Process Of Amyloid Formation Research Articles

On the importance of being amidated: Analysis of the role of the conserved C-terminal amide of amylin in amyloid formation and cytotoxicity

The polypeptide hormone Amylin (also known as islet amyloid polypeptide) plays a role in regulation of glucose metabolism, but forms pancreatic islet amyloid deposits in type 2 diabetes. The process of islet amyloid formation contributes to β-cell dysfunction and the development of the disease. Amylin is produced as a pro-from and undergoes processing prior to secretion. The mature hormone contains an amidated C-terminus. Analysis of an alignment of vertebrate amylin sequences reveals that the processing signal for amidation is strictly conserved. Furthermore, the enzyme responsible for C-terminal amidation is found in all of these organisms. Comparison of the physiologically relevant amidated form to a variant with a free C-terminus (Amylin-COO−) shows that replacement of the C-terminal amide with a carboxylate slows, but does not prevent amyloid formation. Pre-fibrillar species produced by both variants are toxic to cultured β-cells, although hAmylin-COO− is moderately less so. Amyloid fibrils produced by either peptide are not toxic. Prior work (ACS Pharmacol. Translational. Sci. 1, 132–49 (2018)) shows that Amylin- COO− exhibits a 58-fold reduction in activation of the Amylin1 receptor and 20-fold reduction in activation of the Amylin3 receptor. Thus, hAmylin-COO− exhibits significant toxicity, but significantly reduced activity and offers a reagent for studies which aim to decouple hAmylin's toxic effects from its activity. The different behaviours of free and C-terminal amidated Amylin should be considered when designing systems to produce the polypeptide recombinantly.

Modeling transthyretin (TTR) amyloid diseases, from monomer to amyloid fibrils.

ATTR amyloidosis is caused by deposition of large, insoluble aggregates (amyloid fibrils) of cross-β-sheet TTR protein molecules on the intercellular surfaces of tissues. The process of amyloid formation from monomeric TTR protein molecules to amyloid deposits has not been fully characterized and is therefore modeled in this paper. Two models are considered: 1) TTR monomers in the blood spontaneously fold into a β-sheet conformation, aggregate into short proto-fibrils that then circulate in the blood until they find a complementary tissue where the proto-fibrils accumulate to form the large, insoluble amyloid fibrils found in affected tissues. 2) TTR monomers in the native or β-sheet conformation circulate in the blood until they find a tissue binding site and deposit in the tissue or tissues forming amyloid deposits in situ. These models only differ on where the selection for β-sheet complementarity occurs, in the blood where wt-wt, wt-v, and v-v interactions determine selectivity, or on the tissue surface where tissue-wt and tissure-v interactions also determine selectivity. Statistical modeling in both cases thus involves selectivity in fibril aggregation and tissue binding. Because binding of protein molecules into fibrils and binding of fibrils to tissues occurs through multiple weak non-covalent bonds, strong complementarity between β-sheet molecules and between fibrils and tissues is required to explain the insolubility and tissue selectivity of ATTR amyloidosis. Observation of differing tissue selectivity and thence disease phenotypes from either pure wildtype TTR protein or a mix of wildtype and variant molecules in amyloid fibrils evidences the requirement for fibril-tissue complementarity. Understanding the process that forms fibrils and binds fibrils to tissues may lead to new possibilities for interrupting the process and preventing or curing ATTR amyloidosis.

Spider Silk Protein Forms Amyloid-Like Nanofibrils through a Non-Nucleation-Dependent Polymerization Mechanism.

Amyloid fibrils-nanoscale fibrillar aggregates with high levels of order-are pathogenic in some today incurable human diseases; however, there are also many physiologically functioning amyloids in nature. The process of amyloid formation is typically nucleation-elongation-dependent, as exemplified by the pathogenic amyloid-β peptide (Aβ) that is associated with Alzheimer's disease. Spider silk, one of the toughest biomaterials, shares characteristics with amyloid. In this study, it is shown that forming amyloid-like nanofibrils is an inherent property preserved by various spider silk proteins (spidroins). Both spidroins and Aβ capped by spidroin N- and C-terminal domains, can assemble into macroscopic spider silk-like fibers that consist of straight nanofibrils parallel to the fiber axis as observed in native spider silk. While Aβ forms amyloid nanofibrils through a nucleation-dependent pathway and exhibits strong cytotoxicity and seeding effects, spidroins spontaneously and rapidly form amyloid-like nanofibrils via a non-nucleation-dependent polymerization pathway that involves lateral packing of fibrils. Spidroin nanofibrils share amyloid-like properties but lack strong cytotoxicity and the ability to self-seed or cross-seed human amyloidogenic peptides. These results suggest that spidroins´ unique primary structures have evolved to allow functional properties of amyloid, and at the same time direct their fibrillization pathways to avoid formation of cytotoxic intermediates.

Extent of N-Terminus Folding of Semenogelin 1 Cleavage Product Determines Tendency to Amyloid Formation.

It is known that four peptide fragments of predominant protein in human semen Semenogelin 1 (SEM1) (SEM1(86-107), SEM1(68-107), SEM1(49-107) and SEM1(45-107)) are involved in fertilization and amyloid formation processes. In this work, the structure and dynamic behavior of SEM1(45-107) and SEM1(49-107) peptides and their N-domains were described. According to ThT fluorescence spectroscopy data, it was shown that the amyloid formation of SEM1(45-107) starts immediately after purification, which is not observed for SEM1(49-107). Seeing that the peptide amino acid sequence of SEM1(45-107) differs from SEM1(49-107) only by the presence of four additional amino acid residues in the N domain, these domains of both peptides were obtained via solid-phase synthesis and the difference in their dynamics and structure was investigated. SEM1(45-67) and SEM1(49-67) showed no principal difference in dynamic behavior in water solution. Furthermore, we obtained mostly disordered structures of SEM1(45-67) and SEM1(49-67). However, SEM1(45-67) contains a helix (E58-K60) and helix-like (S49-Q51) fragments. These helical fragments may rearrange into β-strands during amyloid formation process. Thus, the difference in full-length peptides' (SEM1(45-107) and SEM1(49-107)) amyloid-forming behavior may be explained by the presence of a structured helix at the SEM1(45-107) N-terminus, which contributes to an increased rate of amyloid formation.

Impact of Ca2+ on membrane catalyzed IAPP amyloid formation and IAPP induced vesicle leakage

Human islet amyloid polypeptide (hIAPP, also known as amylin) is a 37 amino acid pancreatic polypeptide hormone that plays a role in regulating glucose levels, but forms pancreatic amyloid in type-2 diabetes. The process of amyloid formation by hIAPP contributes to β-cell death in the disease. Multiple mechanisms of hIAPP induced toxicity of β-cells have been proposed including disruption of cellular membranes. However, the nature of hIAPP membrane interactions and the effect of ions and other molecules on hIAPP membrane interactions are not fully understood. Many studies have used model membranes with a high content of anionic lipids, often POPS, however the concentration of anionic lipids in the β-cell plasma membrane is low. Here we study the concentration dependent effect of Ca2+ (0 to 50 mM) on hIAPP membrane interactions using large unilamellar vesicles (LUVs) with anionic lipid content ranging from 0 to 50 mol%. We find that Ca2+ does not effectively inhibit hIAPP amyloid formation and hIAPP induced membrane leakage from binary LUVs with a low percentage of POPS, but has a greater effect on LUVs with a high percentage of POPS. Mg2+ had very similar effects, and the effects of Ca2+ and Mg2+ can be largely rationalized by the neutralization of POPS charge. The implications for hIAPP-membrane interactions are discussed.

Crossroads between copper ions and amyloid formation in Parkinson's disease.

Copper (Cu) ion dys-homeostasis and α-synclein amyloid deposits are two hallmarks of Parkinson's disease (PD). Here, I will discuss the connections between these features, with a major focus on the role of Cu in the α-synuclein (aS) amyloid formation process. The structurally disordered aS monomer can bind to both redox states of Cu (i.e., oxidized Cu(II) and reduced Cu(I)) with high affinity in vitro. Notably, the presence of Cu(II) (in absence of aS N-terminal acetylation) and Cu(I) (when in complex with the copper chaperone Atox1) modulate aS assembly into β-structured amyloids in opposite directions in vitro. Albeit the link to biological relevance is not fully unraveled, existing observations clearly emphasize the need for more knowledge on this interplay and its consequences to eventually combat destructive reactions that promote PD.

Relationship between Changes in the Protein Folding Pathway and the Process of Amyloid Formation: The Case of Bovine Carbonic Anhydrase II

Many proteins form amyloid fibrils only under conditions when the probability of transition from a native (structured, densely packed) to an intermediate (labile, destabilized) state is increased. It implies the assumption that some structural intermediates are more convenient for amyloid formation than the others. Hence, if a mutation affects the protein folding pathway, one should expect that this mutation could affect the rate of amyloid formation as well. In the current work, we have compared the effects of amino acid substitutions of bovine carbonic anhydrase II on its unfolding pathway and on its ability to form amyloids at acidic pH and an elevated temperature. Wild-type protein and four mutant forms (L78A, L139A, I208A, and M239A) were studied. We analyzed the change of the protein unfolding pathway by the time-resolved fluorescence technique and the process of amyloid formation by thioflavin T fluorescence assay and electron microscopy. It was revealed that I208A substitution accelerates amyloid formation and affects the structure of the late (molten globule-like)-intermediate state of carbonic anhydrase, whereas the other mutations slow down the growth of amyloids and have either no effect on the unfolding pathway (L78A, L139A) or alter the conformational states arising at the early unfolding stage (M239A).

Differential Effects of Aromatic Residues on Amyloid Formation and Cytotoxicity of Human IAPP.

Islet amyloid polypeptide (IAPP) is a 37-residue polypeptide hormone secreted by the pancreatic β-cells. IAPP plays a role in glycemic regulation, but in the pre-type-2 diabetic state, it aggregates to form an islet amyloid. The process of islet amyloid formation contributes to β-cell dysfunction and disease progression. The features of the IAPP sequence that modulate amyloid formation are still not understood. Human IAPP contains three aromatic residues, F15, F23, and Y37. F15 and Y37 are highly conserved, while F23 is more commonly a Leu or Ile in other species. The role of the aromatic residues in modulating the time course of amyloid formation and the cytotoxicity was examined using aromatic to Leu mutations. All three single and double mutants and the triple mutant were studied. F23 plays a dominant role in both amyloid formation and toxicity. An F15L mutant accelerated amyloid formation, a Y37L mutant had little effect, while an F23L replacement slowed amyloid formation by a factor of 2.6. Double mutants, which contained an F23L replacement, had a larger effect than those that did not, and there are non-additive effects between pairs of aromatic residues. F23 also plays a key role in toxicity. Single or multiple mutants that contain the F23L replacement were noticeably less toxic than the wild-type or mutants which did not include the F23L substitution. In contrast, the F15L mutant was more toxic than the wild-type one. The implications for IAPP amyloid formation and for the design of non-aggregating analogues of IAPP are discussed.

N-terminally truncated Aβ4-x proteoforms and their relevance for Alzheimer’s pathophysiology

BackgroundThe molecular heterogeneity of Alzheimer’s amyloid-β (Aβ) deposits extends well beyond the classic Aβ1-40/Aβ1-42 dichotomy, substantially expanded by multiple post-translational modifications that increase the proteome diversity. Numerous truncated fragments consistently populate the brain Aβ peptidome, and their homeostatic regulation and potential contribution to disease pathogenesis are largely unknown. Aβ4-x peptides have been reported as major components of plaque cores and the limited studies available indicate their relative abundance in Alzheimer’s disease (AD).MethodsImmunohistochemistry was used to assess the topographic distribution of Aβ4-x species in well-characterized AD cases using custom-generated monoclonal antibody 18H6—specific for Aβ4-x species and blind for full-length Aβ1-40/Aβ1-42—in conjunction with thioflavin-S and antibodies recognizing Aβx-40 and Aβx-42 proteoforms. Circular dichroism, thioflavin-T binding, and electron microscopy evaluated the biophysical and aggregation/oligomerization properties of full-length and truncated synthetic homologues, whereas stereotaxic intracerebral injections of monomeric and oligomeric radiolabeled homologues in wild-type mice were used to evaluate their brain clearance characteristics.ResultsAll types of amyloid deposits contained the probed Aβ epitopes, albeit expressed in different proportions. Aβ4-x species showed preferential localization within thioflavin-S-positive cerebral amyloid angiopathy and cored plaques, strongly suggesting poor clearance characteristics and consistent with the reduced solubility and enhanced oligomerization of their synthetic homologues. In vivo clearance studies demonstrated a fast brain efflux of N-terminally truncated and full-length monomeric forms whereas their oligomeric counterparts—particularly of Aβ4-40 and Aβ4-42—consistently exhibited enhanced brain retention.ConclusionsThe persistence of aggregation-prone Aβ4-x proteoforms likely contributes to the process of amyloid formation, self-perpetuating the amyloidogenic loop and exacerbating amyloid-mediated pathogenic pathways.

Differences in interaction lead to the formation of different types of insulin amyloid

Insulin balls, localized insulin amyloids formed at the site of repeated insulin injections in patients with diabetes, cause poor glycemic control and cytotoxicity. Our previous study has shown that insulin forms two types of amyloids; toxic amyloid formed from the intact insulin ((i)-amyloid) and less-toxic amyloid formed in the presence of the reducing reagent TCEP ((r)-amyloid), suggesting insulin amyloid polymorphism. However, the differences in the formation mechanism and cytotoxicity expression are still unclear. Herein, we demonstrate that the liquid droplets, which are stabilized by electrostatic interactions, appear only in the process of toxic (i)-amyloid formation, but not in the less-toxic (r)-amyloid formation process. The effect of various additives such as arginine, 1,6-hexanediol, and salts on amyloid formation was also examined to investigate interactions that are important for amyloid formation. Our results indicate that the maturation processes of these two amyloids were significantly different, whereas the nucleation by hydrophobic interactions was similar. These results also suggest the difference in the formation mechanism of two different insulin amyloids is attributed to the difference in the intermolecular interactions and could be correlated with the cytotoxicity.

Endogenous Human Proteins Interfering with Amyloid Formation.

Amyloid formation is a pathological process associated with a wide range of degenerative disorders, including Alzheimer’s disease, Parkinson’s disease, and diabetes mellitus type 2. During disease progression, abnormal accumulation and deposition of proteinaceous material are accompanied by tissue degradation, inflammation, and dysfunction. Agents that can interfere with the process of amyloid formation or target already formed amyloid assemblies are consequently of therapeutic interest. In this context, a few endogenous proteins have been associated with an anti-amyloidogenic activity. Here, we review the properties of transthyretin, apolipoprotein E, clusterin, and BRICHOS protein domain which all effectively interfere with amyloid in vitro, as well as displaying a clinical impact in humans or animal models. Their involvement in the amyloid formation process is discussed, which may aid and inspire new strategies for therapeutic interventions.

Amyloidosis in Alzheimer's Disease: Pathogeny, Etiology, and Related Therapeutic Directions.

The amyloid hypothesis of Alzheimer’s disease has long been the predominant theory, suggesting that Alzheimer’s disease is caused by the accumulation of amyloid beta protein (Aβ) in the brain, leading to neuronal toxicity in the central nervous system (CNS). Because of breakthroughs in molecular medicine, the amyloid pathway is thought to be central to the pathophysiology of Alzheimer’s disease (AD). Currently, it is believed that altered biochemistry of the Aβ cycle remains a central biological feature of AD and is a promising target for treatment. This review provides an overview of the process of amyloid formation, explaining the transition from amyloid precursor protein to amyloid beta protein. Moreover, we also reveal the relationship between autophagy, cerebral blood flow, ACHE, expression of LRP1, and amyloidosis. In addition, we discuss the detailed pathogenesis of amyloidosis, including oxidative damage, tau protein, NFTs, and neuronal damage. Finally, we list some ways to treat AD in terms of decreasing the accumulation of Aβ in the brain.

Influence of Chaperones on Amyloid Formation of Аβ Peptide.

An extensive study of the folding and stability of proteins and their complexes has revealed a number of problems and questions that need to be answered. One of them is the effect of chaperones on the process of fibrillation of various proteins and peptides. We studied the effect of molecular chaperones, such as GroEL and α-crystallin, on the fibrillogenesis of the Aβ(1-42) peptide using electron microscopy and surface plasmon resonance. Recombinant GroEL and Aβ(1-42) were isolated and purified. It was shown that the assembly of GroEL occurs without the addition of magnesium and potassium ions, as is commonly believed. According to the electron microscopy results, GroEL insignificantly affects the fibrillogenesis of the Aβ(1-42) peptide, while α-crystallin prevents the elongation of the Aβ(1-42) peptide fibrils. We have demonstrated that GroEL interacts nonspecifically with Aβ(1-42), while α-crystallin does not interact with Aβ(1-42) at all using surface plasmon resonance. The data obtained will help us understand the process of amyloid formation and the effect of various components on it.

Implementing Complementary Approaches to Shape the Mechanism of α-Synuclein Oligomerization as a Model of Amyloid Aggregation.

The aggregation of proteins into amyloid fibers is linked to more than forty still incurable cellular and neurodegenerative diseases such as Parkinson’s disease (PD), multiple system atrophy, Alzheimer’s disease and type 2 diabetes, among others. The process of amyloid formation is a main feature of cell degeneration and disease pathogenesis. Despite being methodologically challenging, a complete understanding of the molecular mechanism of aggregation, especially in the early stages, is essential to find new biological targets for innovative therapies. Here, we reviewed selected examples on α-syn showing how complementary approaches, which employ different biophysical techniques and models, can better deal with a comprehensive study of amyloid aggregation. In addition to the monomer aggregation and conformational transition hypothesis, we reported new emerging theories regarding the self-aggregation of α-syn, such as the alpha-helix rich tetramer hypothesis, whose destabilization induce monomer aggregation; and the liquid-liquid phase separation hypothesis, which considers a phase separation of α-syn into liquid droplets as a primary event towards the evolution to aggregates. The final aim of this review is to show how multimodal methodologies provide a complete portrait of α-syn oligomerization and can be successfully extended to other protein aggregation diseases.

Peptide backbone modifications of amyloid \u03b2 (1\u201340) impact fibrillation behavior and neuronal toxicity

Fibril formation of amyloid β (Aβ) peptides is one of the key molecular events connected to Alzheimer’s disease. The pathway of formation and mechanism of action of Aβ aggregates in biological systems is still object of very active research. To this end, systematic modifications of the Phe19–Leu34 hydrophobic contact, which has been reported in almost all structural studies of Aβ40 fibrils, helps understanding Aβ folding pathways and the underlying free energy landscape of the amyloid formation process. In our approach, a series of Aβ40 peptide variants with two types of backbone modifications, namely incorporation of (i) a methylene or an ethylene spacer group and (ii) a N-methylation at the amide functional group, of the amino acids at positions 19 or 34 was applied. These mutations are expected to challenge the inter-β-strand side chain contacts as well as intermolecular backbone β-sheet hydrogen bridges. Using a multitude of biophysical methods, it is shown that these backbone modifications lead, in most of the cases, to alterations in the fibril formation kinetics, a higher local structural heterogeneity, and a somewhat modified fibril morphology without generally impairing the fibril formation capacity of the peptides. The toxicological profile found for the variants depend on the type and extent of the modification.

Under Conditions of Amyloid Formation Bovine Carbonic Anhydrase B Undergoes Fragmentation by Acid Hydrolysis.

The development of many severe human diseases is associated with the formation of amyloid fibrils. Most of the available information on the process of amyloid formation has been obtained from studies of small proteins and peptides, wherein the features of complex proteins’ aggregation remain insufficiently investigated. Our work aimed to research the amyloid aggregation of a large model protein, bovine carbonic anhydrase B (BCAB). It has previously been demonstrated that, when exposed to an acidic pH and elevated temperature, this protein forms amyloid fibrils. Here, we show that, under these conditions and before amyloid formation, BCAB undergoes fragmentation by acid hydrolysis to give free individual peptides and associated peptides. Fragments in associates contain a pronounced secondary structure and act as the main precursor of amyloid fibrils, wherein free peptides adopt mostly unstructured conformation and form predominantly irregular globular aggregates. Reduced acidity decreases the extent of acid hydrolysis, causing BCAB to form amorphous aggregates lacking the thioflavin T binding β-structure. The presented results provide new information on BCAB amyloid formation and show the importance of protein integrity control when working even in mildly acidic conditions.

Transient disorder along pathways to amyloid

High-resolution structures of amyloid fibrils formed from normally-folded proteins have revealed non-native conformations of the polypeptide chains. Attaining these conformations apparently requires transition from the native state via a highly disordered conformation, in contrast to earlier models that posited a role for assembly of partially folded proteins. Modifications or interactions that extend the lifetime or constrain the conformations of these disordered states could act to enhance or suppress amyloid formation. Understanding how the properties of both the folded and transiently disordered structural ensembles influence the process of amyloid formation is a substantial challenge, but research into the properties of intrinsically disordered proteins will deliver important insights.

Effects of the Toxic Metals Arsenite and Cadmium on α-Synuclein Aggregation In Vitro and in Cells.

Exposure to heavy metals, including arsenic and cadmium, is associated with neurodegenerative disorders such as Parkinson’s disease. However, the mechanistic details of how these metals contribute to pathogenesis are not well understood. To search for underlying mechanisms involving α-synuclein, the protein that forms amyloids in Parkinson’s disease, we here assessed the effects of arsenic and cadmium on α-synuclein amyloid formation in vitro and in Saccharomyces cerevisiae (budding yeast) cells. Atomic force microscopy experiments with acetylated human α-synuclein demonstrated that amyloid fibers formed in the presence of the metals have a different fiber pitch compared to those formed without metals. Both metal ions become incorporated into the amyloid fibers, and cadmium also accelerated the nucleation step in the amyloid formation process, likely via binding to intermediate species. Fluorescence microscopy analyses of yeast cells expressing fluorescently tagged α-synuclein demonstrated that arsenic and cadmium affected the distribution of α-synuclein aggregates within the cells, reduced aggregate clearance, and aggravated α-synuclein toxicity. Taken together, our in vitro data demonstrate that interactions between these two metals and α-synuclein modulate the resulting amyloid fiber structures, which, in turn, might relate to the observed effects in the yeast cells. Whilst our study advances our understanding of how these metals affect α-synuclein biophysics, further in vitro characterization as well as human cell studies are desired to fully appreciate their role in the progression of Parkinson’s disease.

All-or-none amyloid disassembly via chaperone-triggered fibril unzipping favors clearance of α-synuclein toxic species

α-synuclein aggregation is present in Parkinson's disease and other neuropathologies. Among the assemblies that populate the amyloid formation process, oligomers and short fibrils are the most cytotoxic. The human Hsc70-based disaggregase system can resolve α-synuclein fibrils, but its ability to target other toxic assemblies has not been studied. Here, we show that this chaperone system preferentially disaggregates toxic oligomers and short fibrils, while its activity against large, less toxic amyloids is severely impaired. Biochemical and kinetic characterization of the disassembly process reveals that this behavior is the result of an all-or-none abrupt solubilization of individual aggregates. High-speed atomic force microscopy explicitly shows that disassembly starts with the destabilization of the tips and rapidly progresses to completion through protofilament unzipping and depolymerization without accumulation of harmful oligomeric intermediates. Our data provide molecular insights into the selective processing of toxic amyloids, which is critical to identify potential therapeutic targets against increasingly prevalent neurodegenerative disorders.

Glycans to improve efficacy and solubility of protein aggregation inhibitors.

Glycans to improve efficacy and solubility of protein aggregation inhibitors.