6th March 2020, 11h30 - 12h30
(Conference room 13 rue Moreau)

Daniele Dell’Orco (Biomedicine and Movement Sciences, University of Verona, Italy)

Invited by: Olivier Goureau

Light sensitivity in photoreceptor cells is finely regulated by negative feedback loops that involve the second messenger guanosine 3´,5´-cyclic monophosphate (cGMP) and Ca2+. The drop of cytoplasmic [Ca2+] following rod illumination is detected by guanylate cyclase-activating proteins (GCAPs), a family of neuronal Ca2+/Mg2+ sensors that control the activity of membrane bound guanylate cyclases (GC) in a Ca2+-dependent manner. GCAPs are quite unique regulators, in that they act as inhibitors of the GC at high (250–800 nM) intracellular [Ca2+], while they switch to an activator of the enzyme at low (20–100 nM) [Ca2+], thereby contributing to the switch-off of the phototransduction cascade.
To date, more than 20 single point mutations in GCAP1 have been associated with cone, cone-rod and macular dystrophies. We present a thorough characterization of altered structure/function relationships in GCAP1 in the presence of disease-associated missense mutations. Interestingly, some of the GCAP1 variants showed a severe disturbance of Ca2+/Mg2+ sensing properties, while some other mutants showed unaltered affinity for Ca2+. However, all the disease-associated GCAP1 variants analysed so far showed a remarkable dysregulation of the GC-activity, leading to constitutive activation of the target enzyme at physiological [Ca2+]. A similar aberrant regulation of the target enzyme must therefore result from a similar perturbation of the GCAP1-GC interaction, which may eventually cause dysregulation of both Ca2+ and cGMP homeostasis and result in retinal degeneration. We corroborate biochemical experimental data with a computational approach based on exhaustive molecular dynamics simulations of GCAP1 in its Mg2+ and Ca2+ loaded states. By applying a Protein Structure Network (PSN) paradigm we could determine the intramolecular communication pathways involved in the specific GC activator/inhibitor switch, which is likely to be altered in disease-associated conditions. Finally, by combining in vitro enzymatic assays with system-level analysis of the phototransduction cascade and in vivo delivery of purified recombinant proteins encapsulated in liposomes we show that extra-delivery of normal GCAP1 can partially compensate for the cGMP signaling dysregulation associated with COD/CORD variants, which could be relevant for protein therapy purposes

1. Marino V, Dal Cortivo G, Oppici E, Maltese PE, D'Esposito F, Manara E, Ziccardi L, Falsini B, Magli A, Bertelli M, Dell'Orco D. Hum Mol Genet. (2018); 27(24):4204-4217
2. Marino V, Scholten A, Koch KW, Dell'Orco D., Hum Mol Genet. (2015); 24(23):6653-66.
3. Marino & Dell'Orco, Sci Rep. (2016); 6:34277.
4. Dell’Orco D. & Dal Cortivo G. (2019); Sci Rep.;9(1):20105.