Abstract
Purpose :
Progressive rod-cone degeneration (PRCD) is an integral membrane protein strongly associated with photoreceptor disc membranes that is crucial for maintaining the structural and functional integrity of photoreceptor outer segments (POS). Previously we showed that the sole cysteine (Cys2) in PRCD is palmitoylated, a post translational lipid modification that is essential for protein stability and trafficking to the OS. However, the precise role of palmitoylation in PRCD remains obscure. The focus of this study is to define how palmitoylation in PRCD supports protein stability and trafficking to the OS using multiple PRCD mice models and cell culture studies.
Methods :
We evaluated visual function by electroretinogram (ERG) at various ages in the PRCD-C2Y animals and littermate controls. To investigate the role of palmitoylation, we made plasmid construct changing palmitoylation to myristoylation (PRCD-C2G) and analyzed protein stability and trafficking by western blotting and immunohistochemistry. Furthermore, PRCD aggregation into mitochondrial membranes and proteasomal insufficiency was investigated in various PRCD-C2Y models. Next, mitochondrial function is analyzed in age matched WT, and PRCD mutant retinas using seahorse flux analyzer. Ultrastructure of PRCD-C2Y retina was performed by transmission electron microscopy.
Results :
Our ERG and ultrastructure studies in PRCD-C2Y animal models show attenuated visual responses, lamellar disorientation, and vesicular profiles in the photoreceptor OS like canine models. Furthermore, substituting myristoylation (PRCD-C2G) for palmitoylation neither alleviated the protein stability nor restored PRCD trafficking to the OS. Interestingly, mutating C-terminal ubiquitin sites (K50R/K54R) enhanced the stability in PRCD-C2Y mutant protein. Also, our data shows dysregulated mitochondrial metabolism in PRCD-C2Y retina.
Conclusions :
Our studies demonstrate that palmitoylation in PRCD is indispensable for PRCD protein stability and trafficking to POS. Substituting myristoylation for palmitoylation does not rescue protein stability or trafficking of PRCD-C2Y mutant. However, mutating ubiquitin signal in palmitoylation deficient PRCD-C2Y led to enhanced protein stability suggesting that both palmitoylation and ubiquitination coordinate to maintain PRCD protein homeostasis.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.