Бесплатный обязательный экземпляр полиграфических и других изданий как один из источников комплектования Латвийской академической библиотеки

Aggregation of amyloid beta (Aβ) into oligomers and fibrils is believed to play an important role in the development of Alzheimer's disease (AD). To gain further insight into the principles of aggregation, we have investigated the induction of β-sheet secondary conformation from disordered native peptide sequences through lipidation, in 1-2% hexafluoroisopropanol (HFIP) in phosphate buffered saline (PBS). Several parameters, such as type and number of lipid chains, peptide sequence, peptide length and net charge, were explored keeping the ratio peptide/HFIP constant. The resulting lipoconjugates were characterized by several physico-chemical techniques: Circular Dichroism (CD), Attenuated Total Reflection InfraRed (ATR-IR), Thioflavin T (ThT) fluorescence, Dynamic Light Scattering (DLS), solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy and Electron Microscopy (EM). Our data demonstrate the generation of β-sheet aggregates from numerous unstructured peptides under physiological pH, independent of the amino acid sequence. The amphiphilicity pattern and hydrophobicity of the scaffold were found to be key factors for their assembly into amyloid-like structures

Discovery and Structure Activity Relationship of Small Molecule Inhibitors of Toxic β-Amyloid-42 Fibril Formation

Increasing evidence implicates Aβ peptides self-assembly and fibril formation as crucial events in the pathogenesis of Alzheimer disease. Thus, inhibiting Aβ aggregation, among others, has emerged as a potential therapeutic intervention for this disorder. Herein, we employed 3-aminopyrazole as a key fragment in our design of non-dye compounds capable of interacting with Aβ42 via a donor-acceptor-donor hydrogen bond pattern complementary to that of the β-sheet conformation of Aβ42. The initial design of the compounds was based on connecting two 3-aminopyrazole moieties via a linker to identify suitable scaffold molecules. Additional aryl substitutions on the two 3-aminopyrazole moieties were also explored to enhance π-π stacking/hydrophobic interactions with amino acids of Aβ42. The efficacy of these compounds on inhibiting Aβ fibril formation and toxicity in vitro was assessed using a combination of biophysical techniques and viability assays. Using structure activity relationship data from the in vitro assays, we identified compounds capable of preventing pathological self-assembly of Aβ42 leading to decreased cell toxicity

Characterization of β-sheet amyloid-like aggregates of Palm1–15.

<p>A) CD spectra at 30 µM peptide Palm1–15 (green) and controls Acetyl1–15 (red) and Aβ1–15 (orange) in 2% HFIP/PBS (v/v). B) Profile of ThT emission with different peptide concentrations in the presence of 24 µM of dye. Fluorescence was measured at 485 nm with excitation at 440 nm. C, D, E) ssNMR of Palm1–15 uniformly labeled at Ala2, Ser8 and Gly9: (¹³C-¹³C) 2D PDSD correlation spectra with mixing times of 20 ms (C) and 150 ms (D); ¹H-¹³C FSLG-HETCOR spectra (E) together with chemical shift predictions based on secondary structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105641#pone.0105641-Wang1" target="_blank">[38]</a> (red for random coil, green for β-sheet, blue for α-helix). F) Electron micrographs of negatively stained Palm1–15 aggregates formed over 24 hour incubation in 2% HFIP/PBS (v/v). Samples were negatively stained with 2% Uranyl Acetate in water. Scale bar 0.1 µm. Magnification 22000 x.</p

Peptide sequences of lipopeptides and controls.

<p>Peptide sequences of lipopeptides and controls.</p

Parameters influencing scaffold conformation.

<p>A) CD spectra and B) ThT fluorescence of tetrapalmitoylated peptides with shortened length, 9 or 5 amino acids (Palm1–9 in blue and Palm1–5 in orange respectively) or different order of amino acids, reverse or scrambled (Palm15-1 in green and scPalm15 in red, respectively) to model sequence Palm1–15. C) CD and D) ThT of tetrapalmitoylated peptides with different isoelectric point with pI (and net charge) value indicated on top of each column (Palm1–15(D7K) in green, Palm1–15(E3A, D7K) in blue, Palm1–15(E3K, D7K) in orange, Palm1–15(E3K, D7K, E11K) in red and Palm1–15 in black). E) CD and F) ThT of peptides with different number/position of palmitic chains (Palm1–15(1C) in orange, Palm1–15(2C) in red, Palm1–15(1N1C) in green, Palm1–15(4C) in blue and Palm1–15 in black). G) CD and H) ThT of peptides acylated with different lipid chain length (Acetyl1–15 in orange, Butyl1–15 in blue, Octyl1–15 in green, Dodecyl1–15 in red and Palm1–15 in black). Peptides were 30 µM in 2% HFIP/PBS (v/v) (CD) and 15 µM in 1% HFIP/PBS (v/v) (ThT, 24 µM). Fluorescence was measured at 485 nm with excitation at 440 nm. Values are the average of 3 replicates, normalized to the Palm1–15 emission (taken as 100%).</p

ACI-35 elicits robust and specific antisera against Tau in wild-type and Tau.P301L mice.

<p>(A) Vaccination schedule with ACI-35 in wild-type mice is shown schematically with s.c. injections represented by the syringes and the bleedings by the letter B with a number. Antisera titers were measured by ELISA on the phosphorylated antigenic sequence incorporated in the vaccine (pTau peptide), and on the non-phosphorylated peptide of the same primary amino acid sequence (Tau peptide) (see text for details). Data are presented as mean± SD. Statistical analysis: one-way ANOVA followed by Bonferroni multiple comparison test (**p<0.01, **** p<0.0001) and by unpaired student's t-test (**** p<0.0001). (B) Similar vaccination with ACI-35 of Tau.P301L mice and analysis by ELISA. Data are presented as mean± SD. Statistical analysis by unpaired student's t-test (** p<0.01; **** p<0.0001) (C) TAUPIR with antisera from ACI-35 vaccinated wild-type mice and Tau.P301L mice demonstrated a specific reaction with neurofibrillary tangles and neuropil threads in forebrain of biGT mice. IHC with Mab AT100 is included for comparison. IHC with sera from Tau.P301L mice injected with PBS or from Tau.P301L mice that were not vaccinated, were devoid of specific antibodies and auto-antibodies against human protein Tau. Scale bars: 50 µm.</p

No increased inflammatory response in ACI-35 treated Tau.P301L mice.

<p>(A) IHC did not reveal marked differences in inflammation-related parameters in forebrain of ACI-35 vaccinated Tau.P301L mice, relative to PBS-injected Tau.P301L mice. IHC reaction with the different specific antibodies, specified in the captions, was analyzed by image analysis (details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072301#s2" target="_blank">Methods</a> section) and presented as mean± SEM. Scale bars: 50 µm. (B) Western blotting for GFAP of total brain homogenates from Tau.P301L mice vaccinated with ACI-35 (n = 34) or injected with PBS (n = 33). Data presented as mean± SEM. (C) ELISPOT analysis of IFN-γ and IL-4 production by T cells isolated from spleens of naive mice or from mice immunized by either ACI-35 or with recombinant protein Tau. Splenocytes were re-stimulated with medium (cells alone), recombinant Tau protein (100 µg/ml) or with the phosphorylated peptide used in the ACI-35 vaccine and with its un-phosphorylated counterpart (1 µg/ml). Results are expressed as the number of foci (spots per million cells) +SD (n = 10 mice). Statistical analysis: two-way ANOVA followed by Bonferroni multiple comparison test (*** p<0.001).</p

CD spectrum of ACI-35 corresponds to β-sheet secondary structure.

<p>The CD spectrum of ACI-35 at (1∶9) dilution in PBS. The spectrum of liposomes lacking the phospho-peptide was subtracted to the signal of ACI-35 for baseline correction. The spectrum shows a maximum around 199.5 nm and a broad minimum around 218 nm.</p

Biochemical analysis of brain from Tau.P301L mice vaccinated with ACI-35.

<p>(A) Fractionation scheme of total brain homogenates from Tau.P301L mice to generate soluble (S1) and sarcosyl insoluble fractions (SInT). (B) Representative western blots of S1 and SInT fractions from forebrain and from brainstem of two untreated terminal Tau.P301L mice (age 10 months) developed for total protein Tau (Mab Tau5), for total human protein Tau (Mab HT7) and for phosphorylated Tau (antibodies pT231, pS396 and pS404 as indicated). (C) Reduction of pS396 in soluble fraction of brainstem and forebrain (p = 0.026 and 0.0523, respectively, Student's t-test) from ACI-35 vaccinated Tau.P301L mice, relative to PBS injected mice. (D) Reduction of pS396 (p = 0.0091, Mann Whitney test) and HT7 (p = 0.0706, Mann Whitney test) in SInT in forebrain by ACI-35 vaccination of Tau.P301L mice. (E) Reduction of tangled neurons, marked by IHC for AT100 or pS422, in the forebrain of ACI-35 vaccinated Tau.P301L mice after 3 months of treatment. Data: mean± SEM.</p

TLR4 and TRIF-dependent stimulation of B lymphocytes by peptide liposomes enables T-cell independent isotype switch in mice

Immunoglobulin class switching from IgM to IgG in response to peptides is generally T cell–dependent and vaccination in T cell–deficient individuals is inefficient. We show that a vaccine consisting of a dense array of peptides on liposomes induced peptide-specific IgG responses totally independent of T-cell help. Independency was confirmed in mice lacking T cells and in mice deficient for MHC class II, CD40L, and CD28. The IgG titers were high, long-lived, and comparable with titers obtained in wild-type animals, and the antibody response was associated with germinal center formation, expression of activation-induced cytidine deaminase, and affinity maturation. The T cell–independent (TI) IgG response was strictly dependent on ligation of TLR4 receptors on B cells, and concomitant TLR4 and cognate B-cell receptor stimulation was required on a single-cell level. Surprisingly, the IgG class switch was mediated by TIR-domain-containing adapter inducing interferon-β (TRIF), but not by MyD88. This study demonstrates that peptides can induce TI isotype switching when antigen and TLR ligand are assembled and appropriately presented directly to B lymphocytes. A TI vaccine could enable efficient prophylactic and therapeutic vaccination of patients with T-cell deficiencies and find application in diseases where induction of T-cell responses contraindicates vaccination, for example, in Alzheimer disease