In Situ Architecture and Cellular Interactions of PolyQ Inclusions.

Expression of many disease-related aggregation-prone proteins results in cytotoxicity and the formation of large intracellular inclusion bodies. To gain insight into the role of inclusions in pathology and the in situ structure of protein aggregates inside cells, we employ advanced cryo-electron tomography methods to analyze the structure of inclusions formed by polyglutamine (polyQ)-expanded huntingtin exon 1 within their intact cellular context. In primary mouse neurons and immortalized human cells, polyQ inclusions consist of amyloid-like fibrils that interact with cellular endomembranes, particularly of the endoplasmic reticulum (ER). Interactions with these fibrils lead to membrane deformation, the local impairment of ER organization, and profound alterations in ER membrane dynamics at the inclusion periphery. These results suggest that aberrant interactions between fibrils and endomembranes contribute to the deleterious cellular effects of protein aggregation. VIDEO ABSTRACT.

Autophagy preferentially degrades non-fibrillar polyQ aggregates.

Aggregation of proteins containing expanded polyglutamine (polyQ) repeats is the cytopathologic hallmark of a group of dominantly inherited neurodegenerative diseases, including Huntington's disease (HD). Huntingtin (Htt), the disease protein of HD, forms amyloid-like fibrils by liquid-to-solid phase transition. Macroautophagy has been proposed to clear polyQ aggregates, but the efficiency of aggrephagy is limited. Here, we used cryo-electron tomography to visualize the interactions of autophagosomes with polyQ aggregates in cultured cells in situ. We found that an amorphous aggregate phase exists next to the radially organized polyQ fibrils. Autophagosomes preferentially engulfed this amorphous material, mediated by interactions between the autophagy receptor p62/SQSTM1 and the non-fibrillar aggregate surface. In contrast, amyloid fibrils excluded p62 and evaded clearance, resulting in trapping of autophagic structures. These results suggest that the limited efficiency of autophagy in clearing polyQ aggregates is due to the inability of autophagosomes to interact productively with the non-deformable, fibrillar disease aggregates.

Binding adaptability of chemical ligands to polymorphic α-synuclein amyloid fibrils.

α-synuclein (α-syn) assembles into structurally distinct fibril polymorphs seen in different synucleinopathies, such as Parkinson's disease and multiple system atrophy. Targeting these unique fibril structures using chemical ligands holds diagnostic significance for different disease subtypes. However, the molecular mechanisms governing small molecules interacting with different fibril polymorphs remain unclear. Here, we investigated the interactions of small molecules belonging to four distinct scaffolds, with different α-syn fibril polymorphs. Using cryo-electron microscopy, we determined the structures of these molecules when bound to the fibrils formed by E46K mutant α-syn and compared them to those bound with wild-type α-syn fibrils. Notably, we observed that these ligands exhibit remarkable binding adaptability, as they engage distinct binding sites across different fibril polymorphs. While the molecular scaffold primarily steered the binding locations and geometries on specific sites, the conjugated functional groups further refined this adaptable binding by fine-tuning the geometries and binding sites. Overall, our finding elucidates the adaptability of small molecules binding to different fibril structures, which sheds light on the diagnostic tracer and drug developments tailored to specific pathological fibril polymorphs.

Structure of biofilm-forming functional amyloid PSMα1 from Staphylococcus aureus.

Biofilm-protected pathogenic Staphylococcus aureus causes chronic infections that are difficult to treat. An essential building block of these biofilms are functional amyloid fibrils that assemble from phenol-soluble modulins (PSMs). PSMα1 cross-seeds other PSMs into cross-ß amyloid folds and is therefore a key element in initiating biofilm formation. However, the paucity of high-resolution structures hinders efforts to prevent amyloid assembly and biofilm formation. Here, we present a 3.5 Å resolution density map of the major PSMα1 fibril form revealing a left-handed cross-ß fibril composed of two C2-symmetric U-shaped protofilaments whose subunits are unusually tilted out-of-plane. Monomeric α-helical PSMα1 is extremely cytotoxic to cells, despite the moderate toxicity of the cross-ß fibril. We suggest mechanistic insights into the PSM functional amyloid formation and conformation transformation on the path from monomer-to-fibril formation. Details of PSMα1 assembly and fibril polymorphism suggest how S. aureus utilizes functional amyloids to form biofilms and establish a framework for developing therapeutics against infection and antimicrobial resistance.

Atomic resolution structure of full-length human insulin fibrils.

Patients with type 1 diabetes mellitus who are dependent on an external supply of insulin develop insulin-derived amyloidosis at the sites of insulin injection. A major component of these plaques is identified as full-length insulin consisting of the two chains A and B. While there have been several reports that characterize insulin misfolding and the biophysical properties of the fibrils, atomic-level information on the insulin fibril architecture remains elusive. We present here an atomic resolution structure of a monomorphic insulin amyloid fibril that has been determined using magic angle spinning solid-state NMR spectroscopy. The structure of the insulin monomer yields a U-shaped fold in which the two chains A and B are arranged in parallel to each other and are oriented perpendicular to the fibril axis. Each chain contains two ß-strands. We identify two hydrophobic clusters that together with the three preserved disulfide bridges define the amyloid core structure. The surface of the monomeric amyloid unit cell is hydrophobic implicating a potential dimerization and oligomerization interface for the assembly of several protofilaments in the mature fibril. The structure provides a starting point for the development of drugs that bind to the fibril surface and disrupt secondary nucleation as well as for other therapeutic approaches to attenuate insulin aggregation.

Is a cure for Parkinson's disease hiding inside us?

α-Synuclein (a-syn) oligomers and fibrils are behind neurodegeneration in Parkinson's disease (PD), but therapeutically targeting them is challenging. Amphipathic and cationic helical peptides inhibit amyloid formation and suppress neurotoxicity by selectively binding the solvent-accessible regions in these toxic species. Can endogenous peptides, like LL-37, constitute a new therapeutic paradigm in PD?

Quantitative NMR analysis of the mechanism and kinetics of chaperone Hsp104 action on amyloid-ß42 aggregation and fibril formation.

The chaperone Hsp104, a member of the Hsp100/Clp family of translocases, prevents fibril formation of a variety of amyloidogenic peptides in a paradoxically substoichiometric manner. To understand the mechanism whereby Hsp104 inhibits fibril formation, we probed the interaction of Hsp104 with the Alzheimer's amyloid-ß42 (Aß42) peptide using a variety of biophysical techniques. Hsp104 is highly effective at suppressing the formation of Thioflavin T (ThT) reactive mature fibrils that are readily observed by atomic force (AFM) and electron (EM) microscopies. Quantitative kinetic analysis and global fitting was performed on serially recorded 1H-15N correlation spectra to monitor the disappearance of Aß42 monomers during the course of aggregation over a wide range of Hsp104 concentrations. Under the conditions employed (50 µM Aß42 at 20 °C), Aß42 aggregation occurs by a branching mechanism: an irreversible on-pathway leading to mature fibrils that entails primary and secondary nucleation and saturating elongation; and a reversible off-pathway to form nonfibrillar oligomers, unreactive to ThT and too large to be observed directly by NMR, but too small to be visualized by AFM or EM. Hsp104 binds reversibly with nanomolar affinity to sparsely populated Aß42 nuclei present in nanomolar concentrations, generated by primary and secondary nucleation, thereby completely inhibiting on-pathway fibril formation at substoichiometric ratios of Hsp104 to Aß42 monomers. Tight binding to sparsely populated nuclei likely constitutes a general mechanism for substoichiometric inhibition of fibrillization by a variety of chaperones. Hsp104 also impacts off-pathway oligomerization but to a much smaller degree initially reducing and then increasing the rate of off-pathway oligomerization.

Cryo-EM structure of amyloid fibril formed by α-synuclein hereditary A53E mutation reveals a distinct protofilament interface.

Synucleinopathies like Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple systems atrophy (MSA), have the same pathologic feature of misfolded α-synuclein protein (α-syn) accumulation in the brain. PD patients who carry α-syn hereditary mutations tend to have earlier onset and more severe clinical symptoms than sporadic PD patients. Therefore, revealing the effect of hereditary mutations to the α-syn fibril structure can help us understand these synucleinopathies' structural basis. Here, we present a 3.38 Å cryo-electron microscopy structure of α-synuclein fibrils containing the hereditary A53E mutation. The A53E fibril is symmetrically composed of two protofilaments, similar to other fibril structures of WT and mutant α-synuclein. The new structure is distinct from all other synuclein fibrils, not only at the interface between proto-filaments, but also between residues packed within the same proto-filament. A53E has the smallest interface with the least buried surface area among all α-syn fibrils, consisting of only two contacting residues. Within the same protofilament, A53E reveals distinct residue re-arrangement and structural variation at a cavity near its fibril core. Moreover, the A53E fibrils exhibit slower fibril formation and lower stability compared to WT and other mutants like A53T and H50Q, while also demonstrate strong cellular seeding in α-synuclein biosensor cells and primary neurons. In summary, our study aims to highlight structural differences - both within and between the protofilaments of A53E fibrils - and interpret fibril formation and cellular seeding of α-synuclein pathology in disease, which could further our understanding of the structure-activity relationship of α-synuclein mutants.

Dissimilar effects of the hydrophilic carbon dots on the amyloid aggregation of two model proteins and the mechanism discussion.

Many proteins could aggregate into amyloid fibrils under certain conditions. However, the aggregation process and morphology of the fibrils may be significantly different because of the distinct protein structure. In this article, the hydrophilic carbon dots (Lys-CA-CDs) were prepared using lysine (Lys) and citric acid (CA) as reactant under the assistance of a microwave. The dissimilar modulation effect of Lys-CA-CDs on the aggregation process of distinct structure protein was further investigated, where bovine serum albumin (BSA) and hen egg white lysozyme (HEWL) were chosen as model proteins. All results showed that Lys-CA-CDs displayed the contrary influence on the aggregation process of BSA and HEWL. Lys-CA-CDs could induce BSA to aggregate into more wormlike fibrils and inhibit the aggregation of HEWL into hair-like fibrils. The influence on the aggregation process of BSA may be assigned to the increased concentration of BSA around the Lys-CA-CDs caused by their interaction. However, inserting of Lys-CA-CDs into the inner structure of HEWL led to the change of protein secondary structure. The change of secondary structure further made it difficult for HEWL to aggregate into fibrils and Lys-CA-CDs showed the inhibition effect on HEWL aggregation.

Exploring the complexity of amyloid-beta fibrils: structural polymorphisms and molecular interactions.

The aggregation of amyloid-beta (Aß) peptides into cross-ß structures forms a variety of distinct fibril conformations, potentially correlating with variations in neurodegenerative disease progression. Recent advances in techniques such as X-ray crystallography, solid-state NMR, and cryo-electron microscopy have enabled the development of high-resolution molecular structures of these polymorphic amyloid fibrils, which are either grown in vitro or isolated from human and transgenic mouse brain tissues. This article reviews our current understanding of the structural polymorphisms in amyloid fibrils formed by Aß40 and Aß42, as well as disease-associated mutants of Aß peptides. The aim is to enhance our understanding of various molecular interactions, including hydrophobic and ionic interactions, within and among cross-ß structures.

Inhibitory effects of extracts from Eucalyptus gunnii on α-synuclein amyloid fibrils.

Amyloid fibril formation is associated with various amyloidoses, including neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Despite the numerous studies on the inhibition of amyloid formation, the prevention and treatment of a majority of amyloid-related disorders are still challenging. In this study, we investigated the effects of various plant extracts on amyloid formation of α-synuclein. We found that the extracts from Eucalyptus gunnii are able to inhibit amyloid formation, and to disaggregate preformed fibrils, in vitro. The extract itself did not lead cell damage. In the extract, miquelianin, which is a glycosylated form of quercetin and has been detected in the plasma and the brain, was identified and assessed to have a moderate inhibitory activity, compared to the effects of ellagic acid and quercetin, which are strong inhibitors for amyloid formation. The properties of miquelianin provide insights into the mechanisms controlling the assembly of α-synuclein in the brain.

The structure of a minimum amyloid fibril core formed by necroptosis-mediating RHIM of human RIPK3.

Receptor-interacting protein kinases 3 (RIPK3), a central node in necroptosis, polymerizes in response to the upstream signals and then activates its downstream mediator to induce cell death. The active polymeric form of RIPK3 has been indicated as the form of amyloid fibrils assembled via its RIP homotypic interaction motif (RHIM). In this study, we combine cryogenic electron microscopy and solid-state NMR to determine the amyloid fibril structure of RIPK3 RHIM-containing C-terminal domain (CTD). The structure reveals a single protofilament composed of the RHIM domain. RHIM forms three ß-strands (referred to as strands 1 through 3) folding into an S shape, a distinct fold from that in complex with RIPK1. The consensus tetrapeptide VQVG of RHIM forms strand 2, which zips up strands 1 and 3 via heterozipper-like interfaces. Notably, the RIPK3-CTD fibril, as a physiological fibril, exhibits distinctive assembly compared with pathological fibrils. It has an exceptionally small fibril core and twists in both handedness with the smallest pitch known so far. These traits may contribute to a favorable spatial arrangement of RIPK3 kinase domain for efficient phosphorylation.

Invited review: Modeling milk stability.

Novel insights into the stability of milk and milk products during storage and processing result from describing caseins near neutral pH as hydrophilic, intrinsically disordered, proteins. Casein solubility is strongly influenced by pH and multivalent ion binding. Solubility is high at a neutral pH or above, but decreases as the casein net charge approaches zero, allowing a condensed casein phase or gel to form, then increases at lower pH. Of particular importance for casein micelle stability near neutral pH is the proportion of free caseins in the micelle (i.e., caseins not bound directly to nanoclusters of calcium phosphate). Free caseins are more soluble and better able to act as molecular chaperones (to prevent casein and whey protein aggregation) than bound caseins. Some free caseins are highly phosphorylated and can also act as mineral chaperones to inhibit the growth of calcium phosphate phases and prevent mineralized deposits from forming on membranes or heat exchangers. Thus, casein micelle stability is reduced when free caseins bind to amyloid fibrils, destabilized whey proteins or calcium phosphate. The multivalent-binding model of the casein micelle quantitatively describes these and other factors affecting the stability of milk and milk protein products during manufacture and storage.

Suppression of amyloid-ß fibril growth by drug-engineered polymorph transformation.

Fibrillization of the protein amyloid ß is assumed to trigger Alzheimer's pathology. Approaches that target amyloid plaques, however, have garnered limited clinical success, and their failures may relate to the scarce understanding of the impact of potential drugs on the intertwined stages of fibrillization. Here, we demonstrate that bexarotene, a T-cell lymphoma medication with known antiamyloid activity both in vitro and in vivo, suppresses amyloid fibrillization by promoting an alternative fibril structure. We employ time-resolved in situ atomic force microscopy to quantify the kinetics of growth of individual fibrils and supplement it with structure characterization by cryo-EM. We show that fibrils with structure engineered by the drug nucleate and grow substantially slower than "normal" fibrils; remarkably, growth remains stunted even in drug-free solutions. We find that the suppression of fibril growth by bexarotene is not because of the drug binding to the fibril tips or to the peptides in the solution. Kinetic analyses attribute the slow growth of drug-enforced fibril polymorph to the distinctive dynamics of peptide chain association to their tips. As an additional benefit, the bexarotene fibrils kill primary rat hippocampal neurons less efficiently than normal fibrils. In conclusion, the suggested drug-driven polymorph transformation presents a mode of action to irreversibly suppress toxic aggregates not only in Alzheimer's but also potentially in myriad diverse pathologies that originate with protein condensation.

Agitation does not induce fibrillation in reduced hen egg-white lysozyme at physiological temperature and pH.

Several proteins and peptides tend to form an amyloid fibril, causing a range of unrelated diseases, from neurodegenerative to certain types of cancer. In the native state, these proteins are folded and soluble. However, these proteins acquired ß-sheet amyloid fibril due to unfolding and aggregation. The conversion mechanism from well-folded soluble into amorphous or amyloid fibril is not well understood yet. Here, we induced unfolding and aggregation of hen egg-white lysozyme (HEWL) by reducing agent dithiothreitol and applied mechanical sheering force by constant shaking (1000 rpm) on the thermostat for 7 days. Our turbidity results showed that reduced HEWL rapidly formed aggregates, and a plateau was attained in nearly 5 h of incubation in both shaking and non-shaking conditions. The turbidity was lower in the shaking condition than in the non-shaking condition. The thioflavin T binding and transmission electron micrographs showed that reduced HEWL formed amorphous aggregates in both conditions. Far-UV circular dichroism results showed that reduced HEWL lost nearly all alpha-helical structure, and ß-sheet secondary structure was not formed in both conditions. All the spectroscopic and microscopic results showed that reduced HEWL formed amorphous aggregates under both conditions.

Chiral and Structural Polymorphism of Fibril Architectures of Homologous Lysozymes.

Amyloid fibrils are fascinating and complex structures with the multilayered chiral organization. Using the multimodal methodology, including VCD, ECD, cryo-EM, and TEM, we characterized in detail different levels of organization (secondary structure/protofilament/mesoscopic structure) of amyloid fibrils prepared from proteins highly homologous in the structure (hen egg white and human lysozymes). Our results demonstrate that small changes in the native protein structure or preparation conditions translate into significant differences in the handedness and architecture of the formed fibrils at various levels of their complexity. In particular, fibrils of hen egg white and human lysozymes obtained in vitro at the same preparation conditions, possess different secondary structure, protofilament twist and ultrastructure. Yet, formed fibrils adopted a relatively similar mesoscopic structure, as observed in high-resolution 3D cryo-EM, scarcely used up to now for fibrils obtained in vitro in denaturing condition. Our results add to other puzzling experiments implicating the indeterministic nature of fibril formation.

Influences of amino-terminal modifications on amyloid fibril formation of human serum amyloid A.

Human serum amyloid A (SAA) is a precursor protein involved in AA amyloidosis. The N-terminal region of the SAA molecule is crucial for amyloid fibril formation, and therefore modifications in this region are considered to influence the pathogenesis of AA amyloidosis. In the present study, using the N-terminal peptide corresponding to the putative first helix region of the SAA molecule, we investigated the influences of N-terminal modifications on amyloid fibril formation. Spectroscopic analyses revealed that carbamoylation of the N-terminal amino group delayed the onset of amyloid fibril formation. From transmission electron microscopic observations, the N-terminal carbamoylated aggregate showed remarkably different morphologies from the unmodified control. In contrast, acetylation of the N-terminal amino group or truncation of N-terminal amino acid(s) considerably diminished amyloidogenic properties. Furthermore, we also tested the cell toxicity of each peptide aggregate on cultured cells by two cytotoxic assays. Irrespective of carbamoylation or acetylation, MTT assay revealed that SAA peptides reduced the reductive activity of MTT on cells, whereas no apparent increase in LDH release was observed during an LDH assay. In contrast, N-terminal truncation did not affect either MTT reduction or LDH release. These results suggest that N-terminal modification of SAA molecules can act as a switch to regulate susceptibility to AA amyloidosis.

Nanomechanical properties of SSTSAA microcrystals are dominated by the inter-sheet packing.

Amyloid fibrils have been associated with human disease for many decades, but it has also become apparent that they play a functional, non-disease-related role in e.g. bacteria and mammals. Moreover, they have been shown to possess interesting mechanical properties that can be harnessed for future man-made applications. Here, the mechanical behaviour of SSTSAA microcrystals has been investigated. The SSTSAA peptide organization in these microcrystals has been related to that in the corresponding amyloid fibrils. Using high-pressure X-ray diffraction experiments, the bulk modulus K, which is the reciprocal of the compressibility ß, has been calculated to be 2.48 GPa. This indicates that the fibrils are tightly packed, although the packing of most native globular proteins is even better. It is shown that the value of the bulk modulus is mainly determined by the compression along the c-axis, that relates to the inter-sheet distance in the fibrils. These findings corroborate earlier data obtained by AFM and molecular dynamics simulations that showed that mechanical resistance varies according to the direction of the applied strain, which can be related to packing and hydrogen bond contributions. Pressure experiments provide complementary information to these techniques and help to acquire a full mechanical characterization of biomolecular assemblies. This article is part of the theme issue 'Exploring the length scales, timescales and chemistry of challenging materials (Part 2)'.

Sequence-independent recognition of the amyloid structural motif by GFP protein family.

Cnidarian fluorescent protein (FP) derivatives such as GFP, mCherry, and mEOS2 have been widely used to monitor gene expression and protein localization through biological imaging because they are considered functionally inert. We demonstrate that FPs specifically bind amyloid fibrils formed from many natural peptides and proteins. FPs do not bind other nonamyloid fibrillar structures such as microtubules or actin filaments and do not bind to amorphous aggregates. FPs can also bind small aggregates formed during the lag phase and early elongation phase of fibril formation and can inhibit amyloid fibril formation in a dose-dependent manner. These findings suggest caution should be taken in interpreting FP-fusion protein localization data when amyloid structures may be present. Given the pathological significance of amyloid-related species in some diseases, detection and inhibition of amyloid fibril formation using FPs can provide insights on developing diagnostic tools.

Parkinson's disease-related phosphorylation at Tyr39 rearranges α-synuclein amyloid fibril structure revealed by cryo-EM.

Posttranslational modifications (PTMs) of α-synuclein (α-syn), e.g., phosphorylation, play an important role in modulating α-syn pathology in Parkinson's disease (PD) and α-synucleinopathies. Accumulation of phosphorylated α-syn fibrils in Lewy bodies and Lewy neurites is the histological hallmark of these diseases. However, it is unclear how phosphorylation relates to α-syn pathology. Here, by combining chemical synthesis and bacterial expression, we obtained homogeneous α-syn fibrils with site-specific phosphorylation at Y39, which exhibits enhanced neuronal pathology in rat primary cortical neurons. We determined the cryo-electron microscopy (cryo-EM) structure of the pY39 α-syn fibril, which reveals a fold of α-syn with pY39 in the center of the fibril core forming an electrostatic interaction network with eight charged residues in the N-terminal region of α-syn. This structure composed of residues 1 to 100 represents the largest α-syn fibril core determined so far. This work provides structural understanding on the pathology of the pY39 α-syn fibril and highlights the importance of PTMs in defining the polymorphism and pathology of amyloid fibrils in neurodegenerative diseases.