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Structure and interaction of BAMs. (A) Structure of BAMs. The macrocyclic ring is connected by two ornithines colored in blue. The Hao residue, which can support intramolecular but not intermolecular hydrogen bonding, is colored in red. All natural amino acids are colored black. In BAM, the recognition strand (upper strand) contains 7 natural aa residues that are replaced with the various amyloidogenic segments in this study. The blocking strand (lower strand) contains 4 natural aa residues to facilitate the folding of the β-conformation and increase the solubility of the molecule. R 10 is sometimes replaced with 4-bromo phenylalanine for phase determination of crystal structures. (B) Three types of potential BAM interactions are shown. (Top) Between blocking strands out of register, four intermolecular hydrogen bonds (colored in orange) can be formed. (Middle) Between recognition strands in register, eight intermolecular hydrogen bonds can be formed. (Lower) Between blocking strands in register, six intermolecular hydrogen bonds can be formed. However, the clash between Hao residues precludes this interaction. (C) Sequences of four different BAMs for out-of-register assembly studies.
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Although aberrant protein aggregation has been conclusively linked to dozens of devastating amyloid diseases, scientists remain puzzled about the molecular features that render amyloid fibrils or small oligomers toxic. Here, we report a previously unobserved type of amyloid fibril that tests as cytotoxic: one in which the strands of the contributin...
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... designed peptides that pack exclusively in out-of- register β-sheets. Our design is based on amyloid β-sheet mimics (34); each is a cyclic molecule of two covalently connected anti- parallel β-strands (now termed BAMs). In the recognition strand, an amyloidogenic sequence is grafted and restrained to maintain its extended β-strand conformation ( Fig. 2A). Each recognition strand is capable of forming an antiparallel β-interaction through hydrogen bonds to the recognition strand of an identical BAM molecule. The second strand in each BAM is termed the blocking strand (35), which contains the nonnatural amino acid Hao flanked by dipeptides. Hao prevents full hydrogen bonding of blocking ...
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... in each BAM is termed the blocking strand (35), which contains the nonnatural amino acid Hao flanked by dipeptides. Hao prevents full hydrogen bonding of blocking strands, forcing the formation of an out-of-register β-strand interface with a neighboring BAM. Only the dipeptides on either side of Hao are available to bond to identical molecules ( Fig. 2B). Therefore, if a BAM forms an amyloid-like oligomer or fi- bril, it must form an out-of-register structure with strong hydro- gen-bonding interfaces between recognition strands and weak interfaces between blocking strands, similar to the out-of-register assembly observed in the KDWSFY fibril (Fig. ...
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... Form Toxic Amyloid-Like Oligomers and Fibrils. We prepared four BAMs with amyloidogenic segments grafted from four dif- ferent disease-related amyloid proteins ( Fig. 2C). These proteins form amyloid-like oligomers and fibrils and exhibit cytotoxicity. Our previous studies showed that macrocyclic peptides form oligomers in solution (36) and mimic amyloid oligomers for structural studies (37). Here, we find that, on incubation at 37 °C for 0.5 h, BAMs form amyloid-like oligomers that are recognized by ...
Citations
... The little influence of structural polymorphism on the estimated solvation energies and our previous studies on isolated transthyretin peptides inform about a potential aggregation pathway (Fig. 5). Our previous xray crystallography study shows that the -strand B forms out-of-register amyloid fibrils, historically associated with oligomerization [35][36][37][38][39] . This strand and a portion of strand A are part of the N-terminal fragment of the mature ATTR fibril. ...
ATTR amyloidosis results from the conversion of transthyretin into amyloid fibrils that deposit in tissues causing organ failure and death. This conversion is facilitated by mutations in ATTRv amyloidosis, or aging in ATTRwt amyloidosis. ATTRv amyloidosis exhibits extreme phenotypic variability, whereas ATTRwt amyloidosis presentation is consistent and predictable. Previously, we found an unprecedented structural variability in cardiac amyloid fibrils from polyneuropathic ATTRv-I84S patients. In contrast, cardiac fibrils from five genotypically-different patients with cardiomyopathy or mixed phenotypes are structurally homogeneous. To understand fibril structure’s impact on phenotype, it is necessary to study the fibrils from multiple patients sharing genotype and phenotype. Here we show the cryo-electron microscopy structures of fibrils extracted from four cardiomyopathic ATTRwt amyloidosis patients. Our study confirms that they share identical conformations with minimal structural variability, consistent with their homogenous clinical presentation. Our study contributes to the understanding of ATTR amyloidosis biopathology and calls for further studies.
One-Sentence Summary: Wild-type cardiac ATTR fibrils are structurally homogeneous.
... Almost all of these oligomers show antiparallel alignment for all of their -bridges; all of the adjacent pairs of -strands are out of register; moreover, the hydrophobic V 306 , I 308 , and Y 310 residues point to the outside of, rather than toward the interior of, the oligomer (SI Appendix, Fig. S9). Notably, the octamers seen here are structurally similar to trimeric and hexameric "cylindrins" first observed in oligomers of the K11V fragment of B-crystallin (56) and to tetramers of an A 30−36 -containing construct (57). Specifically, Liu et al. (57) demonstrate the formation of cylindrin-like tetramers using model constructs designed to aggregate out-of-register and posit a mechanism for toxicity involving membrane disruption from insertion of the cylindrins aided by their exposed hydrophobic residues, structurally similar to the PHF6 oligomers predicted here. ...
... Notably, the octamers seen here are structurally similar to trimeric and hexameric "cylindrins" first observed in oligomers of the K11V fragment of B-crystallin (56) and to tetramers of an A 30−36 -containing construct (57). Specifically, Liu et al. (57) demonstrate the formation of cylindrin-like tetramers using model constructs designed to aggregate out-of-register and posit a mechanism for toxicity involving membrane disruption from insertion of the cylindrins aided by their exposed hydrophobic residues, structurally similar to the PHF6 oligomers predicted here. Whether or not the PHF6 cylindrin octamer has any bearing on the potential toxicity of longer PHF6-containing tau fragments is unclear; however, the unique octamer structure nonetheless provides an interesting hypothesis about potentially functional oligomeric structures of PHF6. ...
The PHF6 (Val-Gln-Ile-Val-Tyr-Lys) motif, found in all isoforms of the microtubule-associated protein tau, forms an integral part of ordered cores of amyloid fibrils formed in tauopathies and is thought to play a fundamental role in tau aggregation. Because PHF6 as an isolated hexapeptide assembles into ordered fibrils on its own, it is investigated as a minimal model for insight into the initial stages of aggregation of larger tau fragments. Even for this small peptide, however, the large length and time scales associated with fibrillization pose challenges for simulation studies of its dynamic assembly, equilibrium configurational landscape, and phase behavior. Here, we develop an accurate, bottom-up coarse-grained model of PHF6 for large-scale simulations of its aggregation, which we use to uncover molecular interactions and thermodynamic driving forces governing its assembly. The model, not trained on any explicit information about fibrillar structure, predicts coexistence of formed fibrils with monomers in solution, and we calculate a putative equilibrium phase diagram in concentration-temperature space. We also characterize the configurational and free energetic landscape of PHF6 oligomers. Importantly, we demonstrate with a model of heparin that this widely studied cofactor enhances the aggregation propensity of PHF6 by ordering monomers during nucleation and remaining associated with growing fibrils, consistent with experimentally characterized heparin–tau interactions. Overall, this effort provides detailed molecular insight into PHF6 aggregation thermodynamics and pathways and, furthermore, demonstrates the potential of modern multiscale modeling techniques to produce predictive models of amyloidogenic peptides simultaneously capturing sequence-specific effects and emergent aggregate structures.
... Increasing evidence demonstrates that low-molecular-weight oligomers are the primary neurotoxic species [7,8]. Oligomers with the β-barrel structure have been found to non-specifically destabilize cellular membranes, leading to ionic leakage and impaired regulation of calcium ions [9][10][11][12]. ...
... We found that Aβ [16][17][18][19][20][21][22] and PHF6/PHF6* peptides co-assemble into β-barrel conformations through the second mode, in which they prefer to form hetero β-barrels. The β-barrel structure has been linked to neurotoxic as it has been reported to destroy cellular membranes [9][10][11][12]. The formation of Aβ 16-22 -PHF6 or Aβ 16-22 -PHF6* hetero β-barrels observed in our simulations suggests their potential neurotoxicity, which may provide an explanation for the experimentally reported neurotoxicity of Aβ-tau co-aggregates [69]. ...
... 78,79 In addition, Aβ16-22 peptide shows different H-bond registers as a function of pH experimentally, 26 indicating that both the in-register and outof-register strands are present in the full conformational spectrum of the oligomers. It is interesting that the crystal structure of hIAPP fragments revealed packing motifs of out-of-register β-sheets, 80 and out-of-register β-sheets have been proposed to be part of a toxic pathway. 81 ...
As a model of self-assembly from disordered monomers to fibrils, the amyloid-β fragment Aβ16-22 was subject to past numerous experimental and computational studies. Because dynamics information between milliseconds and seconds cannot be assessed by both studies, we lack a full understanding of its oligomerization. Lattice simulations are particularly well suited to capture pathways to fibrils. In this study, we explored the aggregation of 10 Aβ16–22 peptides using 65 lattice Monte Carlo simulations, each simulation consisting of 3 × 109 steps. Based on a total of 24 and 41 simulations that converge and do not converge to the fibril state, respectively, we are able to reveal the diversity of the pathways leading to fibril structure and the conformational traps slowing down the fibril formation.
... 17,18 Many studies across different neurodegenerative disease models have shown that the early oligomeric species are toxic while some other studies have proposed fibrils to be toxic. 9,11,19,20 All these characteristic features signify a general progressive pathogenic disease mechanism that might be common to different neurodegenerative diseases. 13 TDP-43 is a multi-domain nucleic acid-binding protein, expressed ubiquitously, that plays a significant role in mRNA and gene regulation. ...
TDP‐43 protein is associated with many neurodegenerative diseases and has been shown to adopt various oligomeric and fibrillar states. However, a detailed kinetic understanding of the structural transformation of the native form of the protein to the fibrillar state is missing. In this study, we delineate the temporal sequence of structural events during the amyloid‐like assembly of the functional nucleic acid‐binding domain of TDP‐43. We kinetically mapped the aggregation process using multiple probes such as tryptophan and Thioflavin T (ThT) fluorescence, circular dichroism (CD), and dynamic light scattering (DLS) targeting different structural events. Our data reveals that aggregation occurs in four distinct steps– very fast, fast, slow and very slow. The ‘very fast’ change results in partially unfolded forms that undergo conformational conversion, oligomerization and bind to ThT in the ‘fast step’ to form higher order intermediates (HOI). The temporal sequence of the formation of ThT binding sites and conformational conversion depends upon the protein concentration. The HOI further undergoes structural rearrangement to form protofibrils in the ‘slow’ step, which, consequently, assembles in the ‘very slow’ step to form an amyloid‐like assembly. The spectroscopic properties of the amyloid‐like assembly across the protein concentration remain similar. Additionally, we observe no lag phase across protein concentration for all the probes studied, suggesting that the aggregation process follows a linear polymerization reaction. Overall, our study demonstrates that the amyloid‐like assembly forms in multiple steps, which is also supported by the temperature dependence of the kinetics. This article is protected by copyright. All rights reserved.
... These proteins form liquid-like condensates that can transition to hydrogels over time [44][45][46] . The inter-peptide β-sheet interactions are then thought to explain transient solidification of, otherwise, liquid-like condensates 33,[35][36][37][38][47][48][49] . Importantly, hundreds of protein sequences capable of such structural transitions, and concomitant enhancement of inter-molecular binding strength, have been identified within the human genome 33 . ...
Biomolecular condensates, some of which are liquid-like during health, can age over time becoming gel-like pathological systems. One potential source of loss of liquid-like properties during ageing of RNA-binding protein condensates is the progressive formation of inter-protein β-sheets. To bridge microscopic understanding between accumulation of inter-protein β-sheets over time and the modulation of FUS and hnRNPA1 condensate viscoelasticity, we develop a multiscale simulation approach. Our method integrates atomistic simulations with sequence-dependent coarse-grained modelling of condensates that exhibit accumulation of inter-protein β-sheets over time. We reveal that inter-protein β-sheets notably increase condensate viscosity but does not transform the phase diagrams. Strikingly, the network of molecular connections within condensates is drastically altered, culminating in gelation when the network of strong β-sheets fully percolates. However, high concentrations of RNA decelerate the emergence of inter-protein β-sheets. Our study uncovers molecular and kinetic factors explaining how the accumulation of inter-protein β-sheets can trigger liquid-to-solid transitions in condensates, and suggests a potential mechanism to slow such transitions down.
... As mentioned previously, mutant and wild type SOD1 might misfold and aggregate into amyloid fibrils under certain conditions [63][64][65] . Several studies have demonstrated the general toxicity of amyloid aggregates 66,67 . We show that recombinant D-DT inhibits the amyloid aggregation of mutant SOD1 G93A , as observed by the ThT assay, and instead changed the aggregation pattern from fibril-like aggregates to amorphous disordered ones, as detected by TEM and validated by turbidity assay and decrease of the soluble SOD1 monomer. ...
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of upper and lower motor neurons. About 20% of familial ALS cases are caused by dominant mutations in SOD1. It has been suggested that toxicity of mutant SOD1 results from its misfolding, however, it is unclear why misfolded SOD1 accumulates within specific tissues. We have demonstrated that macrophage migration inhibitory factor (MIF), a multifunctional protein with cytokine/chemokine and chaperone-like activity, inhibits the accumulation and aggregation of misfolded SOD1. Although MIF homolog, D-dopachrome tautomerase (D-DT/MIF-2), shares structural and genetic similarities with MIF, its biological function is not well understood. In the current study, we investigated, for the first time, the mechanism of action of D-DT in a model of ALS. We show that D-DT inhibits mutant SOD1 amyloid aggregation in vitro, promoting the formation of amorphous aggregates. Moreover, we report that D-DT interacts with mutant SOD1, but does not inhibit misfolded mutant SOD1 accumulation and toxicity in neuronal cells. Finally, we show that D-DT is expressed mainly in liver and kidney, with extremely low expression in brain and spinal cord of adult mice. Our findings contribute to better understanding of D-DT versus MIF function in the context of ALS.
... In addition, antiparallel β-sheets are often observed in metastable oligomers and filamentous species formed transiently during amyloid formation (Yu et al., 2009;Sandberg et al., 2010;Dupuis et al., 2011;Sarroukh et al., 2011;Laganowsky et al., 2012). Direct or indirect conversion from antiparallel to parallel β-sheets has been suggested to be a slow step in fibril nucleation or maturation (Sandberg et al., 2010;Qiang et al., 2012), and antiparallel oligomers and fibrils are often found to be toxic (Sandberg et al., 2010;Laganowsky et al., 2012;Liu et al., 2012;Qiang et al., 2012), suggesting that factors that prolong the lifetime of such assemblies may have a major impact on pathology. Lastly, we note that a recent cryo-EM density map of Aβ(1-40) fibrils (Ghosh et al., 2021), seeded with patient-derived material from Alzheimer's disease cortical tissue, had a parallel in-register core (as in Figure 3A) surrounded by peripheral density that was most consistent with two additional protofilament-like stacks of monomers in an intramolecular βhairpin conformation (as in Figure 3C). ...
... Mature amyloids typically induce membrane distortions via exposed hydrophobics at their ends (Xue et al., 2009;Milanesi et al., 2012;Kollmer et al., 2016), a natural consequence of the pseudo-planar structure of their subunits; however, they can also interact with lipids along their length (Kollmer et al., 2019), or even co-assemble with lipids (Galvagnion et al., 2019). Globular oligomers and certain metastable amyloid fibrils appear to exhibit a generic capacity to transition to an amyloid-like β-barrel state (Jang et al., 2008(Jang et al., , 2010Bellesia and Shea, 2009;Kayed et al., 2009;Tomic et al., 2009;Laganowsky et al., 2012;Liu et al., 2012), and these can act as membrane pores (Quist et al., 2005;Jang et al., 2008Jang et al., , 2010Mustata et al., 2009); in addition, other forms of oligomer-dependent membrane disruption have been documented (Green et al., 2004;Kayed et al., 2004). Apart from membrane interactions, it is worth noting that amyloid fibrils provide a generic mechanism by which functional proteins can undergo a pathological loss of function. ...
Amyloid fibrils are a pathologically and functionally relevant state of protein folding, which is generally accessible to polypeptide chains and differs fundamentally from the globular state in terms of molecular symmetry, long-range conformational order, and supramolecular scale. Although amyloid structures are challenging to study, recent developments in techniques such as cryo-EM, solid-state NMR, and AFM have led to an explosion of information about the molecular and supramolecular organization of these assemblies. With these rapid advances, it is now possible to assess the prevalence and significance of proposed general structural features in the context of a diverse body of high-resolution models, and develop a unified view of the principles that control amyloid formation and give rise to their unique properties. Here, we show that, despite system-specific differences, there is a remarkable degree of commonality in both the structural motifs that amyloids adopt and the underlying principles responsible for them. We argue that the inherent geometric differences between amyloids and globular proteins shift the balance of stabilizing forces, predisposing amyloids to distinct molecular interaction motifs with a particular tendency for massive, lattice-like networks of mutually supporting interactions. This general property unites previously characterized structural features such as steric and polar zippers, and contributes to the long-range molecular order that gives amyloids many of their unique properties. The shared features of amyloid structures support the existence of shared structure-activity principles that explain their self-assembly, function, and pathogenesis, and instill hope in efforts to develop broad-spectrum modifiers of amyloid function and pathology.
... These proteins form liquidlike condensates that can transition to hydrogels over time [43][44][45]. These inter-peptide interactions are then thought to explain transient solidification of, otherwise, liquid-like condensates [33,[35][36][37][38][46][47][48]. Importantly, hundreds of protein sequences capable of such structural transitions, and concomitant enhancement of intermolecular binding strengths, have been identified within the human genome [33]. ...
Biomolecular condensates, some of which are liquid-like during health, can age over time becoming gel-like pathological systems. Ageing of RNA-binding protein condensates can emerge from the progressive accumulation of inter-protein β-sheets. To bridge microscopic understanding of such time-dependent transformation with the modulation of FUS and hnRNPA1 condensate viscoelastic-ity, we develop a multiscale simulation approach. Our method integrates atomistic simulations with sequence-dependent coarse-grained modelling of condensates that age over time due to accumulation of inter-protein β-sheets. We reveal that ageing notably increases condensate viscosity but does not transform the phase diagrams. Strikingly, the network of molecular connections within conden-sates is drastically altered during ageing and culminates in gelation when the network of strong inter-protein β-sheets fully percolates. High concentrations of RNA decelerate the accumulation of inter-protein β-sheets, abrogating the effects of ageing. Our study uncovers molecular and kinetic factors explaining condensate ageing, and suggests a potential mechanism to slow ageing down.
... Interestingly, the intrinsically disordered regions of various phase-separating naturally occurring proteins-including fused in sarcoma (FUS) 75 , TAR DNA-binding Protein of 43 kDa (TDP-43) 76 , heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) 74,79,80 , and nucleoprotein of 98 kDa (NUP-98) 74,109 -which form hydrogels over time 73,110,111 , contain short regions termed Low-complexity Aromatic-Rich Kinked Segments (LARKS) that are prone to form such inter-protein β-sheets 82 . When multiple LARKS meet at the high concentrations found inside condensates, they can assemble into ordered arrays of inter-protein β-sheet structures that stick to one another strongly via π-π bonds and hydrogen bonding between backbone atoms that may lead to gradual solidification of, otherwise, liquid-like condensates 72,74,75,77,87,112 . Importantly, hundreds of protein sequences capable of such disorder-to-order conformational transitions, and concomitant enhancement of intermolecular binding strengths, have been identified in the human genome 74 . ...
Biomolecular condensates formed by the process of liquid–liquid phase separation (LLPS) play diverse roles inside cells, from spatiotemporal compartmentalisation to speeding up chemical reactions. Upon maturation, the liquid-like properties of condensates, which underpin their functions, are gradually lost, eventually giving rise to solid-like states with potential pathological implications. Enhancement of inter-protein interactions is one of the main mechanisms suggested to trigger the formation of solid-like condensates. To gain a molecular-level understanding of how the accumulation of stronger interactions among proteins inside condensates affect the kinetic and thermodynamic properties of biomolecular condensates, and their shapes over time, we develop a tailored coarse-grained model of proteins that transition from establishing weak to stronger inter-protein interactions inside condensates. Our simulations reveal that the fast accumulation of strongly binding proteins during the nucleation and growth stages of condensate formation results in aspherical solid-like condensates. In contrast, when strong inter-protein interactions appear only after the equilibrium condensate has been formed, or when they accumulate slowly over time with respect to the time needed for droplets to fuse and grow, spherical solid-like droplets emerge. By conducting atomistic potential-of-mean-force simulations of NUP-98 peptides—prone to forming inter-protein $$\beta$$ β -sheets—we observe that formation of inter-peptide $$\beta$$ β -sheets increases the strength of the interactions consistently with the loss of liquid-like condensate properties we observe at the coarse-grained level. Overall, our work aids in elucidating fundamental molecular, kinetic, and thermodynamic mechanisms linking the rate of change in protein interaction strength to condensate shape and maturation during ageing.
