Dysfunction of cGMP signalling in photoreceptors by a macular dystrophy-related mutation in the calcium sensor GCAP1.

Macular dystrophy leads to progressive loss of central vision and shows symptoms similar to age-related macular degeneration. Genetic screening of patients diagnosed with macular dystrophy disclosed a novel mutation in the GUCA1A gene, namely a c.526C > T substitution leading to the amino acid substitution p.L176F in the guanylate cyclase-activating protein 1 (GCAP1). The same variant was found in three families showing an autosomal dominant mode of inheritance. For a full functional characterization of the L176F mutant we expressed and purified the mutant protein and measured key parameters of its activating properties, its Ca2+/Mg2+-binding, and its Ca2+-induced conformational changes in comparison to the wildtype protein. The mutant was less sensitive to changes in free Ca2+, resulting in a constitutively active form under physiological Ca2+-concentration, showed significantly higher activation rates than the wildtype (90-fold versus 20-fold) and interacted with an higher apparent affinity with its target guanylate cyclase. However, direct Ca2+-binding of the mutant was nearly similar to the wildtype; binding of Mg2+ occurred with higher affinity. We performed molecular dynamics simulations for comparing the Ca2+-saturated inhibiting state of GCAP1 with the Mg2+-bound activating states. The L176F mutant exhibited significantly lower flexibility, when three Ca2+ or two Mg2+ were bound forming probably the structural basis for the modified GCAP1 function.

Cytotoxic Effects of Tropodithietic Acid on Mammalian Clonal Cell Lines of Neuronal and Glial Origin.

The marine metabolite tropodithietic acid (TDA), produced by several Roseobacter clade bacteria, is known for its broad antimicrobial activity. TDA is of interest not only as a probiotic in aquaculture, but also because it might be of use as an antibacterial agent in non-marine or non-aquatic environments, and thus the potentially cytotoxic influences on eukaryotic cells need to be evaluated. The present study was undertaken to investigate its effects on cells of the mammalian nervous system, i.e., neuronal N2a cells and OLN-93 cells as model systems for nerve cells and glia. The data show that in both cell lines TDA exerted morphological changes and cytotoxic effects at a concentration of 0.3-0.5 µg/mL (1.4-2.4 µM). Furthermore, TDA caused a breakdown of the mitochondrial membrane potential, the activation of extracellular signal-regulated kinases ERK1/2, and the induction of the small heat shock protein HSP32/HO-1, which is considered as a sensor of oxidative stress. The cytotoxic effects were accompanied by an increase in intracellular Ca(2+)-levels, the disturbance of the microtubule network, and the reorganization of the microfilament system. Hence, mammalian cells are a sensitive target for the action of TDA and react by the activation of a stress response resulting in cell death.

CaF2 nanoparticles as surface carriers of GCAP1, a calcium sensor protein involved in retinal dystrophies.

CaF2-based nanoparticles (NP) are promising biocompatible tools for nanomedicine applications. The structure of the NP crystal lattice allows for specific interactions with Ca2+-binding proteins through their EF-hand cation binding motifs. Here we investigated the interaction of 23 nm citrate-coated CaF2 NP with a calcium sensor protein GCAP1 that is normally expressed in photoreceptor cells and involved in the regulation of the early steps of vision. Protein-NP interactions were thoroughly investigated for the wild type (WT) GCAP1 as well as for a variant carrying the Asp 100 to Glu mutation (D100E), which prevents the binding of Ca2+ to the highest affinity site and is linked to cone dystrophy. Circular dichroism and fluorescence spectroscopy showed that protein structure and Ca2+-sensing capability are conserved for both variants upon interaction with the NP surface, although the interaction mode depends on the specific occupation of Ca2+-binding sites. NP binding stabilizes the structure of the bound GCAP1 and occurs with nanomolar affinity, as probed by isothermal titration calorimetry. Surface plasmon resonance revealed a fully reversible binding compatible with physiologically relevant kinetics of protein release whereas biochemical assays indicated a residual capability for NP-dissociated GCAP1 to regulate the target retinal guanylate cyclase. Our study constitutes a proof of concept that CaF2 NP could be optimized to serve as biologically compatible carriers of high amounts of functional GCAP1 in photoreceptors affected by retinal dystrophies.

Retina specific GCAPs in zebrafish acquire functional selectivity in Ca2+-sensing by myristoylation and Mg2+-binding.

Zebrafish photoreceptor cells express six guanylate cyclase-activating proteins (zGCAPs) that share a high degree of amino acid sequence homology, but differ in Ca(2+)-binding properties, Ca(2+)-sensitive target regulation and spatial-temporal expression profiles. We here study a general problem in cellular Ca(2+)-sensing, namely how similar Ca(2+)-binding proteins achieve functional selectivity to control finely adjusted cellular responses. We investigated two parameters of critical importance for the trigger and switch function of guanylate cyclase-activating proteins: the myristoylation status and the occupation of Ca(2+)-binding sites with Mg(2+). All zGCAPs can be myristoylated in living cells using click chemistry. Myristoylation does not facilitate membrane binding of zGCAPs, but it significantly modified the regulatory properties of zGCAP2 and zGCAP5. We further determined for all zGCAPs at least two binding sites exhibiting high affinities for Ca(2+) with KD values in the submicromolar range, whereas for other zGCAPs (except zGCAP3) the affinity of the third binding site was in the micromolar range. Mg(2+) either occupied the low affinity Ca(2+)-binding site or it shifted the affinities for Ca(2+)-binding. Hydrodynamic properties of zGCAPs are more influenced by Ca(2+) than by Mg(2+), although to a different extent for each zGCAP. Posttranslational modification and competing ion-binding can tailor the properties of similar Ca(2+)-sensors.