Ana Méndez
University of Barcelona, Spain
Title: Identification of novel interacting partners of the RetGC1/GCAPs complex responsible for cGMP synthesis in photoreceptor cells of the retina: implications for cell physiology and disease
Biography
Biography: Ana Méndez
Abstract
To gain insight into the organization, assembly and trafficking of the RetGC1/GCAPs complex responsible for cGMP synthesis in rods and cones, as well as its modulation in vivo, we set to identify new interactors of Guanylate Cyclase Activating Protein 1 (GCAP1). Pull-down assays were performed with purified GCAP1 in its Ca2+-bound or Ca2+-free form, from bovine rod outer segment preparations. Bound proteins were identified by liquid chromatography and mass spectrometry (LC-MS/MS). A label-free quantitative proteomic analysis was performed to identify proteins with preferential affinity for one of the GCAP1 conformational forms. Thirty-seven proteins were identified with a fold change >3 for Ca2+-bound over Ca2+-free GCAP1. These proteins included several proteins that could have relevant implications for protein stability, ciliary trafficking and disease. Interestingly, we identified very robustly proteins involved in de novo synthesis of GTP. Co-localization of these enzymes with RetGC1 and GCAP1 was observed at the rod outer segment compartment in bovine retinal sections. Extensive biochemical analysis (size-exclusion chromatography of native tissue, crossed immunoprecipitation and pull-down assays and surface plasmon resonance analysis of the binding kinetics) have further confirmed some of these interactions, which involve direct interactions with RetGC1. Our results unveil an interplay between the RetGC/GCAPs complex responsible for cGMP synthesis and the complex responsible for de novo synthesis of GTP. We speculate that this supraorganization of multienzyme complexes could serve to channel nucleotide metabolism, with an integrated modulation depending on the dark/light physiology of the cell.