Huntington's disease. We have investigated fibrillar aggregates of polyglutamine peptides below, at, and above the toxicity limit of around 37 glutamine residues using solid-state NMR and electron microscopy. Experimental data are consistent with a dry fibril core of at least 70-80 Å in width for all constructs. Solid-state NMR dipolar correlation experiments reveal a largely β-strand character of all samples and point to tight interdigitation of hydrogen-bonded glutamine side chains from different sheets. Two approximately equally frequent populations of glutamine residues with distinct sets of chemical shifts are found, consistent with local backbone dihedral angles compensating for β-strand twist or with two distinct sets of side-chain conformations. Peptides comprising 15 glutamine residues are present as single extended β-strands. Data obtained for longer constructs are most compatible with a superpleated arrangement with individual molecules contributing β-strands to more than one sheet and an antiparallel assembly of strands within β-sheets.
The functional switch of glutamine/asparagine (Q/N)-rich prions and the neurotoxicity of polyQ-expanded proteins involve complex aggregation-prone structural transitions, commonly presumed to be forming β sheets. By analyzing sequences of interaction partners of these proteins, we discovered a recurrent presence of coiled-coil domains both in the partners and in segments that flank or overlap Q/N-rich and polyQ domains. Since coiled coils can mediate protein interactions and multimerization, we studied their possible involvement in Q/N-rich and polyQ aggregations. Using circular dichroism and chemical crosslinking, we found that Q/N-rich and polyQ peptides form α-helical coiled coils in vitro and assemble into multimers. Using structure-guided mutagenesis, we found that coiled-coil domains modulate in vivo properties of two Q/N-rich prions and polyQ-expanded huntingtin. Mutations that disrupt coiled coils impair aggregation and activity, whereas mutations that enhance coiled-coil propensity promote aggregation. These findings support a coiled-coil model for the functional switch of Q/N-rich prions and for the pathogenesis of polyQ-expansion diseases.
Polyglutamine (polyQ) diseases are caused by an abnormal expansion of CAG repeats. While their detailed structure remains unclear, polyQ peptides assume beta-sheet structures when they aggregate. To investigate the conformational ensemble of short, monomeric polyQ peptides, which consist of 15 glutamine residues (Q(15)), we performed replica exchange molecular dynamics (REMD) simulations. We found that Q(15) can assume multiple configurations due to all of the residues affecting the formation of side-chain hydrogen bonds. Analysis of the free energy landscape reveals that Q(15) has a basin for random-coil structures and another for alpha-helix or beta-turn structures. To investigate properties of aggregated polyQ peptides, we performed multiple molecular dynamics (MMD) simulations for monomeric and oligomeric Q(15). MMD revealed that the formation of oligomers stabilizes the beta-turn structure by increasing the number of hydrogen bonds between the main chains.
Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the gene encoding for huntingtin protein. A lot has been learned about this disease since its first description in 1872 and the identification of its causative gene and mutation in 1993. We now know that the disease is characterized by several molecular and cellular abnormalities whose precise timing and relative roles in pathogenesis have yet to be understood. HD is triggered by the mutant protein, and both gain-of-function (of the mutant protein) and loss-of-function (of the normal protein) mechanisms are involved. Here we review the data that describe the emergence of the ancient huntingtin gene and of the polyglutamine trait during the last 800 million years of evolution. We focus on the known functions of wild-type huntingtin that are fundamental for the survival and functioning of the brain neurons that predominantly degenerate in HD. We summarize data indicating how the loss of these beneficial activities reduces the ability of these neurons to survive. We also review the different mechanisms by which the mutation in huntingtin causes toxicity. This may arise both from cell-autonomous processes and dysfunction of neuronal circuitries. We then focus on novel therapeutical targets and pathways and on the attractive option to counteract HD at its primary source, i.e., by blocking the production of the mutant protein. Strategies and technologies used to screen for candidate HD biomarkers and their potential application are presented. Furthermore, we discuss the opportunities offered by intracerebral cell transplantation and the likely need for these multiple routes into therapies to converge at some point as, ideally, one would wish to stop the disease process and, at the same time, possibly replace the damaged neurons.
Protein dynamics on the atomic level and on the microsecond timescale has recently become accessible from both computation and experiment. To validate molecular dynamics (MD) at the submicrosecond timescale against experiment we present microsecond MD simulations in 10 different force-field configurations for two globular proteins, ubiquitin and the gb3 domain of protein G, for which extensive NMR data is available. We find that the reproduction of the measured NMR data strongly depends on the chosen force field and electrostatics treatment. Generally, particle-mesh Ewald outperforms cut-off and reaction-field approaches. A comparison to measured J-couplings across hydrogen bonds suggests that there is room for improvement in the force-field description of hydrogen bonds in most modern force fields. Our results show that with current force fields, simulations beyond hundreds of nanoseconds run an increased risk of undergoing transitions to nonnative conformational states or will persist within states of high free energy for too long, thus skewing the obtained population frequencies. Only for the AMBER99sb force field have such transitions not been observed. Thus, our results have significance for the interpretation of data obtained with long MD simulations, for the selection of force fields for MD studies and for force-field development. We hope that this comprehensive benchmark based on NMR data applied to many popular MD force fields will serve as a useful resource to the MD community. Finally, we find that for gb3, the force-field AMBER99sb reaches comparable accuracy in back-calculated residual dipolar couplings and J-couplings across hydrogen bonds to ensembles obtained by refinement against NMR data.
Polyglutamine (polyQ) diseases are associated with a CAG/polyQ expansion mutation in unrelated proteins. Upon elongation of the glutamine tract, disease proteins aggregate within cells, mainly in the central nervous system (CNS) and this aggregation process is associated with neurotoxicity. However, it remains unclear to what extent and how this aggregation causes neuronal dysfunction in the CNS. Aiming at preventing neuronal dysfunction, it will be crucial to determine the links between aggregation and cellular dysfunction, understand the folding pathway of polyQ proteins and discover the relative neurotoxicity of polyQ protein species formed along the aggregation pathway. Here, we review what is known about conformations of polyQ peptides and proteins in their monomeric state from experimental and modelling data, how conformational changes of polyQ proteins relate to their oligomerisation and morphology of aggregates and which cellular function are impaired by oligomers, in vitro and in vivo. We also summarise the key modulatory cellular mechanisms and co-factors, which could affect the folding pathway and kinetics of polyQ aggregation. Although many studies have investigated the relationship between polyQ aggregation and toxicity, these have mainly focussed on investigating changes in the formation of the classical hallmark of polyQ diseases, i.e. microscopically visible inclusion bodies. However, recent studies in which oligomeric species have been considered start to shed light on the identity of neurotoxic oligomeric species. Initial evidence suggests that conformational changes induced by polyQ expansions and their surrounding sequence lead to the formation of particular oligomeric intermediates that may differentially affect neurotoxicity.
Amyloid fibrils are filamentous protein aggregates that accumulate in diseases such as Alzheimer's or type II diabetes. The amyloid-forming protein is disease specific. Amyloids may also be formed in vitro from many other proteins, after first denaturing them. Unlike the diverse native folds of these proteins, their amyloids are fundamentally similar in being rigid, smooth-sided, and cross-beta-structured, that is, with beta strands running perpendicular to the fibril axis. In the absence of high-resolution fibril structures, increasingly credible models are being derived by integrating data from a crossfire of experimental techniques. Most current models of disease-related amyloids invoke "beta arcades," columnar structures produced by in-register stacking of "beta arches." A beta arch is a strand-turn-strand motif in which the two beta strands interact via their side chains, not via the polypeptide backbone as in a conventional beta hairpin. Crystal structures of beta-solenoids, a class of proteins with amyloid-like properties, offer insight into the beta-arc turns found in beta arches. General conformational and thermodynamic considerations suggest that complexes of 2 or more beta arches may nucleate amyloid fibrillogenesis in vivo. The apparent prevalence of beta arches and their components have implications for identifying amyloidogenic sequences, elucidating fibril polymorphisms, predicting the locations and conformations of beta arcs within amyloid fibrils, and refining existing fibril models.
Aggregation of huntingtin protein with an expanded polyglutamine region is enhanced by its 17-residue N-terminal domain, which binds to itself and to the polyglutamine region. This enhancement is inhibited when the N-terminal domain binds to the chaperonin TRiC.
We describe the testing and release of AutoDock4 and the accompanying graphical user interface AutoDockTools. AutoDock4 incorporates limited flexibility in the receptor. Several tests are reported here, including a redocking experiment with 188 diverse ligand-protein complexes and a cross-docking experiment using flexible sidechains in 87 HIV protease complexes. We also report its utility in analysis of covalently bound ligands, using both a grid-based docking method and a modification of the flexible sidechain technique.
Polyglutamine (polyQ) beta-stranded aggregates constitute the hallmark of Huntington disease. The disease is fully penetrant when Q residues are more than 36-40 ("disease threshold"). Here, based on a molecular dynamics study on polyQ helical structures of different shapes and oligomeric states, we suggest that the stability of the aggregates increases with the number of monomers, while it is rather insensitive to the number of Qs in each monomer. However, the stability of the single monomer does depend on the number of side-chain intramolecular H-bonds, and therefore on the number of Qs. If such number is lower than that of the disease threshold, the beta-stranded monomers are unstable and hence may aggregate with lower probability, consistently with experimental findings. Our results provide a possible interpretation of the apparent polyQ length dependent-toxicity, and they do not support the so-called "structural threshold hypothesis", which supposes a transition from random coil to a beta-sheet structure only above the disease threshold.
Actin filaments have been shown to result from nucleation and consecutive binding of actin monomers to the ends of filaments1. Nucleation is assumed to consist of the aggregation of a few monomers to form a small filament, but apart from the nucleation and the elongation reaction, end-to-end association of filaments has also been reported2. It is not known whether in physiological conditions actin filaments can break spontaneously at subunit contacts along the filament. In view of the possible importance of spontaneous fragmentation in the determination of the length, number and turnover of thin filaments in the cell, I have now studied this reaction by measuring the polymerization kinetics of actin at low total concentrations in physiological conditions (1 mM MgCl2, 100 mM KC1, 37 °C). Quantitative analysis of the polymerization curves suggests that actin filaments break spontaneously and consequently create more nucleation sites as the reaction proceeds. This explains the autocatalytic nature of the polymerization kinetics.
The kinetics of deoxyhemoglobin S gelation have been investigated using photolytic dissociation of the carbon monoxide complex to initiate the process. Measurements over a wide range of times, 10(-3)-10(4) show that both the concentration dependence of the tenth-time (i.e., the time required to complete one-tenth the reaction) and the time dependence of the process decrease as gelation speeds up. In slowly gelling samples, where single domains of polymers are formed in the small sample volumes employed with this technique (1-2 x 10(-9) cm3), there is a marked increase in the variability of the tenth-times. These results are explained by a mechanism in which gelation is initiated by homogeneous nucleation of polymers in the bulk solution phase, followed by heterogeneous nucleation on the surface of existing polymers. At the lowest concentrations, homogeneous nucleation is so improbable that stochastic behavior is observed in the small sample volumes, and heterogeneous nucleation is the dominant pathway for polymer formation, thereby accounting for the high time dependence. At the highest concentrations homogeneous nucleation becomes much more probable, and the time dependence decreases. The decrease in concentration dependence of the tenth-time with increasing concentration results from a decrease in size of both the homogeneous and heterogeneous critical nuclei. The model rationalizes the major observations on the kinetics of gelation of deoxyhemoglobin S, and is readily testable by further experiments.
Four inherited neurodegenerative diseases are linked to abnormally expanded repeats of glutamine residues in the affected proteins. Molecular modeling followed by optical, electron, and x-ray diffraction studies of a synthetic poly(L-glutamine) shows that it forms beta-sheets strongly held together by hydrogen bonds. Glutamine repeats may function as polar zippers, for example, by joining specific transcription factors bound to separate DNA segments. Their extension may cause disease either by increased, nonspecific affinity between such factors or by gradual precipitation of the affected proteins in neurons.