Fast Aggregation Kinetics Research Articles

Modulation of Primary and Secondary Processes in Tau Fibril Formation by Salt-Induced Dynamics.

The initial stages of amyloid fibrilization begin with the monomers populating aggregation-prone conformers. Characterization of such aggregation-prone conformers is crucial in the study of neurodegenerative diseases. The current study characterizes the aggregation pathway of two tau protein constructs that have been recently demonstrated to form Alzheimer's (AD) fibril structures with divalent ions and chronic traumatic encephalopathy (CTE) fibril structures with monovalent ions. The results highlight the involvement of identical residues in both the primary and secondary processes of both AD and CTE fibril propagation. Nuclear magnetic resonance relaxation experiments reveal increased flexibility of the motifs 321KCGS within R3 and 364PGGGN within R4 in the presence of MgCl2/NaCl, correlating with faster aggregation kinetics and indicating efficient primary nucleation. Notably, the seeded aggregation kinetics of the tau monomers in the presence and absence of metal ions are strikingly different. This correlates with the overall sign of the 15N-ΔR2 profile specifying the dominant mechanism involved in the process of aggregation.

Controlled aggregation enhances immunomodulatory potential of mesenchymal stromal cell aggregates.

Human mesenchymal stromal cells (MSCs) are promising candidates for cell therapy due to their ease of isolation and expansion and their ability to secrete antiapoptotic, pro‐angiogenic, and immunomodulatory factors. Three‐dimensional (3D) aggregation “self‐activates” MSCs to augment their pro‐angiogenic and immunomodulatory potential, but the microenvironmental features and culture parameters that promote optimal MSC immunomodulatory function in 3D aggregates are poorly understood. Here, we generated MSC aggregates via three distinct methods and compared them with regard to their (a) aggregate structure and (b) immunomodulatory phenotype under resting conditions and in response to inflammatory stimulus. Methods associated with fast aggregation kinetics formed aggregates with higher cell packing density and reduced extracellular matrix (ECM) synthesis compared to those with slow aggregation kinetics. While all three methods of 3D aggregation enhanced MSC expression of immunomodulatory factors compared to two‐dimensional culture, different aggregation methods modulated cells' temporal expression of these factors. A Design of Experiments approach, in which aggregate size and aggregation kinetics were systematically covaried, identified a significant effect of both parameters on MSCs' ability to regulate immune cells. Compared to small aggregates formed with fast kinetics, large aggregates with slow assembly kinetics were more effective at T‐cell suppression and macrophage polarization toward anti‐inflammatory phenotypes. Thus, culture parameters including aggregation method, kinetics, and aggregate size influence both the structural properties of aggregates and their paracrine immunomodulatory function. These findings underscore the utility of engineering strategies to control properties of 3D MSC aggregates, which may identify new avenues for optimizing the immunomodulatory function of MSC‐based cell therapies.

Different Force Fields Give Rise to Different Amyloid Aggregation Pathways in Molecular Dynamics Simulations.

The progress toward understanding the molecular basis of Alzheimers's disease is strongly connected to elucidating the early aggregation events of the amyloid-β (Aβ) peptide. Molecular dynamics (MD) simulations provide a viable technique to study the aggregation of Aβ into oligomers with high spatial and temporal resolution. However, the results of an MD simulation can only be as good as the underlying force field. A recent study by our group showed that none of the common force fields can distinguish between aggregation-prone and nonaggregating peptide sequences, producing a similar and in most cases too fast aggregation kinetics for all peptides. Since then, new force fields specially designed for intrinsically disordered proteins such as Aβ were developed. Here, we assess the applicability of these new force fields to studying peptide aggregation using the Aβ16-22 peptide and mutations of it as test case. We investigate their performance in modeling the monomeric state, the aggregation into oligomers, and the stability of the aggregation end product, i.e., the fibrillar state. A main finding is that changing the force field has a stronger effect on the simulated aggregation pathway than changing the peptide sequence. Also the new force fields are not able to reproduce the experimental aggregation propensity order of the peptides. Dissecting the various energy contributions shows that AMBER99SB-disp overestimates the interactions between the peptides and water, thereby inhibiting peptide aggregation. More promising results are obtained with CHARMM36m and especially its version with increased protein-water interactions. It is thus recommended to use this force field for peptide aggregation simulations and base future reparameterizations on it.

A Capped Peptide of the Aggregation Prone NAC 71-82 Amino Acid Stretch of α-Synuclein Folds into Soluble β-Sheet Oligomers at Low and Elevated Peptide Concentrations.

Although Lewy bodies and Lewy neurites are hallmarks of Parkinson’s disease (PD) and dementia with Lewy bodies (DLB), misfolded α-synuclein oligomers are nowadays believed to be key for the development of these diseases. Attempts to target soluble misfolded species of the full-length protein have been limited so far, probably due to the fast aggregation kinetics and burial of aggregation prone segments in final cross-β-sheet fibrils. A previous characterisation study of fibrils prepared from a capped peptide of the non-amyloid β-component (NAC) 71–82 amino acid stretch of α-synuclein demonstrated an increased aggregation propensity resulting in a cross-β-structure that is also found in prion proteins. From this, it was suggested that capped NAC 71–82 peptide oligomers would provide interesting motifs with a capacity to regulate disease development. Here, we demonstrated, from a series of circular dichroism spectroscopic measurements and molecular dynamics simulations, the molecular-environment-sensitive behaviour of the capped NAC 71–82 peptide in a solution phase and the formation of β-sheet oligomeric structures in the supernatant of a fibrillisation mixture. These results highlighted the use of the capped NAC 71–82 peptide as a motif in the preparation of oligomeric β-sheet structures that potentially could be used in therapeutic strategies in the fight against progressive neurodegenerative disorders, such as PD and DLB.

Kinetic Insights into Zn2+-Induced Amyloid β-Protein Aggregation Revealed by Stopped-Flow Fluorescence Spectroscopy.

Zn2+ has remarkable impacts on amyloid β-protein (Aβ) aggregation, which is crucial in the pathology of Alzheimer's disease. However, the Zn2+ concentration in human cerebrospinal fluid is commonly too low for interaction with Aβ, and only during neurotransmission is there a transient release of a high concentration of Zn2+. It is difficult to explore the details of the interaction between Zn2+ and Aβ within such a short time scale by using ordinary analytical methods. Herein, stopped-flow fluorescence spectroscopy was used to study the fast aggregation kinetics of Aβ42 in the presence of Zn2+ in the time scale from 1 ms to seconds. It was found that Zn2+ bound to Aβ42 within 1 ms; caused immediate conformational transition around Tyr10, which led to the enhancement of Aβ42 hydrophobicity; and then promoted fast aggregation of Aβ42 through enhanced hydrophobic interactions. Among the two Zn2+-binding sites on Aβ42 (KD = 107 nM and 5.2 μM), the first one of higher affinity had a greater impact on the aggregation of Aβ42. Three kinetic phases were observed in the Zn2+-induced fast aggregation of Aβ42, and the fast phase was extremely accelerated by Zn2+, indicating that accelerated aggregation mainly occurred in the fast phase. The reactions occurring in this phase were closely related to the association of Zn2+ and Aβ42. Moreover, Zn2+ largely broadened the pH range of Aβ42 fast aggregation from pH 5.2-6.2 without Zn2+ to pH 5.2-7.8 in the presence of Zn2+. Besides, the promoting effect of Zn2+ on Aβ42 fast aggregation peaked at pH 6.8-7.8, which includes the pH values of the cerebrospinal fluid (pH 7.3) and hippocampus (pH 7.15-7.35). The findings demonstrate the significant effect of Zn2+ on Aβ aggregation and provide new insight into its mechanisms.

Amyloid β-Protein C-Terminal Fragments: Formation of Cylindrins and β-Barrels

In order to evaluate potential therapeutic targets for treatment of amyloidoses such as Alzheimer's disease (AD), it is essential to determine the structures of toxic amyloid oligomers. However, for the amyloid β-protein peptide (Aβ), thought to be the seminal neuropathogenetic agent in AD, its fast aggregation kinetics and the rapid equilibrium dynamics among oligomers of different size pose significant experimental challenges. Here we use ion-mobility mass spectrometry, in combination with electron microscopy, atomic force microscopy, and computational modeling, to test the hypothesis that Aβ peptides can form oligomeric structures resembling cylindrins and β-barrels. These structures are hypothesized to cause neuronal injury and death through perturbation of plasma membrane integrity. We show that hexamers of C-terminal Aβ fragments, including Aβ(24-34), Aβ(25-35) and Aβ(26-36), have collision cross sections similar to those of cylindrins. We also show that linking two identical fragments head-to-tail using diglycine increases the proportion of cylindrin-sized oligomers. In addition, we find that larger oligomers of these fragments may adopt β-barrel structures and that β-barrels can be formed by folding an out-of-register β-sheet, a common type of structure found in amyloid proteins.

Silver nanoparticles coated with natural polysaccharides as models to study AgNP aggregation kinetics using UV-Visible spectrophotometry upon discharge in complex environments

This study provides quantitative information on the aggregation and dissolution behaviour of silver nanoparticles (AgNPs) upon discharge in fresh and sea waters, represented here as NaCl solutions of increasing ionic strength (up to 1M) and natural fjord waters. Natural polysaccharides, sodium alginate (ALG) and gum Arabic (GA), were used as coatings to stabilize the AgNPs and the compounds acted as models to study AgNP aggregation kinetics. The DLVO theory was used to quantitatively describe the interactions between the AgNPs. The stability of AgNPs was established using UV-Visible spectrophotometry, including unique information collected during the first seconds of the aggregaton process. Alginate coating resulted in a moderate stabilization of AgNPs in terms of critical coagulation concentration (~82mM NaCl) and a low dissolution of <10% total Ag in NaCl solutions up to 1M. Gum Arabic coated AgNPs were more strongly stabilized, with ~7–30% size increase up to 77mM NaCl, but only when the silver ion content initially present in solution was low (<10% total Ag). The ALG and GA coated AgNPs showed a strongly enhanced stability in natural fjord waters (ca. 5h required to reduce the area of the surface plasmon resonance band (SPRB) by two fold) compared with NaCl at an equivalent ionic strength (1–2min period for a two fold SPRB reduction). This is ascribed to a stabilizing effect from dissolved organic matter present in natural fjord waters. Interestingly, for AgNP-GA solutions with 40% of total silver present as unreacted silver ions in the NP stock solution, fast aggregation kinetics were observed in NaCl solutions (SPRB area was reduced by ca. 50% within 40–150min), with even more rapid removal in fjord waters, attributed to the high amount of silver-chloride charged species, that interact with the NP coating and/or organic matter and reduce the NPs stabilization.

Analysis and Control of Protein Crystallization Using Short Peptide Tags That Change Solubility without Affecting Structure, Thermal Stability, and Function

Short peptide tags attached to recombinant proteins are emerging as an important tool for biochemical research. Here, we report the effects of 10 Solubilization Controlling Peptide (SCP) tags on crystallization behavior of a bovine pancreatic trypsin inhibitor (BPTI) variant. The tags did not affect structure, thermodynamics, and activities of BPTI. Moreover, eight of the tagged variants crystallized under the same condition, and six of them diffracted at high resolution. All variants with long-term solubility (LS) between 1 and 6 mg/mL produced crystals that diffracted well, while variants with LS 6 mg/mL did not crystallize, produced poorly diffracting crystals, or crystallized under a different condition. The only exception was a glutamine tagged variant, which had an LS of 5 mg/mL, but fast aggregation kinetics, and produced mere needles unsuitable for further analysis. Crystal structures indicated that most tags were largely invisible, indicating high flexibility, without having interactions...

Differential toxicity, conformation and morphology of typical initial aggregation states of Aβ1-42 and Aβpy3-42 beta-amyloids

Among the different species of water-soluble β-peptides (Aβ1-42, Aβ1-40 and N-terminal truncated Aβ-peptides), Aβpy3-42 is thought to play a relevant role in Alzheimer's pathogenesis due to its abundance, resistance to proteolysis, fast aggregation kinetics, dynamic structure and high neurotoxicity. To evaluate the specific structural characteristics and neurotoxicity of Aβpy3-42, we separated different aggregation states of Aβ1-42 and Aβpy3-42 using fast protein liquid chromatography, isolating in both cases three peaks that corresponded to sa (small), ma (medium) and la (large) aggregates.Conformational analysis, by circular dichroism showed a prevailing random coil conformation for sa and ma, and typical β-sheet conformation for la. AFM and TEM show differential structural features between the three aggregates of a given β-peptide and among the aggregate of the two β-peptides. The potential toxic effects of the different aggregates were evaluated using human neuroblastoma SH-SY5Y cells in the MTT reduction, in the xCELLigence System, and in the Annexin V binding experiments. In the case of Aβ1-42 the most toxic aggregate is la, while in the case of Aβpy3-42 both sa and la are equally toxic. Aβ aggregates were found to be internalized in the cells, as estimated by confocal immunofluorescence microscopy, with a higher effect observed for Aβpy3-42, showing a good correlation with the toxic effects. Together these experiments allowed the discrimination of the intermediate states more responsible of oligomer toxicity, providing new insights on the correlation between the aggregation process and the toxicity and confirming the peculiar role in the pathogenesis of Alzheimer disease of Aβpy3-42 peptide.

Amyloid Beta Peptide: The Influence of Intrinsic Factors on Fibril Formation

Alzheimer's disease (AD) is the most common form of dementia. Studying the amyloid β-peptide (Aβ) involved in the pathogenesis and understanding the mechanism of the disease will hopefully lead to developing drug candidates that could cure or delay the onset of AD.One way of approaching this is to inhibit the fibril formation. Mutating the peptide gives an insight on which amino acids are important in the aggregation mechanism. We have investigated the importance of the aromatic rings in the phenylalanine side chains in position 19 and 20 by substituting these with leucine. This way the hydrophobicity is preserved while aromatic stacking possibilities are removed. Single and double mutants were expressed in E. coli from synthetic genes. The kinetics of fibril formation was followed using a highly reproducible thioflavin T assay and show that Aβ(M1-42)F19L aggregates slower than the wild type, mainly observed as a longer lag time. Also the structural change, monitored by far-UV circular dichroism spectroscopy, differs between wild type and all mutants. This indicates that the phenylalanine rings have a role in the amyloid formation. With cryo-TEM it was confirmed that all peptides form fibrils with similar morphology.The influence of the slower F19L mutant was further studied in co-aggregation experiments together with the wild type. Preliminary results show that the lag time of the mixture ends up in between those of the wild type and the F19L mutant, and that the lag time depends on the ratio of the two peptides as well as the total concentration. Further co-aggregation studies include a familial mutant peptide with faster aggregation kinetics than the wild type, as well as seeding experiments in which wild type is seeded with fibrils from F19L or the familial mutant.1(1)

Fractal-like structures in polystyrene solutions studied by light scattering intensity

By means of a light scattering experiment as a function of scattered wavevector, we have directly studied the aggregation properties of polystyrene particles in water solutions. For different ionic strength the system shows different kinetics with different fractal dimensions. At high strengths a fast aggregation kinetics with a fractal dimension D = 1.75 (diffusion limited aggregation) is obtained, whereas at low strengths the system evidences a slow kinetics D = 2.1 (reaction limited aggregation).