June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Dual Palmitoylation of PRCD, a Photoreceptor-Specific Protein Linked to RP, Alters Protein Stability and Subcellular Localization
Author Affiliations & Notes
  • Boyden Myers
    Ophthalmology, West Virginia University, Morgantown, West Virginia, United States
  • Emily Sechrest
    Ophthalmology, West Virginia University, Morgantown, West Virginia, United States
  • Gabrielle Hamner
    Ophthalmology, West Virginia University, Morgantown, West Virginia, United States
  • Joseph Murphy
    Ophthalmology, West Virginia University, Morgantown, West Virginia, United States
  • Sree Motipally
    Ophthalmology, West Virginia University, Morgantown, West Virginia, United States
  • Saravanan Kolandaivelu
    Ophthalmology, West Virginia University, Morgantown, West Virginia, United States
    Biochemistry, West Virginia University, Morgantown, West Virginia, United States
  • Footnotes
    Commercial Relationships   Boyden Myers None; Emily Sechrest None; Gabrielle Hamner None; Joseph Murphy None; Sree Motipally None; Saravanan Kolandaivelu None
  • Footnotes
    Support  NIH RO1EY028959
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 1793 – F0342. doi:
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      Boyden Myers, Emily Sechrest, Gabrielle Hamner, Joseph Murphy, Sree Motipally, Saravanan Kolandaivelu; Dual Palmitoylation of PRCD, a Photoreceptor-Specific Protein Linked to RP, Alters Protein Stability and Subcellular Localization. Invest. Ophthalmol. Vis. Sci. 2022;63(7):1793 – F0342.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Progressive rod-cone degeneration (PRCD) is a photoreceptor-specific protein essential for maintaining photoreceptor outer segment (OS) structure and rhodopsin packaging densities into disc membranes. Previously, we have shown that PRCD undergoing protein palmitoylation at the second amino acid (Cys2) is crucial for protein stability and proper trafficking to photoreceptor OS. PRCD has several predicted structural domains with unknown significance, with one of the mutations being located in the polybasic region (PBR). This mutation, an arginine to cysteine at the 17th position (R17C), is linked with retinitis pigmentosa (RP). The goal of our study is to determine the structural importance of the PBR in PRCD and to characterize the R17C mutated protein.

Methods : Constructs expressing R17C and a double mutant (C2Y/R17C) were cloned in a pCAG-IRES-EGFP vector with an HA-tag and transiently transfected in hRPE1 cells. Protein stability, membrane association, and subcellular localization were determined by western blot and immunocytochemistry. Palmitoylation status was determined via Acyl-RAC and Acyl-PEG exchange. Localization of R17C and C2Y/R17C was also studied in vivo by subretinal injection in the murine retina.

Results : Protein stability of mutant R17C is significantly reduced by 50% compared to WT, despite having strong membrane association and palmitoylation observed by Acyl-RAC. Interestingly, we distinguished palmitoylation at the Cys17 position with the loss of endogenous palmitoylation (C2Y/R17C), which enhances the protein stability compared to the C2Y protein. Subretinal injection followed by electroporation in murine retina shows C2Y, R17C, and C2Y/R17C double mutant proteins are mislocalized to the subcellular compartment. Immunolocalization in the RPE cell line expressing R17C shows similar phenotypes to WT, and C2Y/R17C shows similarities to the C2Y protein.

Conclusions : Despite being palmitoylated twice and demonstrating strong membrane association, the mutation in the PBR (R17C) affects protein stability and trafficking from the IS. Furthermore, palmitoylation within the PBR alone (C2Y/R17C) does not compensate for protein stability or trafficking. Overall, we demonstrate that the PBR domain in PRCD is indispensable for its function and any defects of the area lead to dysregulation of PRCD protein associated with blinding diseases.

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

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