Biology subjects

Konstantoulea, K.

Publications and source records attributed to Konstantoulea, K..

8 recordsLinked to original sources

bioRxiv2026

An N-terminal ATTR Fibril Segment Promotes Transthyretin Amyloid Nucleation and Polymorphism

ATTR amyloidosis is caused by transthyretin (TTR) amyloid deposition, yet the sequence-encoded events linking TTR misfolding to fibril nucleation and structural polymorphism remain incompletely defined. Here, we exploit the modular organization of patient-derived TTR fibrils to investigate two components of the pathological core: an N-terminal beta-hairpin spanning residues 11-35 (N-TTR) and a larger C-terminal fragment spanning residues 57-123 (C-TTR). Both fragments independently form beta-rich amyloid fibrils, as demonstrated by electron microscopy, circular dichroism, and fluorescence spectroscopy. Yet, their activities differ markedly. N-TTR fibrils promote full-length TTR aggregation and seed in an engineered cellular biosensor platform established to detect templated TTR assembly, whereas C-TTR aggregates show no detectable templating activity. Cryo-electron microscopy reveals two N-TTR polymorphs that preserve structural features of disease-derived folds, while energetic profiling identifies N-TTR as a stabilizing hotspot within ex vivo structures, providing a basis for this templating functionality. These findings reveal a functional hierarchy among amyloidogenic segments of TTR, since distinct regions form fibrils independently, but only those with structural compatibility efficiently template the parent protein. N-TTR therefore represents an autonomous amyloidogenic segment that links local sequence propensity to TTR nucleation, templating, and fibril polymorphism.

biophysics↗

bioRxiv2026

A functional amyloid scaffold shapes insect egg coats

Functional amyloids serve as structural scaffolds across biology, yet the molecular architecture and assembly principles of many remain unresolved. The lepidopteran egg coat, or chorion, presents a striking example: hundreds of paralogous proteins sharing a conserved central domain form a mechanically resilient amyloid matrix essential for embryo protection. Here, combining evolutionary analysis of more than 500 sequences with cryo-electron microscopy and biophysical assays, we determine the atomic structure of chorion amyloid filaments and uncover the principles governing their assembly. Contrary to previous structural predictions, chorion filaments adopt a {beta}-serpentine fold stabilized by a short hexapeptide motif that forms homotypic steric-zipper interfaces, self-assembles autonomously, and seeds full-length filament growth. Amyloid formation proceeds through secondary nucleation, while thermodynamic and structural analyses support a hierarchical assembly mechanism in which motif-driven interactions nucleate filament formation prior to consolidation of the mature yet lalbile protofilament core. These findings establish the molecular basis of insect egg-coat assembly and demonstrate that mechanisms commonly associated with pathological aggregation can also operate within a biologically regulated functional amyloid framework.

biophysics↗

bioRxiv2026

Reverse-engineering amyloid strains with generative protein design

Amyloid fibrils are intrinsically polymorphic protein assemblies that form distinct structural strains linked to diverse biological and pathological outcomes. Yet, the principles governing how sequence encodes diverse fibril architectures, and the extent to which a given fold constrains underlying amino-acid sequence compatibility, remain poorly understood. Here, we apply generative protein design to directly interrogate the sequence-structure relationship of defined fibril architectures, using -synuclein (S), a protein known to form highly polymorphic amyloid fibrils, as a model system. Sampling sequence space under structural constraints reveals a continuous compatibility manifold in which diverse sequences encode a common amyloid architecture. De novo designed sequences assemble into fibrils, often with enhanced aggregation efficiency relative to S. A subset exhibits strain-like behaviour, including similar morphologies, efficient cross-templating, and induction of S cellular propagation, thereby functionally validating structural compatibility with the native fibril fold. Energetic analysis shows that stability is achieved through distinct but compensatory interactions, supporting a non-unique mapping between sequence and structure. Together, our results define a continuous and constrained compatibility landscape underlying amyloid strains, providing a framework for understanding the determinants of polymorphism and establishing generative protein design as a strategy to access this space, interrogate amyloid sequence-structure relationships, and engineer fibrillar protein assemblies and functional biomaterials.

biophysics↗

bioRxiv2025

TAF15 amyloids propagate via defined motifs in a prion-like fashion

TATA-box binding protein-associated factor 15 (TAF15) is an RNA-binding member of the FET family recently identified as the primary fibrillar constituent in a subset of frontotemporal lobar degeneration (FTLD-FET) cases. Although TAF15 is also linked to amyotrophic lateral sclerosis (ALS), the molecular basis and propagation behavior of its aggregates remain unknown. In this work, we show that recombinant TAF15 forms amyloid fibrils under physiological conditions and developed a single-fluorophore TAF15 biosensor to quantitatively monitor their cellular propagation. Using this system, we demonstrate that both recombinant TAF15 fibrils and pathological aggregates extracted from atypical FTLD with ubiquitin inclusions (aFTLD-U) patient brains seed aggregation efficiently and transmit serially between cells, demonstrating hallmark features of prion-like propagation. Seeding was specific to TAF15 and absent for other amyloidogenic proteins, including the homologous protein fused-in-sarcoma (FUS), revealing an unexpected cross-seeding barrier. Occasional colocalization of FUS within TAF15 inclusions was observed upon transient co-expression, suggesting that FUS can be passively recruited rather than acting as an inducer of pathology in FTLD-FET brains. Computational and peptide-based experimental mapping identified multiple aggregation-prone regions within the TAF15 low-complexity domain that coincide with hotspots stabilizing the core of ex vivo TAF15 amyloid fibrils. These short motifs encode the propagation propensity of TAF15 aggregation in vitro and in cells. Together, these findings establish TAF15 as a bona fide amyloid-forming, prion-like protein and define the sequence grammar underlying its self-assembly, providing a mechanistic framework for its role in FTLD-FET and ALS and offering tractable molecular targets for therapeutic intervention.

biophysics↗

bioRxiv2025

Amyloid Explorer: a global atlas of amyloid fibril structures and thermodynamic principles

Amyloid fibrils underpin both functional and pathological protein assemblies and exhibit extensive structural polymorphism. More than 600 high-resolution fibril structures are now available across over 50 protein sequences, yet comparative analysis has been hindered by fragmented resources and inconsistent annotation. Here we present Amyloid Explorer, an open-access platform that integrates the complete fibril archive into a standardized, quality-controlled, and thermodynamically annotated framework. Each entry includes residue- and segment-level stability maps, structural quality metrics, and interactive visualization tools. Global analysis across the dataset reveals conserved rules of amyloid assembly, including cooperative cores, framework polymorphism through reuse of conserved segments in distinct folds, polymorph-specific energetic anchors, and short frustration zones. By combining scale with accessibility, Amyloid Explorer transforms a static archive into a discovery platform for mechanistic insight, classification, and the design of aggregation modulators.

biophysics↗

bioRxiv2025

Phagocytes as Plaque Catalysts: Human Macrophages Actively Generate Pathogenic Aβ42 Fibrils with Seeding and Cross-Seeding Potency

The prevailing view frames microglia and macrophages as guardians against amyloid beta (A{beta}) accumulation in Alzheimers disease (AD). Here, we overturn this paradigm by demonstrating that human phagocytic cells--including differentiated THP-1 macrophages and iPSC-derived microglia--are not merely passive responders but active producers of extracellular, seeding-competent A{beta}42 fibrils, the amyloid species most strongly linked to parenchymal plaque formation and neurodegeneration. These cell-generated aggregates differ structurally and functionally from synthetic fibrils, exhibiting heightened seeding activity and the ability to cross-seed tau aggregation, a key driver of AD progression. Notably, A{beta}42 fibril formation in this system requires active cellular processes and is exacerbated by loss of TREM2, a major AD risk gene. Transcriptomic profiling reveals an early inflammatory response resembling microglial states observed in human AD models, positioning this system as a tractable, human-relevant platform to dissect the interplay between A{beta} aggregation, innate immunity, and genetic susceptibility. Our findings suggest that macrophages and microglia play a dual role in AD, acting both as responders and inadvertent catalysts of pathogenic amyloid formation, with implications for early therapeutic intervention. Significance StatementHow amyloid plaques emerge and spread in Alzheimers disease remains a critical unanswered question. Here, we show that human immune cells--including brain-resident microglia--can actively generate A{beta}42 fibrils, the form of amyloid most strongly linked to neurodegeneration. These cell-produced fibrils not only seed further amyloid buildup but also trigger tau aggregation, a key event in disease progression. We further demonstrate that genetic risk factors like TREM2 amplify this process. Our findings reveal a direct link between immune cell activity, genetic susceptibility, and the earliest stages of Alzheimers pathology, offering new insights into disease mechanisms and potential intervention points.

biochemistry↗

bioRxiv2021

Mapping the sequence specificity of heterotypic amyloid interactions enables the identification of aggregation modifiers

Heterotypic amyloid interactions between related protein sequences have been observed in functional and disease amyloids. While sequence homology seems to favour heterotypic amyloid interactions, we have no systematic understanding of the structural rules determining such interactions nor whether they inhibit or facilitate amyloid assembly. Using structure-based thermodynamic calculations and extensive experimental validation, we performed a comprehensive exploration of the defining role of sequence promiscuity in amyloid interactions. Using this knowledge, we demonstrate, using tau as a model system, that predicted cross-interactions driven by sequence homology indeed can modify nucleation, fibril morphology, kinetic assembly and cellular spreading of aggregates. We also find that these heterotypic amyloid interactions can result in the mis-localisation of brain-expressed protein sequences with prevalent activities in neurodegenerative disorders. Our findings suggest a structural mechanism by which the proteomic background can modulate the aggregation propensity of amyloidogenic proteins and discuss how such sequence-specific proteostatic perturbations could contribute to the selective cellular susceptibility of amyloid disease progression.

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↗