Effects of oxidation,pH and lipids on amyloidogenic peptide structure: implications for fibril formation?

We have performed experimental and computational studies to investigate the influences of phospholipids, methionine oxidation and acidic pH on amyloid fibril formation by a peptide derived from human apolipoprotein C-II (apoC-II), a known component of proteinaceous atherosclerotic plaques. Fibril growth monitored by thioflavin T fluorescence revealed inhibition under lipid-rich and oxidising conditions. We subsequently performed fully-solvated atomistic molecular dynamics (MD) simulations of the peptide monomer to study its conformations under both fibril favouring (neutral and low pH) and inhibiting (lipid-rich and oxidising) conditions. Examination of the chain topology, backbone hydrogen-bonding patterns and aromatic sidechain orientations of the peptide under different conditions reveals that, while the peptide adopts similar structures under the fibril-favouring conditions, significantly different structures are obtained under fibril-disruptive conditions. Based on our results, we advance hypotheses for the roles of peptide conformation on aggregation and fibrillisation propensities.     

In silico investigation on the inhibition of Aβ42 aggregation by Aβ40 peptide by potential of mean force study

Recent experimental data revealed that small, soluble Amyloid beta (Aβ₄₂) oligomers, especially dimers impair synaptic plasticity and memory leading to Alzheimer’s disease. Here, we have studied dimerization of Aβ₄₂/Aβ₄₂ homo-dimer and Aβ₄₀/Aβ₄₂ hetero-dimer in terms of free energy profile by all-atom simulations using the ff99SB force field. We have found that in the presence of Aβ₄₀ peptide, there exists a strong tendency to form a hetero-dimer with Aβ₄₂ peptide, suggesting that a possible co-oligomerization. Furthermore, we have investigated the effects of Aβ₄₀ on the Aβ₄₂ peptide. Our study also shows that in presence of Aβ₄₀, the beta-content of Aβ₄₂ monomer is reduced. Additionally, certain residues important for bending in Aβ₄₂ peptide attained an increased flexibility in the presence of Aβ₄₀. The salt-bridge destabilization also manifested the impact of Aβ₄₀ on Aβ₄₂ peptide as a whole. Based on this, one may expect that Aβ₄₀ inhibits the aggregation propensity of Aβ₄₂. Moreover, the binding free energy obtained by the molecular mechanics–Poisson–Boltzmann surface area method also revealed a strong affinity between the two isoforms thereby suggests that Aβ₄₀ binding induces conformational change in Aβ₄₂. Our results suggest that co-oligomerization of Aβ isoforms may play a substantial role in Alzheimer’s disease.     

Effects of Hydroxylated Carbon Nanotubes on the Aggregation of Aβ16–22 Peptides: A Combined Simulation and Experimental Study

The pathogenesis of Alzheimer’s disease (AD) is associated with the aggregation of amyloid-β (Aβ) peptides into toxic aggregates with β-sheet character. In a previous computational study, we showed that pristine single-walled carbon nanotubes (SWCNTs) can inhibit the formation of β-sheet-rich oligomers in the central hydrophobic core fragment of Aβ (Aβ_16–22). However, the poor solubility of SWCNTs in water hinders their use in biomedical applications and nanomedicine. Here, we investigate the influence of hydroxylated SWCNT, a water-soluble SWCNT derivative, on the aggregation of Aβ_16–22 peptides using all-atom explicit-water replica exchange molecular dynamics simulations. Our results show that hydroxylated SWCNTs can significantly inhibit β-sheet formation and shift the conformations of Aβ_16–22 oligomers from ordered β-sheet-rich structures toward disordered coil aggregates. Detailed analyses of the SWCNT-Aβ interaction reveal that the inhibition of β-sheet formation by hydroxylated SWCNTs mainly results from strong electrostatic interactions between the hydroxyl groups of SWCNTs and the positively charged residue K16 of Aβ_16–22 and hydrophobic and aromatic stacking interactions between SWCNTs and F19 and F20. In addition, our atomic force microscopy and thioflavin T fluorescence experiments confirm the inhibitory effect of both pristine and hydroxylated SWCNTs on Aβ_16–22 fibrillization, in support of our previous and present replica exchange molecular dynamics simulation results. These results demonstrate that hydroxylated SWCNTs efficiently inhibit the aggregation of Aβ_16–22; in addition, they offer molecular insight into the inhibition mechanism, thus providing new clues for the design of therapeutic drugs against amyloidosis.     

Histidine availability is decisive in ROS-mediated cytotoxicity of copper complexes of Aβ1–16 peptide

The binding of metal ions to Aβ peptide plays an important role in the etiology of AD. Copper coordinates chiefly to His residues and produces reactive oxygen species (ROS) upon redox cycling. ROS builds enormous burden on the normal functioning of neuronal cells and results into deleterious effects. Recently, two structurally distinct copper binding sites with contrasting redox properties were characterized. Here, we demonstrate for the first time the effect of binding of two equivalents of Cu²⁺ on redox properties and cytotoxicity of Aβ peptide. Our electrochemical data and ascorbate consumption assay suggest that in the presence of two equivalents of copper; Aβ peptide has higher propensity of H₂O₂ generation. The oxidation of Aβ1–16 peptide due to both gamma radiolysis and metal catalyzed oxidation in the presence of two equivalents of copper is inhibited confirming the binding of both equivalents of copper to peptide. The electrochemical and cytotoxicity study shows that negative shift in the reduction potential is reflected as slightly higher cytotoxicity in SH-SY5Y cell lines for Aβ1–16–Cu²⁺ (1:2) complex.     

Effect of methionine-35 oxidation on the aggregation of amyloid-β peptide

Aggregation of Aβ peptides into amyloid plaques is considered to trigger the Alzheimer’s disease (AD), however the mechanism behind the AD onset has remained elusive. It is assumed that the insoluble Aβ aggregates enhance oxidative stress (OS) by generating free radicals with the assistance of bound copper ions. The aim of our study was to establish the role of Met35 residue in the oxidation and peptide aggregation processes. Met35 can be readily oxidized by H₂O₂. The fibrillization of Aβ with Met35 oxidized to sulfoxide was three times slower compared to that of the regular peptide. The fibrils of regular and oxidized peptides looked similar under transmission electron microscopy. The relatively small inhibitory effect of methionine oxidation on the fibrillization suggests that the possible variation in the Met oxidation state should not affect the in vivo plaque formation. The peptide oxidation pattern was more complex when copper ions were present: addition of one oxygen atom was still the fastest process, however, it was accompanied by multiple unspecific modifications of peptide residues. Addition of copper ions to the Aβ with oxidized Met35 in the presence of H₂O₂, resulted a similar pattern of nonspecific modifications, suggesting that the one-electron oxidation processes in the peptide molecule do not depend on the oxidation state of Met35 residue. Thus, it can be concluded that Met35 residue is not a part of the radical generating mechanism of Aβ–Cu(II) complex.     

Effects of amyloid-β peptide Aβ25–35 on glycolytic and antioxidant enzymes in erythrocytes of different ages

Amyloid-β peptide Aβ_25–35 was shown to cause lysis of rat erythrocytes of different ages. The toxicity of Aβ_25–35 positively correlated with both the erythrocyte age and the peptide concentration. The activity of glycolytic, antioxidant, and Na⁺/K⁺-ATPase enzymes decreased with erythrocyte aging in vivo. In vitro Aβ_25–35 reduced the activity of hexokinase, phosphofructokinase, pyruvate kinase, glutathione peroxidase, and glutathione transferase and increased Na⁺/K⁺-ATPase activity in aged erythrocytes to a greater degree than in young cells.     

Effects of Familial Mutations on the Monomer Structure of Aβ42

Amyloid beta (Aβ) peptide plays an important role in Alzheimer’s disease. A number of mutations in the Aβ sequence lead to familial Alzheimer’s disease, congophilic amyloid angiopathy, or hereditary cerebral hemorrhage with amyloid. Using molecular dynamics simulations of ∼200 μs for each system, we characterize and contrast the consequences of four pathogenic mutations (Italian, Dutch, Arctic, and Iowa) for the structural ensemble of the Aβ monomer. The four familial mutations are found to have distinct consequences for the monomer structure.Amyloid beta (Aβ) peptides have long been thought to play a central role in Alzheimer’s disease (AD). Usually 40 or 42 residues in length, Aβ peptides are proteolytic products of the Aβ precursor protein and they aggregate to form the fibrillar plaques in AD patients’ brains. Besides fibrillar plaques, Aβ oligomers are also neurotoxic. The significance and nature of Aβ oligomerization has recently become a focus of intensive research studies and debates (1,2). Notably, numerous pathogenic mutations have been identified in the Aβ precursor protein sequence and in the enzymes involved in Aβ processing (3). These mutations generally lead to early onset of AD or cerebral amyloid angiopathy. Understanding how the pathogenic mutations alter Aβ oligomerization/aggregation is essential to our understanding of the disease mechanism.Four of these pathogenic mutations (Italian E22K, Dutch E22Q, Arctic E22G, and Iowa D23N) cluster in the region of E22 and D23 in the Aβ sequence (distal from proteolytic cleavage sites) and they have higher neurotoxicity compared to wild-type (WT) Aβ (4). These mutations are thought to modify the physicochemistry of the peptide. For example, kinetic studies (4) show that the E22K and E22Q mutations lead to faster peptide aggregation, whereas the E22G and D23N mutations result in slightly slower aggregation than WT Aβ₄₂ (although the E22G mutation shows increased protofibril formation (5)). Recent solid-state NMR studies also suggest that rather than the in-register β-sheet conformation adopted by WT Aβ, the Iowa D23N mutant forms amyloid fibrils with antiparallel β-sheet structure (6).To understand how the mutations modify the peptide oligomerization/aggregation it is critical to characterize the starting point of the process, the monomers. Unfortunately, investigating the early phase of the oligomerization process experimentally is a challenging task due to the high aggregation propensity of Aβ and its intrinsic disorder. Therefore, a number of computational approaches have been adopted to investigate the consequences of mutations for the monomer structure (7–16). However, due to the high computational demands of explicit-solvent molecular dynamics (MD) simulations to simulate full-length Aβ peptides, most of these computational studies are either on Aβ fragments (to decrease the system size) using explicit-solvent simulations (8–12) or on full-length Aβ using implicit-solvent simulations (which are less computationally demanding and enable longer simulation times, but lack explicit water molecules in the simulations to fully describe water-peptide interactions) (13–15). In a very recent report, explicit-solvent simulations were used to study the effects of the E22Q mutation on full-length Aβ; however, rather limited data (<10 μs) were collected (16). Thus, characterizing full-length Aβ monomers remains quite a daunting task even with simulations.To characterize the effects of mutations on full-length Aβ monomer using explicit-solvent MD simulations, we employed distributed computing (17) to simulate the WT Aβ₄₂, Aβ₄₂-E22K, Aβ₄₂-E22Q, Aβ₄₂-E22G, and Aβ₄₂-D23N monomers. MD simulations of >200 μs were performed for each system and AMBER ff99sb (18) and the tip3p water model (19) were used for force field parameters. Peptide configurations in the MD trajectories were clustered with the root mean-square deviation metric to identify representative conformations (i.e., states) and transitions between these states were counted. Markov state model analysis was then performed where the master equations were solved and the equilibrium population of each state deduced (20). Details of the MD simulation procedures and Markov state model analysis can be found in the Supporting Material.Each of the five Aβ monomer systems exhibits great structural diversity and can only be characterized in an ensemble fashion (rather than described by a handful of representative configurations). This is in accord with the notion that full-length Aβ peptides are intrinsically disordered (21,22). Using the Dictionary of Secondary Structure of Proteins program (23) to assign secondary structure, it is clear that the five Aβ monomer systems are found overall not well structured, although small β-hairpins and α-helices are observed. In Fig. 1 we plot the residue-dependent extended β propensity and α-helix propensity, in the top and bottom panels, respectively, for each Aβ monomer system. Although we are reasonably confident of the convergence behavior of the α-helix propensity, we note that the convergence of the extended β-propensity might be more challenging and demand a much longer sampling time than the current aggregate simulation time of ∼200 μs (24).Open in a separate windowFigure 1Ensemble-averaged %population of β-strand (top) and α-helix (bottom) propensity for all five monomer systems. The sequence of the WT Aβ₄₂ is given on the x axis.We observe in Fig. 1 that all five Aβ monomer systems share a rather similar residue-dependent tendency to form an extended β-structure, although minor differences are present. On the other hand, these pathogenic mutations alter the α-helix propensity quite significantly. The E22K and E22Q mutations increase the α-helix propensity in the region of residues 20–23. All four mutations (E22K, E22Q, E22G, and D23N) decrease the α-helix propensity in the region of residues 33–36.Notably, we find that in all five systems only short stretches of α-helices are formed. That is, when a residue is involved in α-helix formation, it participates in forming mostly short helical segments (consisting of only four helical residues). To provide more insight into the changes of α-helix propensity due to the mutations, in Fig. S1 we plot the tendency of forming short α-helices along the sequence for all five systems. Each data point in Fig. S1 represents the propensity to form an α-helix of four residues in length, ending at the specific residue. For example, in the structural ensemble adopted by the WT peptide, ∼5.5% of the conformations have a short α-helix of size four, involving residues 15–18. We see from Fig. S1 that the E22K and E22Q mutations induce the formation of two short helices in residues 19–22 and 20–23. The higher α-helix propensity in this region for the E22K mutant compared to the WT was previously attributed to the elimination of the electrostatic repulsion between E22 and D23 in the WT by the mutation and the longer aliphatic chain of K22 in the mutant compared to E22 in the WT (9,22). This is consistent with the observation that the E22Q mutation also induces helix formation in this region (by eliminating the electrostatic repulsion between E22 and D23 in the WT) but to a lesser extent, possibly due to the shorter aliphatic chain of Q22 compared to K22.In the E22G mutant, although the mutation eliminates the electrostatic repulsion between E22 and D23 in the WT peptide, glycine is known to be a helix breaker (25), leading to diminished α-helix propensity in the region around residue G22 seen in Fig. S1.In the D23N mutant, although the mutation eliminates the electrostatic repulsion between E22 and D23 in the WT peptide, it does not induce (or rather even slightly decreases) helix formation around residue 23. This may be due to the short aliphatic chain of N23 but it is possible that the mutation induces some nonlocal effects on the peptide structure, disfavoring helix formation in this region.It is worth noting that all four mutations (E22K, E22Q, E22G, and D23N) virtually eliminate the α-helix propensity in the region of residues 33–36. This region is rather far away from the mutation sites in sequence but its α-helix propensity is nonetheless affected. The origin of such a nonlocal effect is less straightforward to explain and further analysis will aid untangling this behavior. Nonetheless, the diminished α-helix propensity in the region of residues 33–36 appears to be a consistent feature across all four mutants.The four mutations studied here (E22K, E22Q, E22G, and D23N) have been thought to modify the physicochemistry of the peptide and alter the oligomerization/aggregation process, leading to higher neurotoxicity. In predicting intrinsic aggregation propensities using peptide sequences, all four mutants are suggested to be more aggregation prone (26). On the other hand, kinetic studies show that only the E22K and E22Q mutants aggregate more quickly, whereas the E22G and D23N mutations result in slightly slower aggregation than WT Aβ₄₂ (4). Our simulation results suggest these pathogenic mutations have complicated effects on the monomer structure—all four mutations decrease helix propensity in residues 33–36, whereas only the E22K and E22Q mutations increase helix propensity in residues 20–23. It is interesting to note that α-helix propensity is generally thought to anticorrelate with aggregation propensity; however, recent studies have suggested an important role of α-helical intermediates in amyloid oligomerization (27–29). Our studies suggest that it would be of great value to investigate how the distinct patterns of α-helix propensity in these five systems may propagate to give rise to different oligomerization kinetics or even mechanisms. The pathogenic mutations studied here have complex effects on the oligomerization of the peptide. The characterization of the monomer structural ensembles reported here should aid understanding of such an important and complicated process.     

Effects of novel acylhydrazones derived from 4-quinolone on the acetylcholinesterase activity and Aβ42 peptide fibrils formation

Acetylcholinesterase inhibitors and compounds that trigger Aβ amyloid oligomerization and fibrillization represent an opportunity to discover new drug candidates to treat Alzheimer’s disease. In this work, we synthesized nine new acylhydrazones and a known one, both employing 3-carboethoxy-4-quinolone derivatives as starting materials with chemical yields ranging from 63% to 90%. We evaluated the effect of these compounds on the acetylcholinesterase (AChE) activity and the fibrillization of Aβ₄₂ peptide. Except for one acylhydrazone, the compounds exhibited good inhibitory effect on AChE (1.2?μM?<?IC₅₀ values?<?17?μM). They also showed a significant decrease in the thioflavin-T fluorescence emission, suggesting an inhibitory effect on the Aβ₄₂ fibril formation.     

SMAD-Independent Down-Regulation of Caveolin-1 by TGF-β: Effects on Proliferation and Survival of Myofibroblasts

Transforming growth factor-β (TGF-β) mediates growth-inhibitory effects on most target cells via activation of the canonical SMAD signaling pathway. This growth-inhibitory activity may be coupled with cellular differentiation. Our studies demonstrate that TGF-β1 inhibits proliferation of primary, non-transformed human lung fibroblasts in association with the induction of myofibroblast differentiation. Differentiated myofibroblasts maintain the capacity to proliferate in response to exogenous mitogenic stimuli and are resistant to serum deprivation-induced apoptosis. These proliferative and anti-apoptotic properties of myofibroblasts are related, in part, to the down-regulation of caveolin-1 (Cav-1) by TGF-β1. Cav-1 down-regulation is mediated by early activation of p38 MAPK and does not require SMAD signaling. In contrast, myofibroblast differentiation is dependent on activation of the SMAD pathway, but not on p38 MAPK. Thus, combinatorial signaling by TGF-β1 of myofibroblast differentiation and down-regulation of Cav-1 by SMAD and p38 MAPK pathways, respectively, confer proliferative and apoptosis-resistant properties to myofibroblasts. Selective targeting of this SMAD-independent, p38-MAPK/Cav-1-dependent pathway is likely to be effective in the treatment of pathological conditions characterized by TGF-β signaling and myofibroblast activation.     

Determination of size of folding nuclei of fibrils formed from recombinant Aβ(1-40) peptide

We have developed a highly efficient method for purification of the recombinant product Aβ(1-40) peptide. The concentration dependence of amyloid formation by recombinant Aβ(1-40) peptide was studied using fluorescence spectroscopy and electron microscopy. We found that the process of amyloid formation is preceded by lag time, which indicates that the process is nucleation-dependent. Further exponential growth of amyloid fibrils is followed by branching scenarios. Based on the experimental data on the concentration dependence, the sizes of the folding nuclei of fibrils were calculated. It turned out that the size of the primary nucleus is one “monomer” and the size of the secondary nucleus is zero. This means that the nucleus for new aggregates can be a surface of the fibrils themselves. Using electron microscopy, we have demonstrated that fibrils of these peptides are formed by the association of rounded ring structures.     

Influence of lβ-, dα- and dβ-Asp isomers of the Asp-76 residue on the properties of αA-crystallin 70–88 peptide

Proteins have been considered to consist exclusively of l-amino acids in living tissues. However, our previous studies showed that two specific aspartyl (Asp) residues in αA- and αB-crystallins from human eye lenses invert to the d-isomers to a high degree during aging. The reaction is also accompanied by isomerization into a form containing β-Asp (isoaspartate) residues. The appearance of d- and β-Asp in a protein potentially induces large changes to the higher order structure of the protein as well as to its function. However, it remains unclear whether the formation of the Asp isomer is the direct trigger of the change to the higher order structure and function. In this study, in order to clarify the effect of the inversion to d-isomers in a protein, we synthesized peptides corresponding to the 70–88 (KFVIFLDVKHFSPEDLTVK) fragment of human αA-crystallin and its corresponding diastereoisomers in which lα-Asp was replaced with lβ-Asp, dα-Asp, and dβ-Asp at position 76 and compared their biochemical properties with that of normal peptide. The peptides containing abnormal isomers (lβ-Asp, dα-Asp, and dβ-Asp residues, respectively) were more hydrophilic than the normal peptide (containing lα-Asp), lost β-sheet structure and changed to random structures. The normal peptide promoted the aggregation of insulin while the other three isomers suppressed the aggregation of insulin. This is the first evidence that a single substitution of an Asp isomer in a peptide induces a large change to the properties of the peptide.     

Experimental study on aggregation of model monopeptide molecules: 6. A model for peptide β-structures?

Homo- and hetero-aggregates of monopeptide molecules Bu^tCONHCHRCONHPrⁱ have been studied by ¹H-n.m.r. Two pairing modes of the molecules are found for both types of aggregate, according to the bulkiness of side chains R. Their hydrogen bond patterns are closely related to the interstrand interactions in β_a (antiparallel) and β_p (parallel) sheets of globular proteins. The pairing mode Γ₁₄ of these molecules, similar to that of the residues in β_a-structures, is the most stable disposition if the side-chains are not C^β or C^γ-branched simultaneously. When both side chains are bulky C^β or C^γ-branched groups, the pairing mode Γ₁₂ found in β_p-structures is favoured. This observation is in agreement with the lower content of β_p compared to β_a-structure in globular proteins and the preferential occurrence of C^β and C^γ-branched residues in β_p structures.     

Use of surface plasmon resonance to study the elongation kinetics and the binding properties of the highly amyloidogenic Aβ(1-42) peptide, synthesized by depsi-peptide technique

A wide variety of human diseases are associated with the formation of highly organized protein aggregates termed amyloid fibrils, whose growth (elongation) is due to the assembly of the basic molecular units (monomers) in a sequential polymerization process. Surface plasmon resonance (SPR) technology has been proposed as a powerful approach to study in detail the fibril elongation of some amyloidogenic peptides. In particular, the injection of monomers over immobilized fibrils allows to follow in real time, and on a very short time-scale, the kinetics of fibril growth. In the present study we confirmed and extended this application of SPR to Aβ(1-42), hampered till now by the very pronounced aggregation propensity of this peptide, involved in Alzheimer disease. We took advantage of a new synthetic strategy ("depsi-peptide" technique) which allows to obtain reliable seed-free solutions (monomers) as well as fibrils of Aβ(1-42). SPR data were consistent with a "dock-and-lock" mechanism underlying Aβ(1-42) elongation process. The setup of an assay monitoring the elongation kinetics is very useful for investigating potential anti-amyloidogenic compounds. Moreover, the possibility to reliably immobilize both Aβ(1-42) monomers and fibrils allows to measure the binding affinities of putative ligands for these different species. The approach applied here to Aβ(1-42) might well be also applied to the study of other fibrillogenic peptides/proteins or to the study of polymerization reactions in general.     

Isotope-assisted vibrational circular dichroism investigations of amyloid β peptide fragment, Aβ(16-22)

Isotope-assisted vibrational circular dichroism (VCD) investigations have been used to probe the site specific local structure of an amyloid peptide for the first time. A seven residue peptide, NH₂-KLVFFAE-COOH, which represents the Aβ(16–22) fragment of the Alzheimer’s amyloid β peptide, was used for these investigations. ¹³C labels were introduced separately at the carbonyl group of leucine (residue 17), alanine (residue 21) and also at both sites together. Since VCD spectra provide structure dependent signs, band shapes and frequencies, the isotope-assisted VCD spectroscopy revealed information on site specific secondary structure of the polypeptide. Isotope dilution VCD experiments provided a means to distinguish between parallel and anti-parallel nature of the β-sheet structure formed by the Aβ(16–22) fragment. The current results establish the usefulness of isotope-assisted VCD analysis in determining the site specific secondary structure of amyloid peptides.     

Parenteral Delivery of HPβCD: Effects on Drug-HSA Binding

It is thought that cyclodextrins, such as 2-hydroxypropyl-β-cyclodextrin (HPβCD), will at high concentration affect pharmacokinetics of drugs through competitive binding with plasma proteins. Albumin is the major component of plasma proteins responsible for plasma protein binding. The purpose of this study was to evaluate in vitro the competitive binding of drugs between human serum albumin (HSA) and HPβCD in isotonic pH 7.4 phosphate buffer saline solution (PBS) at ambient temperature. Eight model drugs were selected based on their physicochemical properties and ability to form complexes with HSA and HPβCD. The drug/HPβCD stability constants (K _1:1) were determined by the phase-solubility method and HSA/HPβCD competitive binding determined by an equilibrium dialysis method. Protein binding of drugs that are both strongly protein bound and have high affinity to HPβCD (i.e., have high K _1:1 value) is most likely to be affected by parenterally administered HPβCD. However, this in vitro study indicates that even for those drugs single parenteral dose of HPβCD has to be as high as 70 g to have detectable effect on their protein binding. Weakly protein bound drugs and drugs with low affinity towards HPβCD are insensitive to the cyclodextrin presence regardless their lipophilic properties.     

Monte Carlo study of the formation and conformational properties of dimers of Aβ42 variants

Small soluble oligomers, and dimers in particular, of the amyloid β-peptide (Aβ) are believed to play an important pathological role in Alzheimer's disease. Here, we investigate the spontaneous dimerization of Aβ42, with 42 residues, by implicit solvent all-atom Monte Carlo simulations, for the wild-type peptide and the mutants F20E, E22G and E22G/I31E. The observed dimers of these variants share many overall conformational characteristics but differ in several aspects at a detailed level. In all four cases, the most common type of secondary structure is intramolecular antiparallel β-sheets. Parallel, in-register β-sheet structure, as in models for Aβ fibrils, is rare. The primary force driving the formation of dimers is hydrophobic attraction. The conformational differences that we do see involve turns centered in the 20-30 region. The probability of finding turns centered in the 25-30 region, where there is a loop in Aβ fibrils, is found to increase upon dimerization and to correlate with experimentally measured rates of fibril formation for the different Aβ42 variants. Our findings hint at reorganization of this part of the molecule as a potentially critical step in Aβ aggregation.     

Selection of peptide inhibitors of soluble Aβ(1-42) oligomer formation by phage display

There have been many reports suggesting that soluble oligomers of amyloid β (Aβ) are neurotoxins causing Alzheimer's disease (AD). Although inhibition of the soluble oligomerization of Aβ is considered to be effective in the treatment of AD, almost all peptide inhibitors have been designed from the β-sheet structure (H14-D23) of Aβ(1-42). To obtain more potent peptides than the known inhibitors of the soluble-oligomer formation of Aβ(1-42), we performed random screening by phage display. After fifth-round panning of a hepta-peptide library against soluble Aβ(1-42), novel peptides containing arginine residues were enriched. These peptides were found to suppress specifically 37/48 kDa oligomer formation and to keep the monomeric form of Aβ(1-42) even after 24 h of incubation, as disclosed by SDS-PAGE and size-exclusion chromatography. Thus we succeeded in acquiring novel efficient peptides for inhibition of soluble 37/48 kDa oligomer formation of Aβ(1-42).     

Effects of integrin α6β1 on migration of hepatocellular carcinoma cells

In this study, we applied specific blocking antibodies for integrin α6 or β1 subunit, and evaluated the in vitro effects of integrins α6β1 on the adhesion, chemotaxis and migration of hepatocellular carcinoma (HCC) cell line SMMC-7721 to type IV collagen. The adhesion force and cell migration, as measured by a micropipette aspiration system and Boyden chamber assay respectively, was dramatically reduced when either integrin subunits was blocked. The chemotaxis, as determined using a dual-micropipette system, was only affected by the antibody against β1 subunit. This study suggests that integrin α6β1 is an important cell surface receptor that mediates the adhesion of SMMC-7721 to type IV collagen. But the α6 subunit has minimal effect on pseudopod formation in response to type IV collagen. Therefore, the integrin α6β1-mediated cell migration is, at least in part, through the regulation on the cell adhesion step.     

Effects of electromagnetic pulse exposure on gelatinase of blood–brain barrier in vitro

The biological effects of electromagnetic pulse (EMP) on the brain have been focused on for years. It was reported that gelatinase played an important role in maintaining brain function through regulating permeability in the blood–brain barrier (BBB). To investigate the effects of EMP on gelatinase of BBB, an in vitro BBB model was established using primary cultured rat brain microvascular endothelial cells (BMVEC), astrocytes and half-contact culture of these cells in a transwell chamber. Cultured supernatant and cells were collected at different time points after exposure to EMP (peak intensity 400 kV/m, rise time 10 ns, pulse width 350 ns, 0.5 pps and 200 pulses). Protein levels of cellular gelatinase MMP-2 and MMP-9, and endogenous inhibitor TIMP-1 and TIMP-2 were detected by Western blot. The activity of gelatinase in culture supernatant was detected by gelatin zymography. It was found that compared with the sham-exposed group, the protein level of MMP-2 was significantly increased at 6 h (p < 0.05), and the protein level of its endogenous inhibitor TIMP-2 did not change after EMP exposure. In addition, the protein levels of MMP-9 and its endogenous inhibitor TIMP-1 did not change after EMP exposure. Gelatin zymography results showed that the activity of MMP-2 in the inner pool and the outer pool of the transwell chamber was significantly increased at 6 h after EMP exposure compared with that of the sham group. These results suggested that EMP exposure could affect the expression and activity of MMP-2 in the BBB model.