Identification of novel interacting proteins of FUZ and GPR161.

Abstract

Proteins modulate signaling pathways through dynamic protein-protein interactions, which provide critical insights into the underlying mechanisms of cellular processes leading to human diseases. Our previous study revealed the biochemical and genetic interactions between FUZ and GPR161 that regulate sonic hedgehog signaling during spinal neural tube development. This study aimed to identify novel co-interacting proteins of FUZ and GPR161 and explore their biochemical and functional associations. We performed affinity-based liquid chromatography-tandem mass spectrometry on immunoprecipitated FUZ and GPR161 proteins. Using this approach, we identified 159 co-interacting proteins, of which 289 proteins that interacted exclusively with FUZ and 617 proteins that interacted exclusively with GPR161. Gene Ontology (GO) analysis of the FUZ and GPR161 co-interactome revealed an enrichment of proteins associated with proteasomal catabolic processes and trafficking. GO analysis of the exclusive FUZ and GPR161 interactomes identified cell cycle progression, mitochondrial membrane, RNA metabolism, receptor complex, and Endoplasmic Reticulum-Golgi transport. These findings were further validated using STRING network analysis. Among the identified proteins, we prioritized FKBP8 and confirmed its biochemical interactions with both FUZ and GPR161 exclusively. In summary, our proteomic profiling uncovered the protein network of FUZ and GPR161, revealing their individual and cooperative functions in multiple cellular processes. Statement of Significance- The functional link between FUZ and GPR161 remains unknown, while our previous study showed their genetic and biochemical link in regulating sonic hedgehog signaling during spinal neural tube development. - This study identified the protein network involving FUZ and GPR161 using affinity- based mass spectrometry combining with gene ontology and STRING analysis, providing the novel molecular regulatory role of FUZ and GPR161. - We prioritized the proteins, FKBP8, as a co-interacting partners of FUZ and GPR161, and validated their biochemical interaction. We identified the FUZ-GPR161-FKBP8 forms protein complex, and their interaction was not exclusive, suggesting those three proteins located in the same signaling pathway potentially regulating spinal neural tube development.