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Kyle Kim, Lance Safarta, Wei-Chieh Chiang, Judith Coppinger, Eun-Jin Lee, Jonathan Lin; Mass Spectrometry Identification of Wild-type and P23H Rhodopsin Protein Interactome. Invest. Ophthalmol. Vis. Sci. 2022;63(7):2581 – F0464.
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© ARVO (1962-2015); The Authors (2016-present)
P23H rho is a common genetic cause of retinitis pigmentosa and encodes for misfolded P23H rhodopsin protein. The mechanisms by which rods recognize and target misfolded rhodopsin for degradation are not well understood. Here, we performed mass spectrometry on P23H rhodopsin immunopurified from rhoP23H/P23H mice retinas to identify interacting proteins and compare with wild-type (WT) rhodopsin from rho+/+ mice.
Monoclonal anti-rhodopsin antibody 1D4 (Santa Cruz Biotechnologies) was used to immunoprecipitate 5mg of WT or P23H rhodopsin protein and its interacting proteins from retinal protein lysates of postnatal day 15 rho+/+ (24 retinas) or rhoP23H/P23H (40 retinas) mice. Liquid chromatography with tandem mass spectrometry (LC-MS-MS) was performed on 3 independent samples of WT or P23H rhodopsin protein (LTQ-Orbitrap mass spectrometer, Thermo Finnigan, Waltman, MA). As a negative control, LC-MS-MS was also performed on immunoprecipitates from mouse embryonic fibroblasts. Spectral counts from WT, P23H, and MEF LC-MS-MS experiments were collected and analyzed by Gene Ontology (GO), Kyoto-Encyclopedia of Genes and Genomes (KEGG), Reactome, and CytoScape.
We found 276 proteins that bound to WT rhodopsin, and 286 proteins bound to P23H rhodopsin of which 173 proteins were unique to P23H rhodopsin. GO, KEGG, and Reactome analysis identified enrichment of multiple unique pathways such as ER-associated protein degradation and ubiquitinoylation in the P23H rhodopsin interactome compared to WT. By contrast, phototransduction and visual perception terms were less enriched for P23H rhodopsin. GO-cellular compartment analysis showed decrease in outer segment, cilium, and inner segment proteins but increase in axonal/synapse proteins in the P23H interactome.
Proteomic analysis reveals numerous differences between P23H and WT rhodopsin protein interactome. The P23H rhodopsin interactome is enriched in translational and post-translational quality control processes. The P23H rhodopsin interactome differs throughout all regions of the rod photoreceptor cell. These proteomic and subcellular changes could correspond to mechanisms rods use to eliminate misfolded rhodopsin. These molecular alterations may contribute to pathogenesis of retinitis pigmentosa caused by P23H rhodopsin.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.
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