Crucial role of nonspecific interactions in amyloid nucleation

Protein oligomers have been implicated as toxic agents in a wide range of amyloid-related diseases. However, it has remained unsolved whether the oligomers are a necessary step in the formation of amyloid fibrils or just a dangerous byproduct. Analogously, it has not been resolved if the amyloid nucleation process is a classical one-step nucleation process or a two-step process involving prenucleation clusters. We use coarse-grained computer simulations to study the effect of nonspecific attractions between peptides on the primary nucleation process underlying amyloid fibrillization. We find that, for peptides that do not attract, the classical one-step nucleation mechanism is possible but only at nonphysiologically high peptide concentrations. At low peptide concentrations, which mimic the physiologically relevant regime, attractive interpeptide interactions are essential for fibril formation. Nucleation then inevitably takes place through a two-step mechanism involving prefibrillar oligomers. We show that oligomers not only help peptides meet each other but also, create an environment that facilitates the conversion of monomers into the β-sheet–rich form characteristic of fibrils. Nucleation typically does not proceed through the most prevalent oligomers but through an oligomer size that is only observed in rare fluctuations, which is why such aggregates might be hard to capture experimentally. Finally, we find that the nucleation of amyloid fibrils cannot be described by classical nucleation theory: in the two-step mechanism, the critical nucleus size increases with increases in both concentration and interpeptide interactions, which is in direct contrast with predictions from classical nucleation theory.During the process of amyloid formation, normally soluble proteins assemble into fibrils that are enriched in β-sheet content and have diameters of a few nanometers and lengths up to several micrometers. This phenomenon has been implicated in a variety of pathogenic processes, including Alzheimer’s and Parkinson’s diseases, type 2 diabetes, and systemic amyloidoses (1–3). The association with human diseases has largely motivated a long-standing effort to probe the assembly process, and numerous studies have aimed at elucidating the mechanism of amyloid aggregation (4). The basic nature of the aggregation reaction has emerged as a nucleation and growth process (5, 6), where the aggregates are created through a not well-understood primary nucleation event and can grow by recruiting additional peptides or proteins to their ends (7, 8). In this paper, we focus on the nature of this primary step in amyloid nucleation and the fundamental initial events that underlie amyloid formation.Amyloidogenic peptides and proteins, when in their nonpathological cellular form, can range in the structures from mainly α-helical to β-sheet and even random coil, whereas the amyloid forms of proteins possess a generic cross–β-structure (9–14). The formation of amyloid is, hence, accompanied by marked changes in the conformations of the peptides and proteins that undergo this process. A pertinent question is whether this conformational change takes place simultaneously with the nucleation process or whether nucleation takes place first and is then followed by conformational change. These two possible scenarios of nucleation have been extensively discussed in the experimental and theoretical literature (5, 8, 15–19). We will refer in this work to the two scenarios simply as one-step nucleation (1SN), in which the β-sheet–enriched nucleus forms directly from the solution, and two-step nucleation (2SN), where soluble monomers first assemble into disordered oligomers, which subsequently convert into a β-sheet nucleus. Disordered oligomers, ranging in size between dimers and micrometer-sized particles, have been observed in some experiments (20–28). These findings highlight a central question regarding the role of disordered oligomers in fibril formation: are such clusters a necessary step in the process of fibril formation or just a byproduct?From a biological and biomedical perspective, it is important to understand the conditions under which oligomeric clusters form, because such species exhibit high cytotoxicity (1, 29–31). Indeed, there is strong evidence that the disordered oligomers rather than fully grown fibrils are the main pathogenic species in protein aggregation diseases (31–33). As such, defining the role of the prefibrillar oligomers during amyloid formation will be crucial to develop intervention strategies that target these species (1, 30, 34, 35).Mutations in the polypeptide sequence and extrinsic changes in the experimental conditions are known to alter the concentrations of aggregated species, their size, and their cytotoxicity (25, 36–39). For instance, mutations that increase hydrophobicity of the Alzheimer’s β-peptide (Aβ1–42) have a pronounced effect on its aggregation behavior and the size distribution of the resulting oligomers (23–26, 40), promoting toxicity and expediting the fibrillization process. In the same spirit, two extra hydrophobic residues in Aβ1–42 are believed to contribute to the more pronounced oligomerization and faster fibrillization compared with its alloform Aβ1–40 (24, 25, 40). Temperature, pH, and concentration of certain metals also affect oligomerization and pathways of fibrillization (41–44).The common feature of the above experiments is that they modify the internal free energy difference between the soluble and the β-sheet–forming state, also called the β-sheet propensity, which has been extensively studied in the literature (45–48). However, they also modify interactions between peptides that aggregate, a crucial contribution that has not yet been systematically addressed.In this paper, we study the effect of nonspecific interactions between peptides on the amyloid nucleation process. Such nonspecific interactions do not depend on the atomistic details of the amino acids involved, allowing us to address question about amyloid aggregation and nucleation using a coarse-grained model. In particular, generic hydrophobic stretches in the sequence of Aβ have been shown to be sufficient to promote aggregation (49, 50). Mutations of nonpolar residues to other nonpolar residues had little or no effect on aggregation, whereas mutations that reduce charge and/or increase hydrophobicity enhanced it (50, 51). Furthermore, atomic force microscopy measurements have shown that the strength of overall interactions between amyloidogenic proteins correlates with their tendency to aggregate (52, 53).We have performed extensive computer simulations that allowed us to observe both the 1SN and the 2SN mechanisms. These simulations reveal that 1SN and 2SN can be viewed as two limits of the same process, something that several previous studies have suspected (16, 18). Importantly, we observe that only 2SN is possible at low peptide concentrations, comparable with the levels that are found in vivo. Another key observation is that fibril nucleation typically does not proceed through the most prevalent oligomeric species but rather, through an oligomer with a size that is only observed as a result of rare fluctuations. As a consequence, such oligomers will be hard to capture experimentally, although their presence is required for nucleation to take place. Our simulations show that the free energy barrier for fibril nucleation through the two-step mechanism decreases with increasing strength of the interpeptide interactions. Furthermore, the critical nucleus size in the two-step mechanism is found to grow with the increase in the peptide concentrations as well as with stronger interpeptide interactions, which is in direct contrast with the classical nucleation. These results imply that weakening the nonspecific interactions between peptide monomers in solution and thereby, simultaneously increasing both the free energy barrier for oligomer formation and the free energy barrier for peptide conversion at a given oligomer size may be a crucial step in preventing amyloid aggregation.     

Linear Addition Polymers and Cyclic Oligomers of N,N‐Diglycidyl Aniline and Amines. Uncrosslinked Epoxide–Amine Addition Polymers, 46

The addition polymerization of N,N‐diglycidyl aniline (DGA) and disecondary diamines leads to linear addition polymers with molecular weights ranging from 2 500 to 9 100 Da respectively. Their relatively broad molecular weight distribution (M̄_w /M̄_n = 5.5 to 17) is caused by the formation of small amounts of cyclic oligomers. Surprisingly, the addition polymerization of primary monoamines and DGA results in the formation of oligomers only. These oligomers have molecular weights between 684 and 1 165 g·mol^–1. ¹³C NMR spectra proof that during addition reaction no side‐reaction took place and that the epoxide end groups were completely consumed. Obviously, the addition products mainly consist of cyclic oligomers. In the MALDI‐TOF mass spectra cyclic oligomers of repeat units between n = 1 and n = 7 were observed. The kinetics of the addition polymerization can be described by both a formal model and the smallest necessary set of elementary reactions. In order to find the optimum parameters, the set of differential equations was solved numerically by multivariate non‐linear regression. The perfect agreement between model calculations and experimental curves allows reliable predictions of the reaction behavior for arbitrary temperature–time profiles.     

Solution‐Processable Donor‐Acceptor‐Donor Oligomers with Cross‐Linkable Functionality

Electron‐acceptor units, combined with bithiophene substituted with flexible chains end‐functionalized with cross‐linkable moieties, provide soluble donor‐acceptor‐donor (DAD) π‐conjugated oligomer‐type molecules with cross‐linking ability and broad absorption in the visible spectrum. A study on the cross‐linking conditions of the new oligomers to yield insoluble polymer networks is presented, including conditions for obtaining polymer films over poly(3,4‐ethylenedioxythiophene):polystyrene sulfonate‐covered substrates. The combination of the DAD molecular design and cross‐linking functionality opens prospects for applications in solution‐processed small‐molecule solar cells with morphologically‐stable organic layers.

     

Dermal, inhalation, and internal exposure to 1,6-HDI and its oligomers in car body repair shop workers and industrial spray painters

Objectives

To study inhalation and dermal exposure to hexamethylene diisocyanate (HDI) and its oligomers as well as personal protection equipment (PPE) use during task performance in conjunction with urinary hexamethylene diamine (HDA) in car body repair shop workers and industrial spray painters.

Methods

Personal task based inhalation samples (n = 95) were collected from six car body repair shops and five industrial painting companies using impingers with di‐n‐butylamine (DBA) in toluene. In parallel, dermal exposure was assessed using nitril rubber gloves. Gloves were submerged into DBA in toluene after sampling. Analysis for HDI and its oligomers was performed by LC‐MS/MS. Urine samples were collected from 55 workers (n = 291) and analysed for HDA by GC‐MS.

Results

Inhalation exposure was strongly associated with tasks during which aerosolisation occurs. Dermal exposure occurred during tasks that involve direct handling of paint. In car body repair shops associations were found between detectable dermal exposure and glove use (odds ratio (OR) 0.22, 95% confidence interval (CI) 0.09 to 0.57) and inhalation exposure level (OR 1.34, 95% CI 0.97 to 1.84 for a 10‐fold increase). HDA in urine could be demonstrated in 36% and 10% of car body repair shop workers and industrial painting company workers respectively. In car body repair shops, the frequency of detectable HDA was significantly elevated at the end of the working day (OR 2.13, 95% CI 1.07 to 4.22 for 3–6 pm v 0–8 am). In both branches HDA was detected in urine of ∼25% of the spray painters. In addition HDA was detected in urine of a large proportion of non‐spray painters in car body repair shops.

Conclusion

Although (spray) painting with lacquers containing isocyanate hardeners results in the highest external exposures to HDI and oligomers, workers that do not perform paint related tasks may also receive a considerable internal dose.     

Insight into amyloid structure using chemical probes

Alzheimer's disease (AD) is a common neurodegenerative disorder characterized by the deposition of amyloids in the brain. One prominent form of amyloid is composed of repeating units of the amyloid-β (Aβ) peptide. Over the past decade, it has become clear that these Aβ amyloids are not homogeneous; rather, they are composed of a series of structures varying in their overall size and shape and the number of Aβ peptides they contain. Recent theories suggest that these different amyloid conformations may play distinct roles in disease, although their relative contributions are still being discovered. Here, we review how chemical probes, such as Congo red, thioflavin T and their derivatives, have been powerful tools for the better understanding of amyloid structure and function. Moreover, we discuss how design and deployment of conformationally selective probes might be used to test emerging models of AD.     

Chain Length Dependence in Diketopyrrolopyrrole/Thiophene Oligomers

Diketopyrrolopyrrole‐based polymers have shown good performance in polymer solar cells. Monodisperse short oligomers, monomer up to tetramer, of one of these polymers, pBBTDPP1, have been prepared and the chain length dependence of their optical and electrochemical properties has been investigated. The optical and electrochemical data obtained in solution lead to the conclusion that conjugation in this system is very limited. The most probable reason for this is a twist in the backbone, caused by steric hindrance of the dodecyl side chains with the sulfur atoms. In thin films, the chain length dependence of the optical properties is much stronger, due to planarization of the oligomer chains upon aggregation.

     

Paramagnetic self‐assembled nanoparticles as supramolecular MRI contrast agents

Nanometer‐sized materials offer a wide range of applications in biomedical technologies, particularly imaging and diagnostics. Current scaffolds in the nanometer range predominantly make use of inorganic particles, organic polymers or natural peptide‐based macromolecules. In contrast we hereby report a supramolecular approach for the preparation of self‐assembled dendritic‐like nanoparticles for applications as MRI contrast agents. This strategy combines the benefits from low molecular weight imaging agents with the ones of high molecular weight. Their in vitro properties are confirmed by in vivo measurements: post injection of well‐defined and meta‐stable nanoparticles allows for high‐resolution blood‐pool imaging, even at very low Gd(III) doses. These dynamic and modular imaging agents are an important addition to the young field of supramolecular medicine using well‐defined nanometer‐sized assemblies. Copyright © 2012 John Wiley & Sons, Ltd.     

A Facile Route to Bio‐Inspired Supramolecular Oligo(Ethylene Glycol) Catecholates

A particularly facile synthetic route to mussel‐inspired oligo(ethylene glycol) catecholates is described, yielding high purity materials with minimal purification. The oligocatecholates show remarkable thermal and rheological properties for their actual chain length, suggesting the operation of non‐covalent interactions between their l ‐DOPA‐bearing chain ends. This end‐group effect is more pronounced in the shorter oligomers (M _n of ethylene glycol chain of 200 and 400 Da), where the end‐group density is higher. Various surfaces (glass, stainless steel) are modified with the oligocatecholates, using a dip‐coating approach from dilute aqueous solutions. Water contact angle measurements and X‐ray photoelectron spectroscopy confirm the presence of a hydrophilic surface coating layer. The potential of these materials for application as antifouling coatings and viscosity modifiers is highlighted.