Biology subjects

Boyer, D. R.

Publications and source records attributed to Boyer, D. R..

11 recordsLinked to original sources

bioRxiv2024

How short peptides can disassemble ultra-stable tau fibrils extracted from Alzheimers disease brain by a strain-relief mechanism

Reducing fibrous aggregates of protein tau is a possible strategy for halting progression of Alzheimers disease (AD). Previously we found that in vitro the D-peptide D-TLKIVWC disassembles tau fibrils from AD brains (AD-tau) into benign segments with no energy source present beyond ambient thermal agitation. This disassembly by a short peptide was unexpected, given that AD-tau is sufficiently stable to withstand disassembly in boiling SDS detergent. To consider D peptide-mediated disassembly as a potential therapeutic for AD, it is essential to understand the mechanism and energy source of the disassembly action. We find assembly of D-peptides into amyloid-like fibrils is essential for tau fibril disassembly. Cryo-EM and atomic force microscopy reveal that these D-peptide fibrils have a right-handed twist and embrace tau fibrils which have a left-handed twist. In binding to the AD-tau fibril, the oppositely twisted D-peptide fibril produces a strain, which is relieved by disassembly of both fibrils. This strain-relief mechanism appears to operate in other examples of amyloid fibril disassembly and provides a new direction for the development of first-in-class therapeutics for amyloid diseases.

neuroscience↗

bioRxiv2023

DNAJB8 oligomerization is mediated by an aromatic-rich motif that is dispensable for substrate activity

J-domain protein (JDP) molecular chaperones have emerged as central players that maintain a healthy proteome. The diverse members of the JDP family function as monomers/dimers and a small subset assemble into micron-sized oligomers. The oligomeric JDP members have eluded structural characterization due to their low-complexity, intrinsically disordered middle domains. This in turn, obscures the biological significance of these larger oligomers in protein folding processes. Here, we identified a short, aromatic motif within DNAJB8, that drives self-assembly through {nu}-{nu} stacking and determined its X-ray structure. We show that mutations in the motif disrupt DNAJB8 oligomerization in vitro and in cells. DNAJB8 variants that are unable to assemble bind to misfolded tau seeds more specifically and retain capacity to reduce protein aggregation in vitro and in cells. We propose a new model for DNAJB8 function in which the sequences in the low-complexity domains play distinct roles in assembly and substrate activity. HIGHLIGHTSDNAJB8 oligomerization is mediated by a short phenylalanine-based motif in the S/T domain Mutation of a single phenylalanine yields a monomeric form of DNAJB8 Monomeric DNABJ8 binds to an aggregation-prone substrate Monomeric DNAJB8 retains substrate aggregation prevention activity

biophysics↗

bioRxiv2022

Structure of a reversible amyloid fibril formed by the CPEB3 prion-like domain reveals a core sequence involved in translational regulation

The cytoplasmic polyadenylation element-binding protein 3 (CPEB3) is a prion-like RNA-binding polypeptide. As a functional prion, CPEB3 is thought to modulate protein synthesis at synapses and enable consolidation of long-term memory in neurons. Here, we report that the prion-like domain 1 of CPEB3 self-assembles into labile amyloid fibrils in vitro. A cryoEM structure of these fibrils reveals an ordered 48-residue core, spanning L103 to F151. CPEB3 constructs lacking this amyloidogenic segment form abnormal puncta in cells when compared to wild type CPEB3, with reduced localization in dormant p-bodies and increased localization in stress granules. Removal of the amyloid core segment in CPEB3 also abolishes its ability to regulate protein synthesis in neurons. Collectively, this evidence suggests that the newly identified amyloidogenic segment within the CPEB3 prion domain is important for its regulated aggregation in cells and suggest its involvement in regulating translational activity and potentially long-term memory formation.

biochemistry↗

bioRxiv2022

Cryo-EM structure of RNA-induced tau fibrils reveals a small C-terminal core that may nucleate fibril formation

In neurodegenerative diseases including Alzheimers and ALS, proteins that bind RNA are found in aggregated forms in autopsied brains. Evidence suggests that RNA aids nucleation of these pathological aggregates; however, the mechanism has not been investigated at the level of atomic structure. Here we present the 3.4 [A] resolution structure of fibrils of full-length recombinant tau protein in the presence of RNA, determined by electron cryo-microscopy (cryoEM). The structure reveals the familiar in-register cross-{beta} amyloid scaffold, but with a small fibril core spanning residues Glu391 to Ala426, a region disordered in the fuzzy coat in all previously studied tau polymorphs. RNA is bound on the fibril surface to the positively charged residues Arg406 and His407 and runs parallel to the fibril axis. The fibrils dissolve when RNAse is added, showing that RNA is necessary for fibril integrity. While this structure cannot exist simultaneously with the tau fibril structures extracted from patients brains, it could conceivably account for the nucleating effects of RNA cofactors followed by remodeling as fibrils mature. Significance statementApplication of cryoEM has greatly expanded our understanding of atomic structures of mature pathological amyloid fibrils, but little is known at the molecular level of the initiation of fibril formation. RNA has been shown to be one cofactor for formation of fibrils of tau protein, and is known also to bind to other proteins, including TDP-43, FUS, and HNRNPA2, which form pathological inclusions. Our cryoEM structure of recombinant tau protein with RNA reveals a 36 residue, C-terminal fibril core bound to RNA which runs parallel to the fibril axis. We speculate that this structure could represent an early step in the formation of tau fibrils.

biophysics↗

bioRxiv2021

Why amyloid fibrils have a limited width

Amyloid fibrils can grow indefinitely long by adding protein chains to the tips of the fibril through {beta}-sheet hydrogen bonding; however, they do not grow laterally beyond [~]10-20 nm. This prevents amyloid fibrils from growing into two-dimensional or three-dimensional arrays. The forces that restrict lateral association of {beta}-sheets in amyloid fibrils are not immediately apparent. We hypothesize that it is the helical symmetry of amyloid fibrils that imposes the limit on fibril width by incurring an increasing separation between helically related molecules as a function of radial distance from the helical axis. The unavoidable consequence is that backbone hydrogen bonds that connect symmetrically related layers of the fibril become weaker towards the edge of the fibril, ultimately becoming too weak to remain ordered. To test our hypothesis, we examined 57 available cryo-EM amyloid fibril structures for trends in interstrand distance and {beta}-sheet hydrogen bonding as a function of radial distance from the helical axis. We find that all fibril structures display an increase in interstrand distance as a function of radius and that most fibril structures have a discernible increase in {beta}-sheet hydrogen bond distances as a function of radius. In addition, we identify a high resolution cryo-EM structure that does not follow our predicted hydrogen bonding trends and perform real space refinement with hydrogen bond distance and angle restraints to restore predicted hydrogen bond trends. This highlights the potential to use our analysis to ensure realistic hydrogen bonding in amyloid fibrils when atomic resolution cryo-EM maps are not available. Significance StatementThe number of amyloid fibril structures determined has exploded in recent years due to advances in structural biology techniques. However, we are still at the beginning stages of understanding amyloid fibril assembly. One important property that is critical to fibril formation and mechanical properties is the fibril width. Despite the diversity of fibril folds discovered, all amyloid fibrils are constrained to a width of 10-20 nm. Here, we use simple geometry and structural analysis to identify that the limited width of amyloid fibrils arises from the helical twist of {beta}-sheets in amyloid fibrils. Our findings provide important considerations for the accurate modeling of hydrogen bonds in amyloid fibrils as well as for the possible prediction and design of amyloid-based nanomaterials.

biochemistry↗

bioRxiv2020

CryoEM reveals how the small molecule EGCG binds to AD brain-derived tau fibrils and initiates fibril disaggregation

EGCG, the most abundant favanol in green tea, is one of the few natural compounds known to inhibit amyloid fibril formation of proteins associated with neurodegeneration, and to disaggregate amyloid fibrils. Little is known of the mechanism of molecular action of EGCG, or how it or other small molecules interact with amyloid fibrils. Here we present a 3.9 [A] resolution cryoEM structure that reveals the site of EGCG binding to Alzheimers disease (AD) brain-derived tau fibrils. The structure suggests that EGCG disaggregates fibrils of AD-tau by wedging into a cleft that is at the interface of two protofilaments of the paired helical filament, and by causing charge repulsions between tau layers of the fibril. In support of this, we observe separation of the protofilaments that EGCG wedges between, and accompanying displacement of the adjacent {beta}-helix domain. By resolving the site of EGCG binding, our structure defines a pharmacophore-like cleft in the AD-tau fibril that will be of use for the discovery of surrogate compounds with more desirable drug-like properties.

biochemistry↗

bioRxiv2020

Cryo-EM structure and inhibitor design of human IAPP (amylin) fibrils

Human islet amyloid polypeptide (hIAPP, or amylin) is a 37 amino acid hormone secreted by pancreatic islet {beta}-cells. Aggregation of hIAPP into amyloid fibrils is found in more than 90% of Type-II Diabetes (T2D) patients and is considered to be associated with T2D pathology. Although different models have been proposed, the high resolution structure of hIAPP fibrils is unknown. Here we report the cryo-EM structure of recombinant full-length hIAPP fibrils. The fibril is composed of two symmetrically-related protofilaments with ordered residues 14-37 that meet at a 14-residue central hydrophobic core. Our hIAPP fibril structure (i) supports the previous hypothesis that residues 20-29, especially 23-29 are the primary amyloid core of hIAPP, (ii) suggests a molecular mechanism for the action of the hIAPP hereditary mutation S20G, (iii) explains why the 6 residue substitutions in rodent IAPP prevent aggregation, and (iv) suggests possible regions responsible for the observed hIAPP cross-seeding with {beta}-amyloid. Furthermore, we performed structure-based inhibitor design to generate potential hIAPP aggregation inhibitors via a capping strategy. Four of the designed candidates delay hIAPP aggregation in vitro, providing a starting point for the development of T2D therapeutics and proof-of-concept that the capping strategy can be used on full-length cryo-EM fibril structures.

biochemistry↗

bioRxiv2020

The cryoEM structure of the fibril-forming low-complexity domain of hnRNPA2 reveals distinct differences from pathogenic amyloid and shows how mutation converts it to the pathogenic form

hnRNPA2 is one of a group of human ribonucleoproteins (RNPs) involved in RNA metabolism which form fibrils both under cellular stress and in mutated form in neurodegenerative conditions. Previous work established that the C-terminal low-complexity domain (LCD) of hnRNPA2 fibrillizes under stress, and that missense mutations in this domain are found in the disease multisystem proteinopathy (MSP) with symptoms indistinguishable from ALS and FTD. However, little is known at the atomic level about the hnRNPA2 LCD structure that is involved in those processes and how disease mutations cause structural change. Here we present the cryo-electron microscopy (cryoEM) structure of hnRNPA2 LCD fibril core and demonstrate its capability to form a reversible hydrogel in vitro containing amyloid-like fibrils. Whereas these fibrils, like pathogenic amyloid, are formed from protein chains stacked into {beta}-sheets by backbone hydrogen bonds, they display distinct structural differences: the chains are kinked, enabling non-covalent cross-linking of fibrils and disfavoring formation of pathogenic steric zippers. Both their reversibility and energetic calculations suggest these fibrils are less stable than pathogenic amyloid. Moreover, the crystal structure of the disease-mutation-containing segment of hnRNPA2 suggests that the replacement fundamentally alters the fibril structure to a more stable energetic state. These findings illuminate how molecular interactions promote protein fibril networks and how mutation can transform fibril structure from functional to pathogenic form.

molecular biology↗

bioRxiv2019

The α-synuclein hereditary mutation E46K unlocks a more stable, pathogenic fibril structure

Aggregation of -synuclein is a defining molecular feature of Parkinsons disease, Lewy Body Dementia, and Multiple Systems Atrophy. Hereditary mutations in -synuclein are linked to both Parkinsons disease and Lewy Body Dementia; in particular, patients bearing the E46K disease mutation manifest a clinical picture of parkinsonism and Lewy Body Dementia, and E46K creates more pathogenic fibrils in vitro. Understanding the effect of these hereditary mutations on -synuclein fibril structure is fundamental to -synuclein biology. We therefore determined the cryoEM structure of -synuclein fibrils containing the hereditary E46K mutation. The 2.5 [A] structure reveals a symmetric double protofilament in which the molecules adopt a vastly re-arranged, lower energy fold compared to wild-type fibrils. We propose that the E46K misfolding pathway avoids electrostatic repulsion between K46 and K80, a residue pair which forms the E46-K80 salt-bridge in the wild-type fibril structure. We hypothesize that under our conditions the wild-type fold does not reach this deeper energy well of the E46K fold because the E46-K80 salt bridge diverts -synuclein into a kinetic trap - a shallower, more accessible energy minimum. The E46K mutation apparently unlocks a more stable and pathogenic fibril structure. Significance StatementParkinsons is the second most prevalent neurodegenerative condition, leading to movement disorders, and dementia in some cases. Because of the strong association of this condition with amyloid aggregates of the protein -synuclein, structural understanding of these amyloid aggregates may be the path to eventual therapies. Our study of the structure of a variant -synuclein inherited in families afflicted with a clinical picture of parkinsonism and Lewy Body Dementia supplements recent structures of the wild type structure, and shows how a single residue change can result in a greatly changed structure that may underlie the inherited form of the disease.

biochemistry↗

bioRxiv2019

Cryo-EM structures of alpha-synuclein fibrils with the H50Q hereditary mutation reveal new polymorphs

Abstract\n\nDeposits of amyloid fibrils of -synuclein are the histological hallmarks of Parkinsons disease, multiple system atrophy, and dementia with Lewy bodies. Although most cases of these diseases are sporadic, autosomal-dominant hereditary mutations have been linked to Parkinsons disease and dementia with Lewy bodies. Seeing the changes to the structure of amyloid fibrils bearing these mutations may help to understand these diseases. To this end, we determined the cryo-EM structures of -synuclein fibrils containing the H50Q hereditary mutation. We find that the H50Q mutation results in two new polymorphs of -synuclein, which we term Narrow and Wide Fibrils. Both polymorphs recapitulate the conserved kernel formed by residues 50-77 observed in wild-type structures; however, the Narrow and Wide Fibrils reveal that H50Q disrupts a key interaction between H50-E57 on the opposing protofilament, abolishing the extensive protofilament interface formed by preNAC residues in the wild-type \"rod\" structure. Instead, the Narrow Fibril is formed from a single protofilament and the two protofilaments of the Wide protofilament are held together by only a pair of atoms - the C{gamma} atoms from the two threonine 59 sidechains. Further, we find that H50Q forms an intramolecular hydrogen bond with K45 leading to the formation of a novel {beta}-arch formed by residues 36-46 that features an extensive hydrogen-bond network between Y39, T44, and E46. The structures of the H50Q polymorphs help to rationalize the faster aggregation kinetics, higher seeding capacity in biosensor cells, and greater cytotoxicity we observe for H50Q compared to wild-type -synuclein.

biochemistry↗

bioRxiv2019

Cryo-EM Structures of Four Polymorphic TDP-43 Amyloid Cores

Summary ParagraphTDP-43 is an essential DNA/RNA processing protein that undergoes both functional and pathogenic aggregation. Functional TDP-43 aggregates are reversible, forming transient species such as nuclear bodies, stress granules, and myo-granules1-3. In contrast pathogenic TDP-43 aggregates are irreversible, forming stable intracellular amyloid-like inclusions4,5. These inclusions are the primary pathology of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP)6. Disease-associated, hereditary mutations in TDP-43 are known to accelerate the deposition of irreversible aggregates in the cytoplasm7. Reversible TDP-43 aggregation has been shown to precede the formation of irreversible amyloid fibrils similar to the behavior of proteins hnRNPA1 and FUS8-10. Still unknown, however, are the structural features of TDP-43 fibrils that confer both reversibility and irreversibility and how hereditary mutations can impose irreversible aggregation. Here, we determined the structures of amyloid fibrils formed by two segments previously reported to be the pathogenic cores of TDP-43 aggregation7,11,12; these are termed SegA (residues 311-360) and SegB A315E (residues 286-331 containing the ALS hereditary mutation A315E). SegA forms three polymorphs, all with dagger-shaped folds. SegB forms R-shaped folds. All four polymorphs have folds confined to two dimensions, and are stabilized by hydrophobic cores and peripheral hydrogen bonds. Energetic analysis suggests that the dagger-shaped polymorphs are examples of the irreversible fibril structures of TDP-43, whereas the SegB polymorph may participate in both reversible and irreversible fibril structure. Our structure suggests how the A315E mutation may convert this polymorph to the irreversible type and lead to mutation-enhanced pathology.

biochemistry↗