Biology subjects

Xue, W.-F.

Publications and source records attributed to Xue, W.-F..

14 recordsLinked to original sources

bioRxiv2026

Structural evolution of a yeast amyloid in vivo is shaped by chaperones

Cryo-electron microscopy (cryo-EM) studies of amyloid fibrils have revealed endpoint structures of disease-relevant filaments and polymorphic intermediates formed during in vitro assembly of prion-like proteins. However, how transmissible prion or prion-like amyloids evolve during de novo formation and maturation in living cells remains unknown. Here, using the yeast prion [PSI+] as a model, we isolated Sup35NM amyloid fibrils from successive stages of [PSI+] maturation in Saccharomyces cerevisiae and characterised their near-atomic structures and population-level structural diversity by combining cryo-EM and atomic force microscopy. We show that intermediate and mature states differ in predominant fibril structure and the regions of the Sup35 sequence incorporated into the core, and that structural diversity decreases during maturation. Curing of [PSI+] at the mature state by guanidine hydrochloride (GdnHCl), which selectively inhibits ATPase activity of the chaperone Hsp104, restored both the predominant intermediate amyloid structure and the broader structural diversity characteristic of the intermediate state. In addition, Hsp104, Ssa1 (Hsp70) and Sis1 (Hsp40) associate differently with fibrils from the two states. Together, these findings provide direct structural evidence for amyloid evolution in vivo and support a chaperone-mediated mechanism of conformer selection within a polymorphic amyloid population.

molecular biology↗

bioRxiv2025

Engineered yeast biofilms deliver plastic degrading enzymes to PET substrates

Enzymes that degrade PET and other plastics are being discovered with increasing pace, and engineering approaches further increase their activity. Most proposed applications for such enzymes require their purification, enabling the regeneration of monomeric subunits suitable for the production of new plastic. However, large amounts of plastics exist as environmental contaminants, and are unlikely to be suitable for standard recycling. Such waste may still be suitable for bioremediation using enzymatic activities, albeit without the economic incentives of a full recycling process. Here we develop a delivery system for PET hydrolases, based on engineered yeast biofilms that grow and secrete enzymes directly on the substrate to be degraded. The system does not require any enzyme purification steps, and the biofilms at least partially assimilate the degradation products into biomass. Our work provides proof-of-principle that demonstrates the potential of biofilm-based approaches.

bioengineering↗

bioRxiv2025

Three-dimensional single-particle reconstruction by atomic force microscopy allows rapid structural-based validation of recombinant SARS-CoV-2 Spike protein from a single topology image

Atomic force microscopy (AFM) is a versatile multi-modal imaging method frequently used for structural characterisation of biological surfaces at the nanoscale. However, AFM-based three-dimensional single-particle reconstruction has hitherto not been possible due to the tip-sample convolution artifact that distorts AFM images of individual molecules, and the disconnect between two-dimensional AFM images of surface deposited molecules and their three-dimensional structures. Here, three-dimensional single-particle analysis was developed for rapid structure-based validation of protein structures using as few as a single AFM topology image, based on contact-point reconstruction AFM (CPR-AFM) in an integrative approach with cryo-electron microscopy maps available in the Electron Microscopy Data Bank by template matching. This approach was demonstrated on the structural validation of recombinant trimeric ectodomain of SARS-CoV-2 Spike glycoprotein to show its immediate utility as a rapid structure-based sample quality control method in the recombinant expression and purification of the Spike protein samples that can be used in vaccines and therapeutics research. These results show that three-dimensional single-particle reconstruction by AFM is possible, that high signal-to-noise AFM imaging offers a rapid and cost-effective way of validation or identification of three-dimensional protein structures at single particle level, and that AFM can be linked to structural data derived from methods such as cryo-electron microscopy, resulting in integrative methodologies with new capabilities for structural biology.

biophysics↗

bioRxiv2025

The architecture of amyloid fibrils formed by a human tau-derived hexapeptide VQIVYK

The sequence 306VQIVYK311 is an aggregation prone region of the tau protein implicated in driving assembly of tau into paired helical filaments. These filaments accumulate as intraneuronal neurofibrillary tangles in Alzheimers disease and a range of tauopathies. Here, we demonstrate that VQIVYK forms highly ordered fibrillar samples after prolonged incubation at room temperature. Remarkably, aligned fibre bundles give rise to unusually detailed and highly oriented X-ray fibre diffraction patterns. Analysis of these patterns provide a model of the core protofilament structure that satisfies the experimental diffraction data. This structural model is consistent with data from X-ray crystallography of microcrystals and conforms to the cross-beta architecture that defines amyloid, but diffraction data analysis shows a highly twisted filamentous protofilament architecture. Analysis of individual fibril envelopes by 3D contact point reconstruction atomic force microscopy reveals a diverse polymorphous population with a major fibril morphology of apparent cylindrical fibrils, and morphological subpopulations of fibrils with clear left-hand twisting patterns, despite the commonality of the core structure indicated by the detailed diffraction pattern. Together, these data suggest that VQIVYK amyloid fibrils form a polymorphous amyloid population by assembly of highly ordered protofilaments and provides molecular information regarding amyloid fibril twist.

biophysics↗

bioRxiv2025

Inhibiting disulphide bonding in truncated tau297-391 results in enhanced self-assembly of tau into seed-competent assemblies.

Tau undergoes fibrillogenesis in a group of neurodegenerative diseases termed tauopathies. Each tauopathy is characterized by tau fibrils with disease-specific conformations, highlighting the complexity of tau self-assembly. This has led to debate surrounding the precise mechanisms that govern the self-assembly of tau in disease, especially the involvement of disulphide bonding (DSB) between cysteine residues. In this study, we use a truncated form of tau, dGAE, capable of forming filaments identical to those in disease. We reveal the impact of DSB in dGAE assembly and propagation by resolving the global mechanisms that dominate its assembly. We found evidence for surface-mediated secondary nucleation and fragmentation being active in dGAE assembly. The inhibition of DSB during dGAE assembly leads to an enhanced aggregation rate through a reduced lag phase, but with no effect on the global assembly mechanisms. We suggest this is due to the formation a dominant, seed-competent species in the absence of DSB that facilitates elongation and secondary nucleation resulting in enhanced assembly. In vitro seeding assays reveal the recruitment of endogenous tau in a cell model only when using dGAE species formed under conditions that inhibit DSB. Our results further support the use of the in vitro dGAE tau aggregation model for investigating the mechanism of tau assembly, the effect of varying conditions on tau assembly and how these conditions affect the resultant species. Further studies may utilise dGAE and its aggregates to investigate tau seeding, propagation and to highlight or test potential targets for therapies that reduce the spread of pathologic tau throughout the brain.

biochemistry↗

bioRxiv2023

Structural reconstruction of individual filaments in Abeta42 fibril populations assembled in vitro reveal rare species that resemble ex vivo amyloid polymorphs from human brains

Structural polymorphism has been demonstrated for both in vitro and ex vivo amyloid fibrils associated with disease. The manner in which different filament structures are assembled from common building blocks remains unclear but the assembly environment is likely to be a key determinant. To address this, three-dimensional reconstruction of individual filament structures was conducted from atomic force microscopy images to map the structural polymorphism landscape of A{beta}42 amyloid fibril populations formed in vitro under most frequently used buffer conditions. The data show sensitivity of A{beta}42 fibril polymorphism to the assembly environment in both the magnitude of heterogeneity and the types of filament species formed. However, some conserved fibril polymorphs were observed across the experimental conditions. Excitingly, by matching individual filament structures to cryo-electron microscopy derived structural data, rare species in these heterogeneous population clouds that show remarkable similarity to A{beta}42 amyloid polymorphs purified from human patient brains were discovered. These results link in vitro experimental approaches with structures formed in vivo, and highlight the polymorph distribution, and the type and magnitude of structural variations within these heterogeneous molecular distributions as important factors in amyloid biology.

biophysics↗

bioRxiv2023

Trace_y: Software algorithms for structural analysis of individual helical filaments by three-dimensional contact point reconstruction atomic force microscopy

Atomic force microscopy (AFM) is a powerful and increasingly accessible technology that has a wide range of imaging applications. AFM is capable of producing detailed three-dimensional topographical height images with a high signal-to-noise ratio. This is a key capability of AFM, which enables the structural features of individual molecules to be studied without the need for ensemble averaging, and could offer structural analysis applications where heterogeneity of molecular populations, structural variations between individual molecules, or population distribution properties in general, hold important information. Here, a software tool called Trace_y designed to reconstruct the three-dimensional (3D) surface envelopes of individual helical filament structures from topographical AFM height images is presented. Workflow using Trace_y is demonstrated on the structural analysis of individual helical amyloid protein fibrils where the assembly mechanism of heterogeneous, complex and diverse fibril populations from a single amino-acid sequence due to structural polymorphism is not fully understood. The software and the workflow presented here allows the structural information encoded in topographical AFM height images to be extracted and understood as 3D contact point clouds. This approach will facilitate the use of AFM in structural biology, in integrated methodologies together with other structural analysis tools to understand the structures and behaviours at individual molecule level.

biophysics↗

bioRxiv2021

Structural identification of individual helical amyloid filaments by integration of cryo-electron microscopy-derived maps in comparative morphometric atomic force microscopy image analysis

The presence of amyloid fibrils is a hallmark of more than 50 human disorders, including neurodegenerative diseases and systemic amyloidoses. A key unresolved challenge in understanding the involvement of amyloid in disease is to explain the relationship between individual structural polymorphs of amyloid fibrils, in potentially mixed populations, and the specific pathologies with which they are associated. Although cryo-electron microscopy (cryo-EM) and solid-state nuclear magnetic resonance (ssNMR) spectroscopy methods have been successfully employed in recent years to determine the structures of amyloid fibrils with high resolution detail, they rely on ensemble averaging of fibril structures in the entire sample or significant subpopulations. Here, we report a method for structural identification of individual fibril structures imaged by atomic force microscopy (AFM) by integration of high-resolution maps of amyloid fibrils determined by cryo-EM in comparative AFM image analysis. This approach was demonstrated using the hitherto structurally unresolved amyloid fibrils formed in vitro from a fragment of tau (297-391), termed dGAE. Our approach established unequivocally that dGAE amyloid fibrils bear no structural relationship to heparin-induced tau fibrils formed in vitro. Furthermore, our comparative analysis resulted in the prediction that dGAE fibrils are closely related structurally to the paired helical filaments (PHFs) isolated from Alzheimers disease (AD) brain tissue characterised by cryo-EM. These results show the utility of individual particle structural analysis using AFM, provide a workflow of how cryo-EM data can be incorporated into AFM image analysis and facilitate an integrated structural analysis of amyloid polymorphism.

biophysics↗

bioRxiv2021

Heterotypic Aβ interactions facilitate amyloid assembly and modify amyloid structure

It is still unclear why pathological amyloid deposition initiates in specific brain regions, nor why specific cells or tissues are more susceptible than others. Amyloid deposition is determined by the self-assembly of short protein segments called aggregation-prone regions (APRs) that favour cross-{beta} structure. Here we investigated whether A{beta} amyloid assembly can be modified by heterotypic interactions between A{beta} APRs and short homologous segments in otherwise unrelated human proteins. We identified heterotypic interactions that accelerate A{beta} assembly, modify fibril morphology and affect its pattern of deposition in vitro. Moreover, we found that co-expression of these proteins in an A{beta} reporter cell line promotes A{beta} amyloid aggregation. Importantly, reanalysis of proteomics data of A{beta} plaques from AD patients revealed an enrichment in proteins that share homologous sequences to the A{beta} APRs, suggesting heterotypic amyloid interactions may occur in patients. Strikingly, we did not find such a bias in plaques from overexpression models in mouse. Based on these data, we propose that heterotypic APR interactions may play a hitherto unrealised role in amyloid-deposition diseases.

neuroscience↗

bioRxiv2020

Amyloid particles facilitate surface-catalyzed cross-seeding by acting as promiscuous nanoparticles

Amyloid seeds are nanometre-sized protein particles that accelerate amyloid assembly, as well as propagate and transmit the amyloid protein conformation associated with a wide range of protein misfolding diseases. However, seeded amyloid growth through templated elongation at fibril ends cannot explain the full range of molecular behaviours observed during cross-seeded formation of amyloid by heterologous seeds. Here, we demonstrate that amyloid seeds can accelerate amyloid formation via a surface catalysis mechanism without propagating the specific amyloid conformation associated with the seeds. This type of seeding mechanism is demonstrated through quantitative characterisation of the cross-seeded assembly reactions involving two non-homologous and unrelated proteins: the human A{beta}42 peptide and the yeast prion-forming protein Sup35NM. Our results suggest experimental approaches to differentiate seeding by templated elongation from non-templated amyloid seeding, and rationalise the molecular mechanism of the cross-seeding phenomenon as a manifestation of the aberrant surface activities presented by amyloid seeds as nanoparticles.

biochemistry↗

bioRxiv2020

Three-dimensional reconstruction of individual helical nano-filament structures from atomic force microscopy topographs

Atomic force microscopy, AFM, is a powerful tool that can produce detailed topographical images of individual nano-structures with a high signal-to-noise ratio without the need for ensemble averaging. However, the application of AFM in structural biology has been hampered by the tip-sample convolution effect, which distorts images of nano-structures, particularly those that are of similar dimensions to the cantilever probe tips used in AFM. Here we show that the tip-sample convolution results in a feature-dependent and non-uniform distribution of image resolution on AFM topographs. We show how this effect can be utilised in structural studies of nano-sized upward convex objects such as spherical or filamentous molecular assemblies deposited on a flat surface, because it causes magnification of such objects in AFM topographs. Subsequently, this enhancement effect is harnessed through contact-point based deconvolution of AFM topographs. Here, the application of this approach is demonstrated through the 3D reconstruction of the surface envelope of individual helical amyloid filaments without the need of cross-particle averaging using the contact-deconvoluted AFM topographs. Resolving the structural variations of individual macromolecular assemblies within inherently heterogeneous populations is paramount for mechanistic understanding of many biological phenomena such as amyloid toxicity and prion strains. The approach presented here will also facilitate the use of AFM for high-resolution structural studies and integrative structural biology analysis of single molecular assemblies.

biophysics↗

bioRxiv2020

Quantification of amyloid fibril polymorphism by nano-morphometry reveals the individuality of filament assembly

Amyloid fibrils are highly polymorphic structures formed by many different proteins. They provide biological function but also abnormally accumulate in numerous human diseases. The physicochemical principles of amyloid polymorphism are not understood due to lack of structural insights at the single-fibril level. To identify and classify different fibril polymorphs and to quantify the level of heterogeneity is essential to decipher the precise links between amyloid structures and their functional and disease associated properties such as toxicity, strains, propagation and spreading. Employing gentle, force-curve based AFM, we produce detailed images, from which the 3D reconstruction of individual filaments in heterogeneous amyloid samples is achieved. Distinctive fibril polymorphs are then classified by hierarchical clustering, and sample heterogeneity is objectively quantified. These data demonstrate the polymorphic nature of fibril populations, provide important information regarding the energy landscape of amyloid self-assembly, and offer quantitative insights into the structural basis of polymorphism in amyloid populations.

biophysics↗

bioRxiv2018

The Division of Amyloid Fibrils

The division of amyloid protein fibrils is required for the propagation of the amyloid state, and is an important contributor to their stability, pathogenicity and normal function. Here, we combine kinetic nano-scale imaging experiments with analysis of a mathematical model to resolve and compare the division stability of amyloid fibrils. Our theoretical results show that the division of any type of filament is uniquely described by a set of three characteristic properties, resulting in convergence to self-similar length distributions distinct to each fibril type and conditions applied. By applying these results to profile the dynamical stability towards breakage for four different amyloid types, we reveal particular differences in the division properties of disease-related amyloid formed from alpha-synuclein compared with non-disease associated model amyloid, the former showing lowered intrinsic stability towards breakage and increased likelihood of shedding smaller particles. Our results enable the comparison of protein filaments intrinsic dynamic stabilities, which are key to unravelling their toxic and infectious potentials.

biophysics↗

bioRxiv2017

A Cell Culture Platform For Cryptosporidium That Enables Long-Term Cultivation And New Tools For The Systematic Investigation Of Its Biology

Cryptosporidium parasites are a major cause of diarrhoea that pose a particular threat to children in developing areas and immunocompromised individuals. Curative therapies and vaccines are lacking. Currently, Cryptosporidium oocysts for research must be freshly produced in animals and cannot be long-term stored. Here, we show that COLO-680N cells infected with two different Cryptosporidium parvum strains (Moredun, Iowa) produce sufficient infectious oocysts to infect subsequent cultures. Oocyst identity was confirmed by specific staining (Crypt-a-glo, Vicia Villosa lectin, Sporo-glo), PCR-based amplification of Cryptosporidium-specific genes, lipidomics fingerprinting, and atomic force microscopy (AFM). Antibody-stained oocysts produced unstained oocysts confirming production of novel oocysts. Infected cultures could be cryoconserved and continued to produce infectious oocysts after resuscitation. Transmission electron microscopy identified all key Cryptosporidium life cycle stages. Infected cultures produced thick-walled (primarily involved in Cryptosporidium transmission between organisms) and thin-walled oocysts (important for Cryptosporidium propagation within a host/tissue) as indicated by DAPI staining (only thin-walled oocysts are permeable to DAPI staining, thus allowing visualisation of sporozoites) and AFM. In conclusion, we present a novel, easy-to-handle cell culture system that enables the propagation, cryopreservation and detailed investigation of Cryptosporidium at a laboratory scale. Its availability will accelerate research on Cryptosporidium and the development of anti-Cryptosporidium drugs.

microbiology↗