Entire Aggregation Process Research Articles

In vitro inhibition of α-Synuclein aggregation and disaggregation of preformed fibers by polyphenol hybrids with 2-conjugated benzothiazole

The misfolding and aggregation of α-Syn play a pivotal role in connecting diverse pathological pathways in Parkinson's disease (PD). Preserving α-Syn proteostasis and functionality by inhibiting its aggregation or disaggregating existing aggregates using suitable inhibitors represents a promising strategy for PD prevention and treatment. In this study, a series of benzothiazole-polyphenol hybrids was designed and synthesized. Three identified compounds exhibited notable inhibitory activities against α-Syn aggregation in vitro, with IC50 values in the low micromolar range. These inhibitors demonstrated sustained inhibitory effects throughout the entire aggregation process, stabilizing α-Syn proteostasis conformation. Moreover, the compounds effectively disintegrated preformed α-Syn oligomers and fibers, potentially by binding to specific domains within the fibers, inducing fibril instability, collapse, and ultimately resulting in smaller-sized aggregates and monomers. These findings offer valuable insights into the therapeutic potential of polyphenol hybrids with 2-conjugated benzothiazole targeting α-Syn aggregation in the treatment of PD.

Therapeutic potential of Coumarin-polyphenolic acid hybrids in PD: Inhibition of α-Syn aggregation and disaggregation of preformed fibrils, leading to reduced neuronal inclusion formation

This study focuses on the discovery of new potential drugs for treating PD by targeting the aggregation of α-Syn. A series of hybrids combining Coumarin and phenolic acid were designed and synthesized. Four particularly promising compounds were identified, showing strong inhibitory effects with IC50 values ranging from low micromolar to submicromolar concentrations, as low as 0.63 μM. These compounds exhibited a higher binding affinity to α-Syn residues and effectively hindered the entire aggregation process, maintaining the proteostasis conformation of α-Syn and preventing the formation of β-sheet aggregates. This approach holds significant promise for PD prevention. Additionally, these candidate compounds demonstrated the ability to break down preformed α-Syn oligomers and fibrils, resulting in the formation of smaller aggregates and monomers. Moreover, the candidate compounds showed impressive effectiveness in inhibiting α-Syn aggregation within nerve cells, thereby reducing the likelihood of α-Syn inclusion formation resembling Lewy bodies, which highlights their potential for treating PD.

The potential of Rhein's aromatic amines for Parkinson's disease prevention and treatment: α-Synuclein aggregation inhibition and disaggregation of preformed fibers

The aggregation of α-Syn is a pivotal mechanism in Parkinson's disease (PD). Effectively maintaining α-Syn proteostasis involves both inhibiting its aggregation and promoting disaggregation. In this study, we developed a series of aromatic amide derivatives based on Rhein. Two of these compounds, 4,5-dihydroxy-N-(3-hydroxyphenyl)-9,10-dioxo-9,10-dihydroanthracene-2-carboxamide (a5) and 4,5-dihydroxy-N-(2-hydroxy-4-chlorophenyl)-9,10-dioxo-9,10-dihydroanthracene-2-carboxamide (a8), exhibited good binding affinities to α-Syn residues, demonstrating promising inhibitory activity against α-Syn aggregation in vitro, with low IC50 values (1.35 and 1.08 μM, respectivly). These inhibitors acted throughout the entire aggregation process by stabilizing α-Syn's conformation and preventing the formation of β-sheet aggregates. They also effectively disassembled preformed α-Syn oligomers and fibrils. Preliminary mechanistic insights indicated that they bound to the specific domain within fibrils, inducing fibril instability, collapse, and the formation of smaller aggregates and monomeric α-Syn units. This research underscores the therapeutic potential of Rhein's aromatic amides in targeting α-Syn aggregation for PD treatment and suggests broader applications in managing and preventing neurodegenerative diseases.

4-Arylidene curcumin derivatives in vitro inhibit α-Synuclein aggregation and disaggregate the preformed fibril

This study focuses on the misfolding and aggregation of α-Syn as a central mechanism linking various pathological processes in PD. Maintaining α-Syn proteostasis through suitable inhibitors emerges as an effective approach to prevent PD. A more efficient strategy for PD treatment involves disintegrating neurotoxic oligomers and fibrils into normal functional α-Syn using inhibitors. To this end, a series of 4-arylidene curcumin derivatives were synthesized with a sheet-like conjugated skeleton and higher binding energies with α-Syn residues. Among these derivatives, three candidate compounds exhibited promising α-Syn aggregation inhibitory activities in vitro, with IC50 values as low as 0.61 μM. The inhibitory action extended throughout the entire aggregation process, stabilizing α-Syn proteostasis conformation and preventing β-sheets aggregation. Furthermore, the candidate compounds demonstrated effective disintegration capabilities against preformed α-Syn oligomers and fibrils. Initial mechanistic investigations indicated that the inhibitors may bind to a specific domain within the fibril, inducing fibril instability and subsequent collapse. This process resulted in the formation of a complex system of aggregates with smaller sizes and monomers. Overall, these findings provide valuable insights into the potential of 4-arylidene curcumin derivatives as therapeutic agents for targeting α-Syn aggregation in PD treatment.

Tracking Internal and Global Diffusive Dynamics During Protein Aggregation by High-Resolution Neutron Spectroscopy.

Proteins can misfold and form either amorphous or organized aggregates with different morphologies and features. Aggregates of amyloid nature are pathological hallmarks in so-called protein conformational diseases, including Alzheimer's and Parkinson's. Evidence prevails that the transient early phases of the reaction determine the aggregate morphology and toxicity. As a consequence, real-time monitoring of protein aggregation is of utmost importance. Here, we employed time-resolved neutron backscattering spectroscopy to follow center-of-mass self-diffusion and nano- to picosecond internal dynamics of lysozyme during aggregation into a specific β-sheet rich superstructure, called particulates, formed at the isoelectric point of the protein. Particulate formation is found to be a one-step process, and protein internal dynamics, to remain unchanged during the entire aggregation process. The time-resolved neutron backscattering spectroscopy approach developed here, in combination with standard kinetics assays, provides a unifying framework in which dynamics and conformational transitions can be related to the different aggregation pathways.

Fibril growth captured by electrical properties of amyloid-β and human islet amyloid polypeptide.

The aggregation of amyloid-β (Aβ) and human islet amyloid polypeptide (hIAPP) proteins have attracted considerable attention because of their involvement in protein misfolding diseases. These proteins have mostly been investigated using atomic force microscopy, transmission electron microscopy, and fluorescence microscopy to study the directional growth of fibrils both perpendicular to and along the fibril axis. Here, we demonstrate the real-time monitoring of the directional growth of fibrils in terms of activation energy of proton transfer using an impedance spectroscopy technique. The activation energy is used to quantify the sensitivity of proton conduction to the different stages of protein aggregation. The decrement (increment) in activation energy is related to the fibril growth along (perpendicular to) the fibril axis in intrinsic protein aggregation. The entire aggregation process shows different phases of the directional growth for Aβ and hIAPP, indicating different pathways for their aggregation. The activation energy for hIAPP is found to be smaller than the activation energy of Aβ during the aggregation process. The oscillatory behavior of the activation energy of hIAPP reflects a rapid change in the directional growth of the protofilaments of hIAPP. The results indicate higher aggregation propensity of Aβ than hIAPP. In the presence of resveratrol, hIAPP exhibits slower aggregation compared to Aβ. Methods of this study may in general be used to reveal the modulated aggregation pathway of proteins in the presence of different ligands.

Label-Free SERS Strategy for In Situ Monitoring and Real-Time Imaging of Aβ Aggregation Process in Live Neurons and Brain Tissues.

The aggregation of Aβ has been reported to closely correlate with Alzheimer's disease (AD). However, clear monitoring of the entire aggregation process of Aβ from monomer to fibril has been scarcely reported until now. Herein, we developed a label-free ratiometric surface enhanced Raman spectroscopic (SERS) platform for real-time monitoring of the entire process of Aβ aggregation in neurons and brain tissues. Different gold nanoparticles, generated in situ with Aβ monomer and fibril as templates separately, were served as effective SERS substrates to achieve a high sensitivity with a limit of detection (LOD) down to 70 ± 4 pM and 3.0 ± 0.5 pM for Aβ40 monomer and fibrils, respectively. Besides, the introduction of ratiometric determination of Aβ monomer and fibril (I1244/I1268) realized real-time monitoring of the entire aggregation process of Aβ monomer with high accuracy and selectivity against other proteins and amino acids. The significant analytical performance of the developed platform, together with good biocompatibility, long-term stability, and remarkable spatial resolution, enabled the present SERS platform imaging and real-time monitoring and imaging of Aβ aggregation influenced by different metal ions (Cu2+, Zn2+, and Fe3+) in neurons and brain tissues at the single cell level. Our results suggested that Cu2+ and Zn2+ ion of low concentration (10 μM) promoted fibril formation, while Fe3+ and Zn2+ of high concentration (100 μM) showed inhibition of fibrosis.

Real-Time Monitoring of Self-Aggregation of β-Amyloid by a Fluorescent Probe Based on Ruthenium Complex.

Self-accumulation of amyloid-β protein (Aβ) into insoluble fibrils is the major hallmark of Alzheimer's disease. Real-time monitoring of fibril growth is essential for clarifying the mechanism underlying aggregation and discovering therapeutic targets. A variety of approaches including NMR, electron microscopy (EM), atomic force microscopy (AFM), and total internal reflection fluorescence microscopy (TIRFM) have been explored to monitor the fibril growth or reveal morphology of Aβ aggregates. However, none of the methods allow real-time observation under physiological conditions while without any perturbations. Here, we present a fluorescent probe [Ru(phen)2(fipc)]2+ (Ru-fipc) (phen = 1,10-phenanthroline, fipc = 5-fluoro-N-(1,10-phenanthrolin-5-yl)-1H-indole-2-carboxamide) that can bind to all the Aβ forms, i.e., monomers, oligomers, and fibrils, while not perturbing aggregation. Using this probe in combination with laser confocal microscopy, the entire aggregation process could be clearly and exactly imaged at the single fibril level. The reliability of Ru-fipc was confirmed based on colocalization with thioflavin T (ThT). Importantly, Ru-fipc can be used to monitor the very early nucleation and oligomerization process, which is thought to be a critical step in the development of neurotoxicity while it cannot be visualized with ThT. To our knowledge, this is the first fluorescent probe developed for real-time monitoring of Aβ aggregation, especially for the very early assembly stage, in solution with minimal perturbation. This method is suitable for in vitro and in vivo studies. We believe this would provide a valuable complementary tool for the study of pathogenesis and discovery of therapeutic targets of neurodegenerative diseases.

Molecular Model for the Surface-Catalyzed Protein Self-Assembly.

The importance of cell surfaces in the self-assembly of proteins is widely accepted. One biologically significant event is the assembly of amyloidogenic proteins into aggregates, which leads to neurodegenerative disorders like Alzheimer's and Parkinson's diseases. The interaction of amyloidogenic proteins with cellular membranes appears to dramatically facilitate the aggregation process. Recent findings indicate that, in the presence of surfaces, aggregation occurs at physiologically low concentrations, suggesting that interaction with surfaces plays a critical role in the disease-prone aggregation process. However, the molecular mechanisms behind the on-surface aggregation process remain unclear. Here, we provide a theoretical model that offers a molecular explanation. According to this model, monomers transiently immobilized to surfaces increase the local monomer protein concentration and thus work as nuclei to dramatically accelerate the entire aggregation process. This physical-chemical theory was verified by experimental studies, using mica surfaces, to examine the aggregation kinetics of amyloidogenic α-synuclein protein and non-amyloidogenic cytosine deaminase APOBEC3G.

Effect of Nitrogen Bases on the Structure of Primary Asphaltene Clusters and Dynamics of Aggregation of Heavy Oil Fractions

Using the method of small-angle X-ray scattering (SAXS), the effect of nitrogen-containing compounds on the structure of primary asphaltene clusters and changes in their structure and size characteristics has been studied. It has been shown that the addition of nitrogen bases leads to a partial degradation of the primary aggregates of asphaltenes and the appearance of a larger number of disordered “non-compact” fragments in the solution, thereby altering the dynamics of the entire aggregation process. Nitrogen bases also provoke a change in the type of packing of the primary particles of asphaltenes, facilitating the formation of a looser secondary structure of asphaltene aggregates.

Surface Ordering in Molecular Clusters by Interactions of Well-Defined Parity Multipoles.

The spontaneous alignment of dipoles in patterns tangential or transversal to the interface of molecular clusters is studied by molecular dynamics simulations throughout the entire aggregation process. Tangential ordering (TANGO) is found to rely on dispersion forces driven by dipolar fluctuations. Transversal ordering (TRANSO) results from the interplay of two conditions: the broken translational invariance at the cluster interface and the interactions of multipoles of opposite parity. In this case, the orientational order parameter at the interface follows a universal scaling law behavior. In the limiting case of strongly interacting particles, a sharp structural transition from a disordered into an ordered state is observed at a critical distance inside the cluster and at a critical magnitude of the dipole moment.

Structural Conversion of Aβ17-42 Peptides from Disordered Oligomers to U-Shape Protofilaments via Multiple Kinetic Pathways.

Discovering the mechanisms by which proteins aggregate into fibrils is an essential first step in understanding the molecular level processes underlying neurodegenerative diseases such as Alzheimer’s and Parkinson's. The goal of this work is to provide insights into the structural changes that characterize the kinetic pathways by which amyloid-β peptides convert from monomers to oligomers to fibrils. By applying discontinuous molecular dynamics simulations to PRIME20, a force field designed to capture the chemical and physical aspects of protein aggregation, we have been able to trace out the entire aggregation process for a system containing 8 Aβ17–42 peptides. We uncovered two fibrillization mechanisms that govern the structural conversion of Aβ17–42 peptides from disordered oligomers into protofilaments. The first mechanism is monomeric conversion templated by a U-shape oligomeric nucleus into U-shape protofilament. The second mechanism involves a long-lived and on-pathway metastable oligomer with S-shape chains, having a C-terminal turn, en route to the final U-shape protofilament. Oligomers with this C-terminal turn have been regarded in recent experiments as a major contributing element to cell toxicity in Alzheimer’s disease. The internal structures of the U-shape protofilaments from our PRIME20/DMD simulation agree well with those from solid state NMR experiments. The approach presented here offers a simple molecular-level framework to describe protein aggregation in general and to visualize the kinetic evolution of a putative toxic element in Alzheimer’s disease in particular.

Simulations of Protein Aggregation

A number of diseases, known as amyloid diseases, are associated with pathological protein folding. Incorrectly or partially folded peptides or proteins can self-assemble into a variety of neurotoxic aggregate species, ranging from small soluble oligomers to amyloid fibrils. I will discuss fully atomic simulations of the early stages of aggregation of the Alzheimer Amyloid-beta peptide implicated in Alzheimer's Disease. I will also introduce an off-lattice coarse-grained peptide model that can be used to simulate the entire aggregation process from monomers to fibrils. The effects of surfaces on the morphology of the aggregates will be discussed.

Effect of calcium chloride and moderate shear on β-lactoglobulin aggregation in processing-like conditions

The influence of two variables (moderate shear and calcium chloride concentration) on the properties of β-lactoglobulin (β-lg) aggregate suspensions obtained by dynamic heat treatment (85°C for 60s) was studied. Variations in calcium chloride concentration from 5.1 to 7.1mM were imposed, and led to major changes in the aggregate properties. The characterization of the aggregate size was performed using three distinct methods: laser granulometer, dynamic light scattering (DLS) and focus beam reflectance measurement (FBRM). Viscosity and residual native fraction were also determined. Statistical analysis of experimental data underlines the important combined effect of the two variables and paves the way for new ways of controlling the aggregate functionalities. The calcium chloride concentration has an effect on the entire aggregation process, from native β-lg to large aggregates. On the other hand, shear only impacts the formation of the large aggregates.

Fractal Behavior of Functionalized Fullerene Aggregates. I. Aggregation of Two-Handed Tetraaminofullerene with DNA

In tris-buffered saline (TBS) with a trace of dimethylformamide (DMF), the homoaggregation process of a functionalized fullerene, the two-handed tetraaminofullerene (TH), and the heteroaggregation process (complex formation) of TH with DNA (pGL3-control plasmid) were studied dynamically by using a combination of static and dynamic laser light scattering measurements. Fractal behavior was investigated in the aggregation process of both TH homoaggregates and TH-DNA heteroaggregates. The stability of aggregates in solution depends on the molar concentration ratio R(M), defined as the molar ratio of moles of TH to moles of the DNA base pair. Higher R(M) values resulted in lower aggregate stability. The transition of the fractal dimension (Df) in TH homoaggregation by rapidly mixing 3.78 microM TH with an equal volume of the blank buffer was found to vary from a value of 1.46 to 2.02. Dynamic light scattering results revealed that, in the aggregation process, the change in the size distribution of aggregates with time could be related to a Df transition. In the Df transition region, the size distribution of homoaggregates displayed a drastic change from a single-mode distribution to a bimodal distribution, which clearly suggested a restructuring process with the formation of large aggregates. When the aggregation process finally reached equilibrium, Df = 2.02, the size of the homoaggregates had a single mode but a broad distribution. However, TH-DNA heteroaggregation showed a Df transition from 1.58 to 1.7, but over a shorter time range of less than 5 min. Then, the Df value fluctuated in the range of 1.7 and finally reached an equilibrium value of Df approximately 1.78, which was independent of molar concentration. There are two main action forces involved in the heteroaggregation process: van der Waals forces and attractive electrostatic forces, with the latter one being stronger and faster than that of the former. Therefore, a two-step action could occur in the heteroaggregation process. In the beginning of mixing, the attractive electrostatic forces dictated the aggregation process, and then van der Waals forces also got involved in the entire aggregation process. By using an initial concentration of 3.78 microM each and R(M) = 1, TH-DNA heteroaggregates showed more stable solution behavior than the homoaggregates. The lower Df value of the heteroaggregates could be related to a looser compact structure. Results from scanning electron microscopy (SEM) also disclosed the different textures between TH homoaggregates and TH-DNA heteroaggregates; the former had a more dense packing than the latter one.

The Aggregation of Thermally Stable Particles in a Premixed Flat Flame Aerosol Reactor

Using a premixed flat flame, the aggregation process of thermally stable particles was studied. Based on the theoretical descriptions of the reaction limited aggregation process (RLAP) and the diffusion limited aggregation process (DLAP), the entire aggregation process was explained by a combination of the RLAP and the DLAP. Experiments were done to test the possibility of this explanation for the dynamic aggregation processes in terms of the parameters that are used in a normal chemical conversion process. By defining the primary particle as the basic unit of the aggregates, the aggregation process was divided into the early stage--when the primary particles are formed--and the stage of aggregation thereafter. Changing the combustion equivalence ratio of the premixed flat flame from 0.7 to 0.98 controlled the sizes of the primary particles. The deficiency of the oxygen in the combustion process controlled the final size of the primary particles in the aggregate. The morphological evolution of particles w...