Abstract
Purpose :
Elovanoids are cell-specific neuroprotective lipid mediators that contribute to photoreceptor cell integrity (Jun et al, 2017. Sci Rep 7(1):5279). Elovanoid 34 (ELV-N34:6), a DHA derivative, protects retinal pigment epithelial (RPE) cells and photoreceptors by promoting the expression of Iduna, Sirt1, and Prohibitin; however, its direct targets remain elusive. We hypothesize that in addition to extracellular targets, ELV-N34:6 acts through intracellular targets to induce pro-homeostatic activity.
Methods :
Our approach to discover the ELV-N34 mechanism of action included limited proteolysis using proteinase K combined with a Mass Spectrometry screening to sort out proteins of RPE cells that underwent conformational change in the presence of ELVs under uncompensated oxidative stress (UOS) conditions. Artificial Intelligence algorithms were applied to determine the targets from the comparison between vehicle
(DMSO) and 250µg ELV-N34:6 in quadruplicate libraries of peptides. A concentration curve was produced to obtain the ED50, and activity was measured using the colorimetric reaction for the target.
Results :
A dose-response curve of ELV-N34:6 (from 0 to 250µg) showed changes in the folding of TXNRD1, suggesting direct interaction between the protein and the lipid mediator. Because there are 3 human variants of TXNRD1, we tested subcellular fractions in the presence or absence of ELV-N34:6. The cytosolic fraction showed a difference between UOS and UOS + ELV-N34:6 treated cells, and moreover the lipid
mediator alone induced a great increase in the TXNRD1 activity in agreement to the results shown in the LiP-MS.
Conclusions :
Here we demonstrated for the first time that ELV-N34:6 induces an activity increase of TXNRD1. Our data uncovers that TXNRD1 is a direct target of ELV-34:6 as a master regulatory system of RPE cell function as well to promote protection in RPE cells against UOS induced apoptosis and senescence programming.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.