Biology subjects

Gadhe, L.

Publications and source records attributed to Gadhe, L..

10 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

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↗

bioRxiv2023

Nucleo-cytoplasmic environment modulates spatio-temporal p53 phase separation

Phase separation of various transcription factors and nucleic acids into biomolecular condensates is known to play an essential role in the regulation of gene expression. Here, we show that p53, a tumor suppressor and transcription factor, phase separates and forms biomolecular condensates in the nucleus of cancer cells as well as when overexpressed in the various cell lines. Although the nuclear condensates of wild-type (WT) p53 maintain their liquid state and are able to bind DNA, cancer-associated mutations not only promote misfolding but also partially rigidify the p53 condensates, which are unable to bind the DNA. Irrespective of WT or mutant form, the cytoplasmic partitioning of p53 with time also results in biomolecular condensate formation, which eventually undergoes rigidification. In vitro, WT p53 core domain (p53C) forms biomolecular condensates, which rigidify with time and the process is further promoted by cancer-associated mutations. Both RNA and non-specific DNA promote LLPS of p53C, but specific DNA promotes the dissolution of p53C condensates. The result suggests that the cellular microenvironment regulates p53 LLPS, material property and its functions.

biophysics↗

bioRxiv2023

p53 amyloid pathology with cancer grades and p53 mutations

p53 mutation and amyloid formation are implicated with cancer pathogenesis, but the direct demonstration of the link between p53 amyloid load and cancer progression is lacking. Using multi-disciplinary techniques and a cohort of 59 tumor tissues (53 from Indian cancer patients and six normal tissues) of oral and stomach cancer types, we showed that p53 amyloid load and cancer grades are highly correlated. Further, next-generation sequencing (NGS) data suggest that not only mutant p53 (e.g., SNVs, deletions, and insertions) but wild-type p53 also formed amyloids either in the nucleus (50%) and/or in the cytoplasm in most cancer tissues. Interestingly, in all these cancer tissues, p53 displays a loss of DNA binding and transcriptional activities, which is highly aggravated with the amyloid load and cancer grades. The p53 amyloids also sequester higher amounts of p63/p73 isoforms in higher-grade of tumor tissues. The data suggest p53 misfolding/aggregation and subsequent amyloid formation lead to loss and gain of p53 tumorigenic function, aggravation of which might determine the cancers grades.

cancer biology↗

bioRxiv2022

Liquid condensate is a common state of proteins and polypeptides at the regime of high intermolecular interactions

Liquid-liquid phase separation (LLPS) has emerged as a crucial biological mechanism for sequestering macromolecules (such as proteins and nucleic acids) into membraneless organelles in cells. Unstructured and intrinsically disordered domains are known to facilitate multivalent interactions driving protein LLPS. We hypothesized that LLPS could be an intrinsic property of proteins/polypeptides at their high intermolecular interaction regime. To examine this, we studied many (a total of 23) proteins/polypeptides with different structures and sequences for LLPS study using molecular crowder polyethylene glycol (PEG-8000). We showed that all proteins and even highly charged polypeptides (under study) can undergo liquid condensate formation, however with different phase space and conditions. Using a single component and combinations of protein multicomponent (co-LLPS) systems, we establish that a variety of intermolecular interactions can drive proteins/polypeptides LLPS.

biophysics↗

bioRxiv2021

Size-dependent secondary nucleation and amplification of α-synuclein amyloid fibrils

The size of the amyloid seeds is known to modulate their autocatalytic amplification and cellular toxicity. However, the seed size-dependent secondary nucleation mechanism, toxicity, and disease-associated biological processes mediated by -synuclein (-Syn) fibrils are largely unknown. Using the cellular model and in vitro reconstitution, we showed that the size of -Syn fibril seeds not only dictates its cellular internalization and associated cell death; but also the distinct mechanisms of fibril amplification pathways involved in the pathological conformational change of -Syn. Specifically, small-sized fibril seeds showed elongation possibly through monomer addition at the fibril termini; whereas longer fibrils template the fibril amplification by surface-mediated nucleation as demonstrated by super-resolution microscopy. The distinct mechanism of fibril amplification, and cellular uptake along with toxicity suggest that breakage of fibrils into different sizes of seeds determine the underlying pathological outcome of synucleinopathies.

neuroscience↗

bioRxiv2021

Co-aggregation and secondary nucleation in the life cycle of human prolactin/galanin functional amyloids

Synergistic-aggregation and cross-seeding by two different amyloid proteins/peptides are well evident in various neurological disorders. However, this phenomenon is not well studied in functional amyloid aggregation. Here, we show Prolactin (PRL) is associated with lactation in mammals and neuropeptide galanin (GAL), which are co-stored in the lactotrophs facilitates the synergic aggregation in the absence of secretory granules helper molecules glycosaminoglycans (GAGS). Interestingly, although each partner possesses homotypic seeding ability, a unidirectional cross-seeding of GAL aggregation can be mediated by PRL seeds. The specificity of co-aggregation by PRL and GAL along with unidirectional cross-seeding suggests tight regulation of functional amyloid formation during co-storage of these hormones in secretory granule biogenesis of female rat lactotrophs. Further mixed fibrils release the constituent functional hormone much faster than the corresponding individual amyloid formed in presence of GAGs, suggesting that co-aggregation of functionally distant hormones might have evolved for efficient storage, synergistic and rapid release of both hormones upon stimulation. The co-aggregation and cross seeding by two different hormones of completely different structures and sequences (PRL and GAL) suggest a novel mechanism of heterologous amyloid formation both in disease and functional amyloids.

biophysics↗

bioRxiv2019

Liquid-liquid phase separation and liquid-to-solid transition mediate α-synuclein amyloid fibril containing hydrogel formation

-Synuclein (-Syn) aggregation and amyloid formation is directly linked with Parkinsons disease (PD) pathogenesis. However, the early events involved in this process remain unclear. Here, using in vitro reconstitution and cellular model, we show that liquid-liquid phase separation (LLPS) of -Syn precedes its aggregation. In particular, in vitro generated -Syn liquid-like droplets eventually undergo a liquid-to-solid transition and form amyloid-hydrogel containing oligomers and fibrillar species. Factors known to aggravate -Syn aggregation such as low pH, phosphomimic substitution, and familial PD mutation also promote -Syn LLPS and its subsequent maturation. We further demonstrate -Syn liquid droplet formation in cells, under oxidative stress. These cellular -Syn droplets eventually transform into perinuclear aggresomes, the process regulated by microtubules. The present work provides detailed insights into the phase separation behavior of natively unstructured -Syn and its conversion to a disease-associated aggregated state, which is highly relevant in PD pathogenesis.

neuroscience↗