Individual Protein Aggregates Research Articles

Nanoscale imaging of individual amyloid aggregates extracted from brains of Alzheimer and Parkinson patients reveals presence of lipids in α-synuclein but not in amyloid β1-42 fibrils.

Abrupt aggregation of misfolded proteins is the underlying molecular cause of Alzheimer disease (AD) and Parkinson disease (PD). Both AD and PD are severe pathologies that affect millions of people around the world. A small 42 amino acid long peptide, known as amyloid β (Aβ), aggregates in the frontal cortex of AD patients forming oligomers and fibrils, highly toxic protein aggregates that cause progressive neuron death. Similar aggregates of α-synuclein (α-Syn), a small protein that facilitates neurotransmitter release, are observed in the midbrain, hypothalamus, and thalamus of people with PD. In this study, we utilized the innovative nano-Infrared imaging technique to investigate the structural organization of individual Aβ and α-syn fibrils postmortem extracted from brains of AD and PD patients, respectively. We observed two morphologically different Aβ and α-Syn fibril polymorphs in each patient's brain. One had twisted topology, whereas another exhibited flat tape-like morphology. We found that both polymorphs shared the same parallel β-sheet-dominated secondary structure. These findings suggested that both fibril polymorphs were built from structurally similar if not identical filaments that coiled forming twisted fibrils or associated side-by-side in the case of straight Aβ and α-Syn fibrils. Nano-Infrared analysis of individual protein aggregates also revealed the presence of lipids in the structure of both twisted and tape-like α-Syn fibrils that were not observed in any of the Aβ fibril polymorphs. These findings demonstrate that lipid membranes can play a critically important role in the onset and progression of PD.

Nanoscale Characterization of Parallel and Antiparallel β-Sheet Amyloid Beta 1-42 Aggregates.

Abrupt aggregation of amyloid beta (Aβ) peptide is strongly associated with Alzheimer's disease. In this study, we used atomic force microscopy-infrared (AFM-IR) spectroscopy to characterize the secondary structure of Aβ oligomers, protofibrils and fibrils formed at the early (4 h), middle (24 h), and late (72 h) stages of protein aggregation. This innovative spectroscopic approach allows for label-free nanoscale structural characterization of individual protein aggregates. Using AFM-IR, we found that at the early stage of protein aggregation, oligomers with parallel β-sheet dominated. However, these species exhibited slower rates of fibril formation compared to the oligomers with antiparallel β-sheet, which first appeared in the middle stage. These antiparallel β-sheet oligomers rapidly propagated into fibrils that were simultaneously observed together with parallel β-sheet fibrils at the late stage of protein aggregation. Our findings showed that aggregation of Aβ is a complex process that yields several distinctly different aggregates with dissimilar toxicities.

PROCESSING–PROPERTY RELATIONSHIPS IN WHEAT PROTEIN–UNVULCANIZED ISOPRENE RUBBER COMPOSITES

ABSTRACT An optimized set of processing parameters was determined for trypsin hydrolyzed gliadin protein (THGd, from wheat) and unvulcanized synthetic isoprene rubber (IR) composite compounds. The compounding temperature, time, and shear dependency of the THGd:IR compounds were investigated with rheology, Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDX). Individual protein aggregates measuring ∼64 × 280 nm, with a length-to-width aspect ratio of ∼4.3, were agglomerated within the IR matrix, as measured in SEM-EDX micrographs. A higher compounding temperature (T = 150 °C) was favorable for good protein aggregate dispersion and deagglomeration, which resulted in IR reinforcement, as demonstrated by up to a 65% increase in storage modulus (G′). The tan δ was lowered by the addition of the THGd protein to IR, indicating less mechanical energy was lost as heat in the composites. As the compounding speed, that is, shear rate, was increased from 30 to 60 rpm at constant temperature (T = 150 °C), the average agglomerate size and G′ were minimally affected, but the loss tangent (tan δ) was increased, indicating an increase in IR degradation with shear rate. By drying the THGd hydrolysate at a slower rate or to a greater solids content, the composite G′ was increased and tan δ was decreased. In the slow-dried THGd hydrolysate, protein aggregates were less resistant to breakup during processing, indicating that the protein preparation was an important processing parameter.

Characterizing Individual Protein Aggregates by Infrared Nanospectroscopy and Atomic Force Microscopy.

The phenomenon of protein misfolding and aggregation results in the formation of highly heterogeneous protein aggregates, which are associated with neurodegenerative conditions such as Alzheimer's and Parkinson's diseases. In particular low molecular weight aggregates, amyloid oligomers, have been shown to possess generic cytotoxic properties and are implicated as neurotoxins in many forms of dementia. We illustrate the use of methods based on atomic force microscopy (AFM) to address the challenging task of characterizing the morphological, structural and chemical properties of these aggregates, which are difficult to study using conventional structural methods or bulk biophysical methods because of their heterogeneity and transient nature. Scanning probe microscopy approaches are now capable of investigating the morphology of amyloid aggregates with sub-nanometer resolution. We show here that infrared (IR) nanospectroscopy (AFM-IR), which simultaneously exploits the high resolution of AFM and the chemical recognition power of IR spectroscopy, can go further and enable the characterization of the structural properties of individual protein aggregates, and thus offer insights into the aggregation mechanisms. Since the approach that we describe can be applied also to the investigations of the interactions of protein assemblies with small molecules and antibodies, it can deliver fundamental information to develop new therapeutic compounds to diagnose or treat neurodegenerative disorders.

Characterizing Individual Protein Aggregates by Infrared Nanospectroscopy and Atomic Force Microscopy

Characterizing Individual Protein Aggregates by Infrared Nanospectroscopy and Atomic Force Microscopy

Extrinsic Amyloid-Binding Dyes for Detection of Individual Protein Aggregates in Solution.

Protein aggregation is a key molecular feature underlying a wide array of neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases. To understand protein aggregation in molecular detail, it is crucial to be able to characterize the array of heterogeneous aggregates that are formed during the aggregation process. We present here a high-throughput method to detect single protein aggregates, in solution, from a label-free aggregation reaction, and we demonstrate the approach with the protein associated with Parkinson’s disease, α-synuclein. The method combines single-molecule confocal microscopy with a range of amyloid-binding extrinsic dyes, including thioflavin T and pentameric formylthiophene acetic acid, and we show that we can observe aggregates at low picomolar concentrations. The detection of individual aggregates allows us to quantify their numbers. Furthermore, we show that this approach also allows us to gain structural insights from the emission intensity of the extrinsic dyes that are bound to aggregates. By analyzing the time evolution of the aggregate populations on a single-molecule level, we then estimate the fragmentation rate of aggregates, a key process that underlies the multiplication of pathological aggregates. We additionally demonstrate that the method permits the detection of these aggregates in biological samples. The capability to detect individual protein aggregates in solution opens up a range of new applications, including exploiting the potential of this method for high-throughput screening of human biofluids for disease diagnosis and early detection.

Optical Structural Analysis of Individual α-Synuclein Oligomers.

Small aggregates of misfolded proteins play a key role in neurodegenerative disorders. Such species have proved difficult to study due to the lack of suitable methods capable of resolving these heterogeneous aggregates, which are smaller than the optical diffraction limit. We demonstrate here an all‐optical fluorescence microscopy method to characterise the structure of individual protein aggregates based on the fluorescence anisotropy of dyes such as thioflavin‐T, and show that this technology is capable of studying oligomers in human biofluids such as cerebrospinal fluid. We first investigated in vitro the structural changes in individual oligomers formed during the aggregation of recombinant α‐synuclein. By studying the diffraction‐limited aggregates we directly evaluated their structural conversion and correlated this with the potential of aggregates to disrupt lipid bilayers. We finally characterised the structural features of aggregates present in cerebrospinal fluid of Parkinson's disease patients and age‐matched healthy controls.

Optical Structural Analysis of Individual α‐Synuclein Oligomers

AbstractSmall aggregates of misfolded proteins play a key role in neurodegenerative disorders. Such species have proved difficult to study due to the lack of suitable methods capable of resolving these heterogeneous aggregates, which are smaller than the optical diffraction limit. We demonstrate here an all‐optical fluorescence microscopy method to characterise the structure of individual protein aggregates based on the fluorescence anisotropy of dyes such as thioflavin‐T, and show that this technology is capable of studying oligomers in human biofluids such as cerebrospinal fluid. We first investigated in vitro the structural changes in individual oligomers formed during the aggregation of recombinant α‐synuclein. By studying the diffraction‐limited aggregates we directly evaluated their structural conversion and correlated this with the potential of aggregates to disrupt lipid bilayers. We finally characterised the structural features of aggregates present in cerebrospinal fluid of Parkinson's disease patients and age‐matched healthy controls.

Automated longitudinal monitoring of in vivo protein aggregation in neurodegenerative disease C. elegans models.

BackgroundWhile many biological studies can be performed on cell-based systems, the investigation of molecular pathways related to complex human dysfunctions – e.g. neurodegenerative diseases – often requires long-term studies in animal models. The nematode Caenorhabditis elegans represents one of the best model organisms for many of these tests and, therefore, versatile and automated systems for accurate time-resolved analyses on C. elegans are becoming highly desirable tools in the field.ResultsWe describe a new multi-functional platform for C. elegans analytical research, enabling automated worm isolation and culture, reversible worm immobilization and long-term high-resolution imaging, and this under active control of the main culture parameters, including temperature. We employ our platform for in vivo observation of biomolecules and automated analysis of protein aggregation in a C. elegans model for amyotrophic lateral sclerosis (ALS). Our device allows monitoring the growth rate and development of each worm, at single animal resolution, within a matrix of microfluidic chambers. We demonstrate the progression of individual protein aggregates, i.e. mutated human superoxide dismutase 1 - Yellow Fluorescent Protein (SOD1-YFP) fusion proteins in the body wall muscles, for each worm and over several days. Moreover, by combining reversible worm immobilization and on-chip high-resolution imaging, our method allows precisely localizing the expression of biomolecules within the worms’ tissues, as well as monitoring the evolution of single aggregates over consecutive days at the sub-cellular level. We also show the suitability of our system for protein aggregation monitoring in a C. elegans Huntington disease (HD) model, and demonstrate the system’s ability to study long-term doxycycline treatment-linked modification of protein aggregation profiles in the ALS model.ConclusionOur microfluidic-based method allows analyzing in vivo the long-term dynamics of protein aggregation phenomena in C. elegans at unprecedented resolution. Pharmacological screenings on neurodegenerative disease C. elegans models may strongly benefit from this method in the near future, because of its full automation and high-throughput potential.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0083-6) contains supplementary material, which is available to authorized users.

Asymmetric Disposal of Individual Protein Aggregates in Escherichia coli, One Aggregate at a Time

Escherichia coli cells employ an asymmetric strategy at division, segregating unwanted substances to older poles, which has been associated with aging in these organisms. The kinetics of this process is still poorly understood. Using the MS2 coat protein fused to green fluorescent protein (GFP) and a reporter construct with multiple MS2 binding sites, we tracked individual RNA-MS2-GFP complexes in E. coli cells from the time when they were produced. Analyses of the kinetics and brightness of the spots showed that these spots appear in the midcell region, are composed of a single RNA-MS2-GFP complex, and reach a pole before another target RNA is formed, typically remaining there thereafter. The choice of pole is probabilistic and heavily biased toward one pole, similar to what was observed by previous studies regarding protein aggregates. Additionally, this mechanism was found to act independently on each disposed molecule. Finally, while the RNA-MS2-GFP complexes were disposed of, the MS2-GFP tagging molecules alone were not. We conclude that this asymmetric mechanism to segregate damage at the expense of aging individuals acts probabilistically on individual molecules and is capable of the accurate classification of molecules for disposal.

Polymorphism of Amyloid Fibrils Formed by a Short Peptide from Yeast Prion Protein Sup35: AFM and Tip Enhanced Raman Scattering Study

Misfolding of prion protein and its subsequent aggregation is the cause of various prion related diseases. Prions are composed of aggregates of a misfolded prion protein. Although primary structure of prion protein is the same, its infectious form, amyloid fibrils, exist as multiple strains. The ability of prions to generate multiple strains poses an immediate health threat, which renders studies of this phenomenon very important. The strains are thought to represent structurally different prion protein molecules packed into amyloid fibrils. The biophysical properties of the fibrils, such as fragility, represent a major mechanism of prion amplification. Here, we demonstrate that variations in environmental conditions such as pH, salt concentration, temperature and mechanical stress (stirring) produces a variety of fibrillar polymorphs for a short peptide CGNNQQNY from yeast prion protein Sup35. The fibrils differ by their length and diameter as well as their ability to bundle together. We have used Tip-Enhanced Raman Scattering (TERS) in combination with AFM to study underlying conformational peculiarities of peptides within individual aggregates. Two types of fibrils were investigated, one formed in water and another one at pH 5.6. These conditions produce morphologically distinct fibrils in terms of their length and diameter. They also exhibited different kinetics of aggregation. The Raman spectra obtained with TERS also revealed the peptide conformational differences between these two types of fibrils. The observed differences are mostly manifested in the positions of the characteristic amide bands (I and III), suggesting that peptides in these two types of fibrils have different conformational states. This study demonstrates potentials of such a combined method as TERS/AFM for structural analysis of individual protein aggregates.

An integrated laser-tweezers instrument for microanalysis of individual protein aggregates

Herein we introduce a new laser-tweezers instrument capable of providing both physical and chemical information of individually trapped protein aggregates. These capabilities were demonstrated by investigating the aggregation process of α-elastin droplets of nanometers to micrometers in diameter. Individual droplets of aggregated elastin molecules were trapped at the focus of a 1064 nm near infrared (NIR) laser in the instrument. The size of these individual elastin aggregates was inferred by recording their thermal motion at a frequency of 100 kHz using a position sensitive photodetector (PSPD). The chemical information of the trapped particles was revealed by confocal fluorescence and confocal Raman spectroscopies using 532-nm laser excitation. Background signals outside trapped particles were eliminated by a pinhole in back reflected geometry. Both physical and confocal fluorescence measurements revealed a transition temperature of 38 °C for a fast process in individual elastin aggregates, a value consistent with the clouding temperature in bulk. Confocal Raman spectroscopy yielded a transition temperature of 35 °C for a slow process characteristic of the formation of elastin aqueous two phase systems (ATPS).