pH-Dependent In-Cell Self-Assembly of Peptide Inhibitors Increases the Anti-Prion Activity While Decreasing the Cytotoxicity

The first step in the conventional approach to self-assembled biomaterials is to develop well-defined nanostructures in vitro, which is followed by disruption of the preformed nanostructures at the inside of the cell to achieve bioactivity. Here, we propose an inverse strategy to develop in-cell gain-of-function self-assembled nanostructures. In this approach, the supramolecular building blocks exist in a unimolecular/unordered state in vitro or at the outside of the cell and assemble into well-defined nanostructures after cell internalization. We used block copolypeptides of an oligoarginine and a self-assembling peptide as building blocks and investigated correlations among the nanostructural state, antiprion bioactivity, and cytotoxicity. The optimal bioactivity (i.e., the highest antiprion activity and lowest cytotoxicity) was obtained when the building blocks existed in a unimolecular/unordered state in vitro and during the cell internalization process, exerting minimal cytotoxic damage to cell membranes, and were subsequently converted into high-charge-density vesicles in the low pH endosome/lysosomes in vivo, thus, resulting in the significantly enhanced antiprion activity. In particular, the in-cell self-assembly concept presents a feasible approach to developing therapeutics against protein misfolding diseases. In general, the in-cell self-assembly provides a novel inverse methodology to supramolecular bionanomaterials

PMCA using cell lysate of RK13SHaPrP.

<p>(A) PK-resistant PrP^Sc amplification of two Syrian hamster-adapted TME prions (HY and DY) with cell lysate (CL) of RK13SHaPrP. The HY and DY seeds were diluted 100–2,500 fold for PMCA. The level of PrP^Sc in pre- (−) and post-PMCA (+) samples was analyzed by Western blotting. (B) Comparison of the PK-resistant PrP^Sc of HY and DY prions generated by PMCA. The HY and DY seeds were diluted 100–62,500 fold for PMCA. Ten % brain homogenate (BH) of HY- and DY-sick Syrian hamsters were used as controls. PrP^Sc in both panels A and B was detected by monoclonal anti-PrP 3F4 antibody.</p

PrP^Sc amplification affected by PrP^C abundance in cell lysate.

<p>PMCA was performed using undiluted and diluted (1∶10 fold) RK13MoPrP cell lysate (CL). Both RML and NBH were used as seeds in dilutions of 100–24,000 fold. PK-treated pre- (−) and post-PMCA (+) samples were analyzed. Western blotting was performed using monoclonal anti-PrP 6H4 antibody.</p

PMCA using lysates of a wide range of cell types.

<p>Cell lysate of neuronal (N2a), prion-free brain mesenchymal (SMB-PS), mixed cerebellar neuronal and glial (CRBL) or fibroblast (NIH 3T3) cells was concentrated to include the PrP^C level of wild brain homogenate. PMCA was performed by seeding with RML-sick (RML) and normal (NBH) brain homogenate. The seed dilution fold was 100–24,000. The PrP^Sc level of PMCA before (−) and after (+) was compared. Monoclonal anti-PrP 6H4 antibody was used for Western blotting.</p

Expression of PrP^C in a variety of cell lines.

<p>(A) Comparison of the PrP^C levels in cell lines and brain homogenate. Western blotting followed by densitometry demonstrated relative differences of the PrP^C levels. Extrapolation of multiplication factors to concentrate cell lysate was based on the amount of protein analyzed and the relative PrP^C levels. PrP^C was detected by D13 (left blot), 6H4 (middle blot), and 3F4 (right blot) antibodies. (B) Fluorescence images of RK13 cells expressing full length mouse PrP^C. Colocalization (yellow, overlay) of PrP^C (green) and GM1 (red) in the lipid rafts of the plasma membrane of RK13MoPrP was shown by confocal microscopy. The nuclei (blue) were stained by Hoechst 33258. Scale was shown by a 30 µm bar.</p