Cell-Penetrable and in vivo activatable mini-chaperones for stabilizing misfolded proteins

Abstract

Purpose : The goal of the study is to synthesize, and characterize cell-penetrable, in vivo activatable minichaperone (CPIAMC) peptides that show increased cellular uptake, exhibit in vivo activation under stress and prevent the formation of cytotoxic protein aggregates.

Methods : Peptide chaperones derived from αA-crystallin chaperone site sequence were synthesized using all L- or all D- amino acids (to increase in vivo stability). The peptides were conjugated to a cell-penetrating peptide sequence, VPTLK or KKKRKV (all L), and had a specific protease cleavage site for the in vivo release of the minichaperone sequence from the precursor in cells under stress to exhibit on-demand chaperone activity. Chimeric peptides having either “always on” or exhibiting “on-demand” chaperone function were tested for their in vitro anti-aggregation activity using alcohol dehydrogenase aggregation assay. The cleavage of CPIAMC to release active minichaperones was tested in vitro using caspases and extracts from cells subjected to H₂O₂ stress and by analyzing the products by mass spec and chaperone assay. Cellular uptake, in vivo activation and anti-apoptotic activities of CPIAMCs, was tested using ARPE-19 and N27 cells. TM cells transfected with a pDsRED2-MUT MYOC plasmid was used to test whether peptide chaperones suppress the cellular aggregation of mutant myocilinY437H.

Results : Chimeric peptides synthesized with KKKRKV was inactive as a chaperone until the action of stress proteases released the minichaperone. Whereas, chimeric peptides with VPTLK was active as a chaperone without cleavage. CPAIMCs readily entered the cells and showed dose-dependent protection of N27 cells from H₂O₂-induced (50 µM) cytotoxicity. The protection offered by all D- minichaperones was higher and lasted for a longer duration. Chimeric peptide pretreated N27 cells showed fewer caspase positive cells when subjected to H₂O₂ stress. CPIAMC-treated TM cells suppressed the aggregation of MYOC(Y437H) mutant protein in cells to a level comparable to the effect observed when phenylbutyrate, a chemical chaperone was used in the assay. Western blot analysis of cell lysates confirmed the decreased accumulation of mutant myocilin in TM cells treated with chaperone peptides.

Conclusions : Our data shows that CPIAMC could help control protein misfolding and associated pathology.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.