Abstract
Purpose :
Oxidative and endoplasmic reticulum (ER) stress-induced retinal degeneration is implicated in ocular pathologies including AMD. Our recent work revealed that a mini-peptide derived from αB-crystallin recombinantly fused with elastin like polypeptides (ELPs) protects RPE cells from stress-induced cell death. In the present study, we compared two ELP fusions and evaluated their potential against tunicamycin, which can induce ER stress responses.
Methods :
Previously, our team fused (mini cry) peptide with multiple elastin-like polypeptide (ELP) motifs. These ELPs consist of pentapeptide sequence, encoded as: (VPGVG)n, at which (n) specifies molecular size and the guest residue ‘X’ determines polymeric hydrophobicity. ELPs phase separate above a tunable transition temperature, Tt, which may facilitate their therapeutic applications. In earlier work, we engineered an amphiphilic mini-cry di-block polymer onto a micelle-forming ELP called SI, encoded as: (VPGSG)48(VPGIG)48, which has two Tt. In contrast, we now have a simpler monoblock ELP, V96 encoded as (VPGVG)96, which displays a single Tt near 37 oC. Physiochemical properties of both constructs (SI and V96) were compared by dynamic light scattering, spectrophotometry, and contrast microscopy. Their comparative cellular release, uptake, and efficacy were tested by Retinal Pigment Epithelial (ARPE-19) cells using confocal microscopy and western blotting. Hydrogen peroxide (H2O2) and tunicamycin challenges were used as oxidative and ER stressors.
Results :
Results indicated successful ELP fusion with sensitive assembly at physiological temperatures. Unlike controls, only cry-ELP-treated groups showed upregulation of BIP/GPR78 and PERK signals in vitro (Figure 1). While both mini cry-ELP conjugates had an increased cellular association under oxidative stress, cry-V96 showed less lysosomal colocalization and faster cellular entry in both normal and stressed cultures.
Conclusions :
Compared to cry-SI, V96 ELP fusion promoted lysosomal release necessary to provide protection against an oxidative stressor; furthermore, in the tunicamycin-induced ER stress model, RPE cells revealed upregulation of protective ER stress markers. These results suggest additional preclinical PK/PD evaluation of these peptide therapeutics is warranted.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.