Oncogenic R248W mutation induced conformational perturbation of the p53 core domain and the structural protection by proteomimetic amyloid inhibitor ADH-6.

The involvement of p53 aggregation in cancer pathogenesis emphasizes the importance of unraveling the mechanisms underlying mutation-induced p53 destabilization. And understanding how small molecule inhibitors prevent the conversion of p53 into aggregation-primed conformations is pivotal for the development of therapeutics targeting p53-aggregation-associated cancers. A recent experimental study highlights the efficacy of the proteomimetic amyloid inhibitor ADH-6 in stabilizing R248W p53 and inhibiting its aggregation in cancer cells by interacting with the p53 core domain (p53C). However, it remains mostly unclear how R248W mutation induces destabilization of p53C and how ADH-6 stabilizes this p53C mutant and inhibits its aggregation. Herein, we conducted all-atom molecular dynamics simulations of R248W p53C in the absence and presence of ADH-6, as well as that of wild-type (WT) p53C. Our simulations reveal that the R248W mutation results in a shift of helix H2 and ß-hairpin S2-S2' towards the mutation site, leading to the destruction of their neighboring ß-sheet structure. This further facilitates the formation of a cavity in the hydrophobic core, and reduces the stability of the ß-sandwich. Importantly, two crucial aggregation-prone regions (APRs) S9 and S10 are disturbed and more exposed to solvent in R248W p53C, which is conducive to p53C aggregation. Intriguingly, ADH-6 dynamically binds to the mutation site and multiple destabilized regions in R248W p53C, partially inhibiting the shift of helix H2 and ß-hairpin S2-S2', thus preventing the disruption of the ß-sheets and the formation of the cavity. ADH-6 also reduces the solvent exposure of APRs S9 and S10, which disfavors the aggregation of R248W p53C. Moreover, ADH-6 can preserve the WT-like dynamical network of R248W p53C. Our study elucidates the mechanisms underlying the oncogenic R248W mutation induced p53C destabilization and the structural protection of p53C by ADH-6.

Structural diversity in the membrane-bound hIAPP dimer correlated with distinct membrane disruption mechanisms.

Amyloid deposits of the human islet amyloid polypeptide (hIAPP) have been identified in 90% of patients with type II diabetes. Cellular membranes accelerate the hIAPP fibrillation, and the integrity of membranes is also disrupted at the same time, leading to the apoptosis of ß cells in pancreas. The molecular mechanism of hIAPP-induced membrane disruption, especially during the initial membrane disruption stage, has not been well understood yet. Herein, we carried out extensive all-atom molecular dynamics simulations investigating the hIAPP dimerization process in the anionic POPG membrane, to provide the detailed molecular mechanisms during the initial hIAPP aggregation stage in the membrane environment. Compared to the hIAPP monomer on the membrane, we observed not only an increase of α-helical structures, but also a substantial increase of ß-sheet structures upon spontaneous dimerization. Moreover, the random coiled and α-helical dimer structures insert deep into the membrane interior with a few inter-chain contacts at the C-terminal region, while the ß-sheet-rich structures reside on the membrane surface accompanied by strong inter-chain hydrophobic interactions. The coexistence of α and ß structures constitutes a diverse structural ensemble of the membrane-bound hIAPP dimer. From α-helical to ß-sheet structures, the degree of membrane disruption decreases gradually, and thus the membrane damage induced by random coiled and α-helical structures precedes that induced by ß-sheet structures. We speculate that insertion of random coiled and α-helical structures contributes to the initial stage of membrane damage, while ß-sheet structures on the membrane surface are more involved in the later stage of fibril-induced membrane disruption.

Elucidating the reversible and irreversible self-assembly mechanisms of low-complexity aromatic-rich kinked peptides and steric zipper peptides.

Many RNA-binding proteins such as fused-in sarcoma (FUS) can self-assemble into reversible liquid droplets and fibrils through the self-association of their low-complexity (LC) domains. Recent experiments have revealed that SYG-rich segments in the FUS LC domains play critical roles in the reversible self-assembly behaviors of FUS. These FUS LC segments alone can self-assemble into reversible kinked fibrils, which are markedly different from the canonical irreversible steric zipper ß-sheet fibrils. However, the molecular determinants underlying the reversible and irreversible self-assembly are poorly understood. Herein we conducted extensive all-atom and coarse-grained molecular dynamics simulations of four representative hexapeptides: two low-complexity aromatic-rich kinked peptides from the amyotrophic lateral sclerosis-related FUS protein, FUS37-42 (SYSGYS) and FUS54-59 (SYSSYG); and two steric zipper peptides from Alzheimer's-associated Aß and Tau proteins, Aß16-21 (KLVFFA) and Tau306-311 (VQIVYK). We dissected their reversible and irreversible self-assembly dynamics, predicted their phase separation behaviors, and elucidated the underpinning molecular interactions. Our simulations showed that alternating stickers (Tyr) and spacers (Gly and Ser) in FUS37-42 and FUS54-59 facilitate the formation of highly dynamic coil-rich oligomers and lead to reversible self-assembly, while consecutive hydrophobic residues of LVFF in Aß16-21 and IVY in Tau306-311 act as hydrophobic patches, favoring the formation of stable ß-sheet-rich oligomers and driving the irreversible self-assembly. Intriguingly, we found that FUS37-42 and FUS54-59 peptides, possessing the same amino acid composition and the same number of sticker and spacer residues, display differential self-assembly propensities. This finding suggests that the self-assembly behaviors of FUS peptides are fine-tuned by the site-specific patterning of spacer residues (Ser and Gly). This study provides significant mechanistic insights into reversible and irreversible peptide self-assembly, which would be helpful for understanding the molecular mechanisms underlying the formation of biological liquid condensates and pathological solid amyloid fibrils.

Structural insights into the co-aggregation of Aß and tau amyloid core peptides: Revealing potential pathological heterooligomers by simulations.

The self-aggregation of amyloid-ß (Aß) and tau proteins are closely implicated in Alzheimer's disease (AD). Recent evidence indicates that Aß and tau proteins can cross-interact to form co-aggregates, which aggravates the development of AD. However, their transient heterooligomer conformations and co-aggregation molecular mechanisms are largely unknown. Herein, we utilize replica exchange molecular dynamics simulations to investigate the conformational ensembles formed by the central hydrophobic core of Aß (Aß16-22) and each of two fibril-nucleating core segments of tau (PHF6* and PHF6). Both PHF6 and PHF6* are found to co-aggregate with Aß16-22 into ß-sheet-rich heterooligomers. Intriguingly, PHF6 and Aß16-22 peptides formed closed ß-barrels, while PHF6* and Aß16-22 formed open ß-barrels, implying their distinct co-aggregation property. Compared to Aß16-22-PHF6*, Aß16-22-PHF6 heterooligomers have higher ß-sheet content, and contain longer ß-strands and larger ß-sheets, indicative of stronger co-aggregation ability of PHF6 with Aß16-22. Further analyses reveal that hydrophobic and π-π stacking interactions between Y310 of PHF6 and Aß16-22 are crucial for the closed ß-barrel/larger ß-sheet formation in Aß16-22-PHF6 heterooligomers. These results highlight the paramount importance of PHF6 fragment, particularly Y310 residue, as a potential target for inhibiting Aß-tau co-aggregation, which could help for effective therapeutic design in mitigating Aß-tau co-aggregation related amyloidogenesis.

Five similar anthocyanidin molecules display distinct disruptive effects and mechanisms of action on Aß1-42 protofibril: A molecular dynamic simulation study.

Alzheimer's disease (AD) is associated with the deposition of amyloid-ß (Aß) fibrillary aggregates. Disaggregation of Aß fibrils is considered as one of the promising AD treatments. Recent experimental studies showed that anthocyanidins, one type of flavonoids abundant in fruits/vegetables, can disaggregate Aß fibrillary aggregates. However, their relative disruptive capacities and underlying mechanisms are largely unknown. Herein, we investigated the detailed interactions between five most common anthocyanidins (cyanidin, aurantinidin, peonidin, delphinidin, and pelargonidin) and Aß protofibril (an intermediate of Aß fibrillization) by performing microsecond molecular dynamic simulations. We found that all five anthocyanidins can destroy F4-L34-V36 hydrophobic core and K28-A42 salt bridge, leading to Aß protofibril destabilization. Aurantinidin exhibits the strongest damage to Aß protofibril (with the most severe disruption on K28-A42 salt bridges), followed by cyanidin (with the most destructive effect on F4-L34-V36 core). Detailed analyses reveal that the protofibril-destruction capacities of anthocyanidins are subtly modulated by the interplay of anthocyanidin-protofibril hydrogen bonding, hydrophobic, aromatic stacking interactions, which are dictated by the number or location of hydroxyl/methyl groups of anthocyanidins. These findings provide important mechanistic insights into Aß protofibril disaggregation by anthocyanidins, and suggest that aurantinidin/cyanidin may serve as promising starting-points for the development of new drug candidates against AD.

Elucidating the Mechanisms of R248Q Mutation-Enhanced p53 Aggregation and Its Inhibition by Resveratrol.

Aggregation of p53 mutants can result in loss-of-function, gain-of-function, and dominant-negative effects that contribute to tumor growth. Revealing the mechanisms underlying mutation-enhanced p53 aggregation and dissecting how small molecule inhibitors prevent the conversion of p53 into aggregation-primed conformations are fundamentally important for the development of novel therapeutics for p53 aggregation-associated cancers. A recent experimental study shows that resveratrol (RSV) has an inhibitory effect on the aggregation of hot-spot R248Q mutant of the p53 core domain (p53C), while pterostilbene (PT) exhibits a relatively poor inhibitory efficacy. However, the conformational properties of the R248Q mutant leading to its enhanced aggregation propensity and the inhibitory mechanism of RSV against p53C aggregation are not well understood. Herein, we performed extensive all-atom molecular dynamics simulations on R248Q p53C in the absence and presence of RSV/PT, as well as wild-type (WT) p53C. Our simulations reveal that loop L3, where the mutation resides, remains compact in WT p53C, while it becomes extended in the R248Q mutant. The extension of loop L3 weakens the interactions between loop L3 and two crucial aggregation-prone regions (APRs) of p53C, leading to impaired interactions within the APRs and their structural destabilization as well as p53C. The destabilized APRs in the R248Q mutant are more exposed than in WT p53C, which is conducive to p53C aggregation. RSV has a higher preference to bind to R248Q p53C than PT. This binding not only stabilizes loop L3 of R248Q mutant to its WT-like conformation, preventing L3-extension-caused APRs' destabilization but also reduces APRs' solvent exposure, thereby inhibiting R248Q p53C aggregation. However, PT exhibits a lower hydrogen-bonding capability and a higher self-association propensity, which would lead to a reduced p53C binding and a weakened inhibitory effect on R248Q mutant aggregation. Our study provides mechanistic insights into the R248Q mutation-enhanced aggregation propensity and RSV's potent inhibition against R248Q p53C aggregation.

Destabilization mechanism of R3-R4 tau protofilament by purpurin: a molecular dynamics study.

The accumulation of tau protein aggregates is a common feature observed in many neurodegenerative diseases. However, the structural characteristics of tau aggregates can vary among different tauopathies. It has been established that the structure of the tau protofilament in Chronic traumatic encephalopathy (CTE) is similar to that of Alzheimer's disease (AD). In addition, a previous study found that purpurin, an anthraquinone, could inhibit and disassemble the pre-formed 306VQIVYK311 isoform of AD-tau protofilament. Herein, we used all-atom molecular dynamic (MD) simulation to investigate the distinctive features between CTE-tau and AD-tau protofilament and the influence of purpurin on CTE-tau protofilament. Our findings revealed notable differences at the atomic level between CTE-tau and AD-tau protofilaments, particularly in the ß6-ß7 angle and the solvent-accessible surface area (SASA) of the ß4-ß6 region. These structural disparities contributed to the distinct characteristics observed in the two types of tau protofilaments. Our simulations substantiated that purpurin could destabilize the CTE-tau protofilament and decrease ß-sheet content. Purpurin molecules could insert the ß4-ß6 region and weaken the hydrophobic packing between ß1 and ß8 through π-π stacking. Interestingly, each of the three rings in purpurin exhibited unique binding preferences with the CTE-tau protofilament. Overall, our study sheds light on the structural distinctions between CTE-tau and AD-tau protofilaments, as well as the destabilizing mechanism of purpurin on CTE-tau protofilament, which may be helpful to the development of drugs to prevent CTE.

Dissecting the Molecular Mechanisms of the Co-Aggregation of Aß40 and Aß42 Peptides: A REMD Simulation Study.

The aggregation of amyloid-ß protein (Aß) into oligomers and amyloid fibrils is closely related to Alzheimer's disease (AD). Aß40 and Aß42, as two most prominent isoforms of Aß peptides, can cross-interact with each other and form co-aggregates, which affect the progression of the disease. However, the molecular determinants underlying Aß40 and Aß42 cross-interaction and the structural details of their co-oligomers remain elusive. Herein, we performed all-atom explicit-solvent replica exchange molecular dynamics simulations on Aß40-Aß42 heterogeneous and Aß40/Aß42 homogeneous dimer systems to dissect the co-aggregation mechanisms of the two isoforms. Our results show that the interpeptide main-chain interaction of Aß40-Aß42 is stronger than that of Aß40-Aß40 and Aß42-Aß42. The positions of hotspot residues in heterodimers and homodimers display high similarity, implying similar molecular recognition sites for both cross-interaction and self-interaction. Contact maps of Aß40-Aß42 heterodimers reveal that residue pairs crucial for cross-interaction are mostly located in the C-terminal hydrophobic regions of Aß40 and Aß42 peptides. Conformational analysis shows that Aß40 and Aß42 monomers can co-assemble into ß-sheet-rich heterodimers with shorter ß-sheets than those in homodimers, which is decremental to monomer addition. Similar molecular recognition sites and ß-sheet distribution of Aß40 and Aß42 peptides are observed in heterodimers and homodimers, which may provide the molecular basis for the two isoforms' co-aggregation and cross-seeding. Our work dissects the co-aggregation mechanisms of Aß40 and Aß42 peptides at the atomic level, which will help for in-depth understanding of the cross-talk between the two Aß isoforms and the pathogenesis of AD.

Probing the Protein Folding Energy Landscape: Dissociation of Amyloid-ß Fibrils by Laser-Induced Plasmonic Heating.

Insoluble amyloid fibrils made from proteins and peptides are difficult to be degraded in both living and artificial systems. The importance of studying their physical stability lies primarily with their association with human neurodegenerative diseases, but also owing to their potential role in multiple bio-nanomaterial applications. Here, gold nanorods (AuNRs) were utilized to investigate the plasmonic heating properties and dissociation of amyloid-ß fibrils formed by different peptide fragments (Aß16-22/Aß25-35/Aß1-42) related to the Alzheimer's disease. It is demonstrated that AuNRs were able to break mature amyloid-ß fibrils from both the full length (Aß1-42) and peptide fragments (Aß16-22/Aß25-35) within minutes by triggering ultrahigh localized surface plasmon resonance (LSPR) heating. The LSPR energy absorbed by the amyloids to unfold and move to higher levels in the protein folding energy landscape can be measured directly and in situ by luminescence thermometry using lanthanide-based upconverting nanoparticles. We also show that Aß16-22 fibrils, with the largest persistence length, displayed the highest resistance to breakage, resulting in a transition from rigid fibrils to short flexible fibrils. These findings are consistent with molecular dynamics simulations indicating that Aß16-22 fibrils possess the highest thermostability due to their highly ordered hydrogen bond networks and antiparallel ß-sheet orientation, hence affected by an LSPR-induced remodeling rather than melting. The present results introduce original strategies for disassembling amyloid fibrils noninvasively in liquid environment; they also introduce a methodology to probe the positioning of amyloids on the protein folding and aggregation energy landscape via nanoparticle-enabled plasmonic and upconversion nanothermometry.

Naphthoquinone-dopamine hybrids disrupt α-synuclein fibrils by their intramolecular synergistic interactions with fibrils and display a better effect on fibril disruption.

α-Synuclein (αSyn) is an intrinsically disordered protein and its abnormal aggregation into amyloid fibrils is the main hallmark of Parkinson's disease (PD). The disruption of preformed αSyn fibrils using small molecules is considered as a potential strategy for PD treatment. Recent experiments have reported that naphthoquinone-dopamine hybrids (NQDA), synthesized by naphthoquinone (NQ) and dopamine (DA) molecules, can significantly disrupt αSyn fibrils and cross the blood-brain barrier. To unravel the fibril-disruptive mechanisms at the atomic level, we performed microsecond molecular dynamics simulations of αSyn fibrils in the absence and presence of NQDA, NQ, DA, or NQ+DA molecules. Our simulations showed that NQDA reduces the ß-sheet content, disrupts K45-E57 and E46-K80 salt-bridges, weakens the inter-protofibril interaction, and thus destabilizes the αSyn fibril structure. NQDA has the ability to form cation-π and H-bonding interactions with K45/K80, and form π-π stacking interactions with Y39/F94. Those interactions between NQDA and αSyn fibrils play a crucial role in disaggregating αSyn fibrils. Moreover, we found that NQDA has a better fibril destabilization effect than that of NQ, DA, and NQ+DA molecules. This is attributed to the synergistic fibril-binding effect between NQ and DA groups in NQDA molecules. The DA group can form strong π-π stacking interactions with aromatic residues Y39/F94 of the αSyn fibril, while the DA molecule cannot. In addition, NQDA can form stronger cation-π interactions with residues K45/K80 than those of both NQ and DA molecules. Our results provide the molecular mechanism underlying the disaggregation of the αSyn fibril by NQDA and its better performance in fibril disruption than NQ, DA, and NQ+DA molecules, which offers new clues for the screening and development of promising drug candidates to treat PD.

The frequency of HKαα allele in silent deletional α-thalassemia carriers in the Yulin region of southern China using the third-generation sequencing.

OBJECTIVES: α-thalassemia is relatively prevalent in Yulin Region in southern China. In order to accurately detect α-globin gene aberrations for genetic counseling, the prevalence of HKαα (Hong Kong αα) allele in this subpopulation of silent deletional α-thalassemia were examined. MATERIALS AND METHODS: A total of 1845 subjects were selected in Yulin Region from January 2021 to March 2021. Peripheral blood was collected from each participant for routine genetic analysis of thalassemia. The HKαα allele was determined using the Single-molecule real-time (SMRT) technology for samples with -α3.7/αα, ßN/ßN genotype. RESULTS: Two samples were identified with HKαα allele from 100 samples with -α3.7/αα, ßN/ßN genotype. The frequency of HKαα allele was 2.0 % (2/100) in -α3.7/αα, ßN/ßN carriers in Yulin Region. One sample was identified with a novel variant of the α-globin gene cluster named αHKαα by SMRT technology. One rare HBA2 variant and six HBB variants were found by SMRT technology, including -α3.7/HBA2:c.300 + 34G > A, HBB:c.316-45G > C/ßN, HBB:c.315 + 180 T > C/ßN, HBB:c.316-179A > C/ßN. CONCLUSION: A certain proportion of HKαα allele had been detected in Yulin Region. SMRT technology plays a crucial role for improving the diagnostic accuracy and positive detection rate of thalassemia. The completion of this study has great meaning for strengthening the prevention and control of thalassemia in Yulin Region.

Dissecting the Effect of ALS Mutation G335D on the Early Aggregation of the TDP-43 Amyloidogenic Core Peptide: Helix-to-ß-Sheet Transition and Conformational Shift.

The aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) into fibrillary deposits is associated with amyotrophic lateral sclerosis (ALS). The 311-360 fragment of TDP-43 (TDP-43311-360), the amyloidogenic core region, can spontaneously aggregate into fibrils, and the ALS-associated mutation G335D has an enhanced effect on TDP-43311-360 fibrillization. However, the molecular mechanism underlying G335D-enhanced aggregation at atomic level remains largely unknown. By utilizing all-atom molecular dynamics (MD) and replica exchange with solute tempering 2 (REST2) simulations, we investigated influences of G335D on the dimerization (the first step of aggregation) and conformational ensemble of the TDP-43311-360 peptide. Our simulations show that G335D mutation increases inter-peptide interactions, especially inter-peptide hydrogen-bonding interactions in which the mutant site has a relatively large contribution, and enhances the dimerization of TDP-43311-360 peptides. The α-helix regions in the NMR-resolved conformation of the TDP-43311-360 monomer (321-330 and 335-343) play an essential role in the formation of the dimer. G335D mutation induces helix unfolding and promotes α-to-ß conversion. G335D mutation alters the conformational distribution of TDP-43311-360 dimers and causes population shift from helix-rich to ß-sheet-rich conformations, which facilitates the fibrillization of the TDP-43311-360 peptide. Our MD and REST2 simulation results suggest that the 321-330 region is of paramount importance to α-to-ß transition and could be the initiation site for TDP-43311-360 fibrillization. Our work reveals the mechanism underlying the enhanced aggregation propensity of the G335D TDP-43311-360 peptide, which provides atomistic insights into the G335D mutation-caused pathogenicity of TDP-43 protein.

Dissecting how ALS-associated D290V mutation enhances pathogenic aggregation of hnRNPA2286-291 peptides: Dynamics and conformational ensembles.

The aggregation of RNA binding proteins, including hnRNPA1/2, TDP-43 and FUS, is heavily implicated in causing or increasing disease risk for a series of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). A recent experimental study demonstrated that an ALS-related D290V mutation in the low complexity domain (LCD) of hnRNPA2 can enhance the aggregation propensity of wild type (WT) hnRNPA2286-291 peptide. However, the underlying molecular mechanisms remain elusive. Herein, we investigated effects of D290V mutation on aggregation dynamics of hnRNPA2286-291 peptide and the conformational ensemble of hnRNPA2286-291 oligomers by performing all-atom molecular dynamic and replica-exchange molecular dynamic simulations. Our simulations demonstrate that D290V mutation greatly reduces the dynamics of hnRNPA2286-291 peptide and that D290V oligomers possess higher compactness and ß-sheet content than WT, indicative of mutation-enhanced aggregation capability. Specifically, D290V mutation strengthens inter-peptide hydrophobic, main-chain hydrogen bonding and side-chain aromatic stacking interactions. Those interactions collectively lead to the enhancement of aggregation capability of hnRNPA2286-291 peptides. Overall, our study provides insights into the dynamics and thermodynamic mechanisms underlying D290V-induced disease-causing aggregation of hnRNPA2286-291, which could contribute to better understanding of the transitions from reversible condensates to irreversible pathogenic aggregates of hnRNPA2 LCD in ALS-related diseases.

ALS-Linked A315T and A315E Mutations Enhance ß-Barrel Formation of the TDP-43307-319 Hexamer: A REST2 Simulation Study.

Pathogenic mutations of transactivation response element DNA-binding protein 43 (TDP-43) are closely linked with amyotrophic lateral sclerosis (ALS). It was recently reported that two ALS-linked familial mutants A315T and A315E of TDP-43307-319 peptides can self-assemble into oligomers including tetramers, hexamers, and octamers, among which hexamers were suggested to form the ß-barrel structure. However, due to the transient nature of oligomers, their conformational properties and the atomic mechanisms underlying the ß-barrel formation remain largely elusive. Herein, we investigated the hexameric conformational distributions of the wild-type (WT) TDP-43307-319 fragment and its A315T and A315E mutants by performing all-atom explicit-solvent replica exchange with solute tempering 2 simulations. Our simulations reveal that each peptide can self-assemble into diverse conformations including ordered ß-barrels, bilayer ß-sheets and/or monolayer ß-sheets, and disordered complexes. A315T and A315E mutants display higher propensity to form ß-barrel structures than the WT, which provides atomic explanation for their enhanced neurotoxicity reported previously. Detailed interaction analysis shows that A315T and A315E mutations increase inter-molecular interactions. Also, the ß-barrel structures formed by the three different peptides are stabilized by distinct inter-peptide side-chain hydrogen bonding, hydrophobic, and aromatic stacking interactions. This study demonstrates the enhanced ß-barrel formation of the TDP-43307-319 hexamer by the pathogenic A315T and A315E mutations and reveals the underlying molecular determinants, which may be helpful for in-depth understanding of the ALS-mutation-induced neurotoxicity of TDP-43 protein.

Molecular Mechanism in the Disruption of Chronic Traumatic Encephalopathy-Related R3-R4 Tau Protofibril by Quercetin and Gallic Acid: Similarities and Differences.

Chronic traumatic encephalopathy (CTE) is a unique progressive neurodegenerative tauopathy pathologically related to the aggregation of the tau protein to neurofibrillary tangles. Disrupting tau oligomers (protofibril) is a promising strategy to prevent CTE. Quercetin (QE) and gallic acid (GA), two polyphenol small molecules abundant in natural crops, were proved to inhibit recombinant tau and the R3 fragment of human full-length tau in vitro. However, their disruptive effect on CTE-related protofibril and the underlying molecular mechanism remain elusive. Cryo-electron microscopy resolution reveals that the R3-R4 fragment of tau forms the core of the CTE-related tau protofibril. In this study, we conducted extensive all-atom molecular dynamics simulations on CTE-related R3-R4 tau protofibril with and without QE/GA molecules. The results disclose that both QE and GA can disrupt the global structure of the protofibril, while GA shows a relatively strong effect. The binding sites, exact binding patterns, and disruptive modes for the two molecules show similarities and differences. Strikingly, both QE and GA can insert into the hydrophobic cavity of the protofibril, indicating they have the potential to compete for the space in the cavity with aggregation cofactors unique to CTE-related protofibril and thus impede the further aggregation of the tau protein. Due to relatively short time scale, our study captures the early disruptive mechanism of CTE-related R3-R4 tau protofibril by QE/GA. However, our research does provide valuable knowledge for the design of supplements or drugs to prevent or delay the development of CTE.

Influence of ALS-linked M337V mutation on the conformational ensembles of TDP-43321-340 peptide monomer and dimer.

The transactive response (TAR) DNA/RNA-binding protein 43 (TDP-43) can self-assemble into both functional stress granules via liquid-liquid phase separation (LLPS) and pathogenic amyloid fibrillary aggregates that are closely linked to amyotrophic lateral sclerosis. Previous experimental studies reported that the low complexity domain (LCD) of TDP-43 plays an essential role in the LLPS and aggregation of the full-length protein, and it alone can also undergo LLPS to form liquid droplets mainly via intermolecular interactions in the 321-340 region. And the ALS-associated M337V mutation impairs LCD's LLPS and facilitates liquid-solid phase transition. However, the underlying atomistic mechanism is not well understood. Herein, as a first step to understand the M337V-caused LLPS disruption of TDP-43 LCD mediated by the 321-340 region and the fibrillization enhancement, we investigated the conformational properties of monomer/dimer of TDP-43321-340 peptide and its M337V mutant by performing extensive all-atom explicit-solvent replica exchange molecular dynamic simulations. Our simulations demonstrate that M337V mutation alters the residue regions with high helix/ß-structure propensities and thus affects the conformational ensembles of both monomer and dimer. M337V mutation inhibits helix formation in the N-terminal Ala-rich region and the C-terminal mutation site region, while facilitating their long ß-sheet formation, albeit with a minor impact on the average probability of both helix structure and ß-structure. Further analysis of dimer system shows that M337V mutation disrupts inter-molecular helix-helix interactions and W334-W334 π-π stacking interactions which were reported to be important for the LLPS of TDP-43 LCD, whereas enhances the overall peptide residue-residue interactions and weakens peptide-water interactions, which is conducive to peptide fibrillization. This study provides mechanistic insights into the M337V-mutation-induced impairment of phase separation and facilitation of fibril formation of TDP-43 LCD.

EGCG attenuates α-synuclein protofibril-membrane interactions and disrupts the protofibril.

The fibrillary aggregates of α-synuclein (α-syn) are closely associated with the etiology of Parkinson's disease (PD). Mounting evidence shows that the interaction of α-syn with biological membranes is a culprit for its aggregation and cytotoxicity. While some small molecules can effectively inhibit α-syn fibrillization in solution, their potential roles in the presence of membrane are rarely studied. Among them, green tea extract epigallocatechin gallate (EGCG) is currently under active investigation. Herein, we investigated the effects of EGCG on α-syn protofibril (an intermediate of α-syn fibril formation) in the presence of a model membrane and on the interactions between α-syn protofibril and the membrane, as well as the underlying mechanisms, by performing microsecond all-atom molecular dynamics simulations. The results show that EGCG has destabilization effects on α-syn protofibril, albeit to a lesser extent than that in solution. Intriguingly, we find that EGCG forms overwhelming H-bonding and cation-π interactions with membrane and thus attenuates protofibril-membrane interactions. Moreover, the decreased protofibril-membrane interactions impede the membrane damage by α-syn protofibril and enable the membrane integrity. These findings provide atomistic understanding towards the attenuation of α-syn protofibril-induced cytotoxicity by EGCG in cellular environment, which is helpful for the development of EGCG-based therapeutic strategies against PD.

Structural Perturbation of Monomers Determines the Amyloid Aggregation Propensity of Calcitonin Variants.

Human calcitonin (hCT) is a polypeptide hormone that participates in calcium-phosphorus metabolism. Irreversible aggregation of 32-amino acid hCT into ß-sheet-rich amyloid fibrils impairs physiological activity and increases the risk of medullary carcinoma of the thyroid. Amyloid-resistant hCT derivatives substituting critical amyloidogenic residues are of particular interest for clinical applications as therapeutic drugs against bone-related diseases. Uncovering the aggregation mechanism of hCT at the molecular level, therefore, is important for the design of amyloid-resistant hCT analogues. Here, we investigated the aggregation dynamics of hCT, non-amyloidogenic salmon calcitonin (sCT), and two hCT analogues with reduced aggregation tendency─TL-hCT and phCT─using long timescale discrete molecular dynamics simulations. Our results showed that hCT monomers mainly adopted unstructured conformations with dynamically formed helices around the central region. hCT self-assembled into helix-rich oligomers first, followed by a conformational conversion into ß-sheet-rich oligomers with ß-sheets formed by residues 10-30 and stabilized by aromatic and hydrophobic interactions. Our simulations confirmed that TL-hCT and phCT oligomers featured more helices and fewer ß-sheets than hCT. Substitution of central aromatic residues with leucine in TL-hCT and replacing C-terminal hydrophobic residue with hydrophilic amino acid in phCT only locally suppressed ß-sheet propensities in the central region and C-terminus, respectively. Having mutations in both central and C-terminal regions, sCT monomers and dynamically formed oligomers predominantly adopted helices, confirming that both central aromatic and C-terminal hydrophobic residues played important roles in the fibrillization of hCT. We also observed the formation of ß-barrel intermediates, postulated as the toxic oligomers in amyloidosis, for hCT but not for sCT. Our computational study depicts a complete picture of the aggregation dynamics of hCT and the effects of mutations. The design of next-generation amyloid-resistant hCT analogues should consider the impact on both amyloidogenic regions and also take into account the amplification of transient ß-sheet population in monomers upon aggregation.

Distinct binding interactions trigger opposite conformational modulations on pathogenic and wildtype Huntingtin exon 1 proteins.

Abnormal elongation of the polyglutamine tract transforms exon 1 of the Huntingtin protein (Htt-exon-1) from wildtype to pathogenic form, and causes Huntington's disease. As an intrinsically disordered protein, the structural ensemble of Htt-exon-1 is highly heterogeneous and the detailed conformation of toxic species is still under debate. Ispinesib, a potential small-molecule drug, has been identified to selectively link the pathogenic Htt-exon-1 into the autophagosome to degrade, thus opening an innovative therapeutic direction. However, the molecular mechanisms behind this selectivity remain largely elusive. Herein, we carry out extensive molecular dynamics simulations with an enhanced sampling method to investigate the ispinesib inducing conformational changes of pathogenic and wildtype Htt-exon-1 and the corresponding binding mechanisms. Our simulations reveal that the ispinesib binding induces opposite conformational changes in pathogenic and wildtype Htt-exon-1, i.e., the 'entropy collapse' with significant reduction of ß-sheets versus the 'entropy expansion' with a slight increase of α-helices. Network analyses further reveal that there are two stable binding sites in the pathogenic Htt-exon-1, while the binding on the wildtype Htt-exon-1 is highly dynamic and non-specific. These dramatic differences originate from the underlying distinct binding interactions. More specifically, stronger hydrogen bonds serve as the specific binding anchors in pathogenic Htt-exon-1, while stronger hydrophobic interactions dominate in the dynamic binding with wildtype Htt-exon-1. Our simulations provide an atomistic mechanism for the ispinesib selective binding on the pathogenic Htt-exon-1, and further shed light on the general mechanisms of small molecule modulation on intrinsically disordered proteins.

Molecular Insights into the Misfolding and Dimerization Dynamics of the Full-Length α-Synuclein from Atomistic Discrete Molecular Dynamics Simulations.

The misfolding and pathological aggregation of α-synuclein forming insoluble amyloid deposits is associated with Parkinson's disease, the second most common neurodegenerative disease in the world population. Characterizing the self-assembly mechanism of α-synuclein is critical for discovering treatments against synucleinopathies. The intrinsically disordered property, high degrees of freedom, and macroscopic timescales of conformational conversion make its characterization extremely challenging in vitro and in silico. Here, we systematically investigated the dynamics of monomer misfolding and dimerization of the full-length α-synuclein using atomistic discrete molecular dynamics simulations. Our results suggested that both α-synuclein monomers and dimers mainly adopted unstructured formations with partial helices around the N-terminus (residues 8-32) and various ß-sheets spanning the residues 35-56 (N-terminal tail) and residues 61-95 (NAC region). The C-terminus mostly assumed an unstructured formation wrapping around the lateral surface and the elongation edge of the ß-sheet core formed by an N-terminal tail and NAC regions. Dimerization enhanced the ß-sheet formation along with a decrease in the unstructured content. The inter-peptide ß-sheets were mainly formed by the N-terminal tail and NACore (residues 68-78) regions, suggesting that these two regions played critical roles in the amyloid aggregation of α-synuclein. Interactions of the C-terminus with the N-terminal tail and the NAC region were significantly suppressed in the α-synuclein dimer, indicating that the interaction of the C-terminus with the N-terminal tail and NAC regions could prevent α-synuclein aggregation. These results on the structural ensembles and early aggregation dynamics of α-synuclein will help understand the nucleation and fibrillization of α-synuclein.