Lysine Ubiquitylation in P23H Rhodopsin Protein Degradation

Abstract

Purpose : Retinitis Pigmentosa (RP) is a group of inherited disorders with loss of rod photoreceptors followed later by cone photoreceptors. The most common point mutation of RP in the United States is a proline to histidine amino acid change at position 23 (P23H). Our previous studies indicated that misfolded P23H rhodopsin activates the inositol-requiring enzyme 1 (IRE1) pathway of the unfolded protein response (UPR) and undergoes endoplasmic reticulum associated degradation (ERAD) by unclear mechanisms. Here, we investigate the role of lysine ubiquitination in P23H and wild-type (WT) rhodopsin protein turnover.

Methods : HEK293FT cells were transfected with a pcDNA3.1 plasmid vector expressing WT or P23H human rhodopsin from a cytomegalovirus (CMV) promoter (Invitrogen). A PrimeStar Mutagenesis site-directed primers using in-frame modifications of 5’-AAG-3’ to 5’-AGG-3’ were used to convert all 11 lysines on human WT and P23H rhodopsin to arginines (K-null). Immunoprecipitation and immunoblots were performed to measure protein levels of rhodopsin and ubiquitin. A cycloheximide chase assay determined t_1/2 of P23H and K-null P23H rhodopsin. RT-qPCR was performed to measure E3 ubiquitin ligase expression in P23H homozygous (Rho^P23H/P23H) mouse retinas. HEK293T cells containing I642G mutant allele were treated with DMF or 1NM-PP1 to artificially activate IRE1.

Results : P23H K-null was significantly less ubiquitylated than intact P23H rhodopsin. P23H K-null had significantly increased protein half-life compared to intact P23H rhodopsin in cycloheximide chase assays. Single lysine-to-arginine point mutations did not significantly change P23H rhodopsin ubiquitination. Additionally, a K-null WT rhodopsin also showed reduced ubiquitination of WT rhodopsin. Lastly, we identified 18 E3 ubiquitin ligase genes expressed in HEK293T cells, regardless of IRE1 activation status with 13 genes upregulated in Rho^P23H/P23H mouse retinas.

Conclusions : Our findings support that lysine ubiquitylation is an important post-translational modification for WT and P23H rhodopsin protein degradation. Multiple lysine residues are likely ubiquitinylated during rhodopsin protein degradation. Strategies to ubiquitinylate P23H rhodopsin could enhance its clearance by autophagy or proteasome degradation and potentially benefit retinitis pigmentosa patients carrying these mutations.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.