Amyloid-related Disorders Research Articles

Antiamyloid Activity of Functionalized Cerium Oxide Nanoparticle on Lysozyme Fibrillation: Spectroscopic and Microscopic Investigation

Accumulation of amyloid fibrils of proteins in different organs is a pathological indication for various neurogenerative disorders. Recently nanotechnology is at the centre of interest to tackle with those disorders. In this study we have chosen hen egg white lysozyme as our model protein which is responsible for hereditary systemic Amyloidosis disease. Here we have reported effect of Cerium oxide nanoparticle (CeONP), folic acid functionalized CeONP (FA-CeONP) and polyacrylic acid functionalized CeONP (PAA-CeONP) (all of them having size ~18 nm) on fibrillation of hen egg white lysozyme (Lyz). The amyloid growth is monitored by Thioflavin T (ThT), Congo Red (CR), 8-anilinonapthalene-1-sulfonic acid (ANS) assay, time resolved fluorescence anisotropy, intrinsic fluorescence, circular dichroism, atomic force microscopy and fluorescence microscopy study. Results demonstrate that all nanoparticle (NP) shows antiamyloid activity by broadening lag phase and suppressing growth phase of Lyz amyloid growth, but PAA-CeONP shows maximum inhibitory effect. These bare and functionalized CeONP shows dose dependent antiamyloid activity. We have also demonstrated that such inhibition of amyloid formation is associated with reduction of s-sheet content of protein. The inhibition efficiency is quantified by half maximal inhibition concentration IC50 values. A mechanistic pathway is explained on the basis of electrostatic interaction between NP and Lyz by ζ- potential measurement. This interaction is also analyzed theoretically by docking analysis. Thus our study provides a basis for use of CeONP as a promising candidate in therapeutic application in amyloid related disorders.

In vitro and in vivo insulin amyloid degradation mediated by Serratiopeptidase

A transition of amyloidogenic protein by alternative folding pathway under certain conditions leads to the formation of protease resistant amyloid fibrils, having predominantly cross β structure. These amyloids are related to various neurodegenerative diseases and clearance of such amyloids may be a therapeutic approach for amyloid-related diseases. Insulin, that can form amyloids, is widely used as a model amyloidogenic protein for the study of various amyloid related diseases. In this study, insulin amyloids were formed in vitro and the potential of Serratiopeptidase (SP), a fibrinolytic-like serine protease, towards the dissociation of insulin amyloids was explored. The dissociation of the amyloids was demonstrated using in vitro and in vivo using zebrafish model. The amyloid dissociation property was compared with a standard amyloid dissociating enzyme nattokinase (NK). SP shows better amyloid dissociation ability than NK and therefore, SP can be considered as amyloid dissociating agent with potential as a drug candidate for different amyloid related disorders.

Prion like behavior of HSA-hydroxylated MWCNT interface

Carbon nanotubes (CNTs) with unique and outstanding properties were expected to revolutionize various aspects of the biomedical sector. Interaction studies of proteins with functionalized CNTs would shed light on their toxicological aspects upon entering the human body. Hyperchromicity of the UV–Visible spectra and declining fluorescence potential of HSA on interaction with CNTs suggested ground state complex to exist between them. Synchronous and 3D spectral features of CNT-HSA system proposed their possible binding site to occur nearby Trp and Tyr residues. FTIR and FT-Raman spectra showed a shift in the amide band region that proportionate the possible alteration to occur in the alpha-helical structures. CD far and near spectra showed loss of alpha-helical structures and shift in the Trp position of the polypeptide backbone. CNT's UV and FTIR band showed shift on interaction with HSA, which conveys the possible aggregation of CNTs in the presence of protein. The promoting role of CNTs against HSA fibril formation has been confirmed by spectroscopic and microscopic evaluations. Secondary conformational changes, besides the existence of increased beta-sheet structures of HSA amyloid fibrils, remain similar to the amyloid behavior of Prion protein. Hence, HSA fibril-CNT interface predominates the possible mechanism for several amyloid-related disorders concerning their toxic accumulations in the body.

Water in Amyloidogenic Intrinsically Disordered Proteins: Interplay of Conformational Preference and Amyloid Aggregation

Water shares an intimate relationship with proteins and strongly influences their dynamics. The behavior of water molecules in and around intrinsically disordered proteins (IDPs) is poorly understood. IDPs have captivated enormous attention in the recent time due to their association with a large number of biological functions and a variety of diseases including amyloid related disorders. The fact that the most pathological amyloids are often formed by IDPs underscores the importance of IDPs in amyloid biology. An enlarging body of data has illuminated the mechanism of coupled conformational switch and aggregation from the disordered monomers to ordered amyloids. However, the role of water in amyloid formation remains elusive. We have embarked upon studies aimed at elucidating the water dynamics during the amyloid transition of a collapsed amyloidogenic IDP, namely bovine κ-casein. Using time-resolved fluorescence spectroscopy, the hydration time was found to be on the nanosecond timescale which is an order of magnitude slower than water present on the protein surface. Thus, our results indicate that the water molecules within the unstructured globules are profoundly restrained and resemble the ordered nanoconfined water cluster (Arya & Mukhopadhyay, J. Phys. Chem. B 2014, 118, 9191). These results are in accordance with our recent simulations and femtosecond time-resolved experiments. It is interesting to note that the nanosecond hydration component is largely absent in another IDP that has much lower propensity to form collapsed globules. The collapsed IDPs containing ordered water are prone to oligomerization that is entropically favored by the release of water molecules upon aggregation. Our recent data indicated that although there is a release of water molecules, but a significant fraction of water remains trapped upon amyloid formation possibly due to hydrogen-bonded wet-interfaces of the cross-β spines.

Synthesis and Characterization of Acylated Polycaprolactone (PCL) Nanospheres and Investigation of Their Influence on Aggregation of Amyloid Proteins

Acylated polycaprolactone (PCL) nanospheres were fabricated and employed to interact with amyloid-β-(25–35) peptides (Aβ25–35), "peptide 11 of the 40 peptide full amyloid-β". The nanospheres were characterized by scanning electron microscopy (SEM), attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, and dynamiclightscattering (DLS), all of which confirmed that the acylated polycaprolactone (Ac-PCL) nanospheres were successfully fabricated. The effect of the nanospheres on the aggregation of Aβ25–35 peptides was investigated by thioflavin T fluorescence measurements. The result showed that, without nanospheres, the Aβ25–35 peptides aggregated gradually from monomers and oligomers to long fibrils with increasing incubation time. In comparison, the nanospheres were effective in interfering with fibrillogenesis and aggregation of amyloid-β. We suggest this study may contribute to the development of new therapeutic strategies against amyloid-related disorders.

Neutralization of TNFSF10 ameliorates functional outcome in a murine model of Alzheimer's disease.

Alzheimer's disease is one of the most common causes of death worldwide, with poor treatment options. A tissue landmark of Alzheimer's disease is accumulation of the anomalous protein amyloid-β in specific brain areas. Whether inflammation is an effect of amyloid-β on the Alzheimer's disease brain, or rather it represents a cause for formation of amyloid plaques and intracellular tangles remains a subject of debate. TNFSF10, a proapoptotic cytokine of the TNF superfamily, is a mediator of amyloid-β neurotoxicity. Here, we demonstrate that blocking TNFSF10 by administration of a neutralizing monoclonal antibody could attenuate the amyloid-β-induced neurotoxicity in a triple transgenic mouse model of Alzheimer's disease (3xTg-AD). The effects of TNFSF10 neutralization on either cognitive parameters, as well as on the expression of TNFSF10, amyloid-β, inflammatory mediators and GFAP were studied in the hippocampus of 3xTg-AD mice. Treatment with the TNFSF10 neutralizing antibody resulted in dramatic improvement of cognitive parameters, as assessed by the Morris water maze test and the novel object recognition test. These results were correlated with decreased protein expression of TNFSF10, amyloid-β, inflammatory mediators and GFAP in the hippocampus. Finally, neutralization of TNFSF10 results in functional improvement and restrained immune/inflammatory response in the brain of 3xTg-AD mice in vivo. Thus, it is plausible to regard the TNFSF10 system as a potential target for efficacious treatment of amyloid-related disorders.

Apigenin modulates the expression levels of pro-inflammatory mediators to reduce the human insulin amyloid-induced oxidant damages in SK-N-MC cells.

Amyloid depositions of proteins play crucial roles in a wide variety of degenerative disorders called amyloidosis. Although the exact mechanisms involved in amyloid-mediated cytotoxicity remain unknown, increased formation of reactive oxygen species (ROS) and nitrogen species and overproduction of pro-inflammatory cytokines are believed to play key roles in the process. In that regard, we investigated the effect of apigenin, a common dietary flavonoid with high antioxidant and anti-inflammatory properties on potential factors involved in cytotoxicity of human insulin amyloids. Pretreatment of SK-N-MC neuroblastoma cells with apigenin increased cell viability and reduced the apoptosis induced by insulin fibrils. In addition, apigenin attenuated insulin fibril-induced ROS production and lipid peroxidation. Our result also demonstrated that pretreatment of the fibril-affected cells with apigenin caused an increase in catalase activity and the intracellular glutathione content along with reduction in nitric oxide production and nuclear factor κB, tumor necrosis factor α, and interleukin 6 gene expression based on real-time polymerase chain reaction evaluation. In accordance with these results, apigenin could be a promising candidate in the design of natural-based drugs for treatment or prevention of amyloid-related disorders.

Gold nanoparticles as amyloid-like fibrillogenesis inhibitors

Amyloid aggregates are one of the likely key factors leading to the development of Alzheimer's disease (AD) and other amyloidosis associated diseases. Several recent studies have shown that some anti-diabetic drugs have a positive therapeutic effect on AD patients by crossing the blood brain barrier (BBB) and preventing or reducing insulin resistance. Nanoparticles (NPs) or nanoscale objects (<600Da.), are able to cross the BBB at low concentrations, and can specifically target amyloidogenic structures. Thus, NPs are fast becoming indispensable tools for directed drug delivery, particularly when targeting structures or regions in the brain. Here, we have explored the inhibitory effect of gold nanoparticles (AuNPs) on the fibrillogenesis process of insulin fibrils. We found that when AuNPs were co-incubated with insulin, the structural transformation into amyloid-like fibrils was delayed by about a week. Further, the fibrils that formed, exhibited altered structure, shape, and dynamics, which further reduced fibril growth, and the stability of available amyloid-like fibrils with cross-β structure for aggregation. Our results demonstrate that AuNPs disrupt insulin amyloid fibrillation resulting in fibrils that are shorter and more compact, and thus may serve a useful role in new therapeutic and diagnostic strategies for amyloid-related disorders.

Vitamin D2 interacts with Human PrPc (90–231) and breaks PrPc oligomerization in vitro

PrPsc, the pathogenic isoform of PrPc, can convert PrPc into PrPsc through direct interactions. PrPc oligomerization is a required processing step before PrPsc formation, and soluble oligomers appear to be the toxic species in amyloid-related disorders. In the current study, direct interactions between vitamin D2 and human recombinant PrPc (90–231) were observed by Biacore assay, and 3F4 antibody, specific for amino acid fragment 109–112 of PrPc, inhibited this interaction. An ELISA study using3F4 antibody showed that PrPc (101–130), corresponding sequence to human PrP, was affected by vitamin D2, supporting the results of Biacore studies and suggesting that the PrPc sequence around the 3F4 epitope was responsible for the interaction with vitamin D2. Furthermore, the effects of vitamin D2 on disruption of PrPc (90–231) oligomerization were elucidated by dot blot analysis and differential protease k susceptibilities. While many chemical compounds have been proposed as potential therapeutic agents for the treatment of scrapie, most of these are toxic. However, given the safety and blood brain barrier permeability of vitamin D2, we propose that vitamin D2 may be a suitable agent to target PrPc in the brain and therefore is a potential therapeutic candidate for prion disease.

Ion Mobility Spectrometry-Mass Spectrometry of Intrinsically Unfolded Proteins: Trying to Put Order into Disorder

Intrinsically disordered proteins do not adopt well-defined native structures and therefore present an intriguing challenge in terms of structural elucidation as they are relatively inaccessible to traditional approaches such as NMR and X-ray crystallography. Many members of this important group of proteins have a distinct biological function and frequently undergo a conformational change on binding to their physiological targets which can in turn modulate their function. Furthermore, many intrinsically unstructured proteins are associated with a wide range of major diseases including cancer and amyloid-related disorders. Here, electrospray ionisation-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) has been used to probe the conformational characteristics of two intrinsically disordered proteins: apo-cytochrome c and apo-osteocalcin. Both proteins are structured in their holo-states when bound to their respective substrates, but disordered in their apo-states. Here, the conformational properties of the holo- and the apo-protein forms for both species have been analysed and their mass spectral data and ion mobility spectrometry-derived collision cross-sectional areas, indicative of their physical size, compared to study the relationship between substrate binding and tertiary structure. In both cases, the intrinsically unstructured apo-states populated multiple conformations with larger cross-sectional areas than their holo-analogues, suggesting that intrinsic disorder in proteins does not preclude the formation of preferred conformations. Additionally, analysis of truncated analogues of osteocalcin has located the region of the protein responsible for the conformational changes detected upon metal cation binding. Together, the data illustrate the scope and utility of ESI-IMS-MS for studying the characteristics and properties of intrinsically disordered proteins whose analysis by other techniques is limited.

Characterization of Oligomers of Heterogeneous Size as Precursors of Amyloid Fibril Nucleation of an SH3 Domain: An Experimental Kinetics Study

Understanding the earliest molecular events during nucleation of the amyloid aggregation cascade is of fundamental significance to prevent amyloid related disorders. We report here an experimental kinetic analysis of the amyloid aggregation of the N47A mutant of the α-spectrin SH3 domain (N47A Spc-SH3) under mild acid conditions, where it is governed by rapid formation of amyloid nuclei. The initial rates of formation of amyloid structures, monitored by thioflavine T fluorescence at different protein concentrations, agree quantitatively with high-order kinetics, suggesting an oligomerization pre-equilibrium preceding the rate-limiting step of amyloid nucleation. The curves of native state depletion also follow high-order irreversible kinetics. The analysis is consistent with the existence of low-populated and heterogeneous oligomeric precursors of fibrillation that form by association of partially unfolded protein monomers. An increase in NaCl concentration accelerates fibrillation but reduces the apparent order of the nucleation kinetics; and a double mutant (K43A, N47A) Spc-SH3 domain, largely unfolded under native conditions and prone to oligomerize, fibrillates with apparent first order kinetics. On the light of these observations, we propose a simple kinetic model for the nucleation event, in which the monomer conformational unfolding and the oligomerization of an amyloidogenic intermediate are rapidly pre-equilibrated. A conformational change of the polypeptide chains within any of the oligomers, irrespective of their size, is the rate-limiting step leading to the amyloid nuclei. This model is able to explain quantitatively the initial rates of aggregation and the observed variations in the apparent order of the kinetics and, more importantly, provides crucial thermodynamic magnitudes of the processes preceding the nucleation. This kinetic approach is simple to use and may be of general applicability to characterize the amyloidogenic intermediates and oligomeric precursors of other disease-related proteins.

Non-specific peroxidase activity and catalase-inhibitory behavior of fibrillar aggregates after interaction with heme: relevance to the etiology of amyloid-related neurodegenerative disorders using the experimental-based evidences

The exact mechanism of cell death in neurodegenerative diseases remains obscure, but the aberrant assembly of proteins into fibrillar aggregates is accused to be the primary cause of pathogenesis. Furthermore, the structural determinants of protein fibrils that are responsible for cell dysfunction are not yet clear. In the present study, using α-crystallin-/α-chymotrypsin-based experimental systems, we reported non-specific peroxidase behavior of “heme–amyloid fibril” complex using DNA, serotonin and DOPA as potential substrates. Also, amyloid-mediated inhibition of catalase activity was documented. More importantly, we suggested that uncontrollable peroxidase activity may be involved in neurodegenerative cell toxicity via several independent (mechanistic) routes. According to the results and literature, it is reasonable to assume that oxidative stress plays at least partially a causative rather than merely bystander role in the neurofibrillary pathology in AD/PD in vivo. Since the consequence of heme–amyloid interaction has yet to be identified, additional in vitro/in vivo data on it may help us to manage amyloid aggregation processes and postpone/attenuate the onset/extent of irreversible part of neurodegenerative pathogenesis.

The amyloid stretch hypothesis: Recruiting proteins toward the dark side

A detailed understanding of the molecular events underlying the conversion and self-association of normally soluble proteins into amyloid fibrils is fundamental to the identification of therapeutic strategies to prevent or cure amyloid-related disorders. Recent investigations indicate that amyloid fibril formation is not just a general property of the polypeptide backbone depending on external factors, but that it is strongly modulated by amino acid side chains. Here, we propose and address the validation of the premise that the amyloidogenicity of a protein is indeed localized in short protein stretches (amyloid stretch hypothesis). We demonstrate that the conversion of a soluble nonamyloidogenic protein into an amyloidogenic prone molecule can be triggered by a nondestabilizing six-residue amyloidogenic insertion in a particular structural environment. Interestingly enough, although the inserted amyloid sequences clearly cause the process, the protease-resistant core of the fiber also includes short adjacent sequences from the otherwise soluble globular domain. Thus, short amyloid stretches accessible for intermolecular interactions trigger the self-assembly reaction and pull the rest of the protein into the fibrillar aggregate. The reliable identification of such amyloidogenic stretches in proteins opens the possibility of using them as targets for the inhibition of the amyloid fibril formation process.

Beta-hairpin conformation of fibrillogenic peptides: structure and alpha-beta transition mechanism revealed by molecular dynamics simulations.

Understanding the conformational transitions that trigger the aggregation and amyloidogenesis of otherwise soluble peptides at atomic resolution is of fundamental relevance for the design of effective therapeutic agents against amyloid-related disorders. In the present study the transition from ideal alpha-helical to beta-hairpin conformations is revealed by long timescale molecular dynamics simulations in explicit water solvent, for two well-known amyloidogenic peptides: the H1 peptide from prion protein and the Abeta(12-28) fragment from the Abeta(1-42) peptide responsible for Alzheimer's disease. The simulations highlight the unfolding of alpha-helices, followed by the formation of bent conformations and a final convergence to ordered in register beta-hairpin conformations. The beta-hairpins observed, despite different sequences, exhibit a common dynamic behavior and the presence of a peculiar pattern of the hydrophobic side-chains, in particular in the region of the turns. These observations hint at a possible common aggregation mechanism for the onset of different amyloid diseases and a common mechanism in the transition to the beta-hairpin structures. Furthermore the simulations presented herein evidence the stabilization of the alpha-helical conformations induced by the presence of an organic fluorinated cosolvent. The results of MD simulation in 2,2,2-trifluoroethanol (TFE)/water mixture provide further evidence that the peptide coating effect of TFE molecules is responsible for the stabilization of the soluble helical conformation.

Poly- l-lysine Dissolves Fibrillar Aggregation of the Alzheimer β-Amyloid Peptide in Vitro

β-Amyloid peptide (βA) is a major fibrillar component of neuritic plaques in Alzheimer's disease (AD) brains and is related to the pathogenesis of the disease. In this study, using electron microscopy, we describe herein the results concerning the efficacy of compounds that can dissolve preformed βA fibrils in vitro. For such a purpose, two hydrosoluble and biocompatible polymers such as polyethylene glycol and poly- l-lysine were used. The poly- l-lysine appears as a potent dissolver of preformed βA fibrils in vitro. Its efficiency is instantaneous. Poly- l-lysine can be used as a universal dissolver of all types of oligomeric β-sheet conformation, precursor of the fibrils. This finding provides the basis for future investigation of the therapeutic potential of poly- l-lysine in terms of preventing and/or retarding amyloidogenesis in AD and other types of amyloid-related disorders.

4′-Iodo-4′-Deoxydoxorubicin Disrupts the Fibrillar Structure of Transthyretin Amyloid

Transthyretin (TTR) is a tetrameric protein synthesized mainly by the liver and the choroid plexus, from where it is secreted into the plasma and the cerebrospinal fluid, respectively. Some forms of polyneuropathy, vitreopathy, and cardiomyopathy are caused by the deposition of normal and/or mutant TTR molecules in the form of amyloid fibrils. Familial amyloidotic polyneuropathy is the most common form of TTR amyloidosis related to the V30M variant. It is still unclear the process by which soluble proteins deposit as amyloid. The treatment of amyloid-related disorders might attempt the stabilization of the soluble protein precursor to retard or inhibit its deposition as amyloid; or aim at the resorption of the deposited amyloid. The anthracycline 4'-iodo-4'-deoxydoxorubicin (I-DOX) has been shown to reduce the amyloid load in immunoglobulin light-chain amyloidosis. We investigated 1) whether I-DOX has affinity for TTR amyloid in tissues, 2) determined the I-DOX binding constants to TTR synthetic fibrils, and 3) determined the nature of the effect of I-DOX on TTR fibrils. We report that 1) I-DOX co-localizes with amyloid deposits in tissue sections of patients with familial amyloidotic polyneuropathy; 2) I-DOX strongly interacts with TTR amyloid fibrils and presents two binding sites with k(d) of 1.5 x 10(-11) mol/L and 5.6 x 10(-10) mol/L, respectively; and 3) I-DOX disrupts the fibrillar structure of TTR amyloid into amorphous material, as assessed by electron microscopy but does not solubilize the fibrils as confirmed by filter assays. These data support the hypothesis that I-DOX and less toxic derivatives can prove efficient in the treatment of TTR-related amyloidosis.