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A coumarin based turn-on sensor for the selective detection of Hg(ii) through oxidation of diselenide. The sensing mechanism supported with single crystal XRD of monoselenide. Sensitivity of the probe for the detection of Hg(ii) confirmed with imaging in MCF-7 cells.

Biomolecules are known to interact with metals and produce nanostructured hybrid materials with diverse morphologies and functions. In spite of the great advancement in the principles of biomimetics for designing complex nano-bio structures, the interplay between the physical properties of biomolecules such as sequence, charge, and hydrophobicity with predictable morphology of the resulting nanomaterials is largely unknown. Here, using various amyloidogenic proteins/peptides and their corresponding fibrils in combination with different pH, we show defined principle for gold nanocrystal growth into triangular and supra-spheres with high prediction. Using a combination of different biophysical and structural techniques, we establish the mechanism of nucleation and crystal growth of gold nanostructures and show the effective isolation of intact nanostructures from amyloid templates using protein digestion. This study will significantly advance our design principle for bioinspired materials for specific functions with great predictability.

Transcription factor p53 (also known as TP53) has been shown to aggregate into cytoplasmic and nuclear inclusions, compromising its native tumor suppressive functions. Recently, p53 has been shown to form amyloids, which play a role in conferring cancerous properties to cells, leading to tumorigenesis. However, the exact pathways involved in p53 amyloid-mediated cellular transformations are unknown. Here, using an in cellulo model of full-length p53 amyloid formation, we demonstrate the mechanism of loss of p53 tumor-suppressive function with concomitant oncogenic gain of functions. Global gene expression profiling of cells suggests that p53 amyloid formation dysregulates genes associated with the cell cycle, proliferation, apoptosis and senescence along with major signaling pathways. This is further supported by a proteome analysis, showing a significant alteration in levels of p53 target proteins and enhanced metabolism, which enables the survival of cells. Our data indicate that specifically targeting the key molecules in pathways affected by p53 amyloid formation, such as cyclin-dependent kinase-1, leads to loss of the oncogenic phenotype and induces apoptosis of cells. Overall, our work establishes the mechanism of the transformation of cells due to p53 amyloids leading to cancer pathogenesis. This article has an associated First Person interview with the first author of the paper.

The size of 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 α-synuclein (α-Syn) fibrils are largely unknown. Using the cellular model and

Protein aggregation into amyloid fibrils is a key feature of a multitude of neurodegenerative diseases such as Alzheimer's, Parkinson's, and Prion disease. To detect amyloid fibrils, fluorophores with high sensitivity and better efficiency coupled with the low toxicity are in high demand even to date. In this pursuit, we have unveiled two benzimidazole-based fluorescence sensors ([C15 H15 N3 ] (C1) and [C16 H16 N3 O2 ] (C2), which possess exceptional affinity toward different amyloid fibrils in its submicromolar concentration (8 × 10-9 M), whereas under a similar concentration, the gold standard Thioflavin-T (ThT) fails to bind with amyloid fibrils. These fluorescent markers bind to α-Syn amyloid fibrils as well as amyloid fibrils forming other proteins/peptides including Aβ42 amyloid fibrils. The 1 H-15 N heteronuclear quantum correlation spectroscopy nuclear magnetic resonance data collected on wild-type α-Syn monomer with and without the fluorophores (C1 and C2) reveal that there is weak or no interactions between C1 or C2 with residues in α-Syn monomer, which indirectly reflects the specific binding ability of C1 and C2 to the α-Syn amyloid fibrils. Detailed studies further suggest that C1 and C2 can detect/bind with the α-Syn amyloid fibril as low as 100 × 10-9 M. Extremely low or no cytotoxicity is observed for C1 and C2 and they do not interfere with α-Syn fibrillation kinetics, unlike ThT. Both C1/C2 not only shows selective binding with amyloid fibrils forming various proteins/peptides but also displays excellent affinity and selectivity toward α-Syn amyloid aggregates in SH-SY5Y cells and Aβ42 amyloid plaques in animal brain tissues. Overall, our data show that the developed dyes could be used for the detection of amyloid fibrils including α-Syn and Aβ42 amyloids with higher sensitivity as compared to currently used ThT.

Synucleinopathies are a class of neurodegenerative diseases, including Parkinson's disease (PD), Dementia with Lewy bodies (DLB), and Multiple System Atrophy (MSA). The common pathological hallmark of synucleinopathies is the filamentous α-synuclein (α-Syn) aggregates along with membrane components in cytoplasmic inclusions in the brain. β-Synuclein (β-Syn), an isoform of α-Syn, inhibits α-Syn aggregation and prevents its neurotoxicity, suggesting the neuroprotective nature of β-Syn. However, this notion changed with the discovery of disease-associated β-Syn mutations, V70M and P123H, in patients with DLB. It is still unclear how these missense mutations alter the structural and amyloidogenic properties of β-Syn, leading to neurodegeneration. Here, we characterized the biophysical properties and investigated the effect of mutations on β-Syn fibrillation under different conditions. V70M and P123H show high membrane binding affinity compared to wild-type β-Syn, suggesting their potential role in membrane interactions. β-Syn and its mutants do not aggregate under normal physiological conditions; however, the proteins undergo self-polymerization in a slightly acidic microenvironment and/or in the presence of an inducer, forming long unbranched amyloid fibrils similar to α-Syn. Strikingly, V70M and P123H mutants exhibit accelerated fibrillation compared to native β-Syn under these conditions. NMR study further revealed that these point mutations induce local perturbations at the site of mutation in β-Syn. Overall, our data provide insight into the biophysical properties of disease-associated β-Syn mutations and demonstrate that these mutants make the native protein more susceptible to aggregation in an altered microenvironment.

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

All aerobic cells contain reactive oxygen species (ROSs) in balance with biochemical antioxidants. Oxidative stress is developed when this balance gets disturbed because of excessive production of ROSs or depletion of antioxidants. Here, in this work, we have developed the first cyclic diselenide BODIPY-based (organoselenium-containing) probe for the selective detection of superoxide. The probe demonstrates excellent selective response for superoxide over other ROSs with nine-fold increase in fluorescence intensity. The detection limit was found to be 0.924 μM. The plausible “turn-on” mechanism has been proposed based on the spectroscopic and quantum chemical data. Usefulness of the probe for selective detection of superoxide was confirmed in mammalian breast cancer cell lines.

α-Synuclein (α-Syn) amyloids in synucleinopathies are suggested to be structurally and functionally diverse, reminiscent of prion-like strains. The mechanism of how the aggregation of the same precursor protein results in the formation of fibril polymorphs remains elusive. Here, we demonstrate the structure-function relationship of two polymorphs, pre-matured fibrils (PMFs) and helix-matured fibrils (HMFs), based on α-Syn aggregation intermediates. These polymorphs display the structural differences as demonstrated by solid-state NMR and mass spectrometry studies and also possess different cellular activities such as seeding, internalization, and cell-to-cell transfer of aggregates. HMFs, with a compact core structure, exhibit low seeding potency but readily internalize and transfer from one cell to another. The less structured PMFs lack transcellular transfer ability but induce abundant α-Syn pathology and trigger the formation of aggresomes in cells. Overall, the study highlights that the conformational heterogeneity in the aggregation pathway may lead to fibril polymorphs with distinct prion-like behavior.

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.

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 α-synuclein (α-Syn) fibrils are largely unknown. Using the cellular model and in vitro reconstitution, we showed that the size of α-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 α-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.

Synergistic-aggregation and cross-seeding by two different proteins/peptides in the amyloid aggregation are well evident in various neurological disorders including Alzheimer's disease. Here, we show co-storage of human Prolactin (PRL), which is associated with lactation in mammals, and neuropeptide galanin (GAL) as functional amyloids in secretory granules (SGs) of the female rat. Using a wide variety of biophysical studies, we show that irrespective of the difference in sequence and structure, both hormones facilitate their synergic aggregation to amyloid fibrils. Although each hormone possesses homotypic seeding ability, a unidirectional cross-seeding of GAL aggregation by PRL seeds and the inability of cross seeding by mixed fibrils suggest tight regulation of functional amyloid formation by these hormones for their efficient storage in SGs. Further, the faster release of functional hormones from mixed fibrils compared to the corresponding individual amyloid, suggests a novel mechanism of heterologous amyloid formation in functional amyloids of SGs in the pituitary.The formation of plaques of proteins called ‘amyloids’ in the brain is one of the hallmark characteristics of both Alzheimer’s and Parkinson’s disease, but amyloids can form in many tissues and organs, often disrupting normal activity. A lot of the research into amyloids has focused on their role in disease, but it turns out that amyloids can also appear in healthy tissues. For example, some protein hormones form amyloids that act as storage depots, helping cells to release the hormone when it is needed. Normally, amyloids are made mostly of a single type of protein or protein fragment associated with a particular disease like Alzheimer's. Often, this type of amyloid promotes plaque formation in other proteins, which aggravates other diseases (for example, the amyloids that form in Alzheimer’s can lead to Parkinson’s disease or type II diabetes getting worse).The plaques start growing from small amyloid fragments called seeds. In mixed amyloids – amyloids made of two types of proteins – seeds made of one protein can trigger the formation of amyloids of the other protein. This raises the question, is this true for hormones? The body often releases more than one hormone at a time from the same tissue; for example, the pituitary gland releases prolactin and galanin simultaneously. However, these hormones have completely different structures, so whether they can form a mixed amyloid is unclear. To answer this question, Chatterjee et al. first determined that, within the pituitary gland of female rats, prolactin and galanin could be found together in the same cells, forming mixed amyloids. To understand out how this happens, Chatterjee et al. tried seeding new amyloids using either prolactin or galanin. This revealed that only prolactin seeds were able to trigger the formation of galanin amyloids. Chatterjee et al. also found that the mixed amyloids could release the hormones faster than amyloids made from either protein alone. Together, these results suggest that the collaboration between these two proteins may help maintain hormone balance in the body. Problems with hormone storage and release lead to various human diseases, including prolactinoma. Understanding amyloid storage depots could reveal new ways to control hormone levels. Further research could also help to explain more about well-studied diseases linked to amyloids, like Alzheimer's.

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.

The global transcription factor, p53, is a master regulator of gene expression in cells. Mutations in the TP53 gene promote unregulated cell growth through the inactivation of downstream effectors of the p53 pathway. In fact, mutant p53 is highly prone to misfolding and frequently resides inside the cell as large aggregates, causing loss of physiological function of the tumor-suppressor protein. Here, we review the plausible reasons for functional loss of p53, including amyloid formation leading to unhindered cancer progression. We discuss previous as well as recent findings regarding the amyloid formation of p53 in vitro and in vivo. We elaborate on prion-like properties of p53 amyloids and their possible involvement in cancer progression. Because the p53 pathway is historically most targeted for the development of anticancer therapeutics, we have also summarized some of the recent approaches and advances in reviving the antiproliferative activities of wild-type p53. In this Perspective, we provide insight into understanding p53 as a prion-like protein and propose cancer to be recognized as an amyloid or prion-like disease.

A monoselenium containing diBODIPY-based probe was synthesized. The probe was found to be selective and sensitive for the detection of superoxide (KO2) over other reactive oxygen species through fluorescence turn-on event with 11-fold increase in fluorescence intensity. The detection limit was found to be 4.42 μM. The utility of the probe for detection of intracellular superoxide was demonstrated in a mammalian breast cancer cell line (MCF-7) through fluorescence microscopy.

Biomaterials mimicking extracellular matrices (ECM) for three-dimensional (3D) cultures have gained immense interest in tumor modeling and in vitro organ development. Here, we introduce versatile, thixotropic amyloid hydrogels as a bio-mimetic ECM scaffold for 3D cell culture as well as high-throughput tumor spheroid formation using a drop cast method. The unique cross-β-sheet structure, sticky surface, and thixotropicity of amyloid hydrogels allow robust cell adhesion, survival, proliferation, and migration, which are essential for 3D tumor modeling with various cancer cell types. The spheroids formed show overexpression of the signature cancer biomarkers and confer higher drug resistance compared to two-dimensional (2D) monolayer cultures. Using breast tumor tissue from mouse xenograft, we showed that these hydrogels support the formation of tumor spheroids with a well-defined necrotic core, cancer-associated gene expression, higher drug resistance, and tumor heterogeneity reminiscent of the original tumor. Altogether, we have developed a rapid and cost-effective platform for generating in vitro cancer models for the screening of anti-cancer therapeutics and developing personalized medicines.

Tumor suppressor p53 mutations are associated with more than 50% of cancers. Aggregation and amyloid formation of p53 is also implicated in cancer pathogenesis, but direct evidence for aggregated p53 amyloids acting as an oncogene is lacking. Here, we conclusively demonstrate that wild-type p53 amyloid formation imparts oncogenic properties to non-cancerous cells. p53 amyloid aggregates were transferred through cell generations, contributing to enhanced survival, apoptotic resistance with increased proliferation and migration. The tumorigenic potential of p53 amyloid-transformed cells was further confirmed in mouse xenografts, wherein the tumors showed p53 amyloids. p53 disaggregation rescued the cellular transformation and inhibited tumor development in mice. We propose that wild-type p53 amyloid formation contributes to tumorigenesis and can be a potential target for therapeutic intervention. This article has an associated First Person interview with the first author of the paper.

Phenylselenide substituted BOPHY probes (BOPHY-SePh and PhSe-BOPHY-SePh) were synthesized and characterized by NMR spectroscopy and single-crystal XRD. Both the probes selectively detect HOCl in water with high sensitivity over other reactive oxygen species. A fluorescence “turn-on” event was attained due to cease of the PET process through transformation of selenide to selenoxide. Both the probes react with HOCl in less than 1 s. PhSe-BOPHY-SePh probe depicted low background fluorescence due to presence of two phenylselenide groups at BOPHY. PhSe-BOPHY-SePh probe has a low detection limit (0.63 μM) than BOPHY-SePh probe (1.08 μM). The bioimaging studies of both the probes were carried out in MCF 7 cells. Both the probes exhibited a good fluorescence response for HOCl in vitro and in mammalian cells. In addition, the probes showed reversibility with all bio-thiols, which was validated in MCF 7 cells using GSH.

Tumor suppressor p53 mutations, with subsequent loss-of-tumor suppressive function and gain-of oncogenic functions, are associated with more than 50% of human cancers. Aggregation and amyloid formation are also mechanisms by which wild type and mutant p53 might be involved in cancer, but the direct evidence of how aggregated p53 acts as an oncogene is lacking. In this study, we directly demonstrate that wild-type p53 amyloid formation imparts oncogenic properties to normal cells. Cells with p53 amyloids show enhanced survival, apoptotic resistance with increased proliferation and migration rates. The tumorigenic potential of p53 amyloid transformed cells is further confirmed in a mice xenograft model, wherein the tumor showed p53 amyloid aggregates. Gene-expression analysis and proteomic profiling suggest that p53 amyloid formation triggers aberrant expression of pro-oncogenes while downregulating the tumor-suppressive genes. Interestingly, disaggregating p53 rescues the cellular transformation and also inhibits tumor development in mice. We propose that wild-type p53 amyloid formation can potentially contribute to the initiation of tumor development.

α-Synuclein (α-Syn) amyloid fibrils in synucleinopathies (such as Parkinson’s disease (PD), multiple system atrophy (MSA)) are structurally and functionally different, reminiscent of prion-like strains. However, how a single protein can form different fibril polymorphs in various synucleinopathies is not known. Here, we demonstrate the structure-function relationship of two distinct α-Syn fibril polymorphs, the pre-matured fibrils (PMF) and helix-matured fibrils (HMF) based on α-Syn aggregation intermediates. These polymorphs not only display the structural differences, including their fibril core structure as demonstrated by solid-state nuclear magnetic resonance (NMR) spectroscopy and H/D-exchange coupled with mass spectrometry but also possess different cellular activities such as seeding, cellular internalization, and cell-to-cell transmission. The HMF with a compact core structure exhibits low seeding potency in cells but readily internalizes and transmits from one cell to another. Whereas the less structured PMF lacks the cell-to-cell transmission ability but induces abundant α-Syn pathology and triggers the formation of aggresomes in cells. Overall, the study highlights how the conformational heterogeneity in the aggregation pathway may lead to fibril polymorphs with distinct prion-like behavior in PD.

Amyloid formation of α-synuclein (α-Syn) and its familial mutations are directly linked with Parkinson's disease (PD) pathogenesis. Recently, a new familial α-Syn mutation (A53E) was discovered, associated with an early onset aggressive form of PD, which delays α-Syn aggregation. When we overexpressed wild-type (WT) and A53E proteins in cells, showed neither toxicity nor aggregate formation, suggesting merely overexpression may not recapitulate the PD phenotype in cell models. We hypothesized that cells expressing the A53E mutant might possess enhanced susceptibility to PD-associated toxicants compared to that of the WT. When cells were treated with PD toxicants (dopamine and rotenone), cells expressing A53E showed more susceptibility to cell death along with compromised mitochondrial potential and an increased production of reactive oxygen species. The higher toxicity of A53E could be due to more oligomers being formed in cells as confirmed by a dot blot assay using amyloid specific OC and A11 antibody and using an in vitro aggregation study. The cellular model presented here suggests that along with familial mutation, environmental and other cellular factors might play a crucial role in dictating PD pathogenesis.

Aggregation is the cause of numerous protein conformation diseases. A common facet of these maladies is the transition of a protein from its functional native state into higher order forms, such as oligomers and amyloid fibrils. p53 is an essential tumor suppressor that is prone to such conformational transitions, resulting in its compromised ability to avert cancer. This work explores the biophysical properties of early-, mid-, and late-stage p53 core domain (p53C) aggregates. Atomistic and coarse-grained molecular dynamics (MD) simulations suggest that early- and mid-stage p53C aggregates have a polymorphic topology of antiparallel and parallel β-sheets that localize to the core amyloidogenic sequence. Both topologies involve similar extents of interstrand mainchain hydrogen bonding, while sidechain interactions could play a role in regulating strand orientation. The free energy difference between the antiparallel and parallel states was within statistical uncertainty. Negative stain electron microscopy of mature fibrils shows a wide distribution of fiber widths, indicating that polymorphism may extend to the quaternary structure level. Circular dichroism of the fibrils was indicative of β-sheet rich structures in atypical conformations. The Raman spectrum of aggregated p53C was consistent with a mixture of arranged β-sheets and heterogeneous structural elements, which is compatible with the MD findings of an ordered β-sheet nucleus flanked by disordered structure. Structural polymorphism is a common property of amyloids; however, because certain polymorphs of the same protein can be more harmful than others, going forward it will be pertinent to establish correlations between p53C aggregate structure and pathology.

The study of binding of drug with protein is an important process in determining the activity and consequences of the drug. However, the current screening methods to investigate the drug–protein interaction are still pervaded with some disadvantages such as large requirement of sample volume and the need of expensive instrumentation. Here, we present a proof of concept for a method to study the interaction of different kinds of drugs with protein on a microwell array, to enable the understanding of pharmacokinetics in a fast and facile manner. To this end, we demonstrated the applicability of present method by analysing the interaction of fluorescently labelled bovine serum albumin (FITC-BSA) with sulphanilamide (SMO) and sulfamethoxazole (SMZ) drugs on lotus leaf-based microwells. The quenching mechanism of FITC-BSA in the presence of SMO and SMZ is visualized through time-lapse fluorescence imaging for the first time in femto-litre sized microwells. The binding parameters extracted from the present method are found to be in good agreement with the fluorescent spectroscopy measurements. The interaction between protein and drugs was further corroborated by molecular docking analysis and FT-IR technique. Finally, the bio-inspired microwell array was integrated into a magnetic digital microfluidic device to facilitate investigation of drug–protein interaction in power-free and cost-effective manner. A substantial decrease in sample volume and relatively short processing times when compared to other bulk methods makes the present method attractive. The results presented here therefore open up the possibility of rapid measurement of binding parameters in protein–ligand interaction studies in low-resource environments.

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

SUMMARYα-Synuclein (α-Syn) aggregation and amyloid formation is directly linked with Parkinson’s 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 α-Syn precedes its aggregation. In particular, in vitro generated α-Syn liquid-like droplets eventually undergo a liquid-to-solid transition and form amyloid-hydrogel containing oligomers and fibrillar species. Factors known to aggravate α-Syn aggregation such as low pH, phosphomimic substitution, and familial PD mutation also promote α-Syn LLPS and its subsequent maturation. We further demonstrate α-Syn liquid droplet formation in cells, under oxidative stress. These cellular α-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 α-Syn and its conversion to a disease-associated aggregated state, which is highly relevant in PD pathogenesis.

Altered intestinal permeability has been correlated with Parkinson's pathophysiology in the enteric nervous system, before manifestations in the central nervous system (CNS). The inflammatory endotoxin or lipopolysaccharide (LPS) released by gut bacteria is known to modulate α-synuclein amyloidogenesis through the formation of intermediate nucleating species. Here, biophysical techniques in conjunction with microscopic images revealed the molecular interaction between lipopolysaccharide and α-synuclein that induce rapid nucleation events. This heteromolecular interaction stabilizes the α-helical intermediates in the α-synuclein aggregation pathway. Multitude NMR studies probed the residues involved in the LPS-binding structural motif that modulates the nucleating forms, affecting the cellular internalization and associated cytotoxicity. Collectively, our data characterizes this heteromolecular interaction associated with an alternative pathway in Parkinson's disease progression.

Microwell platforms show great promise in single-cell studies and protein measurements because of their low volume sampling, rapid analysis and high throughput screening ability. However, the existing actuation mechanisms to manipulate the target samples and fabrication procedures involved in the microwell-based microfluidic devices are complex, resource-intensive and require an external power source. In this work, we present proof of concept of a simple, power-free and low-cost closed magnet digital microfluidics device for isolating biological entities in femtoliter-sized microwells. The target biological entities were encapsulated in magnetic liquid marbles and shuttled back and forth between micropatterned top and bottom plates in the microdevice to obtain high loading efficiency and short processing time. The microdevice performance was studied through fluorescent detection of three different entities: microbeads, bovine serum albumin (BSA) and Escherichia coli, captured in the microwell array. Almost 80% of the microwells were loaded with single microbeads in five shuttling cycles, in less than a minute. Further, a low volume of BSA was compartmentalized in the microwell array over a two order range of concentration. The microdevice exhibits two unique features: lotus leaf stamps were used to fabricate micropatterns (microwells and micropillars) on top and bottom plates to impart functionality and cost-effectiveness, and the target samples were actuated by a permanent magnet to make the microdevice power-free and simple in operation. The developed biomimetic microdevice is therefore capable of capturing a multitude of biological entities in low-resource settings.

The involvement of α-synuclein (α-Syn) amyloid formation in Parkinson's disease (PD) pathogenesis is supported by the discovery of α-Syn gene (SNCA) mutations linked with familial PD, which are known to modulate the oligomerization and aggregation of α-Syn. Recently, the A53V mutation has been discovered, which leads to late-onset PD. In this study, we characterized for the first time the biophysical properties of A53V, including the aggregation propensities, toxicity of aggregated species, and membrane binding capability, along with those of all familial mutations at the A53 position. Our data suggest that the A53V mutation accelerates fibrillation of α-Syn without affecting the overall morphology or cytotoxicity of fibrils compared to those of the wild-type (WT) protein. The aggregation propensity for A53 mutants is found to decrease in the following order: A53TA53VWTA53E. In addition, a time course aggregation study reveals that the A53V mutant promotes early oligomerization similar to the case for the A53T mutation. It promotes the largest amount of oligomer formation immediately after dissolution, which is cytotoxic. Although in the presence of membrane-mimicking environments, the A53V mutation showed an extent of helix induction capacity similar to that of the WT protein, it exhibited less binding to lipid vesicles. The nuclear magnetic resonance study revealed unique chemical shift perturbations caused by the A53V mutation compared to those caused by other mutations at the A53 site. This study might help to establish the disease-causing mechanism of A53V in PD pathology.

Aggregation of α-synuclein (α-Syn) into neurotoxic oligomers and amyloid fibrils is suggested to be the pathogenic mechanism for Parkinson's disease (PD). Recent studies have indicated that oligomeric species of α-Syn are more cytotoxic than their mature fibrillar counterparts, which are responsible for dopaminergic neuronal cell death in PD. Therefore, the effective therapeutic strategies for tackling aggregation-associated diseases would be either to prevent aggregation or to modulate the aggregation process to minimize the formation of toxic oligomers during aggregation. In this work, we showed that arginine-substituted α-Syn ligands, based on the most aggregation-prone sequence of α-Syn, accelerate the protein aggregation in a concentration-dependent manner. To elucidate the mechanism by which Arg-substituted peptides could modulate α-Syn aggregation kinetics, we performed surface plasmon resonance (SPR) spectroscopy, nuclear magnetic resonance (NMR) studies, and all-atom molecular dynamics (MD) simulation. The SPR analysis showed a high binding potency of these peptides with α-Syn but one that was nonspecific in nature. The two-dimensional NMR studies suggest that a large stretch within the C-terminus of α-Syn displays a chemical shift perturbation upon interacting with Arg-substituted peptides, indicating C-terminal residues of α-Syn might be responsible for this class of peptide binding. This is further supported by MD simulation studies in which the Arg-substituted peptide showed the strongest interaction with the C-terminus of α-Syn. Overall, our results suggest that the binding of Arg-substituted ligands to the highly acidic C-terminus of α-Syn leads to reduced charge density and flexibility, resulting in accelerated aggregation kinetics. This may be a potentially useful strategy while designing peptides, which act as α-Syn aggregation modulators.

Aggregation of α-synuclein (α-Syn) into toxic oligomers and fibrils leads to Parkinson's disease (PD) pathogenesis. Molecules that can inhibit the fibrillization and oligomerization of α-Syn have potential therapeutic value. Here, we studied four selective amyloid inhibitors: dopamine (Dopa), amphotericin-B (Amph), epigallocatechingallate (EGCG), and quinacrinedihydrochloride (Quin) for their effect on oligomerization, fibrillization, and preformed fibrils of α-Syn. The aggregation kinetics of α-Syn using ThT fluorescence and conformational transition by circular dichroism (CD) in the presence and absence of these four compounds suggest that, except Quin, the remaining three molecules inhibit α-Syn aggregation in a concentration dependent manner. Consistent with the aggregation kinetics data, the morphological study of aggregates formed in the presence of these compounds showed corresponding decrease in fibrillar size. The analysis of cell viability using MTT assay showed reduction in toxicity of α-Syn aggregates formed in the presence of these compounds, which also correlates with reduction of exposed hydrophobic surface as studied by ANS binding. Additionally, these inhibitors, except Quin, demonstrated reduction in size as well as the toxicity of oligomeric/fibrillar aggregates of α-Syn. The residue specific interaction to low molecular weight (LMW) species of α-Syn by 2D NMR study revealed that, the region and extent of binding are different for all these molecules. Furthermore, fibril-binding data using SPR suggested that there is no direct relationship between the binding affinity and fibril inhibition by these compounds. The present study suggests that sequence based interaction of small molecules with soluble α-Syn might dictate their inhibition or modulation capacity, which might be helpful in designing modulators of α-Syn aggregation.

The transcriptional regulator p53 has an essential role in tumor suppression. Almost 50% of human cancers are associated with the loss of p53 functions, where p53 often accumulates in the nucleus as well as in cytoplasm. Although it has been previously suggested that amyloid formation could be a cause of p53 loss-of-function in subset of tumors, the characterization of these amyloids and its structure-function relationship is not yet established. In the current study, we provide several evidences for the presence of p53 amyloid formation (in human and animal cancer tissues); along with its isolation from human cancer tissues and the biophysical characterization of these tissue-derived fibrils. Using amyloid seed of p53 fragment (P8, p53(250-257)), we show that p53 amyloid formation in cells not only leads to its functional inactivation but also transforms it into an oncoprotein. The in vitro studies further show that cancer-associated mutation destabilizes the fold of p53 core domain and also accelerates the aggregation and amyloid formation by this protein. Furthermore, we also show evidence of prion-like cell-to-cell transmission of different p53 amyloid species including full-length p53, which is induced by internalized P8 fibrils. The present study suggests that p53 amyloid formation could be one of the possible cause of p53 loss of function and therefore, inhibiting p53 amyloidogenesis could restore p53 tumor suppressor functions.