Formation Of Amyloid Aggregates Research Articles

Distinct Modes of Action of IAPP Oligomers on Membranes.

Islet amyloid polypeptide (IAPP, also known as amylin) is a peptide hormone that is co-secreted with insulin by pancreatic β-cells and forms amyloid aggregates in type II diabetes. Various lines of evidence indicate that oligomers of this peptide may induce toxicity by disrupting or forming pores in cell membranes, but the structure of these pores is unknown. Here, we create models of pores for both helical and β-structured peptides using implicit membrane modeling and test their stability using multimicrosecond all-atom simulations. We find that the helical peptides behave similarly to antimicrobial peptides; they remain stably inserted in a highly tilted or partially unfolded configuration creating a narrow water channel. Parallel helix orientation creates a somewhat larger pore. An octameric β barrel of parallel β-hairpins is highly stable in the membrane, whereas the corresponding barrel made of antiparallel hairpins is not. We propose that certain experiments probe the helical pore state while others probe the β-structured pore state; this provides a possible explanation for lack of correlation that is sometimes observed between in vivo toxicity and in vitro liposome permeabilization experiments.

Elevated IgG Antibody to Aluminum Bound to Human Serum Albumin in Patients with Crohn’s, Celiac and Alzheimer’s Disease

Aluminum is in our water and food, and is used as an adjuvant in vaccines. About 40% of the ingested dose accumulates within the intestinal mucosa, making the gut the main target of inflammation and autoimmunity; about 1% accumulates in the skeletal system and brain, inducing the cross-linking of amyloid-β-42 peptide and the formation of amyloid aggregates associated with Alzheimer’s disease. To examine whether the accumulation of aluminum in the gut and brain tissues results in neoantigen formation, we bound aluminum compounds to human serum albumin. We used ELISA to measure IgG antibody in 94 different sera from healthy controls and 47 sera from each group of patients: anti-Saccharomyces cerevisiae antibody-positive (Crohn’s), and positive for deamidated α-gliadin and transglutaminase-2 IgA antibodies (celiac disease), autoimmune disorders associated with intestinal tissue antigens. Because earlier studies have shown that aluminum exposure is linked to Alzheimer’s disease etiology, and high aluminum content is detected in Alzheimer’s patients’ brain tissue, we also measured aluminum antibody in the blood of these patients. Additionally, we measured aluminum antibody in the sera of mixed connective tissue disease patients who were positive for antinuclear antibodies, and used them as disease controls. We found significant IgG antibody elevation against all three aluminum compounds in the sera of patients with Crohn’s, celiac and Alzheimer’s disease, but not in patients with mixed connective tissue disease. We concluded that aluminum ingestion and absorption from the GI tract and brain may contribute to Crohn’s, celiac and Alzheimer’s disease, but not to mixed connective tissue disease.

Purification and characterization of an amyloidogenic repeat domain from the functional amyloid Pmel17

The pre-melanosomal protein (Pmel17) is a human functional amyloid that supports melanin biosynthesis within melanocytes. This occurs in the melanosome, a membrane-bound organelle with an acidic intraluminal pH. The repeat region of Pmel17 (RPT, residues 315–444) has been previously shown to form amyloid aggregates under acidic melanosomal conditions, but not under neutral cytosolic conditions, when expressed and purified using a C-terminal hexa-histidine tag (RPT-His). Given the importance of protonation states in RPT-His aggregation, we questioned whether the histidine tag influenced the pH-dependent behavior. In this report, we generated a tagless RPT by inserting a tobacco etch virus (TEV) protease recognition sequence (ENLYGQ(G/S)) immediately upstream of a native glycine residue at position 312 in Pmel17. After purification of the fusion construct using a histidine tag, cleavage with TEV protease generated a fully native RPT (nRPT) spanning resides 312–444. We characterized the aggregation of nRPT, which formed amyloid fibrils under acidic conditions (pH ≤ 6) but not at neutral pH. Characterizing the morphologies of nRPT aggregates using transmission electron microscopy revealed a pH-dependent maturation from short, curved structures at pH 4 to paired, rod-like fibrils at pH 6. This was accompanied by a secondary structural transition from mixed random coil/β-sheet at pH 4 to canonical β-sheet at pH 6. We also show that pre-formed nRPT fibrils undergo disaggregation upon dilution into pH 7 buffer. More broadly, this strategy can be utilized to generate native amyloidogenic domains from larger proteins by utilizing intrinsic N-terminal glycine or serine residues.

Insulin Fibril Formation Caused by Mechanical Shock and Cavitation.

Cavitation can occur when liquids are exposed to pressure waves of sufficient amplitude, producing rapidly expanding and collapsing gas bubbles that generate localized regions of high energy dissipation. When vials containing insulin were subjected to mechanical shock or when ultrasound was applied to the vials, the resulting cavitation events induced formation of insulin amyloid fibril nuclei that were detected by transmission electron microscopy and quantified by fluorescence spectroscopy following staining with the amyloid-sensitive dye thioflavin-T. Dropping insulin solutions in glass vials produced only minute amounts of insulin fibril nuclei, which could be detected by allowing the nuclei to grow. Cavitation-induced formation of amyloid aggregates may be relevant for iatrogenic insulin deposition disease, where insulin fibrils formed in vitro prior to administration to patients could serve as nuclei for growing fibril deposits in vivo.

Amyloid Structural Changes Studied by Infrared Microspectroscopy in Bigenic Cellular Models of Alzheimer's Disease.

Alzheimer’s disease affects millions of lives worldwide. This terminal disease is characterized by the formation of amyloid aggregates, so-called amyloid oligomers. These oligomers are composed of β-sheet structures, which are believed to be neurotoxic. However, the actual secondary structure that contributes most to neurotoxicity remains unknown. This lack of knowledge is due to the challenging nature of characterizing the secondary structure of amyloids in cells. To overcome this and investigate the molecular changes in proteins directly in cells, we used synchrotron-based infrared microspectroscopy, a label-free and non-destructive technique available for in situ molecular imaging, to detect structural changes in proteins and lipids. Specifically, we evaluated the formation of β-sheet structures in different monogenic and bigenic cellular models of Alzheimer’s disease that we generated for this study. We report on the possibility to discern different amyloid signatures directly in cells using infrared microspectroscopy and demonstrate that bigenic (amyloid-β, α-synuclein) and (amyloid-β, Tau) neuron-like cells display changes in β-sheet load. Altogether, our findings support the notion that different molecular mechanisms of amyloid aggregation, as opposed to a common mechanism, are triggered by the specific cellular environment and, therefore, that various mechanisms lead to the development of Alzheimer’s disease.

Membrane-dependent amyloid aggregation of human BAX α9 (173-192).

Mitochondrial outer membrane permeabilization, which is a critical step in apoptosis, is initiated upon transmembrane insertion of the C-terminal α-helix (α9) of the proapoptotic Bcl-2 family protein BAX. The isolated α9 fragment (residues 173-192) is also competent to disrupt model membranes, and the structures of its membrane-associated oligomers are of interest in understanding the potential roles of this sequence in apoptosis. Here, we used ultrafast two-dimensional infrared (2D IR) spectroscopy, thioflavin T binding, and transmission electron microscopy to show that the synthetic BAX α9 peptide (α9p) forms amyloid aggregates in aqueous environments and on the surfaces of anionic small unilamellar vesicles. Its inherent amyloidogenicity was predicted by sequence analysis, and 2D IR spectra reveal that vesicles modulate the β-sheet structures of insoluble aggregates, motivating further examination of the formation or suppression of BAX amyloids in apoptosis.

Amyloid Self-Assembly of Lysozyme in Self-Crowded Conditions: The Formation of a Protein Oligomer Hydrogel.

A method is designed to quickly form protein hydrogels, based on the self-assembly of highly concentrated lysozyme solutions in acidic conditions. Their properties can be easily modulated by selecting the curing temperature. Molecular insights on the gelation pathway, derived by in situ FTIR spectroscopy, are related to calorimetric and rheological results, providing a consistent picture on structure–property correlations. In these self-crowded samples, the thermal unfolding induces the rapid formation of amyloid aggregates, leading to temperature-dependent quasi-stationary levels of antiparallel cross β-sheet links, attributed to kinetically trapped oligomers. Upon subsequent cooling, thermoreversible hydrogels develop by the formation of interoligomer contacts. Through heating/cooling cycles, the starting solutions can be largely recovered back, due to oligomer-to-monomer dissociation and refolding. Overall, transparent protein hydrogels can be easily formed in self-crowding conditions and their properties explained, considering the formation of interconnected amyloid oligomers. This type of biomaterial might be relevant in different fields, along with analogous systems of a fibrillar nature more commonly considered.

In Cellulo Analysis of Huntingtin Inclusion Bodies by Cryogenic Nanoprobe SAXS

AbstractHuntington's disease (HD) is one of nine neurodegenerative disorders associated with an extension of polyglutamine (polyQ) in proteins. In HD, the polyQ tract in the huntingtin protein is extended beyond a threshold of 38 amino acids leading to the formation of amyloidal structures in the cytoplasm and nucleus. We investigated here the structure of Htt (Huntingtin) amyloid fibrils in cellulo with nanoprobe small angle X‐ray scattering. As these measurements were performed under cryogenic conditions, the information is obtained on the aggregates in their natural, hydrated environment without the need of staining and chemical fixation. We also could show the presence of oligomer structures not visible in fluorescence microscopy. Structural information on repetitive units inside of Htt inclusion bodies was determined from the SAXS data and compared to electron microscopy images. The results suggest that nanoprobe cryo‐SAXS can serve as powerful tool to investigate the kinetics of amyloid aggregate formation inside cells and to understand how fibril formation can be influenced by drugs and other external stimuli.

P53, A Victim of the Prion Fashion.

Simple SummaryThe tumor suppressor gene TP53 is found mutated in around half of human cancers. The accumulation of the mutant p53 protein in the form of aggregates in cancer cells have led to the emergence of the “prion p53” hypothesis which states that mutant p53 is able to drive the wild-type form of the protein into an alternate conformation, thereby contributing to tumor progression. This report challenges the “prion p53” hypothesis by reviewing evidence of p53 behavior in light of our current knowledge regarding amyloid proteins, prionoids and prions.Identified in the late 1970s as an oncogene, a driving force leading to tumor development, p53 turned out to be a key tumor suppressor gene. Now p53 is considered a master gene regulating the transcription of over 3000 target genes and controlling a remarkable number of cellular functions. The elevated prevalence of p53 mutations in human cancers has led to a recurring questioning about the roles of mutant p53 proteins and their functional consequences. Both mutants and isoforms of p53 have been attributed dominant-negative and gain of function properties among which is the ability to form amyloid aggregates and behave in a prion-like manner. This report challenges the ongoing “prion p53” hypothesis by reviewing evidence of p53 behavior in light of our current knowledge regarding amyloid proteins, prionoids and prions.

Distinct impact of glycation towards the aggregation and toxicity of murine and human amyloid-β.

Glycation of human Aβ (hAβ) is implicated to induce the deposition of amyloid aggregates found in the Alzheimer's disease (AD)-affected brain. Murine Aβ (mAβ) differs from hAβ in three different amino acid residues (Gly5, Phe10, and Arg13) and is less likely to form amyloid aggregates. Herein, we report that the advanced glycated end products of mAβ40 over hAβ40 are distinctly generated. The different glycation between the two peptides can govern their aggregation kinetics, structural transition, and cytotoxicity.

Inhibition of human amylin aggregation by Flavonoid Chrysin: An in-silico and in-vitro approach.

Islet amyloid polypeptide (amylin), consecrated by the pancreatic β-cells with insulin, has a significant role to play in maintaining homeostasis of islet cell hormones. Alzheimer's disease is the predominant source of dementia. However, its etiology remains uncertain; it appears that type 2 diabetes mellitus and other prediabetic states of insulin resistance contribute to the intermittent Alzheimer's disease presence. Amylin is abnormally elevated in Type II diabetes patients, accumulated into amylin aggregates, and ultimately causes apoptosis of the β-cells, and till date, its mechanism remains unclear. Several flavonoids have inhibitory effects on amylin amyloidosis, but its inhibition mechanisms are unknown. Screening a collection of traditional compounds revealed the flavone Chrysin, a potential lead compound. Chrysin inhibits amyloid aggregate formation according to Thioflavin T binding, turbidimetry assay. We report results of molecular interaction analysis of Chrysin with amylin which shows potent binding affinity against amylin. Pharmacokinetics and Drug likeness studies of Chrysin also suggest that it is a potential lead compound. Therefore, Chrysin prevented amylin aggregation.

The possible effect of silver nanoparticles on glyceraldehyde‐3‐phosphate dehydrogenase activity and formation of amyloid‐like aggregates in MCF‐7 cell line

Silver nanoparticles (AgNPs) are widely used in medicine, however, the underlying mechanisms of their action on cellular signaling have not been completely determined, and fundamental studies are required to clarify them. We aimed to investigate AgNPs effects on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as both the internal control gene and the redox-sensitive enzyme involved in apoptosis-related pathways and the formation of amyloid aggregates. To achieve this purpose, MCF-7 cells were treated with different concentrations (0, 3, 22, and 200 μg/ml) of AgNPs and then cell viability, generation of reactive oxygen species (ROS), induction of apoptosis, expression of GAPDH gene, the formation of amyloid aggregates, and GAPDH activity were assessed. The results indicated that treatment with AgNPs significantly reduced cell viability and increased apoptosis in a dose-dependent manner. The ROS levels increased at lower concentrations of AgNPs (up to 22 μg/ml) and during short-term exposure (30 min). The level of GAPDH gene expression was significantly upregulated by 1.22, 1.47, and 1.56 fold, in the concentrations of 3, 22, and 200 μg/ml, respectively. The amount of amyloid aggregates was significantly increased in a dose-dependent manner. The results of enzyme activity showed that AgNPs were affected on the activity of GAPDH protein, however, it has fluctuated that could not be interpreted by our limited data. In conclusion, our results suggested that AgNPs could affect the GAPDH gene expression and enzyme activity, therefore the selection of GAPDH as a gene and protein internal control in the (AgNPs)-related studies requires careful consideration. Additionally, AgNPs may cause apoptosis due to the increase in the production of amyloid aggregates.

Amyloid aggregation of spin-labeled β-lactoglobulin. Part I: Influence of spin labeling on amyloid aggregation

Site-directed spin labeling (SDSL) of the natural food protein β-lactoglobulin (β-lg) was established with the aim of characterizing amyloid aggregation while explicitly avoiding the usual manipulation of primary protein structure. For its successful application, spin labels must not alter secondary protein structure or the formation of β-lg amyloid aggregates. The two spin labels—the MTSSL (flexible S–S binding) and Iodoacetamido-proxyl spin label (IPSL) (more rigid C–S binding)—were used for amyloid aggregation at pH 2 and pH 3.5. At pH 3.5, IPSL caused minor changes in the secondary protein structure, where it reduced intra- and intermolecular β-sheets as determined by ATR-FTIR. Analysis of the extent of amyloid aggregation using thioflavin T fluorescence indicated that the spin probes interfered with the binding of the fluorescent probes to the β-sheets. Non-amyloid and amyloid fractions were obtained from the amyloid aggregated system by ultrafiltration (300 kDa), which also proved to be equivalent and independent of the spin labeling process. Atomic force microscopy and size-exclusion chromatography results suggest the same building blocks and morphologies between unlabeled and spin-labeled proteins; therefore, substantial changes in the behavior of β-lg during amyloid aggregation can be excluded. Ultimately, 10–17% of the β-lg molecules were labeled so that the SDSL approach can be used to label the natural food protein at these low concentrations without affecting protein conformation or the formation of amyloid aggregates, which may subsequently provide deep insights into the aggregation mechanism of food proteins under processing conditions.

An amyloidogenic hexapeptide from the cataract-associated γD-crystallin is a model for the full-length protein and is inhibited by naphthoquinone-tryptophan hybrids

The eye lens is rich in proteins called crystallins, whose native conformation is crucial for preserving its transparency. With aging, crystallins may be exposed to environmental changes, which could lead to their aggregation and eventually to cataract development. Human γD-crystallin, among the most abundantly expressed γ-crystallins in the lens, was shown to form amyloid aggregates under denaturing conditions in vitro. However, the exact mechanism of aggregation remains to be clearly defined. Here, using prediction algorithms and biophysical methods, we identified a hexapeptide 41GCWMLY46 as a most aggregative fragment in human γD-crystallin. Two aromatic naphthoquinone-tryptophan hybrid molecules (NQTrp and Cl-NQTrp), inhibited the in vitro aggregation of this hexapeptide as well as full-length γD-crystallin in a dose-dependent manner, plausibly facilitated by hydrogen bonding and aromatic contacts with the hydrophobic residues. The two compounds had no toxic effect toward retinal cell culture and reduced the cytotoxicity induced by aggregates of the hexapeptide. In addition, NQTrp and Cl-NQTrp were able to disassemble pre-formed aggregates of the hexapeptide and the full-length γD-crystallin. Our results indicate that the amyloidogenic hexapeptide is a useful model for screening inhibitors of γD-crystallin and that the NQTrp hybrid scaffolds may serve as leads for developing new drugs for treating cataract.

The Impact of Natural Compounds on S-Shaped Aβ42 Fibril: From Molecular Docking to Biophysical Characterization.

The pursuit for effective strategies inhibiting the amyloidogenic process in neurodegenerative disorders, such as Alzheimer’s disease (AD), remains one of the main unsolved issues, and only a few drugs have demonstrated to delay the degeneration of the cognitive system. Moreover, most therapies induce severe side effects and are not effective at all stages of the illness. The need to find novel and reliable drugs appears therefore of primary importance. In this context, natural compounds have shown interesting beneficial effects on the onset and progression of neurodegenerative diseases, exhibiting a great inhibitory activity on the formation of amyloid aggregates and proving to be effective in many preclinical and clinical studies. However, their inhibitory mechanism is still unclear. In this work, ensemble docking and molecular dynamics simulations on S-shaped Aβ42 fibrils have been carried out to evaluate the influence of several natural compounds on amyloid conformational behaviour. A deep understanding of the interaction mechanisms between natural compounds and Aβ aggregates may play a key role to pave the way for design, discovery and optimization strategies toward an efficient destabilization of toxic amyloid assemblies.

Probing the fibrillation of lysozyme by nanoscale-infrared spectroscopy

Amyloid fibrillation is the root cause of several neuro as well as non-neurological disorders. Understanding the molecular basis of amyloid aggregate formation is crucial for deciphering various neurodegenerative diseases. In our study, we have examined the lysozyme fibrillation process using nano-infrared spectroscopy (nanoIR). NanoIR enabled us to investigate both structural and chemical characteristics of lysozyme fibrillar species concurrently. The spectroscopic results indicate that lysozyme transformed into a fibrillar structure having mainly parallel β-sheets, with almost no antiparallel β-sheets. Features such as protein stiffness have a good correlation with obtained secondary structural information showing the state of the protein within the fibrillation state. The structural and chemical details were compared with transmission electron microscopy (TEM) and circular dichroism (CD). We have utilized nanoIR and measured infrared spectra to characterize lysozyme amyloid fibril structures in terms of morphology, molecular structure, secondary structure content, stability, and size of the cross-β core. We have shown that the use of nanoIR can complement other biophysical studies to analyze the aggregation process and is particularly useful for studying proteins involved in aggregation to help in designing molecules against amyloid aggregation. Specifically, the nanoIR spectra afford higher resolution information and a characteristic fingerprint for determining states of aggregation. Communicated by Ramaswamy H. Sarma

Understanding the self-assembly pathways of a single chain variant of monellin: A first step towards the design of sweet nanomaterials

Peptides and proteins possess an inherent tendency to self-assemble, prompting the formation of amyloid aggregates from their soluble and functional states. Amyloids are linked to many devastating diseases, but self-assembling proteins can also represent formidable tools to produce new and sustainable biomaterials for biomedical and biotechnological applications. The mechanism of fibrillar aggregation, which influences the morphology and the properties of the protein aggregates, depend on factors such as pH, ionic strength, temperature, agitation, and protein concentration. We have here used intensive mechanical agitation, with or without beads, to prompt the aggregation of the single-chain derivative of the plant protein monellin, named MNEI, which is a well characterized sweet protein. Transmission electron microscopy confirmed the formation of fibrils several micrometers long, morphologically different from the previously characterized fibers of MNEI. Changes in the protein secondary structures during the aggregation process were monitored by Fourier transform infrared spectroscopy, which detected differences in the conformation of the final aggregates obtained under mechanical agitation. Moreover, soluble oligomers could be detected in the early phases of aggregation by polyacrylamide gel-electrophoresis. These findings emphasize the existence of multiple pathways of fibrillar aggregation for MNEI, which could be exploited for the design of innovative protein-based biomaterials.

Oligomers Are Promising Targets for Drug Development in the Treatment of Proteinopathies.

Currently, there is no effective treatment of proteinopathies, as well as their diagnosis in the early stages of the disease until the first clinical symptoms appear. The proposed model of fibrillation of the Aβ peptide and its fragments not only describes molecular rearrangements, but also offers models of processes that occur during the formation of amyloid aggregates. Since this model is also characteristic of other proteins and peptides, a new potential target for drug development in the treatment of Alzheimer’s disease (AD) and other proteinopathies is proposed on the basis of this model. In our opinion, it is oligomers that are promising targets for innovative developments in the treatment of these diseases.

Probing structural changes during amyloid aggregation of the sweet protein MNEI.

Protein self-assembly is a ubiquitous phenomenon, traditionally studied for its links to amyloid pathologies, which has also gained attention as its physiological roles and possible biotechnological applications emerged over time. It is also known that varying the conditions to which proteins are exposed can lead to aggregate polymorphism. To understand the factors that trigger aggregation and/or direct it toward specific outcomes, we performed a multifaceted structural characterization of the thermally induced self-assembly process of MNEI, a model protein able to form amyloid aggregates under nondenaturing conditions. MNEI is also known for its extreme sweetness which, combined with a considerable thermal stability, makes the protein a promising alternative sweetener. Fourier-transformed infrared spectroscopy and electron microscopy data showed that the presence of NaCl accelerates the kinetics of fibrillar aggregation, while disfavoring the population of off-pathway states that are instead detected by native gel electrophoresis at low ionic strength. NMR studies revealed how NaCl modulates the self-assembling mechanism of MNEI, switching the process from soluble oligomeric forms to fibrils. Comparative analysis demonstrated that the presence of NaCl induces local differences in the protein dynamics and surface accessibility, without altering the native fold. We identified the regions most affected by the presence of NaCl, which control the aggregation process, and represent hot spots on the protein surface for the rational design of new mutants with controlled aggregation propensity.

Binding and Reactivity of Copper to R1 and R3 Fragments of tau Protein.

Tau protein is present in significant amounts in neurons, where it contributes to the stabilization of microtubules. Insoluble neurofibrillary tangles of tau are associated with several neurological disorders known as tauopathies, among which is Alzheimer's disease. In neurons, tau binds tubulin through its microtubule binding domain which comprises four imperfect repeats (R1-R4). The histidine residues contained in these fragments are potential binding sites for metal ions and are located close to the regions that drive the formation of amyloid aggregates of tau. In this study, we present a detailed characterization through potentiometric and spectroscopic methods of the binding of copper in both oxidation states to R1 and R3 peptides, which contain one and two histidine residues, respectively. We also evaluate how the redox cycling of copper bound to tau peptides can mediate oxidation that can potentially target exogenous substrates such as neuronal catecholamines. The resulting quinone oxidation products undergo oligomerization and can competitively give post-translational peptide modifications yielding catechol adducts at amino acid residues. The presence of His-His tandem in the R3 peptide strongly influences both the binding of copper and the reactivity of the resulting copper complex. In particular, the presence of the two adjacent histidines makes the copper(I) binding to R3 much stronger than in R1. The copper-R3 complex is also much more active than the copper-R1 complex in promoting oxidative reactions, indicating that the two neighboring histidines activate copper as a catalyst in molecular oxygen activation reactions.