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Amyloid fibrils are structurally heterogeneous protein aggregates that are implicated in a wide range of neurodegenerative and other proteopathic diseases. These fibrils exist in a variety of different tertiary and higher-level structures, and this exhibited polymorphism greatly complicates any structural study of amyloid fibrils. In this work, we demonstrate a method of using polarization-resolved microscopy to directly observe the structural heterogeneity of individual amyloid fibrils using amyloid-bound fluorophores. We formulate a mathematical quantity, helical anisotropy, which utilizes the polarized emission of amyloid-bound fluorophores to report on the local structure of individual fibrils. Using this method, we show how model amyloid fibrils generated from short peptides exhibit diverse structural properties both between different fibrils and within a single fibril, in a manner that is replicated for fibrils assembled from longer proteins. Our method represents an accessible and easily adaptable technique by which polymorphism in the structure of amyloid fibrils can be probed. Additionally, the methodology we describe here can be easily extended to the study of other fibrillar and otherwise ordered supramolecular structures.

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.

Amyloid formation is a generic property of many protein/polypeptide chains. A broad spectrum of proteins, despite having diversity in the inherent precursor sequence and heterogeneity present in the mechanism of aggregation produces a common cross β-spine structure that is often associated with several human diseases. However, a general modeling framework to interpret amyloid formation remains elusive. Herein, we propose a data-driven mathematical modeling approach that elucidates the most probable interaction network for the aggregation of a group of proteins (α-synuclein, Aβ42, Myb, and TTR proteins) by considering an ensemble set of network models, which include most of the mechanistic complexities and heterogeneities related to amyloidogenesis. The best-fitting model efficiently quantifies various timescales involved in the process of amyloidogenesis and explains the mechanistic basis of the monomer concentration dependency of amyloid-forming kinetics. Moreover, the present model reconciles several mutant studies and inhibitor experiments for the respective proteins, making experimentally feasible non-intuitive predictions, and provides further insights about how to fine-tune the various microscopic events related to amyloid formation kinetics. This might have an application to formulate better therapeutic measures in the future to counter unwanted amyloidogenesis. Importantly, the theoretical method used here is quite general and can be extended for any amyloid-forming protein., Amyloid formation is a generic property of many protein/polypeptide chains.

In the pituitary gland, hormones are stored in a functional amyloid state within acidic secretory granules before they are released into the blood. To gain a detailed understanding of the structure–function relationship of amyloids in hormone secretion, the three-dimensional (3D) structure of the amyloid fibril of the human hormone β-endorphin was determined by solid-state NMR. We find that β-endorphin fibrils are in a β-solenoid conformation with a protonated glutamate residue in their fibrillar core. During exocytosis of the hormone amyloid the pH increases from acidic in the secretory granule to neutral level in the blood, thus it is suggested—and supported with mutagenesis data—that the pH change in the cellular milieu acts through the deprotonation of glutamate 8 to release the hormone from the amyloid. For amyloid disassembly in the blood, it is proposed that the pH change acts together with a buffer composition change and hormone dilution. In the pituitary gland, peptide hormones can be stored as amyloid fibrils within acidic secretory granules before release into the blood stream. Here, we use solid-state NMR to determine the 3D structure of the amyloid fiber formed by the human hormone β-endorphin. We find that β-endorphin fibrils are in a β-solenoid conformation that is generally reminiscent of other functional amyloids. In the β-endorphin amyloid, every layer of the β-solenoid is composed of a single peptide and protonated Glu8 is located in the fibrillar core. The secretory granule has an acidic pH but, on exocytosis, the β-endorphin fibril would encounter neutral pH conditions (pH 7.4) in the blood; this pH change would result in deprotonation of Glu8 to release the hormone peptide from the amyloid. Analyses of β-endorphin variants carrying mutations in Glu8 support the role of the protonation state of this residue in fibril disassembly, among other environmental changes. The solid-state NMR structure of an amyloid fiber formed by human hormone β-endorphin reveals a protonated Glu8 in the fibrillar core. Under neutral conditions, deprotonation of Glu8 would contribute to fibril disassembly and hormone peptide release.

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.

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.

Liquid-liquid phase separation (LLPS) and subsequent liquid-to-solid transition is implicated in membraneless organelles formation as well as disease associated protein aggregation. However, how liquid-to-solid transition is initiated inside a liquid droplet remains unclear. Here, using studies at single droplet resolution, we show that liquid-to-solid transition of α-synuclein (α-Syn) liquid droplets is associated with significant changes in the local microenvironment as well as secondary structure of the protein, which is prominently observed at the center of the liquid droplets. With the ageing of liquid droplets, the “structured” core at the center gradually expands and propagates over entire droplets. Further, during droplet fusion, smaller, homogeneous droplets progressively dissolve and supply proteins to the larger, heterogeneous droplets containing solid-like core at their center. The present study will significantly help to understand the physical mechanism of LLPS and liquid-to-solid transition in biological compartmentalization as well as in protein aggregation associated with human neurodegenerative disorders.Abstract Figure

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.

Abnormal accumulation of aggregated α-synuclein (α-Syn) is seen in a variety of neurodegenerative diseases, including Parkinson’s disease (PD), multiple system atrophy (MSA), dementia with Lewy body (DLB), Parkinson’s disease dementia (PDD), and even subsets of Alzheimer’s disease (AD) showing Lewy-body-like pathology. These synucleinopathies exhibit differences in their clinical and pathological representations, reminiscent of prion disorders. Emerging evidence suggests that α-Syn self-assembles and polymerizes into conformationally diverse polymorphs in vitro and in vivo, similar to prions. These α-Syn polymorphs arising from the same precursor protein may exhibit strain-specific biochemical properties and the ability to induce distinct pathological phenotypes upon their inoculation in animal models. In this review, we discuss clinical and pathological variability in synucleinopathies and several aspects of α-Syn fibril polymorphism, including the existence of high-resolution molecular structures and brain-derived strains. The current review sheds light on the recent advances in delineating the structure–pathogenic relationship of α-Syn and how diverse α-Syn molecular polymorphs contribute to the existing clinical heterogeneity in synucleinopathies.

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.

Phase separation of biomolecules could be mediated by both specific and non-specific interactions. How the interplay between non-specific and specific interactions along with polymer entropy influences phase separation is an open question. We address this question by simulating self-associating molecules as polymer chains with a short core stretch that forms the specifically interacting functional interface, and longer non-core regions that participate in non-specific/promiscuous interactions. Our results show that the interplay of specific (strength, ϵsp) and non-specific interactions (strength, ϵns) could result in phase separation of polymers and its transition to solid-like aggregates (mature state). In the absence of ϵns, the polymer chains do not dwell long enough in the vicinity of each other to undergo phase separation and transition into a mature state. On the other hand, in the limit of strong ϵns, the assemblies cannot transition into the mature state and form a non-specific assembly, suggesting an optimal range of interactions favoring mature multimers. In the scenario where only a fraction (Nfrac) of the non-core regions participate in attractive interactions, we find that a slight modifications to either ϵns or Nfrac can result in a dramatically altered self-assembled states. Using a combination of heterogeneous and homogeneous mix of polymers, we establish how this interplay between interaction energies dictates the propensity of biomolecules to find the correct binding partner at dilute concentrations in crowded environments.Author SummaryBiological function relies on the ability of biomolecules to bind to specific interaction partners. In the crowded cellular milieu, the process of biomolecules binding to specific interaction partners to carry out a function is non-trivial. A mere diffusion-limited meeting of interaction partners in space does not ensure biomolecular function. Rather, even when in contact these molecules have to find the correct orientations to carry out function. Further, for dynamic biomolecules such as polymers, the functional configuration is often one of several possible configurations. In this scenario, assuming the functional configuration after binding involves overcoming of a significant entropic barrier. Therefore, the interacting biomolecules have to dwell in contact long enough before they reorganize to find the functional orientation! While functional contacts offer an enthalpic gain, they are often only a small fraction of all protein-protein interactions. Therefore, in this paper, we study the role played by non-functional, promiscuous interactions in shaping the thermodynamics and kinetics of formation of specific interactions. Our results suggest that there exists an optimal range of non-specific interaction strengths which promotes the process of biomolecular complexes finding the functional configurations.

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.

Phase separation of intrinsically disordered proteins (IDPs) is a remarkable feature of living cells to dynamically control intracellular partitioning. Despite the numerous new IDPs that have been identified, progress towards rational engineering in cells has been limited. To address this limitation, we systematically scanned the sequence space of native IDPs and designed artificial IDPs (A-IDPs) with different molecular weights and aromatic content, which exhibit variable condensate saturation concentrations and temperature cloud points in vitro and in cells. We created A-IDP puncta using these simple principles, which are capable of sequestering an enzyme and whose catalytic efficiency can be manipulated by the molecular weight of the A-IDP. These results provide a robust engineered platform for creating puncta with new, phase-separation-mediated control of biological function in living cells.

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.

With the aim of controlling helicity of self-assembled fibers, a pair of water soluble perylene derivatives has been designed and synthesized. Intermolecular hydrogen bonding interactions and pi-pi stacking, combined with the effect of molecular chirality, have been found to guide the aggregation that eventually led to gelation in water upon decreasing pH. This work has demonstrated a new paradigm of simple and non-toxic perylene derivatives for biological applications.

In the pituitary gland, hormones are stored in a functional amyloid state within acidic secretory granules before they are released into the blood. To gain a detailed understanding of the structure-function relationship of amyloids in hormone secretion, the three-dimensional (3D) structure of the amyloid fibril of the human hormone β-endorphin was determined by solid-state NMR. We find that β-endorphin fibrils are in a β-solenoid conformation with a protonated glutamate residue in their fibrillar core. During exocytosis of the hormone amyloid the pH increases from acidic in the secretory granule to neutral level in the blood, thus it is suggested-and supported with mutagenesis data-that the pH change in the cellular milieu acts through the deprotonation of glutamate 8 to release the hormone from the amyloid. For amyloid disassembly in the blood, it is proposed that the pH change acts together with a buffer composition change and hormone dilution. In the pituitary gland, peptide hormones can be stored as amyloid fibrils within acidic secretory granules before release into the blood stream. Here, we use solid-state NMR to determine the 3D structure of the amyloid fiber formed by the human hormone β-endorphin. We find that β-endorphin fibrils are in a β-solenoid conformation that is generally reminiscent of other functional amyloids. In the β-endorphin amyloid, every layer of the β-solenoid is composed of a single peptide and protonated Glu8 is located in the fibrillar core. The secretory granule has an acidic pH but, on exocytosis, the β-endorphin fibril would encounter neutral pH conditions (pH 7.4) in the blood; this pH change would result in deprotonation of Glu8 to release the hormone peptide from the amyloid. Analyses of β-endorphin variants carrying mutations in Glu8 support the role of the protonation state of this residue in fibril disassembly, among other environmental changes.

Loss of p53 function is largely responsible for the occurrence of cancer in humans. Aggregation of mutant p53 has been found in multiple cancer cell types, suggesting a role of aggregation in loss of p53 function and cancer development. The p53 protein has recently been hypothesized to possess a prion-like conformation, although experimental evidence is lacking. Here, we report that human p53 can be inactivated upon exposure to preformed fibrils containing an aggregation-prone sequence-specific peptide, PILTIITL, derived from p53, and the inactive state was found to be stable for many generations. Importantly, we provide evidence of a prion-like transmission of these p53 aggregates. This study has significant implications for understanding cancer progression due to p53 malfunctioning without any loss-of-function mutation or occurrence of transcriptional inactivation. Our data might unlock new possibilities for understanding the disease and will lead to rational design of p53 aggregation inhibitors for the development of drugs against cancer.

Proteins often get misfolded into highly ordered amyloid aggregates that are found to be associated with various neurodegenerative diseases. However, the dynamical events that lead to such self organization of different types of proteins are still poorly understood due to the experimentally untractable complex molecular mechanism, which governs the process of amyloidogenesis. Herein, we propose a generic and data-driven mathematical modeling approach that enables us to decipher a most probable molecular mechanism responsible for amyloidogensis in a context dependent manner. The preferred model efficiently elucidates various aspects of amyloid forming kinetics as a function of initial protein concentration, and qualitatively predicts the dynamics of amyloidogenesis for point mutated proteins. Importantly, the model analysis reveals that intermediate aggregates formed just after the primary necleation steps plays a vital role in dictating the nature of amyloid forming kinetics. Moreover, our model makes experimentally feasible insightful predictions to formulate better therapeutic measures in future to counter unwanted amyloidogenesis by just fine-tuning the molecular interactions related to amyloidogenesis.Author SummaryIn cells, proteins mostly function by being in a native folded conformation. However, under certain circumstances, some of these proteins undergo high degree of aggregation and eventually misfold to form highly stable amyloid aggregates that are often involved in different neurodegenerative disease progression and pathogenesis. To develop appropriate therapeutic strategies to resist such oligomerazation of protein, it is imperative to unravel the molecular interactions that essentially organize the dynamical events of amyloidogenesis. We have proposed a generic computational modeling framework to elucidate the important molecular events that govern the dynamics of amyloid formation. Our model reconciles diverse set of experimental observations including different mutant phenotyes from a dynamical perspective, and predicts possible ways to control the kinetics of amyloid formation experimentally. In future, these insights will be helpful to design drugs to treat patients with neurodegenerative disorders. Importantly, our model, in general, will be quite useful to figure out the important molecular events that are orchestrating the amyloidogenesis of different amyloid forming proteins.

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.

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.

Amyloids are highly ordered, cross-β-sheet-rich protein/peptide aggregates associated with both human diseases and native functions. Given the well established ability of amyloids in interacting with cell membranes, we hypothesize that amyloids can serve as universal cell-adhesive substrates. Here, we show that, similar to the extracellular matrix protein collagen, amyloids of various proteins/peptides support attachment and spreading of cells via robust stimulation of integrin expression and formation of integrin-based focal adhesions. Additionally, amyloid fibrils are also capable of immobilizing non-adherent red blood cells through charge-based interactions. Together, our results indicate that both active and passive mechanisms contribute to adhesion on amyloid fibrils. The present data may delineate the functional aspect of cell adhesion on amyloids by various organisms and its involvement in human diseases. Our results also raise the exciting possibility that cell adhesivity might be a generic property of amyloids.

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.

Parkinson's disease is mainly a sporadic disorder in which both environmental and cellular factors play a major role in the initiation of this disease. Glycosaminoglycans (GAG) are integral components of the extracellular matrix and are known to influence amyloid aggregation of several proteins, including α-synuclein (α-Syn). However, the mechanism by which different GAGs and related biological polymers influence protein aggregation and the structure and intercellular spread of these aggregates remains elusive. In this study, we used three different GAGs and related charged polymers to establish their role in α-Syn aggregation and associated biological activities of these aggregates. Heparin, a representative GAG, affected α-Syn aggregation in a concentration-dependent manner, whereas biphasic α-Syn aggregation kinetics was observed in the presence of chondroitin sulfate B. Of note, as indicated by 2D NMR analysis, different GAGs uniquely modulated α-Syn aggregation because of the diversity of their interactions with soluble α-Syn. Moreover, subtle differences in the GAG backbone structure and charge density significantly altered the properties of the resulting amyloid fibrils. Each GAG/polymer facilitated the formation of morphologically and structurally distinct α-Syn amyloids, which not only displayed variable levels of cytotoxicity but also exhibited an altered ability to internalize into cells. Our study supports the role of GAGs as key modulators in α-Syn amyloid formation, and their distinct activities may regulate amyloidogenesis depending on the type of GAG being up- or down-regulated in vivo.

Graphene-based hybrid nanomaterials have been shown to have great potential in various biotechnology applications including enzyme sensing and bone tissue engineering. Harnessing the unique properties of graphene and material strength of amyloids, a graphene-amyloid composite film is developed that can self-organize into periodic troughs and crests without the need of lithographic techniques or etching. The topographies generated by the film provide powerful modulators of contact and guidance to neural precursor cells, enabling efficient cellular polarization and differentiation. Using molecular dynamic simulations and high-resolution atomic force microscopy, the amyloidogenicity and handedness of matured amyloid nanofibrils to the micropatterns generated on the dried film are also correlated. These insights provide principles for peptide designing for generation of micropatterned, cell adhesive conductive substrates for optimal cell alignment and differentiation.

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.

Amyloid based hydrogels can mimic the extracellular matrix and serve as matrices for tissue engineering both in vitro and in vivo. A pH responsive self-assembled amyloid hydrogel system is used to encapsulate various growth factors for driving stem cell differentiation toward neuronal lineage. Diffusion studies with fluorescence recovery after photobleaching and bulk release with the model protein fluorescein isothiocyanate-bovine serum albumin show that encapsulated protein molecules can be released in a sustained fashion from the hydrogel over a considerable period of time (30 d). Moreover, by modulating the porosity of the hydrogel by the simple addition of salt, the encapsulated protein molecules can be retained for a longer period of time within the hydrogel. Mesenchymal stem cells, when cultured in 3D amyloid hydrogels with growth factors fibroblast growth factor 8 and sonic hedgehog, show more neuron specific differentiation as compared to hydrogel alone. This higher differentiation potential of growth factor encapsulated amyloid hydrogels can be due to concomitant exposure of cells to biomechanical as well as biochemical cues during the course of differentiation. The present study suggests that amyloid based hydrogel can be exploited for controlled growth factor delivery as well as directed stem cell differentiation to neuron.

Loss of p53 function is largely responsible for the occurrence of cancer in humans. Aggregation of mutant p53 has been found in multiple cancer cell types, suggesting a role of aggregation in loss of p53 function and cancer development. The p53 protein has recently been hypothesized to possess a prion-like conformation, although experimental evidence is lacking. Here, we report that human p53 can be inactivated upon exposure to preformed fibrils containing an aggregation-prone sequence-specific peptide, PILTIITL, derived from p53, and the inactive state was found to be stable for many generations. Importantly, we provide evidence of a prion-like transmission of these p53 aggregates. This study has significant implications for understanding cancer progression due to p53 malfunctioning without any loss-of-function mutation or occurrence of transcriptional inactivation. Our data might unlock new possibilities for understanding the disease and will lead to rational design of p53 aggregation inhibitors for the development of drugs against cancer.

Parkinson's disease (PD) is a neurological disorder marked by the presence of cytoplasmic inclusions, Lewy bodies (LBs) and Lewy neurites (LNs) as well as the degeneration of dopamine producing neurons in the substantia nigra region of the brain. The LBs and LNs in PD are mainly composed of aggregated form of a presynaptic protein, alpha-synuclein (alpha-Syn). However, the mechanisms of alpha-Syn aggregation and actual aggregated species responsible for the degeneration of dopaminergic neurons have not yet been resolved. Despite the fact that alpha-Syn aggregation in LBs and LNs is crucial and mutations of alpha-Syn are associated with early onset PD, it is really a challenging task to establish a correlation between alpha-Syn aggregation rate and PD pathogenesis. Regardless of strong genetic contribution, PD is mostly sporadic and familial forms of the disease represent only a minor part (

The storage of protein/peptide hormones within subcellular compartments and subsequent release are crucial for their native function, and hence these processes are intricately regulated in mammalian systems. Several peptide hormones were recently suggested to be stored as amyloids within endocrine secretory granules. This leads to an apparent paradox where storage requires formation of aggregates, and their function requires a supply of non-aggregated peptides on demand. The precise mechanism behind amyloid formation by these hormones and their subsequent release remain an open question. To address this, we examined aggregation and fibril reversibility of a cyclic peptide hormone somatostatin (SST)-14 using various techniques. After proving that SST gets stored as amyloid in vivo, we investigated the role of native structure in modulating its conformational dynamics and self-association by disrupting the disulfide bridge (Cys(3)-Cys(14)) in SST. Using two-dimensional NMR, we resolved the initial structure of somatostatin-14 leading to aggregation and further probed its conformational dynamics in silico. The perturbation in native structure (S-S cleavage) led to a significant increase in conformational flexibility and resulted in rapid amyloid formation. The fibrils formed by disulfide-reduced noncyclic SST possess greater resistance to denaturing conditions with decreased monomer releasing potency. MD simulations reveal marked differences in the intermolecular interactions in SST and noncyclic SST providing plausible explanation for differential aggregation and fibril reversibility observed experimentally in these structural variants. Our findings thus emphasize that subtle changes in the native structure of peptide hormone(s) could alter its conformational dynamics and amyloid formation, which might have significant implications on their reversible storage and secretion.

It has been suggested that conjugated charged polymers are amyloid imaging agents and promising therapeutic candidates for neurological disorders. However, very less is known about their efficacy in modulating the amyloid aggregation pathway. Here, we studied the modulation of Parkinson's disease associated α-synuclein (AS) amyloid assembly kinetics using conjugated polyfluorene polymers (PF, cationic; PFS, anionic). We also explored the complexation of these charged polymers with the various AS aggregated species including amyloid fibrils and oligomers using multidisciplinary biophysical techniques. Our data suggests that both polymers irrespective of their different charges in the side chains increase the fibrilization kinetics of AS and also remarkably change the morphology of the resultant amyloid fibrils. Both polymers were incorporated/aligned onto the AS amyloid fibrils as evident from electron microscopy (EM) and atomic force microscopy (AFM), and the resultant complexes were structurally distinct from their pristine form of both polymers and AS supported by FTIR study. Additionally, we observed that the mechanism of interactions between the polymers with different species of AS aggregates were markedly different.

Pancreastatin (PST), a chromogranin A-derived peptide, is a potent physiological inhibitor of glucose-induced insulin secretion. PST also triggers glycogenolysis in liver and reduces glucose uptake in adipocytes and hepatocytes. Here, we probed for genetic variations in PST sequence and identified two variants within its functionally important carboxyl terminus domain: E287K and G297S. To understand functional implications of these amino acid substitutions, we tested the effects of wild-type (PST-WT), PST-287K, and PST-297S peptides on various cellular processes/events. The rank order of efficacy to inhibit insulin-stimulated glucose uptake was: PST-297S > PST-287K > PST-WT. The PST peptides also displayed the same order of efficacy for enhancing intracellular nitric oxide and Ca2+ levels in various cell types. In addition, PST peptides activated gluconeogenic genes in the following order: PST-297S ≈ PST-287K > PST-WT. Consistent with these in vitro results, the common PST variant allele Ser-297 was associated with significantly higher (by ∼17 mg/dl, as compared with the wild-type Gly-297 allele) plasma glucose level in our study population (n = 410). Molecular modeling and molecular dynamics simulations predicted the following rank order of α-helical content: PST-297S > PST-287K > PST-WT. Corroboratively, circular dichroism analysis of PST peptides revealed significant differences in global structures (e.g. the order of propensity to form α-helix was: PST-297S ≈ PST-287K > PST-WT). This study provides a molecular basis for enhanced potencies/efficacies of human PST variants (likely to occur in ∼300 million people worldwide) and has quantitative implications for inter-individual variations in glucose/insulin homeostasis.

Background: Copper is an essential trace element required for the proper functioning of various enzymes present in the central nervous system. An imbalance in the copper homeostasis results in the pathology of various neurodegenerative disorders including Parkinson's Disease. Hence, residue specific interaction of Cu2+ to alpha-Syn along with the familial mutants H50Q and G51D needs to be studied in detail. Methods: We investigated the residue specific mapping of Cu2+ binding sites and binding strength using solution-state NMR and ITC respectively. The aggregation kinetics, secondary structural changes, and morphology of the formed fibrils in the presence and absence of Cu2+ were studied using fluorescence, CD, and AFM respectively. Results: Copper binding to alpha-Syn takes place at three different sites with a higher affinity for the region 48-53. While one of the sites got abolished in the case of H50Q the mutant G51D showed a binding pattern similar to WT. The aggregation kinetics of these proteins in the presence of Cu2+ showed an enhanced rate of fibril formation with a pronounced effect for G51D. Conclusion: Cu2+ binding results in the destabilization of long-range tertiary interactions in alpha-Syn leading to the exposure of highly amyloidogenic NAC region which results in the increased rate of fibril formation. Although the residues 48-53 have a stronger affinity for Cu2+ in case of WT and G51D, the binding is not responsible for enhancing the rate of fibril formation in case of H50Q. General Significance: These findings will help in the better understanding of Cu2+ catalyzed aggregation of synucleins. (C) 2016 Elsevier B.V. All rights reserved.

Alpha-synuclein (α-Syn) aggregation into oligomers and fibrils is associated with dopaminergic neuron loss occurring in Parkinson’s disease (PD) pathogenesis. Compounds that modulate α-Syn aggregation and interact with preformed fibrils/oligomers and convert them to less toxic species could have promising applications in the drug development efforts against PD. Curcumin is one of the Asian food ingredient which showed promising role as therapeutic agent against many neurological disorders including PD. However, the instability and low solubility makes it less attractive for the drug development. In this work, we selected various curcumin analogs and studied their toxicity, stability and efficacy to interact with different α-Syn species and modulation of their toxicity. We found a subset of curcumin analogs with higher stability and showed that curcumin and its various analogs interact with preformed fibrils and oligomers and accelerate α-Syn aggregation to produce morphologically different amyloid fibrils in vitro. Furthermore, these curcumin analogs showed differential binding with the preformed α-Syn aggregates. The present data suggest the potential role of curcumin analogs in modulating α-Syn aggregation.