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 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.

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.

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.

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.

Label-Free Confocal Raman Mapping of Transportan in Melanoma Cells.

Cell-penetrating peptides (CPPs) are promising vectors for the intracellular delivery of a variety of membrane-impermeable bioactive compounds. The mechanisms by which CPPs cross the cell membrane, and the effects that CPPs may have on cell function, still remain to be fully clarified. In this work, we employed confocal Raman microscopy (CRM) and atomic force microscopy (AFM) to study the infiltration and physiological effects of the amphipathic CPP transportan (Tp) on the metastatic melanoma cell line SK-Mel-2. CRM enabled the detection of label-free Tp within the cells. Raman maps of live cells revealed rapid entry (within 5 min) and widespread distribution of the peptide throughout the cytoplasm and the presence of the peptide within the nucleus after ∼20 min. Principal component analysis of the CRM data collected from Tp-treated and untreated cells showed that Tp Raman bands were not positively correlated with lipid Raman bands, indicating that Tp entered the cells via a nonendocytic mechanism. Analysis of intracellularly recovered Tp by mass spectrometry showed that Tp remained intact in SK-Mel-2 cells for up to 24 h. The Raman spectroscopic data also showed that, although Tp was predominantly unstructured (random coil) in aqueous solution, it accumulated to high densities within the cells with mostly ß-sheet and α-helical structures. AFM was employed to measure the effect of Tp treatment on cell stiffness. These data showed that Tp induced a significant increase in cell stiffness within the first hour of treatment, which was partially abated after 2 h. It is hypothesized that the increase in cell stiffness was the result of cytoskeletal changes triggered by Tp.

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.

TNF-α and IFN-γ promote lymphocyte adhesion to endothelial junctional regions facilitating transendothelial migration.

Inflammatory conditions induce redistribution of junctional adhesion receptors toward the apical regions of endothelial cells promoting lymphocyte TEM. Much of the molecular structures of TEM have been revealed; however, the biophysical mechanisms underlying this process remain to be fully elucidated. Here, we used immunofluorescence microscopy and AFM to study endothelial distribution of adhesion molecules upon lymphocyte activation and transmigration. Our immunofluorescence results revealed redistribution of JAM-A and PECAM-1 but not ICAM-1 or VCAM-1 toward the apical junctional regions of HUVECs following a 6-h stimulation with TNF-α and IFN-γ. Consistently, our SCFS studies revealed that Jurkat cell adhesion to stimulated HUVEC monolayers was significantly greater in junctional regions. Enhanced adhesion was mediated mostly by JAM-A receptors. Further AFM adhesion mapping of the homophilic JAM-A/JAM-A interaction on the surfaces of HUVECs revealed a greater number of JAM-A receptors available for binding along junctional regions after TNF-α and IFN-γ stimulation. Our data reveal for the first time that adhesion "hot spots" of JAM-A receptors are involved in initiating lymphocyte TEM under inflammatory conditions.

Poly(4-styrenesulfonate) as an inhibitor of Aß40 amyloid fibril formation.

The formation of amyloid, a cross-ß-sheet fibrillar aggregate of proteins, is associated with a variety of neurodegenerative diseases. Amyloidogenic proteins such as ß-amyloid (Aß) are known to exist with a large amount of polyelectrolyte macromolecules in vivo. The exact nature of Aß-polyelectrolyte interactions and their roles in Aß-aggregation are largely unknown. In this regard, we report the inhibiting effect of an anionic polyelectrolyte poly(4-styrenesulfonate) (PSS) on the aggregation of Aß40 peptide. The results demonstrate the strong inhibition potential of PSS on the aggregation of Aß40 and imply the dominant role of hydrophobicity of the polyelectrolyte in reducing the propensity of Aß40 amyloid formation. Additional studies with poly(vinyl sulfate) (PVS) and p-toluenesulfonate (PTS), which share similar charge density with PSS except the former lacking the nonpolar aromatic side chain and the latter the aliphatic hydrocarbon backbone, reveal that the presence of both aliphatic backbone and aromatic side chain group in PSS is essential for its Aß-aggregation inhibition activity. The interactions involved in the Aß40-PSS complex were further investigated using molecular dynamics (MD) simulation. Our results provide new insights into the structural interplay between polyelectrolytes and Aß peptide, facilitating the ultimate understanding of amyloid formation in Alzheimer's disease. The results should assist in developing novel polyelectrolytes as potential chemical tools to study amyloid aggregation.

LFA-1 binding destabilizes the JAM-A homophilic interaction during leukocyte transmigration.

Leukocyte transendothelial migration into inflamed areas is regulated by the integrity of endothelial cell junctions and is stabilized by adhesion molecules including junctional adhesion molecule-A (JAM-A). JAM-A has been shown to participate in homophilic interactions with itself and in heterophilic interactions with leukocyte function-associated antigen-1 (LFA-1) via its first and second immunoglobulin domains, respectively. Using competitive binding assays in conjunction with atomic force microscopy adhesion measurements, we provide compelling evidence that the second domain of JAM-A stabilizes the homophilic interaction because its deletion suppresses the dynamic strength of the JAM-A homophilic interaction. Moreover, binding of the LFA-1 inserted domain to the second domain of JAM-A reduces the dynamic strength of the JAM-A homophilic interaction to the level measured with the JAM-A domain 2 deletion mutant. This finding suggests that LFA-1 binding cancels the stabilizing effects of the second immunoglobulin domain of JAM-A. Finally, our atomic force microscopy measurements reveal that the interaction of JAM-A with LFA-1 is stronger than the JAM-A homophilic interaction. Taken together, these results suggest that LFA-1 binding to JAM-A destabilizes the JAM-A homophilic interaction. In turn, the greater strength of the LFA-1/JAM-A complex permits it to support the tension needed to disrupt the JAM-A homophilic interaction, thus allowing transendothelial migration to proceed.

Atomic force microscopy measurements of lens elasticity in monkey eyes.

PURPOSE: To demonstrate the feasibility of measuring the elasticity of intact crystalline lenses using atomic force microscopy (AFM). METHODS: AFM elasticity measurements were performed on intact lenses from 18 fresh cynomolgus monkey cadaver eyes (4-10 years old, <1 day postmortem) that had been left attached to their zonule-ciliary body-sclera framework. The eyes were prepared by bonding a plastic ring on the sclera after removal of the conjunctival, adipose, and muscle tissues. The posterior pole was sectioned, with the excess vitreous removed, and the eye's anterior section was placed on a Teflon slide to protect the posterior pole of the lens. The cornea and iris were then sectioned. The lens-zonule-ciliary body-sclera section was then placed in a Petri dish filled with balanced salt solution in an AFM system designed for force measurements. Next, the central pole of the anterior surface of the intact lens was probed with the AFM cantilever tip. The recorded AFM cantilever deflection-indentation curves were used to derive force-indentation curves for the lens after factoring out the deflection of the cantilever on a hard surface. Young's modulus of the lens was calculated from the force-indentation relation using the Hertz model. RESULTS: Young's modulus was 1,720+/-880 Pa (range: 409-3,210 Pa) in the 18 cynomolgus monkey lenses. CONCLUSIONS: AFM can be used to provide measurements of the elasticity of the whole lens including the capsule. Values obtained using AFM on cynomolgus monkey lenses are similar to published values obtained using dynamic mechanical analysis on young human lenses.

Force spectroscopy of LFA-1 and its ligands, ICAM-1 and ICAM-2.

Single-molecule measurements of the interaction of leukocyte function-associated antigen-1 (LFA-1), expressed on Jurkat T cells, with intercellular adhesion molecules-1 and -2 (ICAM-1 and ICAM-2) were conducted using atomic force microscopy (AFM). The force spectra (i.e., unbinding force versus loading rate) of both the LFA-1/ICAM-1 and LFA-1/ICAM-2 interactions were acquired at a loading rate range covering 3 orders of magnitude (50-60,000 pN/s) and revealed a fast loading regime and a slow loading regime. This indicates that the dissociation of both complexes involves overcoming a steep inner and a wide outer activation barrier. LFA-1 binding to ICAM-1 and ICAM-2 was strengthened in the slow loading regime by the addition of Mg(2+). Differences in the dynamic strength of the LFA-1/ICAM-1 and LFA-1/ICAM-2 interactions can be attributed to the presence of wider barriers in the ICAM-2 complex, making it more responsive to a pulling force than the ICAM-1 complex.

Dynamic adhesion of T lymphocytes to endothelial cells revealed by atomic force microscopy.

The recruitment of T lymphocytes to lymphoid organs or sites of inflammation is a crucial step in adaptive immunity. These processes require endothelial activation and expression of adhesion molecules, including E- and P-selectins, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1). However, the complete characterization of the adhesion strength and dynamics between lymphocytes and endothelial cells has been hampered by the lack of sensitive quantitative techniques. Here we report on the application of atomic force microscopy to characterize the interaction between individual pairs of living T lymphocytes (i.e., Jurkat cells) and human umbilical vein endothelial cells (HUVECs). The detachment of individual cell-cell conjugates was a complex process involving several step-like rupture events and the viscoelastic deformation of cells on the scale of several microns. Adhesion between Jurkat cells and activated endothelial cells increased with compression force and contact time, with the most dramatic changes occurring within the first half second of contact. After 0.25 sec of contact, E-selectin, ICAM-1, and VCAM-1 contributed to 18%, 39%, and 41% of total adhesion strength, respectively, suggesting that ICAM-1 and VCAM-1 contributed more than the selectins in supporting cell attachment.

Force and Compliance Measurements on Living Cells Using Atomic Force Microscopy (AFM).

We describe the use of atomic force microscopy (AFM) in studies of cell adhesion and cell compliance. Our studies use the interaction between leukocyte function associated antigen-1 (LFA-1)/intercellular adhesion molecule-1 (ICAM-1) as a model system. The forces required to unbind a single LFA-1/ICAM-1 bond were measured at different loading rates. This data was used to determine the dynamic strength of the LFA-1/ICAM-1 complex and characterize the activation potential that this complex overcomes during its breakage. Force measurements acquired at the multiple- bond level provided insight about the mechanism of cell adhesion. In addition, the AFM was used as a microindenter to determine the mechanical properties of cells. The applications of these methods are described using data from a previous study.

Contributions of molecular binding events and cellular compliance to the modulation of leukocyte adhesion.

The interaction of leukocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) is central to the regulation of adhesion in leukocytes. In this report, we investigated the mechanisms by which phorbol myristate acetate (PMA) promotes LFA-1-dependent cell adhesion. The adhesion of PMA-stimulated cells to immobilized ICAM-1 was quantified in direct force measurements acquired by atomic force microscopy (AFM). Enhanced adhesion of PMA-stimulated cells to immobilized ICAM-1 stemmed from an increase in the number of LFA-1-ICAM-1 complexes formed between the two apposing surfaces on contact, rather than by affinity modulation of LFA-1. Single molecule force measurements revealed that the force spectrum of the LFA-1-ICAM-1 complex formed by PMA-stimulated cells is identical to the force spectrum of the complex formed by resting cells. Thus, PMA stimulation does not modify the mechanical strength of the individual LFA-1-ICAM-1 interaction. Instead, the enhanced cell adhesion of PMA-stimulated cells appears to be a complex process that correlates with changes in the mechanical properties of the cell. We estimate that changes in the elasticity of the cell gave rise to a more than 10-fold increase in cell adhesion.