Effect of phospholipid liposomes on prion fragment (106-128) amyloid formation.

Misfolding and aggregation of cellular prion protein (PrPc) is a major molecular process involved in the pathogenesis of prion diseases. Here, we studied the aggregation properties of a prion fragment peptide PrP(106-128). The results show that the peptide aggregates in a concentration-dependent manner in an aqueous solution and that the aggregation is sensitive to pH and the preformed amyloid seeds. Furthermore, we show that the zwitterionic POPC liposomes moderately inhibit the aggregation of PrP(106-128), whereas POPC/cholesterol (8:2) vesicles facilitate peptide aggregation likely due to the increase of the lipid packing order and membrane rigidity in the presence of cholesterol. In addition, anionic lipid vesicles of POPG and POPG/cholesterol above a certain concentration accelerate the aggregation of the peptide remarkably. The strong electrostatic interactions between the N-terminal region of the peptide and POPG may constrain the conformational plasticity of the peptide, preventing insertion of the peptide into the inner side of the membrane and thus promoting fibrillation on the membrane surface. The results suggest that the charge properties of the membrane, the composition of the liposomes, and the rigidity of lipid packing are critical in determining peptide adsorption on the membrane surface and the efficiency of the membrane in catalyzing peptide oligomeric nucleation and amyloid formation. The peptide could be used as an improved model molecule to investigate the mechanistic role of the crucial regions of PrP in aggregation in a membrane-rich environment and to screen effective inhibitors to block key interactions between these regions and membranes for preventing PrP aggregation.

Effects of Charged Polyelectrolytes on Amyloid Fibril Formation of a Tau Fragment.

The microtubule-associated protein tau is involved in more than 20 different neurological disorders characterized by aberrant intracellular aggregation of tau in the brain. Here, we investigated the aggregation of a novel 20-residue model peptide, tau298-317, which is derived from the key microtubule binding domain of the full sequence tau. Our results show that tau298-317 highly mimics the physical and aggregation properties of tau. Under normal physiological conditions, the peptide maintains a disordered random coil without aggregation. The presence of polyanionic heparin (Hep) significantly promotes the aggregation of this peptide to form amyloid fibrils. The Hep-induced aggregation is sensitive to the ionic strength of the solution and the introduction of the negatively charged phosphate group on a serine (Ser305) residue in the sequence, suggesting an important role of electrostatic interactions in the mechanism of Hep-mediated aggregation. In addition, two positively charged polysaccharides, chitosan (CHT) and its quaternary derivative N-trimethyl chitosan (TMC), were found to effectively inhibit Hep-induced aggregation of tau298-317 in a concentration-dependent manner. Attractive electrostatic interactions between the positively charged moieties in CHT/TMC and the negatively charged residues of Hep play a critical role in inhibiting Hep-peptide interactions and suppressing peptide aggregation. Our results suggest that positively charged polyelectrolytes with optimized charged groups and charge distribution patterns can serve as effective molecular candidates to block tau-Hep interactions and prevent aggregation of tau induced by Hep and other polyanions.

Effects of zinc and carnosine on aggregation kinetics of Amyloid-ß40 peptide.

The accumulation and amyloid formation of amyloid-ß (Aß) peptides is closely associated with the pathology of Alzheimer's disease. The physiological environment wherein Aß aggregation happens is crowded with a large variety of metal ions including Zn2+. In this study, we investigated the role of Zn2+ in regulating the aggregation kinetics of Aß40 peptide. Our results show that Zn2+ can shift a typical single sigmoidal aggregation kinetics of Aß40 to a biphasic aggregation process. Zn2+ aids in initiating the rapid self-assembly of monomers to form oligomeric intermediates, which further grow into amyloid fibrils in the first aggregation phase. The presence of Zn2+ also retards the appearance of the second aggregation phase in a concentration dependent manner. In addition, our results show that a natural dipeptide, carnosine, can greatly alleviate the effect of Zn2+ on Aß aggregation kinetics, most likely by coordinating with the metal ion to form chelates. These results suggest a potential in vivo protective effect of carnosine against the cytotoxicity of Aß by suppressing Zn2+-induced rapid formation of Aß oligomers.

Tau liquid-liquid phase separation: At the crossroads of tau physiology and tauopathy.

Abnormal deposition of tau in neurons is a hallmark of Alzheimer's disease and several other neurodegenerative disorders. In the past decades, extensive efforts have been made to explore the mechanistic pathways underlying the development of tauopathies. Recently, the discovery of tau droplet formation by liquid-liquid phase separation (LLPS) has received a great deal of attention. It has been reported that tau condensates have a biological role in promoting and stabilizing microtubule (MT) assembly. Furthermore, it has been hypothesized that the transition of phase-separated tau droplets to a gel-like state and then to fibrils is associated with the pathology of neurodegenerative diseases. In this review, we outline LLPS, the structural disorder that facilitates tau droplet formation, the effects of posttranslational modification of tau on condensate formation, the physiological function of tau droplets, the pathways from droplet to toxic fibrils, and the therapeutic strategies for tauopathies that might evolve from toxic droplets. We expect a deeper understanding of tau LLPS will provide additional insights into tau physiology and tauopathies.

Mucin-Type O-Glycosylation Proximal to ß-Secretase Cleavage Site Affects APP Processing and Aggregation Fate.

The amyloid-ß precursor protein (APP) undergoes proteolysis by ß- and γ-secretases to form amyloid-ß peptides (Aß), which is a hallmark of Alzheimer's disease (AD). Recent findings suggest a possible role of O-glycosylation on APP's proteolytic processing and subsequent fate for AD-related pathology. We have previously reported that Tyr681-O-glycosylation and the Swedish mutation accelerate cleavage of APP model glycopeptides by ß-secretase (amyloidogenic pathway) more than α-secretase (non-amyloidogenic pathway). Therefore, to further our studies, we have synthesized additional native and Swedish-mutated (glyco)peptides with O-GalNAc moiety on Thr663 and/or Ser667 to explore the role of glycosylation on conformation, secretase activity, and aggregation kinetics of Aß40. Our results show that conformation is strongly dependent on external conditions such as buffer ions and solvent polarity as well as internal modifications of (glyco)peptides such as length, O-glycosylation, and Swedish mutation. Furthermore, the level of ß-secretase activity significantly increases for the glycopeptides containing the Swedish mutation compared to their nonglycosylated and native counterparts. Lastly, the glycopeptides impact the kinetics of Aß40 aggregation by significantly increasing the lag phase and delaying aggregation onset, however, this effect is less pronounced for its Swedish-mutated counterparts. In conclusion, our results confirm that the Swedish mutation and/or O-glycosylation can render APP model glycopeptides more susceptible to cleavage by ß-secretase. In addition, this study sheds new light on the possible role of glycosylation and/or glycan density on the rate of Aß40 aggregation.

Therapeutic strategies for tauopathies and drug repurposing as a potential approach.

Tauopathies are neurodegenerative diseases characterized by the deposition of abnormal tau in the brain. To date, there are no disease-modifying therapies approved by the U.S. Food and Drug Administration (US FDA) for the treatment of tauopathies. In the past decades, extensive efforts have been provided to develop disease-modifying therapies to treat tauopathies. Specifically, exploring existing drugs with the intent of repurposing for the treatment of tauopathies affords a reasonable alternative to discover potent drugs for treating these formidable diseases. Drug repurposing will not only reduce formulation and development stage effort and cost but will also take a key advantage of the established toxicological studies, which is one of the main causes of clinical trial failure of new molecules. In this review, we provide an overview of the current treatment strategies for tauopathies and the recent progress in drug repurposing as an alternative approach to treat tauopathies.

Gallium nanoparticles as novel inhibitors of Aß40 aggregation.

Alzheimer's disease (AD) has been consistently related to the formation of senile amyloid plaques mainly composed of amyloid ß (Aß) peptides. The toxicity of Aß aggregates has been indicated to be responsible for AD pathology. One scenario to decrease Aß toxicity is the development of effective inhibitors against Aß amyloid formation. In this study, we investigate the effect of gallium nitride nanoparticles (GaN NPs) as inhibitors of Aß40 amyloid formation using a combination of biophysical approaches. Our results show that the lag phase of Aß40 aggregation kinetics is significantly retarded by GaN NPs in a concentration dependent manner, implying the activity of GaN NPs in interfering with the formation of the crucial nucleus during Aß aggregation. Our results also show that GaN NPs can reduce the amyloid fibril elongation rate in the course of the aggregation kinetics. It is speculated that the high polarization characteristics of GaN NPs may provoke a strong interaction between the particles and Aß40 peptide and in this way decrease self-association of the peptide monomers to form amyloids.

Effects of the Hydrophilic N-Terminal Region on Aß-Mediated Membrane Disruption.

Amyloidogenesis of amyloid-ß (Aß) peptides is intimately related to pathological neurodegeneration in Alzheimer's disease. Here, we investigated the membrane damage activity of Aß40 and its derivatives that contain mutation at the N-terminal charged residues using a membrane leakage assay. A model 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) phospholipid vesicle encapsulating the fluorescent dye carboxyfluorescein was used in the study. Our results show that the mutations of the N-terminal charged residues of Aß40 significantly affect the peptide-induced membrane leakage. The results suggest that favorable electrostatic interactions of the N-terminal charged residues and the phosphatidylcholine membrane surface are crucial in Aß-mediated membrane permeation. The flexible and charge-rich N-terminal region may play a critical role in directing Aß self-association on the membrane surface and in anchoring and stabilizing the peptide aggregates inserted in the phospholipid membrane, which are closely related with membrane disruption activity of Aß. The results provide new mechanistic insight into the Aß-mediated membrane damage process, which may be critical for understanding the mechanism of Aß neurotoxicity in Alzheimer's disease.

High-energy square-wave pulses generated in a 1/1.5-µm dual-band mode-locked fiber laser.

The generation of square-wave pulses in a 1/1.5-µm dual-band mode-locked fiber laser is experimentally demonstrated. The laser is based upon a peculiar "figure-θ" architecture that exploits a single active fiber to realize dual-band operation. High-energy square-wave pulses are simultaneously generated in both the 1-µm and the 1.5-µm spectral band using the laser. The 1-µm pulse maintains wave-breaking-free operation during the increase of the pump power and finally achieves energy as high as 88.6 nJ, while the 1.5-µm pulse achieves energy up to 1.5 µJ before it ultimately collapses into second-order mode locking. To the best of our knowledge, this is the first report on the formation of square-wave pulses in dual-band mode-locked fiber lasers.

Flavonoids with Vicinal Hydroxyl Groups Inhibit Human Calcitonin Amyloid Formation.

Human calcitonin (hCT) is a 32-residue peptide hormone that can aggregate into amyloid fibrils and cause cellular toxicity. In this study, we investigated the inhibition effects of a group of polyphenolic molecules on hCT amyloid formation. Our results suggest that the gallate moiety in epigallocatechin-3-gallate (EGCG), a well-recognized amyloid inhibitor, is not critical for its inhibition function in the hCT amyloid formation. Our results demonstrate that flavonoid compounds, such as myricetin, quercetin, and baicalein, that contain vicinal hydroxyl groups on the phenyl ring effectively prevent hCT fibrillization. This structural feature may also be applied to non-flavonoid polyphenolic inhibitors. Moreover, our results indicate a plausible mechanistic role of these vicinal hydroxyl groups which might include the oxidation to form a quinone and the subsequent covalent linkage with amino acid residues such as lysine or histidine in hCT. This may further disrupt the crucial electrostatic and aromatic interactions involved in the process of hCT amyloid fibril formation. The inhibition activity of the polyphenolic compounds against hCT fibril formation may likely be attributed to a combination of factors such as covalent linkage formation, aromatic stacking, and hydrogen bonding interactions.

Effects of disulfide bond and cholesterol derivatives on human calcitonin amyloid formation.

Human calcitonin (hCT) is a 32-residue peptide that aggregates to form amyloid fibrils under appropriate conditions. In this study, we investigated the effect of the intramolecular disulfide bond formed at the N-terminal region of the peptide in the aggregation kinetics of hCT. Our results indicate that the presence of the disulfide bond in hCT plays a crucial role in forming the critical nucleus needed for fibril formation, facilitating the rate of hCT amyloidogenesis. Furthermore, we reported for the first time the effects of cholesterol, cholesterol sulfate, and 3ß-[N-(dimethylaminoethane)carbamoyl]-cholesterol (DC-cholesterol) on the amyloid formation of oxidized hCT. Our results show that while cholesterol does not affect amyloidogenesis of oxidized hCT, high concentrations of cholesterol sulfate exhibits a moderate inhibiting activity on hCT amyloid formation. In particular, our results show that DC-cholesterol strongly inhibits amyloidogenesis of oxidized hCT in a dose-dependent manner. Further studies at different pH conditions imply the crucial impact of electrostatic and hydrogen bonding interactions in mediating the interplay of hCT and the surface of DC-cholesterol vesicles and the inhibiting function of DC-cholesterol on hCT fibrillization.

Vibrational Approach to the Dynamics and Structure of Protein Amyloids.

Amyloid diseases, including neurodegenerative diseases such as Alzheimer's and Parkinson's, are linked to a poorly understood progression of protein misfolding and aggregation events that culminate in tissue-selective deposition and human pathology. Elucidation of the mechanistic details of protein aggregation and the structural features of the aggregates is critical for a comprehensive understanding of the mechanisms of protein oligomerization and fibrillization. Vibrational spectroscopies, such as Fourier transform infrared (FTIR) and Raman, are powerful tools that are sensitive to the secondary structure of proteins and have been widely used to investigate protein misfolding and aggregation. We address the application of the vibrational approaches in recent studies of conformational dynamics and structural characteristics of protein oligomers and amyloid fibrils. In particular, introduction of isotope labelled carbonyl into a peptide backbone, and incorporation of the extrinsic unnatural amino acids with vibrational moieties on the side chain, have greatly expanded the ability of vibrational spectroscopy to obtain site-specific structural and dynamic information. The applications of these methods in recent studies of protein aggregation are also reviewed.

Residue-Specific Dynamics and Local Environmental Changes in Aß40 Oligomer and Fibril Formation.

Elucidating local dynamics of protein aggregation is crucial for understanding the mechanistic details of protein amyloidogenesis. Herein, we studied the residue-specific dynamics and local environmental changes of Aß40 along the course of aggregation by using para-cyanophenylalanine (PheCN ) as a fluorescent and vibrational probe. Our results show that the PheCN residues introduced at various positions all exhibited an immediate decay of fluorescence intensity, indicating a relatively synergistic process in early oligomer formation. The fast decreases in the fluorescence intensities of residues 19 and 20 in the central hydrophobic core region and residue 10 in the N-terminal region suggest that they play crucial roles in the formation of the oligomeric core. The PheCN 4 residue exhibits a remarkably slower decrease in fluorescence intensity, implicating its dynamic conformational characteristics in oligomer and fibril formation. Our results also suggest that the N-terminal residues in fibrils are surrounded by a relatively hydrophobic local environment, as opposed to being solvated.

N-Terminal Charged Residues of Amyloid-ß Peptide Modulate Amyloidogenesis and Interaction with Lipid Membrane.

Interactions of amyloid-ß (Aß) peptides and cellular membranes are proposed to be closely related with Aß neurotoxicity in Alzheimer's disease. In this study, we systematically investigated the effect of the N-terminal hydrophilic region of Aß40 on its amyloidogenesis and interaction with supported phospholipid bilayer. Our results show that modulation of the charge properties of the dynamic N-terminal region dramatically influences the aggregation properties of Aß. Furthermore, our results demonstrate that the N-terminal charged residues play a crucial role in driving the early adsorption and latter remobilization of the peptide on membrane bilayer, and mediating the rigidity and viscoelasticity properties of the bound Aß40 at the membrane interface. The results provide new mechanistic insight into the early Aß-membrane interactions and binding, which may be critical for elucidating membrane-mediated Aß amyloidogenesis in a physiological environment and unravelling the origin of Aß neurotoxicity.

Gold Nanoparticles as a Probe for Amyloid-ß Oligomer and Amyloid Formation.

The process of amyloid-ß (Aß) amyloid formation is pathologically linked to Alzheimer's disease (AD). The identification of Aß amyloids and intermediates that are crucial players in the pathology of AD is critical for exploring the underlying mechanism of Aß aggregation and the diagnosis of the disease. Herein, we performed a gold nanoparticle (AuNP)-based study to detect the formation of Aß amyloid fibrils and oligomers. Our results demonstrate that the intensity of the surface plasmon resonance (SPR) absorption band of the AuNPs is sensitive to the quantity of Aß40 amyloids. This allows the SPR assay to be used for detection and semi-quantification of Aß40 amyloids, and characterization of the kinetics of Aß amyloid formation. Furthermore, our study demonstrates that the SPR band intensity of the AuNPs is sensitive to the presence of oligomers of both Aß40 and an Aß40 mutant, which forms more stable oligomers. The kinetics of the stable oligomer formation of the Aß40 mutant can also be monitored following the SPR band intensity change of AuNPs. Our results indicate that this nanoparticle based method can be used for mechanistic studies of early protein self-assembly and fibrillogenesis.

Effects of Charged Cholesterol Derivatives on Aß40 Amyloid Formation.

Understanding of the mechanistic progess of amyloid-ß peptide (Aß) aggregation is critical for elucidating the underlying pathogenesis of Alzheimer's disease (AD). Herein, we report for the first time the effects of two cholesterol derivatives, negatively charged cholesterol sulfate (cholesterol-SO4) and positively charged 3ß-[N-(dimethylaminoethane)carbamoyl]-cholesterol (DC-cholesterol), on the fibrillization of Aß40. Our results demonstrate that both of the nonvesicular forms of cholesterol-SO4 and DC-cholesterol moderately accelerate the aggregation rate of Aß40. This effect is similar to that observed for unmodified cholesterol, indicating the importance of hydrophobic interactions in binding of Aß40 to these steroid molecules. Furthermore, we show that the vesicles formed at higher concentrations of anionic cholesterol-SO4 facilitate Aß40 aggregation rate markedly. In contrast, the cationic DC-cholesterol vesicles show the ability to inhibit Aß40 fibril formation under appropriate experimental conditions. The results suggest that the electrostatic interactions between Aß40 and the charged vesicles can be of great importance in regulating Aß40-vesicle interaction. Our results also indicate that the structural properties of the aggregates of the cholesterol derivatives, including the surface charge and the size of the vesicles, are critical in regulating the effects of these vesicles on Aß40 aggregation kinetics.

Positively Charged Chitosan and N-Trimethyl Chitosan Inhibit Aß40 Fibrillogenesis.

Amyloid fibrils, formed by aggregation of improperly folded or intrinsically disordered proteins, are closely related with the pathology of a wide range of neurodegenerative diseases. Hence, there is a great deal of interest in developing molecules that can bind and inhibit amyloid formation. In this regard, we have investigated the effect of two positively charged polysaccharides, chitosan (CHT) and its quarternary derivative N-trimethyl chitosan chloride (TMC), on the aggregation of Aß40 peptide. Our aggregation kinetics and atomic force microscopy (AFM) studies show that both CHT and TMC exhibit a concentration-dependent inhibiting activity on Aß40 fibrillogenesis. Systematic pH-dependent studies demonstrate that the attractive electrostatic interactions between the positively charged moieties in CHT/TMC and the negatively charged residues in Aß40 play a key role in this inhibiting activity. The stronger inhibiting activity of TMC than CHT further suggests the importance of charge density of the polymer chain in interacting with Aß40 and blocking the fibril formation. The possible interactions between CHT/TMC and Aß40 are also revealed at the atomic level by molecular docking simulation, showing that the Aß40 monomer could be primarily stabilized by electrostatic interactions with charged amines of CHT and quaternary amines of TMC, respectively. Binding of CHT/TMC on the central hydrophobic core region of Aß40 peptide may be responsible for blocking the propagation of the nucleus to form fibrillar structures. These results suggest that incorporation of sugar units such as d-glucosamine and N-trimethyl-d-glucosamine into polymer structural template may serve as a new strategy for designing novel antiamyloid molecules.

Site-specific dynamics of amyloid formation and fibrillar configuration of Aß(1-23) using an unnatural amino acid.

We identify distinct site-specific dynamics over the time course of Aß1-23 amyloid formation by using an unnatural amino acid, p-cyanophenylalanine, as a sensitive fluorescent and Raman probe. Our results also suggest the key role of an edge-to-face aromatic interaction in the conformational conversion to form and stabilize ß-sheet structure.

Aggregation gatekeeper and controlled assembly of Trpzip ß-hairpins.

Protein and peptide aggregation is an important issue both in vivo and in vitro. Herein, we examine the aggregation behaviors of two well-studied ß-hairpins, Trpzip1 and Trpzip2. Previous studies suggested that Trpzip2 remains monomeric up to a concentration of ~15 mM whereas Trpzip1 readily aggregates at micromolar concentrations at acidic or neutral pH. This disparity is puzzling considering that these two peptides differ only in their turn sequences (i.e., GN vs NG). We hypothesize that these peptides can aggregate from their folded states via native edge-to-edge interactions and that the Lys8 residue in Trpzip2 is a more effective aggregation gatekeeper, because of a more favorable orientation. In support of this hypothesis, we find that increasing the pH to 13 or replacing Lys8 with a hydrophobic and photolabile Lys analogue, Lys(nvoc), leads to a significant increase in the aggregation propensity of Trpzip2, and that the aggregation of this Trpzip2 mutant can be reversed upon restoring the native Lys side chain via photocleavage of the nvoc moiety. In addition, we find that while both Trpzip1 and Trpzip2 form parallel ß-sheet aggregates, the Lys(nvoc) Trpzip2 mutant forms antiparallel ß-sheets and more stable fibrils. Taken together, these findings provide another example showing how sensitive peptide and protein aggregation is to minor sequence variation and that it is possible to use a photolabile non-natural amino acid, such as Lys(nvoc), to tune the rate of peptide aggregation and to control fibrillar structure.

Mechanisms of transthyretin inhibition of ß-amyloid aggregation in vitro.

Tissue-specific overexpression of the human systemic amyloid precursor transthyretin (TTR) ameliorates Alzheimer's disease (AD) phenotypes in APP23 mice. TTR-ß-amyloid (Aß) complexes have been isolated from APP23 and some human AD brains. We now show that substoichiometric concentrations of TTR tetramers suppress Aß aggregation in vitro via an interaction between the thyroxine binding pocket of the TTR tetramer and Aß residues 18-21 (nuclear magnetic resonance and epitope mapping). The K(D) is micromolar, and the stoichiometry is <1 for the interaction (isothermal titration calorimetry). Similar experiments show that engineered monomeric TTR, the best inhibitor of Aß fibril formation in vitro, did not bind Aß monomers in liquid phase, suggesting that inhibition of fibrillogenesis is mediated by TTR tetramer binding to Aß monomer and both tetramer and monomer binding of Aß oligomers. The thousand-fold greater concentration of tetramer relative to monomer in vivo makes it the likely suppressor of Aß aggregation and disease in the APP23 mice.