Recent developments and applications of photothermal AFM-IR in characterization of amyloids and amyloids aggregation processes: Mini-review.

The process of protein aggregation has attracted a great deal of research attention, as aggregates are first of all a nuisance to preparation of high quality protein and secondly used as novel materials. In the latter case, the process of protein aggregation needs to be controlled. Here, we show how arginine (Arg) regulates the process of heat-induced protein aggregation. Dynamic light scattering and transmission electron microscopy revealed that heat-induced aggregation of lysozyme at around the isoelectric point occurred in a two-step process: formation of start aggregates, followed by further growth mediated by their sticking with diffusion-limited cluster-cluster aggregation. In the presence of Arg, the diffusion-limited regime changed to reaction-limited cluster-cluster aggregation. The data indicated that the solution additives that coexisted with proteins would affect the property of the formed product, such as morphology and mechanic strength.

This thesis aims to provide structural and biophysical insights on the molecular mechanism of amyloid aggregation. Three different molecular models were chosen for these studies; all of them being able to form fibrillar aggregates, even though starting from different conformation, therefore likely following different mechanisms. The main model is represented by amyloid beta peptides, in particular the 40 and the 42 residues peptides (Abeta40 and Abeta42), which are related to Alzheimer Disease. The ability to switch from alfa-helical conformation in membrane and apolar environment to beta-sheet-based fibril structure in aqueous solution characterizes these peptides. The second model is hemopressin, a short peptide that shows very promising pharmacological application, hampered by its aggregation propensity; this peptide is unstructured in aqueous buffers, but it forms amyloid fibrils in some pH conditions. The third model is an engineered protein, called Y65R-MNEI, derived from monellin, very interesting for its potential biotechnological application as ipocaloric sweetener. Its structures is very close to that of the parent protein MNEI which, at native state, is a soluble globular protein rich in beta-sheet. Like MNEI, it is able to form fibrils, and may represent a useful model for protein folding and aggregation. Still no aggregation study was performed on Y65R-MNEI. These molecules were studied at different levels of deepness in literature, with several different biophysical techniques. Therefore, the choice of these molecules allowed me to study the amyloid aggregation process at different stages. In particular, the effect of different physico-chemical parameters (temperature, ionic strength, pH and solvent polarity), as well as the influence of lipid interaction and of a non-enzymatic post-translational modification on the aggregation properties of these models was investigated. An integrated experimental approach was used, including biophysical techniques such as Fluorescence, CD, EPR, NMR spectroscopies and AFM, in order to fill some gaps present in literature on the aggregation process of these molecules.

α‐Synuclein (αS) is a presynaptic small protein that has attracted much interest because its aggregation and accumulation in the form of amyloid fibrils is the hallmark of a range of neurodegenerative disorders, collectively referred to as synucleinopathies. Despite intense research on this protein since it was first identified two decades ago as the major component of the proteinaceous intracellular inclusion characteristics of Parkinson disease, there is still no consensus on the physiological function of the protein and much remains to be established on the molecular basis of its toxicity. Recently, important steps have been undertaken to identify the different conformational states that this protein is able to adopt and elucidate their role in physiological and pathological conditions. Key Concepts The physiological function(s) of α‐synuclein remains controversial, although recent evidences suggest a major regulatory role in synapsis. At physiological conditions, α‐synuclein is in a dynamic equilibrium between a membrane‐bound α‐helical (likely multimeric) conformation and a cytosolic intrinsically disordered (monomeric) conformation. α‐Synuclein aggregation and fibril formation likely play a central role in Parkinson disease and other neurodegenerative disorders. Different strains or fibril polymorphs of α‐synuclein have different degrees of infectivity and might be associated with distinct types of pathologies. Different mechanisms of formation of α‐synuclein amyloid aggregates have been observed in vitro , but their relative relevance in vivo remains unknown. Recent studies support the idea that multiple aggregated species of α‐synuclein can be generated through diverse misfolding pathways during the process of amyloid aggregation and could play distinct roles during the development of disease. Combined methods to target specifically different α‐synuclein conformations could potentially prevent α‐synuclein‐associated toxicity.

Understanding the early events during amyloid aggregation processes is crucial to single out the involved molecular mechanisms and for designing ad hoc strategies to prevent and reverse amyloidogenic disorders. Here, we show that, in conditions in which the protein is positively charged and its conformational flexibility is enhanced, Concanavalin A leads to fibril formation via a non-conventional aggregation pathway. Using a combination of light scattering, circular dichroism, small angle X-ray scattering, intrinsic (Tryptophan) and extrinsic (ANS) fluorescence and confocal and 2-photon fluorescence microscopy we characterize the aggregation process as a function of the temperature. We highlight a multi-step pathway with the formation of an on-pathway long-lived intermediate and a subsequent coagulation of such “crinkled” precursors into amyloid-like fibrils. The process results in a temperature-dependent aggregation-coagulation pathway, with the late phase of coagulation determined by the interplay between hydrophobic and electrostatic forces. Our data provide evidence for the complex aggregation pathway for a protein with a highly flexible native conformation. We demonstrate the possibility to generate a long-lived intermediate whose proportion and occurrence are easily tunable by experimental parameters (i.e. temperature). As a consequence, in the case of aggregation processes developing through well-defined energy barriers, our results can open the way to new strategies to induce more stable in vitro on-pathway intermediate species through a minute change in the initial conformational flexibility of the protein. This will allow isolating and experimentally studying such transient species, often indicated as relevant in neurodegenerative diseases, both in terms of structural and cytotoxic properties.

The process of protein aggregation, due to an incorrect protein folding, has very important implications in medicine. An increasing number of pathologies, including Alzheimer's and Parkinson's diseases, are associated unambiguously with the formation of large aggregates of misfolded proteins. Yet are the causes and conditions for which aggregates become pathogenic unclear. Increasing evidences point towards the early stages of fibril formation. Of interest are hence the early dynamic events in the aggregation process. The scope of this thesis is the study of two types of optical components, based on periodic multilayer structures, and aimed at the early detection of protein aggregation. These are 1) a Bloch surface wave (BSW) sensor and 2) a type of narrow-band wire-grid polarisers (WGP) for the mid-infrared range. In the first part, the computational methods used to investigate these components theoretically are presented. A derivation of approximate analytical results regarding BSW sensing in Kretschmann configuration is proposed. These results are suggested as guidelines for the efficient design of BSW sensing structures. The second part details the experimental application of BSW sensing to the detection of protein aggregation. As an example, two types of proteins are studied : Hen Egg White Lysozyme (HEWL) and Alzheimer's amyloid beta 1-42 (Aβ1-42). Measurements on HEWL are shown to demonstrate the ability of the BSW sensing scheme to detect aggregates from monomeric proteins in a label-free way. Then, dynamic measurements conducted on Aβ1-42 reveal the ability of the sensor to observe the early events of the aggregation process. In the last part, the study of multilayer WGP as cost-effective filtering components in the mid-infrared (MIR) is presented. The presence of specific protein absorption bands in the MIR is a potential mean for selective label-free detection using BSW sensing. A simple modal model describing a particular class of multilayer WGP is proposed. A suitable fabrication method is proposed. The developed methods are illustrated by an example designed at a wavelength λ = 4.36 µm. Measurements on the transmission characteristics of the structures show good agreement with the theoretical model.

Misfolding and amyloid aggregation of intrinsically disordered proteins (IDPs) are implicated in a variety of diseases. Studies have shown that membrane plays important roles on the formation of intermediate structures of IDPs that can initiate (and/or speed-up) amyloid aggregation to form fibers. The process of amyloid aggregation also disrupts membrane to cause cell death in amyloid diseases like Alzheimer's disease and type-2 diabetes. On the other hand, recent studies reported the membrane fusion properties of amyloid fibers. Remarkably, amyloid-fibril formation by short peptide fragments of highly abundant prostatic acidic-phosphatase (PAP) in human semen and are capable of boosting the rate of HIV infection up to 400,000-fold during sexual contact. Unlike the least toxic fully matured fibers of most amyloid proteins, the semen-derived enhancer of virus infection (SEVI) amyloid-fibrils of PAP peptide fragments are highly potent in rendering the maximum rate of HIV infection. This unusual property of amyloid fibers has witnessed increasing number of studies on the biophysical aspects of fiber formation and fiber-membrane interactions. NMR studies have reported a highly disordered partial helical structure in a membrane environment for the intrinsically disordered PAP peptide that promotes the fusion of the viral membrane with that of host cells. The purpose of this review article is to unify and integrate biophysical and immunological research reported in the previous studies on SEVI. Specifically, amyloid aggregation, dramatic HIV infection enhancing properties, membrane fusion properties, high resolution NMR structure, and approaches to eliminate the enhancement of HIV infection of SEVI peptides are discussed.

Chapter 20 - Amyloid-Like Protofibrils with Different Physical Properties

The process of amyloid aggregation is quite complex and poorly understood. In this work, having summarized previously obtained results on the aggregation of the multidomain smooth muscle protein titin, an attempt has been made to expand understanding of this process, and a new possible mechanism by which amyloid aggregation of titin may occur is delineated. Our main conclusion is that the ability of titin to form amorphous aggregates seems to be the only possible way of aggregation of this protein. Most likely, only separate parts of the molecules, but not the whole protein, are involved in the formation of the amyloid structure in amorphous aggregates of smooth muscle titin. This feature, given the large size of the protein molecule, distinguishes titin from other amyloid or amyloid-like proteins. The paper discusses the potential energy landscape underlying the formation of titin amyloid aggregates.

Chapter 11 - Nucleation of polyglutamine amyloid fibres modelling using molecular dynamics

Protein function and flexibility is directly related to the native distribution of its structural elements and any alteration in protein architecture leads to several abnormalities and accumulation of misfolded proteins. This phenomenon is associated with a range of increasingly common human disorders, including Alzheimer and Parkinson diseases, type II diabetes, and a number of systemic amyloidosis characterized by the accumulation of amyloid aggregates both in the extracellular space of tissues and as intracellular deposits. Post-translational modifications are known to have an active role in the in vivo amyloid aggregation as able to affect protein structure and dynamics. Among them, a key role seems to be played by non-enzymatic glycation, the most unwanted irreversible modification of the protein structure, which strongly affects long-living proteins throughout the body. This study provided an overview of the molecular effects induced by glycation on the amyloid aggregation process of several protein models associated with misfolding diseases. In particular, we analyzed the role of glycation on protein folding, kinetics of amyloid formation, and amyloid cytotoxicity in order to shed light on the role of this post-translational modification in the in vivo amyloid aggregation process.

BackgroundAlzheimer’s Disease (AD) is a neurodegenerative proteinopathy in which Aβ can misfold and aggregate into seeds that structurally corrupt native proteins, mimicking a prion‐like process. These amyloid aggregation and propagation processes are influenced by three factors: the origin of the Aβ seed, time of incubation and host. However, the mechanism underlying the differential effect of each factor is poorly known. Previous studies have shown that the Aβ source is relevant for the amyloid process, since its pathogenicity is different according to its origin. Furthermore, recent evidence suggests that microglia plays a key role in the amyloidogenic event, and can modulate the propagation and aggregation process. Here, we seek to perform a comparative study to determine whether Aβ seeds from humans vs a familial AD line (the 3xTg‐AD model) are more efficient to generate amyloid aggregates, as well as the role of the microglia in the propagation process.MethodAmyloid seeds from AD patient (stage C for amyloid; from the Alzheimer’s Disease Research Center at UCI) and 25 mo‐3xTg‐AD mice were injected into the hippocampus of 7‐8‐month‐old 3xTg‐AD mice. They were analyzed 10 months post‐surgery for amyloid and microglia markers.ResultsOur findings demonstrated that amyloid seeds from the human patient seem to induce a more aggressive amyloid pathology compared to seeds from aged 3xTg‐AD mice. Moreover, human and mice seeds differentially affect the presence of plaque‐associated microglia in 3xTg‐AD mice.ConclusionThese results suggest that seeds from human patients seem to be more amyloidogenic than from aged 3xTg‐AD mice, and also microglia cells may play a key role in this differential effect. Therefore, more profound understanding these factors will provide key insight on how amyloid pathology progresses in AD.Funding: This study was supported by Minister of Science and Innovation grant PID2019‐108911RA‐100 (D.B.V.), Alzheimer’s Association grant AARG‐22‐928219 (D.B.V), Beatriz Galindo program BAGAL18/00052 (D.B.V.) and Institute of Health Carlos III (ISCiii) grant PI18/01557 (A.G.) co‐financed by FEDER funds from European Union.

BackgroundAlzheimer’s Disease (AD) is a neurodegenerative proteinopathy in which Aβ can misfold and aggregate into seeds that structurally corrupt native proteins, mimicking a prion‐like process. These amyloid aggregation and propagation processes are influenced by three factors: the origin of the Aβ seed, time of incubation and host. However, the mechanism underlying the differential effect of each factor is poorly known. Previous studies have shown that the Aβ source is relevant for the amyloid process, since its pathogenicity is different according to its origin. Furthermore, recent evidence suggests that microglia plays a key role in the amyloidogenic event, and can modulate the propagation and aggregation process. Here, we seek to perform a comparative study to determine whether Aβ seeds from humans vs a familial AD line (the 3xTg‐AD model) are more efficient to generate amyloid aggregates, as well as the role of the microglia in the propagation process.MethodAmyloid seeds from AD patient (stage C for amyloid; from the Alzheimer’s Disease Research Center at UCI) and 25 mo‐3xTg‐AD mice were injected into the hippocampus of 7‐8‐month‐old 3xTg‐AD mice. They were analyzed 10 months post‐surgery for amyloid and microglia markers.ResultOur findings demonstrated that amyloid seeds from the human patient seem to induce a more aggressive amyloid pathology compared to seeds from aged 3xTg‐AD mice. Moreover, human and mice seeds differentially affect the presence of plaque‐associated microglia in 3xTg‐AD mice.ConclusionThese results suggest that seeds from human patients seem to be more amyloidogenic than from aged 3xTg‐AD mice, and also microglia cells may play a key role in this differential effect. Therefore, more profound understanding these factors will provide key insight on how amyloid pathology progresses in AD. Grant support: This study was supported by Minister of Science and Innovation grant PID2019‐108911RA‐100 (D.B.V.), Alzheimer’s Association grant AARG‐22‐928219 (D.B.V), Beatriz Galindo program BAGAL18/00052 (D.B.V.) and Institute of Health Carlos III (ISCiii) grant PI18/01557 (A.G.) co‐financed by FEDER funds from European Union.

Chalcone derivatives as dual-sensitive fluorescent probe for protein aggregation investigation

Aggregation of amyloidogenic proteins is an abnormal biological process implicated in neurodegenerative disorders. Whereas the aggregation process of amyloid-forming proteins has been studied extensively, the mechanism of aggregate removal is poorly understood. We recently demonstrated that proteasomes could fragment filamentous aggregates into smaller entities, restricting aggregate size [1]. Here, we show in vitro that UBE2W can modify the N-terminus of both α-synuclein and a tau tetra-repeat domain with a single ubiquitin. We demonstrate that an engineered N-terminal ubiquitin modification changes the aggregation process of both proteins, resulting in the formation of structurally distinct aggregates. Single-molecule approaches further reveal that the proteasome can target soluble oligomers assembled from ubiquitin-modified proteins independently of its peptidase activity, consistent with our recently reported fibril-fragmenting activity. Based on these results, we propose that proteasomes are able to target oligomers assembled from N-terminally ubiquitinated proteins. Our data suggest a possible disassembly mechanism by which N-terminal ubiquitination and the proteasome may together impede aggregate formation.

Amyloid β peptide (Aβ) is the crucial protein component of extracellular plaques in Alzheimer's disease. The plaques also contain gangliosides lipids, which are abundant in membranes of neuronal cells and in cell-derived vesicles and exosomes. When present at concentrations above its critical micelle concentration (cmc), gangliosides can occur as mixed micelles. Here, we study the coassembly of the ganglioside GM1 and the Aβ peptides Aβ40 and 42 by means of microfluidic diffusional sizing, confocal microscopy, and cryogenic transmission electron microscopy. We also study the effects of lipid-peptide interactions on the amyloid aggregation process by fluorescence spectroscopy. Our results reveal coassembly of GM1 lipids with both Aβ monomers and Aβ fibrils. The results of the nonseeded kinetics experiments show that Aβ40 aggregation is delayed with increasing GM1 concentration, while that of Aβ42 is accelerated. In seeded aggregation reactions, the addition of GM1 leads to a retardation of the aggregation process of both peptides. Thus, while the effect on nucleation differs between the two peptides, GM1 may inhibit the elongation of both types of fibrils. These results shed light on glycolipid-peptide interactions that may play an important role in Alzheimer's pathology.