Process Of Fibril Formation Research Articles

Characterization and kinetics of whey protein isolate amyloid fibril aggregates under moderate electric fields

Food proteins solutions enable the controlled flow of electrical current when subjected to a Moderate electric field (MEF). This application results in internal heat generation, known as ohmic heating, accompanied by electrochemical reactions. These effects play a significant role in influencing the formation of protein amyloid fibril aggregates (AFA), which have broad applications in food and biomedical fields, ranging from improving food texture to biomedical applications such as drug delivery and disease treatment. This study focused on investigating the formation of β-Lactoglobulin (β-lg) AFA under MEF conditions using WPI as protein source and various characterization techniques (e.g., Thioflavin T fluorescence, circular dichroism, and electron microscopy). The results indicated that MEF prolonged the lag phase of AFA formation. Furthermore, nucleation and fibril growth showed higher activation coefficients and cooperativity level (n > 2) compared to conventional heating method. MEF treatment resulted in unique fibril clustering and the presence of unexpected higher-order AFA networks confirmed by electron microscopy. These findings highlight the significant role of MEF in influencing the aggregation kinetics of β-lg AFA. Exploring further into the electrochemical and thermal effects during protein aggregation processes holds the key to unlocking deeper insights fibril formation process.

A Cationic Zn-Phthalocyanine Turns Alzheimer's Amyloid β Aggregates into Non-Toxic Oligomers and Inhibits Neurotoxicity in Culture.

Amyloid β peptide (Aβ) aggregation and deposition are considered the main causes of Alzheimer's disease. In a previous study, we demonstrated that anionic Zn-phthalocyanine (ZnPc) can interact with the Aβ peptide and inhibit the fibril-formation process. However, due to the inability of anionic ZnPc to cross the intact blood-brain barrier, we decided to explore the interaction of cationic methylated Zn-phthalocyanine (cZnPc) with the peptide. Using a ThT fluorescence assay, we observed that cZnPc dose-dependently and time-dependently inhibited Aβ1-42 fibril levels under in vitro fibril-formation conditions. Electron microscopy revealed that it caused Aβ1-42 peptides to form small aggregates. Western blotting and dot immunoblot oligomer experiments demonstrated that cZnPc increased rather than decreased the levels of oligomers from the very early stages of incubation. A binding assay confirmed that cZnPc could bind with the peptide. Docking simulations indicated that the oligomer species of Aβ1-42 had a higher ability to interact with cZnPc. ANS fluorescence assay results indicated that cZnPc did not affect the hydrophobicity of the peptide. However, cZnPc significantly increased intrinsic tyrosine fluorescence of the peptide after 8 h of incubation in fibril-formation conditions. Importantly, cell culture experiments demonstrated that cZnPc did not exhibit any toxicity up to a concentration of 10 µM. Instead, it protected a neuronal cell line from Aβ1-42-induced toxicity. Thus, our results suggest that cZnPc can affect the aggregation process of Aβ1-42, rendering it non-toxic, which could be crucial for the therapy of Alzheimer's disease.

Understanding alpha-synuclein aggregation propensity in animals and humans

Alpha-synuclein (α-syn) aggregation plays a critical role in the pathogenicity of Parkinson's Disease (PD). This study aims to evaluate the aggregation propensity of α-syn fragment peptides designed using the variability found in humans and animals. Thioflavin T (ThT) and transmission electron microscopy (TEM) were used to validate the formation of fibrils to identify important amino acid residues. Human α-syn fragments 51–75, 37–61, 62–86, 76–100, and 116–140 demonstrate a significantly higher tendency to aggregate compared to fragments 1–25, 26–50, and 91–115. All species analyzed of the α-syn 37–61 and 62–86 regions were shown to form fibrils on both ThT and TEM. The α-syn 37–61 and 62–86 fragment regions exhibited a high susceptibility to aggregation, with fibril formation observed in all species. The A53T mutation in several α-syn 37–61 fragments may enhance their propensity for aggregation, suggesting a correlation between this mutation and the capacity for fibril formation. Furthermore, the presence of the non-amyloid-β component (NAC) region, specifically in α-syn 62–86, was consistently observed in several fragments that displayed fibril formation, indicating a potential correlation between the NAC region and the process of fibril formation in α-syn. Finally, the combination of a high quantity of valine and a low quantity of acidic amino acids in these fragments may serve as indicators of α-syn fibril formation.

A molecular synergy of non-covalent interactions facilitated via theobromine modifying the self-assembly process of type I collagen

Type I Collagen is a multifunctional fibrous protein present significantly in the connective tissues. Generally, fibril formation process of an interstitial collagen begins with a triple helix alignment to form a pentafibril, which sequentially self-assembles into a microfibril. Typically, this sequential formation can be achieved under an in vitro condition as well by maintaining certain specific physiological parameters to mimic the interstitial microenvironment of collagen. The necessity to study such a kinetically driven process is due to its implications in template fabrication particularly as a scaffold to enhance the growth of specific stem cells or accelerating nanoparticle synthesis. Drawing inspiration from this, theobromine, a methylxanthine that contributes towards the hydrogen bond network via dimerization and stacking interaction was used as a small molecule in modifying the molecular level interactions of a self-assembled collagen. Initially, rheological studies revealed certain variations in the viscous properties of collagen on addition of theobromine. Turbidity assay showed an increase in the rate of fibril formation and its topographical changes were observed through the high-resolution scanning electron microscope. The stability and probable physico-chemical interactions between collagen and theobromine were comprehended from the vibrational frequency changes recorded using (FT-IR) (ATR) and conformational changes using Circular Dichroism (CD) studies. Our results show possible multibinding sites on theobromine, which would compete to bind with the polar and non-polar residues exposed over the monomeric surfaces of triple helix causing a local conformational fluctuation modifying protein-protein interaction.

S100A9 inhibits and redirects prion protein 89-230 fragment amyloid aggregation

Protein aggregation in the form of amyloid fibrils has long been associated with the onset and development of various amyloidoses, including Alzheimer's, Parkinson's or prion diseases. Recent studies of their fibril formation process have revealed that amyloidogenic protein cross-interactions may impact aggregation pathways and kinetic parameters, as well as the structure of the resulting aggregates. Despite a growing number of reports exploring this type of interaction, they only cover just a small number of possible amyloidogenic protein pairings. One such pair is between two neurodegeneration-associated proteins: the pro-inflammatory S100A9 and prion protein, which are known to co-localize in vivo. In this study, we examined their cross-interaction in vitro and discovered that the fibrillar form of S100A9 modulated the aggregation pathway of mouse prion protein 89-230 fragment, while non-aggregated S100A9 also significantly inhibited its primary nucleation process. These results complement previous observations of the pro-inflammatory protein's role in amyloid aggregation and highlight its potential role against neurodegenerative disorders.

Peptide Sequence Variations Govern Hydrogel Stiffness: Insights from a Multi-Scale Structural Analysis of H-FQFQFK-NH2 Peptide Derivatives.

Throughout the past decades, amphipathic peptide-based hydrogels have proven to be promising materials for biomedical applications. Amphipathic peptides are known to adopt β-sheet configurations that self-assemble into fibers that then interact to form a hydrogel network. A fundamental understanding of how the peptide sequence alters the structural properties of the hydrogels would allow for a more rational design of novel peptides for a variety of biomedical applications in the future. Therefore, the current work investigates how changing the type of amino acid, the amphipathic pattern, and the peptide length affects the secondary structure, fiber characteristics, and stiffness of peptide-based hydrogels. Hereto, seven amphipathic peptides of different sequence and length, four of which have not been previously reported, based on and including the hexapeptide H-Phe-Gln-Phe-Gln-Phe-Lys-NH2, are synthesized and thoroughly characterized by circular dichroism (CD), Fourier Transform Infrared (FTIR) spectroscopy, Wide Angle X-ray Scattering (WAXS), Small Angle X-ray Scattering (SAXS), Transmission Electron Microscopy (TEM), and Thioflavin T (ThT) fibrillization assays. The results show that a high amount of regularly spaced β-sheets, a high amount of fibers, and fiber bundling contribute to the stiffness of the hydrogel. Furthermore, a study of the time-dependent fibril formation process reveals complex transient dynamics. The peptide strands structure through an intermediate helical state prior to β-sheet formation, which is found to be concentration- andtime-dependent.

Prediction of the Feasibility of Using the ≪Gold Standard≫ Thioflavin T to Detect Amyloid Fibril in Acidic Media.

Ordered protein aggregates, amyloid fibrils, form toxic plaques in the human body in amyloidosis and neurodegenerative diseases and provide adaptive benefits to pathogens and to reduce the nutritional value of legumes. To identify the amyloidogenic properties of proteins and study the processes of amyloid fibril formation and degradation, the cationic dye thioflavin T (ThT) is the most commonly used. However, its use in acidic environments that induce amyloid formation in vitro can sometimes lead to misinterpretation of experimental results due to electrostatic repulsion. In this work, we show that calculating the net charge per residue of amyloidogenic proteins or peptides is a simple and effective approach for predicting whether their fibrils will interact with ThT at acidic pH. In particular, it was shown that at pH 2, proteins and peptides with a net charge per residue > +0.18 are virtually unstained by this fluorescent probe. The applicability of the proposed approach was demonstrated by predicting and experimentally confirming the absence of ThT interaction with amyloids formed from green fluorescent (sfGFP) and odorant-binding (bOBP) proteins, whose fibrillogenesis was first carried out in an acidic environment. Correct experimental evidence that the inability to detect these fibrils under acidic conditions is precisely because of the lack of dye binding to amyloids (and not their specific structure or the low fluorescence quantum yield of the bound dye) and that the number of ThT molecules associated with fibrils increases with decreasing acidity of the medium was obtained by using the equilibrium microdialysis approach.

Formation and Physicochemical Properties of Freeze-Dried Amyloid-Like Fibrils From Pinto Bean Protein: Amyloid-Like Fibrils From Pinto Bean Protein.

Amyloid nanofibrils are long and thin strands with cross β structures associated by hydrogen bonds. These structures can be formed under suitable conditions commonly at low pH and high temperatures. Fibrillated pinto bean protein isolate (FPBPI) was made by heating pinto bean protein at 85°C in an acidic condition while gently stirring at initial protein solution concentrations of 4 mg/mL, 13 mg/mL, and 21 mg/mL. Freeze-dried FPBPI's physicochemical, structural, and thermal characteristics were assessed, and they were compared with a native pinto bean protein isolate (PBPI) as a control. An increase in Congo red spectral absorption at 544 nm was observed following the fibril formation process. The largest concentration of freeze-dried fibrillated protein exhibited the highest Congo red spectral absorption. Fibrillar proteins' Fourier transform infrared (FTIR) spectrograms with lower wave numbers were seen than the native protein. For native PBPI, transmission electron microscopy (TEM) images were globular in shape, but they changed to long and curly morphologies in fibrillated proteins. FPBPI has a lower melting enthalpy than native protein when measured by differential scanning calorimetry (DSC). With the rising initial protein content, the enthalpy rose. Concurrently, semicrystalline structure for native and fibrillated pinto bean proteins was revealed by X-ray diffraction (XRD) findings. As the original protein concentration grew, so did the crystallinity intensity. Water-holding capacity (WHC) and oil-holding capacity (OHC) of freeze-dried FPBPI were higher than those of native protein. So, fibrillation of pinto bean protein helped it to serve as a good thickener in food industries.

Cyclic and differential pulse voltammetric measurements on fibrils formation of alpha synuclein in Parkinson’s disease by a gold interdigitated tetraelectrodes

Parkinson's disease is a neurodegenerative disorder characterized by the aggregation and deposition of alpha-synuclein protein, which are pathological hallmarks. To understand the fibril formation of alpha-synuclein in Parkinson's disease, this study uses cyclic and differential pulse voltammetric measurements. These measurements analyze the electrochemical properties and behavior of alpha-synuclein during its fibril formation process. By applying a potential sweep or pulse to the alpha-synuclein sample, it is possible to gain insights into its redox activity and structural changes during fibril formation. This could lead to the development of therapeutic strategies to prevent or disrupt this pathological event in Parkinson's disease. To detect Parkinson's disease, a 15 nm sized gold conjugated antibody was used as the probe and seeded on gold interdigitated tetraelectrodes (AuIDTE). Alpha synuclein variations (fibriled and non-fibriled) were detected using phosphate-buffer saline and glycine buffer based on cyclic voltammetry and differential pulse voltammetry techniques. Discriminated by Tau protein measurement that was employed as a control. The linear regression for detecting alpha synuclein aggregation using differential pulse voltammetry was R2 = 0.9987 [y = 9E-06x - 4E-07], with a limit of detection of 10 aM, on a linear range of 1 aM-1 pM. Cyclic voltammetry determined the limit of detection of aggregated alpha synuclein to be 100 aM, with a linear relationship of R2 = 0.9939 [y = 7E-06x - 2E-06]. The sensor has excellent analytical performance in terms of detection limit, sensitivity, selectivity, repeatability, and stability.

PAP(248-286) Conformational Changes during the Lag Phase of Amyloid Fibril Formation.

The initial stage of fibril formation of C-terminal region PAP(248-286) of human seminal plasma protein prostatic acid phosphatase was considered. Amyloid fibrils from the peptide PAP(248-286) are termed as a semen-derived enhancer of viral infection (SEVI) found in abundant quantities in semen. The kinetics of the amyloid fibril formation process consists of two characteristic phases (lag phase/nucleation phase and growth phase/elongation phase). The lag phase can be caused by the presence of mature amyloid fibrils (seeds) in protein solution, so-called secondary nucleation. The secondary nucleation includes interaction of protein monomers with the mature fibril surface that leads to protein spatial structural changes for further amyloid fibril formation. In this work, changes of the PAP(248-286) spatial structure were obtained during the secondary nucleation phase. Pulsed-field gradient (PFG) NMR was used to characterize the behavior of monomeric PAP(248-286) in water solution after PAP(248-286) seed addition. The self-diffusion coefficient showed compactization of the peptide monomer due to fibril-monomer interactions. PAP(248-286) spatial structural changes were detected with the help of high-resolution NMR spectroscopy and molecular dynamics (MD) simulation. The folding of PAP(248-286) occurs due to backbone chain bending in the region of H270 and T275 amino acid residues. Obtained folded conformation of PAP(248-286) emerging in the secondary nucleation process is energetically favorable and retains after monomer-amyloid interaction. The structural changes are associated with localization of PAP(248-286) hydrophobic surface regions, which are probably responsible for peptide monomer-amyloid interactions.

Conformation-Specific Association of Prion Protein Amyloid Aggregates with Tau Protein Monomers.

Protein aggregation into amyloid fibrils is associated with several amyloidoses, including neurodegenerative Alzheimer's and Parkinson's diseases. Despite years of research and numerous studies, the process is still not fully understood, which significantly impedes the search for cures of amyloid-related disorders. Recently, there has been an increase in reports of amyloidogenic protein cross-interactions during the fibril formation process, which further complicates the already intricate process of amyloid aggregation. One of these reports displayed an interaction involving Tau and prion proteins, which prompted a need for further investigation into the matter. In this work, we generated five populations of conformationally distinct prion protein amyloid fibrils and examined their interaction with Tau proteins. We observed that there was a conformation-specific association between Tau monomers and prion protein fibrils, which increased the aggregate self-association and amyloidophilic dye binding capacity. We also determined that the interaction did not induce the formation of Tau protein amyloid aggregates, but rather caused their electrostatic adsorption to the prion protein fibril surface.

Characterization of Pairs of Toxic and Nontoxic Misfolded Protein Oligomers Elucidates the Structural Determinants of Oligomer Toxicity in Protein Misfolding Diseases.

The aberrant misfolding and aggregation of peptides and proteins into amyloid aggregates occurs in over 50 largely incurable protein misfolding diseases. These pathologies include Alzheimer's and Parkinson's diseases, which are global medical emergencies owing to their prevalence in increasingly aging populations worldwide. Although the presence of mature amyloid aggregates is a hallmark of such neurodegenerative diseases, misfolded protein oligomers are increasingly recognized as of central importance in the pathogenesis of many of these maladies. These oligomers are small, diffusible species that can form as intermediates in the amyloid fibril formation process or be released by mature fibrils after they are formed. They have been closely associated with the induction of neuronal dysfunction and cell death. It has proven rather challenging to study these oligomeric species because of their short lifetimes, low concentrations, extensive structural heterogeneity, and challenges associated with producing stable, homogeneous, and reproducible populations. Despite these difficulties, investigators have developed protocols to produce kinetically, chemically, or structurally stabilized homogeneous populations of protein misfolded oligomers from several amyloidogenic peptides and proteins at experimentally ameneable concentrations. Furthermore, procedures have been established to produce morphologically similar but structurally distinct oligomers from the same protein sequence that are either toxic or nontoxic to cells. These tools offer unique opportunities to identify and investigate the structural determinants of oligomer toxicity by a close comparative inspection of their structures and the mechanisms of action through which they cause cell dysfunction.This Account reviews multidisciplinary results, including from our own groups, obtained by combining chemistry, physics, biochemistry, cell biology, and animal models for pairs of toxic and nontoxic oligomers. We describe oligomers comprised of the amyloid-β peptide, which underlie Alzheimer's disease, and α-synuclein, which are associated with Parkinson's disease and other related neurodegenerative pathologies, collectively known as synucleinopathies. Furthermore, we also discuss oligomers formed by the 91-residue N-terminal domain of [NiFe]-hydrogenase maturation factor from E. coli, which we use as a model non-disease-related protein, and by an amyloid stretch of Sup35 prion protein from yeast. These oligomeric pairs have become highly useful experimental tools for studying the molecular determinants of toxicity characteristic of protein misfolding diseases. Key properties have been identified that differentiate toxic from nontoxic oligomers in their ability to induce cellular dysfunction. These characteristics include solvent-exposed hydrophobic regions, interactions with membranes, insertion into lipid bilayers, and disruption of plasma membrane integrity. By using these properties, it has been possible to rationalize in model systems the responses to pairs of toxic and nontoxic oligomers. Collectively, these studies provide guidance for the development of efficacious therapeutic strategies to target rationally the cytotoxicity of misfolded protein oligomers in neurodegenerative conditions.

AL(light chain)-amyloidogenesis by mesangial cells involves active participation of lysosomes: An ultrastructural study

Amyloid formation by cells is a stepwise process that occurs in macrophages and cells capable of transforming into a macrophage phenotype. One such cell is the mesangial cell in the kidney. It has been shown that mesangial cells are engaged in AL (light chain associated)- amyloidogenesis after transforming phenotypically from a smooth muscle to a macrophage phenotype.The actual process of amyloid fibril formation has not been dissected. This ultrastructural study which includes the examination of lysosomal gradient specimens addresses this issue by analyzing the sequence of events that takes place as fibrils are formed in endosomes and lysosomes.The findings indicate that fibrillogenesis begins in endosomes but is completed and most pronounced in the lysosomal compartment. As early as 10 min after incubation of human mesangial cells with AL-LCs, amyloid fibrils are formed in endosomes but mostly occurs in the mature lysosomal compartment. This is the first time that fibril formation is demonstrated experimentally occurring inside human mesangial cells and the entire sequence of events taking place is elucidated.

Modulation of Aβ42 Aggregation Kinetics and Pathway by Low-Molecular-Weight Inhibitors.

The aggregation of amyloid-β 42 (Aβ42) is directly related to the pathogenesis of Alzheimer's disease. Here, we have investigated the early stages of the aggregation process, during which most of the cytotoxic species are formed. Aβ42 aggregation kinetics, characterized by the quantification of Aβ42 monomer consumption, were tracked by real-time solution NMR spectroscopy (RT-NMR) allowing the impact that low-molecular-weight (LMW) inhibitors and modulators exert on the aggregation process to be analysed. Distinct differences in the Aβ42 kinetic profiles were apparent and were further investigated kinetically and structurally by using thioflavin T (ThT) and transmission electron microscopy (TEM), respectively. LMW inhibitors were shown to have a differential impact on early-state aggregation. Insight provided here could direct future therapeutic design based on kinetic profiling of the process of fibril formation.

Peptide-based inhibitors of amyloid beta aggregation and toxicity.

Neurotoxic aggregates of amyloid beta (Aβ) peptide contribute to the etiology of Alzheimer's disease. Aβ1-42 forms oligomeric structures that undergo further aggregation into protofibrils and fibrils. Previous work has shown that seven overlapping fragments of Aβ1-42 can disrupt the process of fibril formation, possibly through intercalation into the growing aggregate. The potential neuroprotective properties of these fragments were not directly examined. In this project, Aβ1-42 was allowed to aggregate in aqueous buffer for 2, 4, or 24 h in the absence and presence of an Aβ fragment, at 1:2 Aβ1-42/fragment molar ratio. Neuronal PC12 cells were then incubated with the samples for 50 h before conducting a cell viability assay. Thioflavin-T fluorescence assays to detect the fibrillar form of Aβ1-42 were also conducted. There was an inverse correlation between the time span of Aβ1-42 aggregation and cellular toxicity, with the 2 h sample exhibiting the greatest level of toxicity. Aβ fragments displayed varying effects on the inhibition of Aβ1-42 fibrillogenesis and suppression of its toxicity. Three fragments completely neutralized the toxicity of the 2 h aggregate, while two others conferred weak cellular protection against Aβ1-42 toxicity. One fragment enhanced Aβ1-42 toxicity at all stages of aggregation. Every fragment disrupted fibril formation to some extent, but there was no obvious correlation between the inhibition of Aβ1-42 aggregation and the inhibition of cellular toxicity. This suggests that fragment intercalation into the growing fibril may block further aggregation but not the toxicity of oligomeric Aβ1-42. Fragments that block both Aβ1-42 aggregation and toxicity represent potential targets for therapeutic development.

Influence of denaturants on amyloid β42 aggregation kinetics.

Amyloid formation is linked to devastating neurodegenerative diseases, motivating detailed studies of the mechanisms of amyloid formation. For Aβ, the peptide associated with Alzheimer’s disease, the mechanism and rate of aggregation have been established for a range of variants and conditions in vitro and in bodily fluids. A key outstanding question is how the relative stabilities of monomers, fibrils and intermediates affect each step in the fibril formation process. By monitoring the kinetics of aggregation of Aβ42, in the presence of urea or guanidinium hydrochloride (GuHCl), we here determine the rates of the underlying microscopic steps and establish the importance of changes in relative stability induced by the presence of denaturant for each individual step. Denaturants shift the equilibrium towards the unfolded state of each species. We find that a non-ionic denaturant, urea, reduces the overall aggregation rate, and that the effect on nucleation is stronger than the effect on elongation. Urea reduces the rate of secondary nucleation by decreasing the coverage of fibril surfaces and the rate of nucleus formation. It also reduces the rate of primary nucleation, increasing its reaction order. The ionic denaturant, GuHCl, accelerates the aggregation at low denaturant concentrations and decelerates the aggregation at high denaturant concentrations. Below approximately 0.25 M GuHCl, the screening of repulsive electrostatic interactions between peptides by the charged denaturant dominates, leading to an increased aggregation rate. At higher GuHCl concentrations, the electrostatic repulsion is completely screened, and the denaturing effect dominates. The results illustrate how the differential effects of denaturants on stability of monomer, oligomer and fibril translate to differential effects on microscopic steps, with the rate of nucleation being most strongly reduced.

Temporal and Sex-Linked Protein Expression Dynamics in a Familial Model of Alzheimer’s Disease

Mouse models of Alzheimer’s disease (AD) show progression through stages reflective of human pathology. Proteomics identification of temporal and sex-linked factors driving AD-related pathways can be used to dissect initiating and propagating events of AD stages to develop biomarkers or design interventions. In the present study, we conducted label-free proteome measurements of mouse hippocampus tissue with variables of time (3, 6, and 9 months), genetic background (5XFAD versus WT), and sex (equal males and females). These time points are associated with well-defined phenotypes with respect to the following: Aβ42 plaque deposition, memory deficits, and neuronal loss, allowing correlation of proteome-based molecular signatures with the mouse model stages. Our data show 5XFAD mice exhibit increases in known human AD biomarkers as amyloid-beta peptide, APOE, GFAP, and ITM2B are upregulated across all time points/stages. At the same time, 23 proteins are here newly associated with Alzheimer’s pathology as they are also dysregulated in 5XFAD mice. At a pathways level, the 5XFAD-specific upregulated proteins are significantly enriched for DNA damage and stress-induced senescence at 3-month only, while at 6-month, the AD-specific proteome signature is altered and significantly enriched for membrane trafficking and vesicle-mediated transport protein annotations. By 9-month, AD-specific dysregulation is also characterized by significant neuroinflammation with innate immune system, platelet activation, and hyper-reactive astrocyte–related enrichments. Aside from these temporal changes, analysis of sex-linked differences in proteome signatures uncovered novel sex and AD-associated proteins. Pathway analysis revealed sex-linked differences in the 5XFAD model to be involved in the regulation of well-known human AD-related processes of amyloid fibril formation, wound healing, lysosome biogenesis, and DNA damage. Verification of the discovery results by Western blot and parallel reaction monitoring confirm the fundamental conclusions of the study and poise the 5XFAD model for further use as a molecular tool for understanding AD.

NMR spectroscopy, excited states and relevance to problems in cell biology - transient pre-nucleation tetramerization of huntingtin and insights into Huntington's disease.

Solution nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for analyzing three-dimensional structure and dynamics of macromolecules at atomic resolution. Recent advances have exploited the unique properties of NMR in exchanging systems to detect, characterize and visualize excited sparsely populated states of biological macromolecules and their complexes, which are only transient. These states are invisible to conventional biophysical techniques, and play a key role in many processes, including molecular recognition, protein folding, enzyme catalysis, assembly and fibril formation. All the NMR techniques make use of exchange between sparsely populated NMR-invisible and highly populated NMR-visible states to transfer a magnetization property from the invisible state to the visible one where it can be easily detected and quantified. There are three classes of NMR experiments that rely on differences in distance, chemical shift or transverse relaxation (molecular mass) between the NMR-visible and -invisible species. Here, I illustrate the application of these methods to unravel the complex mechanism of sub-millisecond pre-nucleation oligomerization of the N-terminal region of huntingtin, encoded by exon-1 of the huntingtin gene, where CAG expansion leads to Huntington's disease, a fatal autosomal-dominant neurodegenerative condition. I also discuss how inhibition of tetramerization blocks the much slower (by many orders of magnitude) process of fibril formation.

HSP10 as a Chaperone for Neurodegenerative Amyloid Fibrils.

Neurodegenerative diseases (NDs) are associated with accumulated misfolded proteins (MPs). MPs oligomerize and form multiple forms of amyloid fibril polymorphs that dictate fibril propagation and cellular dysfunction. Protein misfolding processes that impair protein homeostasis are implicated in onset and progression of NDs. A wide variety of molecular chaperones safeguard the cell from MP accumulation. A rather overlooked molecular chaperone is HSP10, known as a co-chaperone for HSP60. Due to the ubiquitous presence in human tissues and protein overabundance compared with HSP60, we studied how HSP10 alone influences fibril formation in vitro of Alzheimer’s disease-associated Aβ1–42. At sub-stoichiometric concentrations, eukaryotic HSP10s (human and Drosophila) significantly influenced the fibril formation process and the fibril structure of Aβ1–42, more so than the prokaryotic HSP10 GroES. Similar effects were observed for prion disease-associated prion protein HuPrP90–231. Paradoxically, for a chaperone, low concentrations of HSP10 appeared to promote fibril nucleation by shortened lag-phases, which were chaperone and substrate dependent. Higher concentrations of chaperone while still sub-stoichiometric extended the nucleation and/or the elongation phase. We hypothesized that HSP10 by means of its seven mobile loops provides the chaperone with high avidity binding to amyloid fibril ends. The preserved sequence of the edge of the mobile loop GGIM(V)L (29–33 human numbering) normally dock to the HSP60 apical domain. Interestingly, this segment shows sequence similarity to amyloidogenic core segments of Aβ1–42, GGVVI (37–41), and HuPrP90-231 GGYML (126–130) likely allowing efficient competitive binding to fibrillar conformations of these MPs. Our results propose that HSP10 can function as an important molecular chaperone in human proteostasis in NDs.

Influence of Cortisol on the Fibril Formation Kinetics of Aβ42 Peptide: A Multi-Technical Approach.

Amyloid-β peptide (Aβ) aggregates are known to be correlated with pathological neurodegenerative diseases. The fibril formation process of such peptides in solution is influenced by several factors, such as the ionic strength of the buffer, concentration, pH, and presence of other molecules, just to mention a few. In this paper, we report a detailed analysis of in vitro Aβ42 fibril formation in the presence of cortisol at different relative concentrations. The thioflavin T fluorescence assay allowed us to monitor the fibril formation kinetics, while a morphological characterization of the aggregates was obtained by atomic force microscopy. Moreover, infrared absorption spectroscopy was exploited to investigate the secondary structure changes along the fibril formation path. Molecular dynamics calculations allowed us to understand the intermolecular interactions with cortisol. The combined results demonstrated the influence of cortisol on the fibril formation process: indeed, at cortisol-Aβ42 concentration ratio (ρ) close to 0.1 a faster organization of Aβ42 fragments into fibrils is promoted, while for ρ = 1 the formation of fibrils is completely inhibited.