p53 amyloid pathology is correlated with higher cancer grade irrespective of the mutant or wild-type form.

p53 (also known as TP53) mutation and amyloid formation are long associated with cancer pathogenesis; however, the direct demonstration of the link between p53 amyloid load and cancer progression is lacking. Using multi-disciplinary techniques and 59 tissues (53 oral and stomach cancer tumor tissue samples from Indian individuals with cancer and six non-cancer oral and stomach tissue samples), we showed that p53 amyloid load and cancer grades are highly correlated. Furthermore, next-generation sequencing (NGS) data suggest that not only mutant p53 (e.g. single-nucleotide variants, deletions, and insertions) but wild-type p53 also formed amyloids either in the nucleus (50%) and/or in the cytoplasm in most cancer tissues. Interestingly, in all these cancer tissues, p53 displays a loss of DNA-binding and transcriptional activities, suggesting that the level of amyloid load correlates with the degree of loss and an increase in cancer grades. The p53 amyloids also sequester higher amounts of the related p63 and p73 (also known as TP63 and TP73, respectively) protein in higher-grade tumor tissues. The data suggest p53 misfolding and/or aggregation, and subsequent amyloid formation, lead to loss of the tumor-suppressive function and the gain of oncogenic function, aggravation of which might determine the cancer grade.

Glycosaminoglycans have variable effects on α-synuclein aggregation and differentially affect the activities of the resulting amyloid fibrils.

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.

Comparison of Kinetics, Toxicity, Oligomer Formation, and Membrane Binding Capacity of α-Synuclein Familial Mutations at the A53 Site, Including the Newly Discovered A53V Mutation.

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: A53T > A53V > WT > A53E. 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.

Amyloid fibril-based thixotropic hydrogels for modeling of tumor spheroids in vitro.

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 a new class of amyloid fibril-based peptide hydrogels as a versatile biomimetic ECM scaffold for 3D cell culture and homogenous tumor spheroid modeling. We show that these amyloid fibril-based hydrogels are thixotropic and allow cancer cell adhesion, proliferation, and migration. All seven designed hydrogels support 3D cell culture with five different cancer cell lines forming spheroid with necrotic core and upregulation of the cancer biomarkers. We further developed the homogenous, single spheroid using the drop cast method and the data suggest that all hydrogels support the tumor spheroid formation but with different necrotic core diameters. The detailed gene expression analysis of MCF7 spheroid by microarray suggested the involvement of pro-oncogenes and significant regulatory pathways responsible for tumor spheroid formation. Further, using breast tumor tissue from a mouse xenograft model, we show that selected amyloid 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 an easy-to-use, rapid, cost-effective, and scalable platform for generating in vitro cancer models for the screening of anti-cancer therapeutics and developing personalized medicine.

Intermolecular interactions underlie protein/peptide phase separation irrespective of sequence and structure at crowded milieu.

Liquid-liquid phase separation (LLPS) has emerged as a crucial biological phenomenon underlying the sequestration of 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 but with distinct phase regimes irrespective of their sequence and structure. To examine this, we studied many (a total of 23) proteins/polypeptides with different structures and sequences for LLPS study in the presence and absence of 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 regimes and intermolecular interactions. We further demonstrated that electrostatic, hydrophobic, and H-bonding or a combination of such intermolecular interactions plays a crucial role in individual protein/peptide LLPS.

Direct Demonstration of Seed Size-Dependent α-Synuclein Amyloid Amplification.

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 in vitro reconstitution, we showed that the size of α-Syn fibril seeds dictates not only their 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 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 seeds of different sizes determines the underlying pathological outcome of synucleinopathies.

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

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.

Effect of Disease-Associated P123H and V70M Mutations on ß-Synuclein Fibrillation.

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. ß-Synuclein (ß-Syn), an isoform of α-Syn, inhibits α-Syn aggregation and prevents its neurotoxicity, suggesting the neuroprotective nature of ß-Syn. However, this notion changed with the discovery of disease-associated ß-Syn mutations, V70M and P123H, in patients with DLB. It is still unclear how these missense mutations alter the structural and amyloidogenic properties of ß-Syn, leading to neurodegeneration. Here, we characterized the biophysical properties and investigated the effect of mutations on ß-Syn fibrillation under different conditions. V70M and P123H show high membrane binding affinity compared to wild-type ß-Syn, suggesting their potential role in membrane interactions. ß-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 ß-Syn under these conditions. NMR study further revealed that these point mutations induce local perturbations at the site of mutation in ß-Syn. Overall, our data provide insight into the biophysical properties of disease-associated ß-Syn mutations and demonstrate that these mutants make the native protein more susceptible to aggregation in an altered microenvironment.

α-Synuclein aggregation nucleates through liquid-liquid phase separation.

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.

Amyloid Fibrils with Positive Charge Enhance Retroviral Transduction in Mammalian Cells.

Amyloid fibrils are cross-ß-sheet-rich protein/peptide fibrils that are typically associated with neurodegenerative diseases such as Parkinson's and Alzheimer's disease. Recently, functional amyloids have been discovered where amyloids are implicated in performing normal physiological functions of the host organism rather than creating diseases. The ability of amyloids to interact with the cell membrane and other small biomolecules exhibits its great potential to be used as a biomaterial for cell adhesion and gene delivery system. Given the established ability of semen-derived amyloids to concentrate HIV in semen and that of charged polymers as an enhancer of retroviral gene transfer, we hypothesized that charged amyloid fibrils can augment virus-mediated delivery system. We show that amyloids of α-synuclein formed in the presence and absence of cationic polymers chitosan and amyloid of poly-l-lysine can interact with lentiviral particles and enhance transduction efficiency in cells. The amyloid nanofibrils increase transduction efficiency up to ∼4 fold similar to widely used cationic polymer Polybrene. This study shows that amyloid nanofibril scaffolds may be used as targeted gene delivery systems.