α‑Casein Inhibits Insulin Amyloid Formation by Preventing the Onset of Secondary Nucleation Processes

α-Casein is known to inhibit the aggregation of several proteins, including the amyloid β-peptide, by mechanisms that are not yet completely clear. We studied its effects on insulin, a system extensively used to investigate the properties of amyloids, many of which are common to all proteins and peptides. In particular, as for other proteins, insulin aggregation is affected by secondary nucleation pathways. We found that α-casein strongly delays insulin amyloid formation, even at extremely low doses, when the aggregation process is characterized by secondary nucleation. At difference, it has a vanishing inhibitory effect on the initial oligomer formation, which is observed at high concentration and does not involve any secondary nucleation pathway. These results indicate that an efficient inhibition of amyloid formation can be achieved by chaperone-like systems, by sequestering the early aggregates, before they can trigger the exponential proliferation brought about by secondary nucleation mechanisms

Figure 3

<p>Effect of temperature on aggregation. <b>A)</b> Light scattered intensity at 90° angle registered during the temperature scan. Data relative to different proteins are normalized by dividing for the respective initial values of scattered intensity. <b>B)</b> Weight-average radius of the largest species, as obtained by CONTIN analysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000111#pone.0000111-Provencher1" target="_blank">[38]</a>, followed during the temperature scan.</p

Figure 1

<p>Comparison of the intensity-weighted size distribution of GST (black line), GST-Q22 (red line) and GST-Q41 (green line) at 90° scattering angle. The measurements were carried out on 20 µM protein samples and at 20°C.</p

Figure 5

<p>The effect of temperature on the far-UV CD spectra. <b>A)</b> Far-UV CD spectra of GST-Q41 recorded at different temperatures: 20°C (continuous black line), sample incubated at 51°C for 30 minutes (dashed line), and then incubated at 68°C for 1 hour and 20 minutes (grey line). Protein concentration was 4 µM. <b>Inset</b>: Far-UV CD spectra of GST (dashed line), GST-Q22 (dotted line), and GST-Q41 (continuous line) recorded at 20°C. <b>B)</b> Far-UV CD thermal scans of GST (continuous black line), GST-Q22 (dotted line), and GST-Q41 (grey line) recorded at 222 nm with a heating rate of 1°C/min. The curves were normalized to the intensity of the GST sample at 20°C for comparison purposes. The cooling profile of GST (dashed line) was recorded with the same rate and is added as an example of irreversibility. A similar behaviour was observed for the other two proteins. Protein concentration was 20 µM in all samples.</p

Figure 2

<p>Effect of an antioxidant on aggregation. <b>A)</b> Comparison between the intensity-weighted size distributions obtained in the absence and in the presence of DTT. <b>B)</b> Time course of light scattered intensity at 90° angle upon addition of DTT to fresh protein samples. GST, GST-Q22 and GST-Q41 are shown using black, red and green lines, respectively. The measurements shown were carried out at 20°C using 20 µM samples.</p

Minimalism in Radiation Synthesis of Biomedical Functional Nanogels

A scalable, single-step, synthetic approach for the manufacture of biocompatible, functionalized micro- and nanogels is presented. In particular, poly­(<i>N</i>-vinyl pyrrolidone)-grafted-(aminopropyl)­methacrylamide microgels and nanogels were generated through e-beam irradiation of PVP aqueous solutions in the presence of a primary amino-group-carrying monomer. Particles with different hydrodynamic diameters and surface charge densities were obtained at the variance of the irradiation conditions. Chemical structure was investigated by different spectroscopic techniques. Fluorescent variants were generated through fluorescein isothiocyanate attachment to the primary amino groups grafted to PVP, to both quantify the available functional groups for bioconjugation and follow nanogels localization in cell cultures. Finally, a model protein, bovine serum albumin, was conjugated to the nanogels to demonstrate the attachment of biologically relevant molecules for targeting purposes in drug delivery. The described approach provides a novel strategy to fabricate biohybrid nanogels with a very promising potential in nanomedicine

Blue Native Polyacrylamide Gel Electrophoresis: Hsp60 at various concentrations.

<p>Blue Native PAGE (4–16%) image of naïve Hsp60 in the concentration range 1.9–4.8 µM. The pattern reveals that the protein exists in two oligomeric forms independently of the concentration.</p

Static light scattering characterization.

<p>Scattered light intensities from solutions of naïve Hsp60 protein at different concentrations, expressed in terms of the Rayleigh ratio <i>R_90°</i> at 18.7 µm⁻¹ normalized by the concentration values <i>c</i>. Together with the total scattered intensity (empty circles), the intensity contribution by species with diameter size lower than 70 nm (filled circles) is also reported. The lines represent the dependence of <i>R_90°/c</i> on concentration predicted for Hsp60 protein totally assembled in tetradecamers (solid) or heptamers (dashed). The experimental values of intensity scattered by Hsp60 species always fall between those predicted for totally tetradecameric or heptameric populations.</p

Size exclusion chromatography results.

<p>Size exclusion chromatography experiments on the naïve Hsp60 at different concentrations (0.8 µM: red, 1.6 µM: black, 2.2 µM: magenta, 6.4 µM: green, 16.0 µM: turquoise) compared with GroEL at 7.0 µM (blue) and BSA (dotted line). The vertical line drawn across the Hsp60 peak helps to highlight the independency of the retention time from protein concentration. The value of the retention time is consistent with that expected for heptameric and tetradecameric species in equilibrium.</p

SAXS profiles of GroEL and Hsp60.

<p>SAXS profiles of GroEL (red) and naïve Hsp60 (green). GroEL is at c = 52.5 µM, while Hsp60 is at c = 79.5 µM. Experimental curves are scaled for the sake of clarity. SAXS spectrum for Hsp60 is quite different from that of GroEL, which corresponds to a more well-defined structure.</p