Vibrational circular dichroism of tetraphenylporphyrin in peptide complexes? A computational study

The Raman and absorption spectra of tetraphenylporphyrin (TPP) were calculated and compared to experiment. The computation was based on the harmonic molecular force field and electric tensors obtained ab initio at the BPW91/6-31G* level. Good agreement was found between experimental and calculated frequencies and intensities. In order to estimate whether induced optical activity in chiral complexes interferes with the signal of peptide vibrations, the vibrational circular dichroism (VCD) spectra of TPP were simulated. The magnetic field perturbation theory (MFP) and the gauge-invariant atomic orbitals (GIAO) were used for the simulation. Such spectra were compared to theoretical VCD intensities of a model tripeptide as well to experimental spectra of a complex of the peptide and tetrakis(p-sulfonatophenyl)porphyrin (TSPP). No significant contribution to VCD signal from the TPP residue was found in experimental spectra. Thus, possible peptide conformational changes occurring during the complexation can be monitored directly in the amide I frequency region.     

α-Secretase-derived fragment of cellular prion, N1, protects against monomeric and oligomeric amyloid β (Aβ)-associated cell death

In physiological conditions, both β-amyloid precursor protein (βAPP) and cellular prion (PrP(c)) undergo similar disintegrin-mediated α-secretase cleavage yielding N-terminal secreted products referred to as soluble amyloid precursor protein-α (sAPPα) and N1, respectively. We recently demonstrated that N1 displays neuroprotective properties by reducing p53-dependent cell death both in vitro and in vivo. In this study, we examined the potential of N1 as a neuroprotector against amyloid β (Aβ)-mediated toxicity. We first show that both recombinant sAPPα and N1, but not its inactive parent fragment N2, reduce staurosporine-stimulated caspase-3 activation and TUNEL-positive cell death by lowering p53 promoter transactivation and activity in human cells. We demonstrate that N1 also lowers toxicity, cell death, and p53 pathway exacerbation triggered by Swedish mutated βAPP overexpression in human cells. We designed a CHO cell line overexpressing the London mutated βAPP (APP(LDN)) that yields Aβ oligomers. N1 protected primary cultured neurons against toxicity and cell death triggered by oligomer-enriched APP(LDN)-derived conditioned medium. Finally, we establish that N1 also protects neurons against oligomers extracted from Alzheimer disease-affected brain tissues. Overall, our data indicate that a cellular prion catabolite could interfere with Aβ-associated toxicity and that its production could be seen as a cellular protective mechanism aimed at compensating for an sAPPα deficit taking place at the early asymptomatic phase of Alzheimer disease.     

Determination of regions involved in amyloid fibril formation for Aβ(1-40) peptide

The studies of amyloid structures and the process of their formation are important problems of biophysics. One of the aspects of such studies is to determine the amyloidogenic regions of a protein chain that form the core of an amyloid fibril. We have theoretically predicted the amyloidogenic regions of the Aβ(1-40) peptide capable of forming an amyloid structure. These regions are from 16 to 21 and from 32 to 36 amino acid residues. In this work, we have attempted to identify these sites experimentally by the method of tandem mass spectrometry. As a result, we show that regions of the Aβ(1-40) peptide from 16 to 22 and from 28 to 40 amino acid residues are resistant to proteases, i.e. they are included in the core of amyloid fibrils. Our results correlate with the results of the theoretical prediction.     

Carbon nanotube inhibits the formation of β-sheet-rich oligomers of the Alzheimer's amyloid-β(16-22) peptide

Alzheimer's disease is associated with the abnormal self-assembly of the amyloid-β (Aβ) peptide into toxic β-rich aggregates. Experimental studies have shown that hydrophobic nanoparticles retard Aβ fibrillation by slowing down the nucleation process; however, the effects of nanoparticles on Aβ oligomeric structures remain elusive. In this study, we investigate the conformations of Aβ(16-22) octamers in the absence and presence of a single-walled carbon nanotube (SWCNT) by performing extensive all-atom replica exchange molecular-dynamics simulations in explicit solvent. Our simulations starting from eight random chains demonstrate that the addition of SWCNT into Aβ(16-22) solution prevents β-sheet formation. Simulation starting from a prefibrillar β-sheet octamer shows that SWCNT destabilizes the β-sheet structure. A detailed analysis of the Aβ(16-22)/SWCNT/water interactions reveals that both the inhibition of β-sheet formation and the destabilization of prefibrillar β-sheets by SWCNT result from the same physical forces: hydrophobic and π-stacking interactions (with the latter playing a more important role). By analyzing the stacking patterns between the Phe aromatic rings and the SWCNT carbon rings, we find that short ring–centroid distances mostly favor parallel orientation, whereas large distances allow all other orientations to be populated. Overall, our computational study provides evidence that SWCNT is likely to inhibit Aβ(16-22) and full-length Aβ fibrillation.     

Pyroglutamate amyloid β (Aβ) aggravates behavioral deficits in transgenic amyloid mouse model for Alzheimer disease

Pyroglutamate-modified Aβ peptides at amino acid position three (Aβ(pE3-42)) are gaining considerable attention as potential key players in the pathogenesis of Alzheimer disease (AD). Aβ(pE3-42) is abundant in AD brain and has a high aggregation propensity, stability and cellular toxicity. The aim of the present work was to study the direct effect of elevated Aβ(pE3-42) levels on ongoing AD pathology using transgenic mouse models. To this end, we generated a novel mouse model (TBA42) that produces Aβ(pE3-42). TBA42 mice showed age-dependent behavioral deficits and Aβ(pE3-42) accumulation. The Aβ profile of an established AD mouse model, 5XFAD, was characterized using immunoprecipitation followed by mass spectrometry. Brains from 5XFAD mice demonstrated a heterogeneous mixture of full-length, N-terminal truncated, and modified Aβ peptides: Aβ(1-42), Aβ(1-40), Aβ(pE3-40), Aβ(pE3-42), Aβ(3-42), Aβ(4-42), and Aβ(5-42). 5XFAD and TBA42 mice were then crossed to generate transgenic FAD42 mice. At 6 months of age, FAD42 mice showed an aggravated behavioral phenotype compared with single transgenic 5XFAD or TBA42 mice. ELISA and plaque load measurements revealed that Aβ(pE3) levels were elevated in FAD42 mice. No change in Aβ(x)(-42) or other Aβ isoforms was discovered by ELISA and mass spectrometry. These observations argue for a seeding effect of Aβ(pE-42) in FAD42 mice.     

Vacuum ultraviolet circular dichroism of (1 → 6)-β-d-glucan

V.u.c.d. spectra recorded for freshly prepared aqueous solutions of (1 → 6)-β)-D-glucan(pustulan) contained a single positive band near 177 nm. This band was similar in position and magnitude to the single positive band observed in the spectrum of (1 → 6)-α-D-glucan (dextran). Pustulan solutions (20 mg/ml) were observed to gel with time at 10 C. Concurrently, a negative band at 190 nm developed in the pustulan v.u.c.d. spectrum followed by a blue shift of both bands with continued aging. Crystalline films of pustulan yield spectra which resembled the blue shifted spectra of aged gels. The time dependent development of the negative band was attributed to pustulan attaining a helical conformation in solution, and the blue shift to aggregation of helices, Na⁺ and Ca²⁺ were found to accelerate gelation presumably by decreasing the activity of the aqueous solvent.     

Spontaneous formation of twisted Aβ(16-22) fibrils in large-scale molecular-dynamics simulations

Protein aggregation is associated with fatal neurodegenerative diseases, including Alzheimer's and Parkinson's. Mapping out kinetics along the aggregation pathway could provide valuable insights into the mechanisms that drive oligomerization and fibrillization, but that is beyond the current scope of computational research. Here we trace out the full kinetics of the spontaneous formation of fibrils by 48 Aβ_16-22 peptides, following the trajectories in molecular detail from an initial random configuration to a final configuration of twisted protofilaments with cross-β-structure. We accomplish this by performing large-scale molecular-dynamics simulations based on an implicit-solvent, intermediate-resolution protein model, PRIME20. Structural details such as the intersheet distance, perfectly antiparallel β-strands, and interdigitating side chains analogous to a steric zipper interface are explained by and in agreement with experiment. Two characteristic fibrillization mechanisms—nucleation/templated growth and oligomeric merging/structural rearrangement—emerge depending on the temperature.     

Small amphipathic molecules modulate secondary structure and amyloid fibril-forming kinetics of Alzheimer disease peptide Aβ(1-42)

Amyloid fibril formation is associated with a number of debilitating systemic and neurodegenerative diseases. One of the most prominent is Alzheimer disease in which aggregation and deposition of the Aβ peptide occur. Aβ is widely considered to mediate the extensive neuronal loss observed in this disease through the formation of soluble oligomeric species, with the final fibrillar end product of the aggregation process being relatively inert. Factors that influence the aggregation of these amyloid-forming proteins are therefore very important. We have screened a library of 96 amphipathic molecules for effects on Aβ(1-42) aggregation and self-association. We find, using thioflavin T fluorescence and electron microscopy assays, that 30 of the molecules inhibit the aggregation process, whereas 36 activate fibril formation. Several activators and inhibitors were subjected to further analysis using analytical ultracentrifugation and circular dichroism. Activators typically display a 1:10 peptide:detergent stoichiometry for maximal activation, whereas the inhibitors are effective at a 1:1 stoichiometry. Analytical ultracentrifugation and circular dichroism experiments show that activators promote a mixture of unfolded and β-sheet structures and rapidly form large aggregates, whereas inhibitors induce α-helical structures that form stable dimeric/trimeric oligomers. The results suggest that Aβ(1-42) contains at least one small molecule binding site, which modulates the secondary structure and aggregation processes. Further studies of the binding of these compounds to Aβ may provide insight for developing therapeutic strategies aimed at stabilizing Aβ in a favorable conformation.     

Self-assembly pathways of E22Δ-type amyloid β peptide mutants generated from non-aggregative O-acyl isopeptide precursors

The recently identified E22Δ-type amyloid β peptide (Aβ) mutants are reported to favor oligomerization over fibrillization and to exhibit more-potent synaptotoxicity than does wild-type (WT) Aβ. Aβ(E22Δ) mutants can thus be expected to serve as tools for clarifying the impact of Aβ oligomers in Alzheimer's disease (or Alzheimer's-type dementia). However, the biochemical and biophysical properties of Aβ(E22Δ) have not been conclusively determined. Here, we evaluated the self-assembly pathways of Aβ(E22Δ) mutants generated from water-soluble, non-aggregative O-acyl isopeptide precursors. Circular dichroism spectroscopy, Western blot analysis, and thioflavin-T fluorescence intensity and cellular toxicity assays suggest that the self-assembly pathways of Aβ(E22Δ) differed from those of Aβ(WT). Aβ1-40(E22Δ) underwent a rapid random coil→β-sheet conformational change in its monomeric or low-molecular-weight oligomeric states, whereas Aβ1-40(WT) self-assembled gradually without losing its propensity to form random coil structures. The Aβ1-42(E22Δ) monomer formed β-sheet-rich oligomers more rapidly than did Aβ1-42(WT). Additionally, the Aβ1-42(E22Δ) oligomers appear to differ from Aβ1-42(WT) oligomers in size, shape, or both. These results should provide new insights into the functions of Aβ(E22Δ) mutants.     

Effects of oxidation on copper-binding properties of Aβ1-16 peptide: A pulse radiolysis study

Abstract

The reaction of hydroxyl radicals (^?OH) with Aβ1-16 peptide was carried out using pulse radiolysis to understand the effect of oxidation of peptide on its copper-binding properties. This reaction produced oxidized, dimeric and trimeric Aβ1-16 peptide species. The formation of these products was established with the help of fluorescence spectroscopy and mass spectrometry. The mass spectral data indicate that the major site of oxidation is at His6, while the site for dimerization is at Tyr10. Diethyl pyrocarbonate-treated Aβ1-16 peptide did not produce any trimeric species upon oxidation with ^?OH. The quantitative chemical modification studies indicated that one of the three histidine residues is covalently modified during pulse radiolysis. The copper-binding studies of the oxidized peptide revealed that it has similar copper-binding properties as the unoxidized peptide. Further, the cytotoxicity studies point out that both oxidized and unoxidized Aβ1-16 peptide are equally efficient in producing free radicals in presence of copper and ascorbate that resulted in comparable cell death.     

Membrane interactions and the effect of metal ions of the amyloidogenic fragment Aβ(25-35) in comparison to Aβ(1-42)

Aβ(1−42) peptide, found as aggregated species in Alzheimer's disease brain, is linked to the onset of Alzheimer's disease. Many reports have linked metals to inducing Aβ aggregation and amyloid plaque formation. Aβ(25-35), a fragment from the C-terminal end of Aβ(1−42), lacks the metal coordinating sites found in the full-length peptide and is neurotoxic to cortical cortex cell cultures. We report solid-state NMR studies of Aβ(25-35) in model lipid membrane systems of anionic phospholipids and cholesterol, and compare structural changes to those of Aβ(1-42). When added after vesicle formation, Aβ(25-35) was found to interact with the lipid headgroups and slightly perturb the lipid acyl-chain region; when Aβ(25-35) was included during vesicle formation, it inserted deeper into the bilayer. While Aβ(25-35) retained the same β-sheet structure irrespective of the mode of addition, the longer Aβ(1-42) appeared to have an increase in β-sheet structure at the C-terminus when added to phospholipid liposomes after vesicle formation. Since the Aβ(25-35) fragment is also neurotoxic, the full-length peptide may have more than one pathway for toxicity.     

Triazole-containing BODIPY dyes as novel fluorescent probes for soluble oligomers of amyloid Aβ1-42 peptide

A straightforward functionalization of BODIPY dyes via incorporation of a triazole moiety produced fluorescent dyes that were capable of distinguishing between secondary structure conformations of soluble oligomeric species of Aβ1-42 peptide. Small concentrations of the dyes, relative to Aβ1-42, provided up to an 8-fold and 35-fold fluorescence increase in the presence of the unordered and ordered, β-sheet-rich conformations of soluble Aβ1-42 oligomers, respectively. These triazole-containing dyes could prove to be useful probes for monitoring amyloid conformational transitions in vitro.     

Lysine 624 of the amyloid precursor protein (APP) is a critical determinant of amyloid β peptide length: support for a sequential model of γ-secretase intramembrane proteolysis and regulation by the amyloid β precursor protein (APP) juxtamembrane region

γ-Secretase is a multiprotein intramembrane cleaving aspartyl protease (I-CLiP) that catalyzes the final cleavage of the amyloid β precursor protein (APP) to release the amyloid β peptide (Aβ). Aβ is the primary component of senile plaques in Alzheimer's disease (AD), and its mechanism of production has been studied intensely. γ-Secretase executes multiple cleavages within the transmembrane domain of APP, with cleavages producing Aβ and the APP intracellular domain (AICD), referred to as γ and ε, respectively. The heterogeneous nature of the γ cleavage that produces various Aβ peptides is highly relevant to AD, as increased production of Aβ 1-42 is genetically and biochemically linked to the development of AD. We have identified an amino acid in the juxtamembrane region of APP, lysine 624, on the basis of APP695 numbering (position 28 relative to Aβ) that plays a critical role in determining the final length of Aβ peptides released by γ-secretase. Mutation of this lysine to alanine (K28A) shifts the primary site of γ-secretase cleavage from 1-40 to 1-33 without significant changes to ε cleavage. These results further support a model where ε cleavage occurs first, followed by sequential proteolysis of the remaining transmembrane fragment, but extend these observations by demonstrating that charged residues at the luminal boundary of the APP transmembrane domain limit processivity of γ-secretase.     

Effects of Hydrophobic Macromolecular Crowders on Amyloid β (16–22) Aggregation

In Alzheimer’s disease (AD), the amyloid β (Aβ) peptide aggregates in the brain to form progressively larger oligomers, fibrils, and plaques. The aggregation process is strongly influenced by the presence of other macromolecular species, called crowders, that can exert forces on the proteins. One very common attribute of macromolecular crowders is their hydrophobicity. We examined the effect of hydrophobic crowders on protein aggregation by using discontinuous molecular dynamics (DMD) simulations in combination with an intermediate resolution protein model, PRIME20. The systems considered contained 48 Aβ (16–22) peptides and crowders with diameters of 5 Å, 20 Å, and 40 Å, represented by hard spheres or spheres with square-well/square-shoulder interactions, at a crowder volume fraction of ϕ = 0.10. Results show that low levels of crowder hydrophobicity are capable of increasing the fibrillation lag time and high levels of crowder hydrophobicity can fully prevent the formation of fibrils. The types of structures that remain during the final stages of the simulations are summarized in a global phase diagram that shows fibril, disordered oligomer, or β-sheet phases in the space spanned by crowder size and crowder hydrophobicity. In particular, at high levels of hydrophobicity, simulations with 5 Å crowders result in only disordered oligomers and simulations with 40 Å crowders result in only β-sheets. The presence of hydrophobic crowders reduces the antiparallel β-sheet content of fibrils, whereas hard sphere crowders increase it. Finally, strong hydrophobic crowders alter the secondary structure of the Aβ (16–22) monomers, bending them into a shape that is incapable of forming ordered β-sheets or fibrils. These results qualitatively agree with previous theoretical and experimental work.     

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.     

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).     

The extracellular domain of Bri2 (ITM2B) binds the ABri peptide (1-23) and amyloid β-peptide (Aβ1-40): Implications for Bri2 effects on processing of amyloid precursor protein and Aβ aggregation

In Alzheimer’s disease the amyloid β-peptide (Aβ) aggregates in brain tissue and arteries. Aβ is proteolytically cleaved out from amyloid precursor protein (APP) by different secretases. Recently, the transmembrane protein ITM2B/Bri2, which is expressed in neurons and associated with familial British and Danish dementia, was shown to inhibit APP processing in transfected cells as well as in transgenic mice. Several mechanisms by which Bri2 can interfere with Aβ production and aggregation have been proposed. Herein, we studied recombinant human Bri2 (residues 90-236) containing the extracellular Brichos domain without the ABri23 peptide. Bri2(90-236) binds to ABri23, which suggests that these two parts interact during Bri2 biosynthesis, in line with proposed functions of Brichos domains in other proteins. Moreover, Bri2(90-236) binds Aβ1-40 and inhibits its aggregation and fibril formation. These data suggest a model for how the processing of Bri2 and APP are interrelated.     

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

Misfolding and aggregation of cellular prion protein (PrP^c) 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.