Altering Peptide Fibrillization by Polymer Conjugation

A strategy is presented that exploits the ability of synthetic polymers of different nature to disturb the strong self-assembly capabilities of amyloid based β-sheet forming peptides. Following a convergent approach, the peptides of interest were synthesized via solid-phase peptide synthesis (SPPS) and the polymers via reversible addition–fragmentation chain transfer (RAFT) polymerization, followed by a copper­(I) catalyzed azide–alkyne cycloaddition (CuAAC) to generate the desired peptide–polymer conjugates. This study focuses on a modified version of the core sequence of the β-amyloid peptide (Aβ), Aβ(16–20) (KLVFF). The influence of attaching short poly­(<i>N</i>-isopropylacrylamide) and poly­(hydroxyethylacrylate) to the peptide sequences on the self-assembly properties of the hybrid materials were studied via infrared spectroscopy, TEM, circular dichroism and SAXS. The findings indicate that attaching these polymers disturbs the strong self-assembly properties of the biomolecules to a certain degree and permits to influence the aggregation of the peptides based on their β-sheets forming abilities. This study presents an innovative route toward targeted and controlled assembly of amyloid-like fibers to drive the formation of polymeric nanomaterials

Slow-Release RGD-Peptide Hydrogel Monoliths

We report on the formation of hydrogel monoliths formed by functionalized peptide Fmoc-RGD (Fmoc: fluorenylmethoxycarbonyl) containing the RGD cell adhesion tripeptide motif. The monolith is stable in water for nearly 40 days. The gel monoliths present a rigid porous structure consisting of a network of peptide fibers. The RGD-decorated peptide fibers have a β-sheet secondary structure. We prove that Fmoc-RGD monoliths can be used to release and encapsulate material, including model hydrophilic dyes and drug compounds. We provide the first insight into the correlation between the absorption and release kinetics of this new material and show that both processes take place over similar time scales

Interaction between a Cationic Surfactant-like Peptide and Lipid Vesicles and Its Relationship to Antimicrobial Activity

We investigate the properties of an antimicrobial surfactant-like peptide (Ala)₆(Arg), A₆R, containing a cationic headgroup. The interaction of this peptide with zwitterionic (DPPC) lipid vesicles is investigated using a range of microscopic, X-ray scattering, spectroscopic, and calorimetric methods. The β-sheet structure adopted by A₆R is disrupted in the presence of DPPC. A strong effect on the small-angle X-ray scattering profile is observed: the Bragg peaks from the DPPC bilayers in the vesicle walls are eliminated in the presence of A₆R and only bilayer form factor peaks are observed. All of these observations point to the interaction of A₆R with DPPC bilayers. These studies provide insight into interactions between a model cationic peptide and vesicles, relevant to understanding the action of antimicrobial peptides on lipid membranes. Notably, peptide A₆R exhibits antimicrobial activity without membrane lysis

Cell Adhesion Motif-Functionalized Lipopeptides: Nanostructure and Selective Myoblast Cytocompatibility

The conformation and self-assembly of four lipopeptides, peptide amphiphiles comprising peptides conjugated to lipid chains, in aqueous solution have been examined. The peptide sequence in all four lipopeptides contains the integrin cell adhesion RGDS motif, and the cytocompatibility of the lipopeptides is also analyzed. Lipopeptides have either tetradecyl (C14, myristyl) or hexadecyl (C16, palmitoyl) lipid chains and peptide sequence WGGRGDS or GGGRGDS, that is, with either a tryptophan-containing WGG or triglycine GGG tripeptide spacer between the bioactive peptide motif and the alkyl chain. All four lipopeptides self-assemble above a critical aggregation concentration (CAC), determined through several comparative methods using circular dichroism (CD) and fluorescence. Spectroscopic methods [CD and Fourier transform infrared (FTIR) spectroscopy] show the presence of β-sheet structures, consistent with the extended nanotape, helical ribbon, and nanotube structures observed by cryogenic transmission electron microscopy (cryo-TEM). The high-quality cryo-TEM images clearly show the coexistence of helically twisted ribbon and nanotube structures for C14-WGGRGDS, which highlight the mechanism of nanotube formation by the closure of the ribbons. Small-angle X-ray scattering shows that the nanotapes comprise highly interdigitated peptide bilayers, which are also present in the walls of the nanotubes. Hydrogel formation was observed at sufficiently high concentrations or could be induced by a heat/cool protocol at lower concentrations. Birefringence due to nematic phase formation was observed for several of the lipopeptides, along with spontaneous flow alignment of the lyotropic liquid crystal structure in capillaries. Cell viability assays were performed using both L929 fibroblasts and C2C12 myoblasts to examine the potential uses of the lipopeptides in tissue engineering, with a specific focus on application to cultured (lab-grown) meat, based on myoblast cytocompatibility. Indeed, significantly higher cytocompatibility of myoblasts was observed for all four lipopeptides compared to that for fibroblasts, in particular at a lipopeptide concentration below the CAC. Cytocompatibility could also be improved using hydrogels as cell supports for fibroblasts or myoblasts. Our work highlights that precision control of peptide sequences using bulky aromatic residues within “linker sequences” along with alkyl chain selection can be used to tune the self-assembled nanostructure. In addition, the RGDS-based lipopeptides show promise as materials for tissue engineering, especially those of muscle precursor cells

Effect of IDEQ and ATP upon the kinetic of Aβ aggregation and seeding.

<p>(<b>A</b>) Turbidity profiles of Aβ1-42 at 15 μM alone in working buffer (▴) or in the presence of IDEQ at the indicated molar ratios (IDEQ:Aβ), from top to bottom: 1∶200 (□), 1∶100 (Δ), 1∶10 (◯) and 1∶10 containing 0.5 mM ATP (<b>•</b>). Light scattering at 340 nm was measured every 30 min using a TECAN GENios multi-well reader (for clarity, only the points every other 90 min are shown). The bracket encloses the curves obtained after co-incubation of Aβ1-42 with IDEQ at the indicated conditions. Results are expressed as mean ± S.E.M. of at least two independent experiments in duplicate. (<b>B</b>) Representative TEM images of samples at steady state of Aβ1-42 alone (top) or with IDEQ at 1∶10 molar ratio in the presence of ATP (bottom). Bars = 100 nm. (<b>C</b>) Time course of Aβ1-42 aggregation alone (▴), in the presence of seeds previously formed with IDEQ (◯), or after the addition of pure Aβ1-42 seeds (□). (<b>D</b>) Kinetics of aggregation of Aβ1-42 after the addition of IDEQ (◯) or the same volume of working buffer (▴) to Aβ1-42 after 48 h of self-assembly, as indicated by the arrow.</p

Effect of IDEQ upon solubility and secondary structure of Aβ aggregates.

<p>(<b>A</b>) samples containing Aβ1-42 before incubation (“dead time”) and after incubation for 5 days with or without IDEQ were centrifuged at 3,000× g for 5 min and supernatants and pellets analyzed by Western blots with anti-Aβ 6E10 and 4G8. Arrowheads indicate: H, high molecular mass oligomers, T, Aβ tetramers and <i>t</i>, Aβ trimers. (<b>B</b>) Densitometric quantification of Aβ1-42 obtained from Western blots shown in panel (<b>A</b>). Bars represent the mean ± SEM of total Aβ immunoreactivity in arbitrary units (AU). * p<0.05, Student's <i>t</i> test. (<b>C</b>) Far UV-CD spectra recorded at “dead time” of Aβ1-42 (solid black line), IDEQ alone (dotted line) and Aβ1-42 co-incubated with IDEQ (solid gray line) at a 1∶300 molar ratio (IDEQ:Aβ). (<b>D</b>) Far UV-spectra recorded after 6 days of incubation of Aβ1-42 alone or Aβ1-42 with IDEQ at 1∶300 molar ratio. Samples were centrifuged as described above and supernatants analyzed. IDEQ alone, dotted line; Aβ1-42 alone, solid black line; Aβ1-42 co-incubated with IDEQ, solid gray line.</p

Aβ1-42 oligomers formed in the presence of IDEQ are not neurotoxic.

<p>(<b>A</b>) Representative AFM images showing the size and morphology of Aβ1-42 neurotoxic species. Left, Aβ1-42 incubated alone for 4 days. Approximately spherical species of ∼20–30 nm are indicated by arrowheads. Rods and short protofibrils are depicted by arrows. Inset: a larger Aβ1-42 protofibril is shown. Right; Aβ1-42 incubated in the presence of IDEQ at a 1∶10 molar ratio (IDEQ: Aβ) showing larger aggregates of 50–60 nm (arrowheads) and rods with lengths of ∼100–120 nm (arrows). (<b>B</b>) Representative immunofluorescence of primary differentiated neurons exposed to vehicle, Aβ1-42 alone or Aβ1-42 pre-incubated with IDEQ from top to bottom, as indicated. White arrows point at neuronal processes. Bars  = 30 μM. (<b>C</b>) Analysis of neuronal processes under the conditions as shown in panel (<b>A</b>). Bars represent the mean ± SEM of processes' lengths as measured from the centre of the neuronal body * p<0.01, one-way ANOVA, Tukey post-hoc test. (<b>D</b>) Viability of mature primary neurons after the indicated treatments as assessed by MTT reduction. * p<0.05, one-way ANOVA, Tukey post-hoc test. Results are shown for three independent experiments.</p

IDEQ does not modify insulin conformation.

<p>(<b>A</b>) <i>Circular dichroism (CD) spectra</i> of 10 μM insulin (ins.) in working buffer (solid black line), insulin with IDEQ at 1∶100 molar ratio, enzyme:insulin (solid gray line) and IDEQ alone (dotted line) with no prior incubation. (<b>B</b>) Same samples as in panel (<b>A</b>) after incubation for 24 h at 25°C. Insulin alone (solid black line), insulin with IDEQ (solid gray line) and IDEQ alone (dotted line). (<b>C</b>) Western blot with anti-phospho-Akt and anti-total Akt of U-87 cell lysates. Cells were exposed for 30 min with insulin alone, insulin previously co-incubated with IDEwt or IDEQ, as indicated. Wortmannin (wort) was incubated at 10 nM for 30 min before treatments.</p

Time-course, amount and pH-dependence of the formation of complexes between Aβ1-42 and IDEwt or IDEQ.

<p>(<b>A</b>) Western blots with anti-Aβ 6E10 showing the ∼120 kDa band corresponding to IDE-AβSCx (IDE-Aβ stable complex) as a function of the incubation time. Top panel, IDEQ; lower panel, IDEwt. Both PVDF membranes were developed simultaneously with a STORM 860 scanner. Below each Western blot, the same membranes stained with Coomassie blue, show IDEwt or IDEQ loading. (<b>B</b>) Densitometric data from Western blots for IDEQ (◯) and IDEwt (▴) were fitted to a single exponential equation using Graph Pad Prism v.4 software. Points represent the mean ± SEM from two independent experiments in duplicate. (<b>C</b>) IDEQ-AβSCx formation is partially competed by pre-incubation for 1 h with insulin at the indicated molar excess before the addition of Aβ1-42. Data are expressed as the percentage of the remaining Aβ-positive band at ∼120 kDa, in arbitrary units, as a function of insulin concentration. Each point represents the mean ± SEM of two independent experiments in duplicate. Inset: a representative Western blot of IDEQ-AβSCx developed with 6E10. (<b>D</b>) Densitometry of IDEQ-AβSCx at the indicated range of pH as determined by Western blot with anti-Aβ. Bars represent the mean ± SEM of three separate experiments. Inset: top, representative Western blot with anti-Aβ of IDEQ-AβSCx; bottom, Coomassie blue of IDEQ loaded in each lane. In panels (<b>A</b>), (<b>C</b>) and (<b>D</b>), IDEwt or IDEQ-AβSCxs are indicated by arrowheads.</p