Calcium sensor proteins in hearing and sight. Biochemical investigation of diseases-associated variants.

Calcium is a cation which plays a pivotal role as second messenger, thus its concentration in cells needs to be finely regulated. Many systems work for that purpose, including Ca2+ sensor proteins, which undergo conformational changes upon Ca2+ coordination via EF-hands. Ca2+ sensors can be ubiquitous or tissue specific. Examples in this sense are represented by Guanylate Cyclase Activating Protein 1 (GCAP1) and Calcium- and Integrin-Binding Protein 2 (CIB2), involved in sight and hearing respectively. Missense point mutations in GCAP1 and CIB2 were found to be associated with genetic diseases characterized by retinal dystrophies and/or deafness. During my PhD, I focused my attention on the characterization of two point mutations namely p.Glu111Val (E111V) in GCAP1, leading to Cone/Rod dystrophy in an Italian family, and p.Glu64Asp (E64D) in CIB2, linked to Usher syndrome type 1J (USH1J), a rare disease characterized by the copresence of blindness and deafness. In particular, I spent the first part of the PhD investigating the role of CIB2 which is still under debate, finding that it is per se uncapable to work as a Ca2+ sensor under physiological conditions and that the conservative mutation linked to USH1J perturbs an allosteric communication between pseudo-EF1 and EF3, thus blocking the protein in an unfunctional conformation. Then, I characterized E111V GCAP1, finding that it is incapable of regulating its molecular target (Guanylate Cyclase), leading to a constitutive active enzyme and thus a progressively high concentrations of Ca2+ and cGMP in cells, which may explain the pathological phenotype. Looking for a potential therapeutic approach for Cone-Rod dystrophies, we found that the well-established Ca2+-relay model, explaining the gradual activation of Guanylate Cyclase by multiple GCAP molecules following gradual changes in intracellular Ca2+ concentrations, seems to be species-specific, since it apparently does not work in the same way in humans as in mouse and bovine photoreceptors. Finally, we identified a general method for the characterization of the interaction between a ubiquitous Ca2+ sensor protein (calmodulin) and inorganic CaF2 nanoparticles, suggesting their suitability as devices for nanomedicine applications

Normal GCAPs partly compensate for altered cGMP signaling in retinal dystrophies associated with mutations in GUCA1A

Missense mutations in the GUCA1A gene encoding guanylate cyclase-activating protein 1 (GCAP1) are associated with autosomal dominant cone/cone-rod (CORD) dystrophies. The nature of the inheritance pattern implies that a pool of normal GCAP proteins is present in photoreceptors together with the mutated variant. To assess whether human GCAP1 and GCAP2 may similarly regulate the activity of the retinal membrane guanylate cyclase GC-1 (GC-E) in the presence of the recently discovered E111V-GCAP1 CORD-variant, we combined biochemical and in silico assays. Surprisingly, human GCAP2 does not activate GC1 over the physiological range of Ca2+ whereas wild-type GCAP1 significantly attenuates the dysregulation of GC1 induced by E111V-GCAP1. Simulation of the phototransduction cascade in a well-characterized murine system, where GCAP2 is able to activate the GC1, suggests that both GCAPs can act in a synergic manner to mitigate the effects of the CORD-mutation. We propose the existence of a species-dependent compensatory mechanism. In murine photoreceptors, slight increases of wild-type GCAPs levels may significantly attenuate the increase in intracellular Ca2+ and cGMP induced by E111V-GCAP1 in heterozygous conditions. In humans, however, the excess of wild-type GCAP1 may only partly attenuate the mutant-induced dysregulation of cGMP signaling due to the lack of GC1-regulation by GCAP2

Calcium- and Integrin-Binding Protein 2 (CIB2) in physiology and disease: bright and dark sides

Calcium- and integrin-binding protein 2 (CIB2) is a small EF-hand protein capable of binding Mg2+ and Ca2+ ions. While its biological function remains largely unclear, an increasing number of studies have shown that CIB2 is an essential component of the mechano-transduction machinery that operates in cochlear hair cells. Mutations in the gene encoding CIB2 have been associated with non-syndromic deafness. In addition to playing an important role in the physiology of hearing, CIB2 has been implicated in a multitude of very different processes, ranging from integrin signaling in platelets and skeletal muscle to autophagy, suggesting extensive functional plasticity. In this review, we summarize the current understanding of biochemical and biophysical properties of CIB2 and the biological roles that have been proposed for the protein in a variety of processes. We also highlight the many molecular aspects that remain unclarified and deserve further investigation

Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors

Among its many molecular targets, the ubiquitous calcium sensor protein calmodulin (CaM) recognizes and regulates the activity of ryanodine receptors type 1 (RyR1) and 2 (RyR2), mainly expressed in skeletal and cardiac muscle, respectively. Such regulation is essential to achieve controlled contraction of muscle cells. To unravel the molecular mechanisms underlying the target recognition process, we conducted a comprehensive biophysical investigation of the interaction between two calmodulin variants associated with congenital arrhythmia, namely N97I and Q135P, and a highly conserved calmodulin-binding region in RyR1 and RyR2. The structural, thermodynamic, and kinetic properties of protein-peptide interactions were assessed together with an in-depth structural and topological investigation based on molecular dynamics simulations. This integrated approach allowed us to identify amino acids that are crucial in mediating allosteric processes, which enable high selectivity in molecular target recognition. Our results suggest that the ability of calmodulin to discriminate between RyR1 an RyR2 targets depends on kinetic discrimination and robust allosteric communication between Ca2+-binding sites (EF1-EF3 and EF3-EF4 pairs), which is perturbed in both N97I and Q135P arrhythmia-associated variants

Alterations in calmodulin-cardiac ryanodine receptor molecular recognition in congenital arrhythmias

Calmodulin (CaM), a ubiquitous and highly conserved Ca2+-sensor protein involved in the regulation of over 300 molecular targets, has been recently associated with severe forms of lethal arrhythmia. Here, we investigated how arrhythmia-associated mutations in CaM localized at the C-terminal lobe alter the molecular recognition with Ryanodine receptor 2 (RyR2), specifically expressed in cardiomyocytes. We performed an extensive structural, thermodynamic, and kinetic characterization of the variants D95V/H in the EF3 Ca2+-binding motif and of the D129V and D131H/E variants in the EF4 motif, and probed their interaction with RyR2. Our results show that the specific structural changes observed for individual CaM variants do not extend to the complex with the RyR2 target. Indeed, some common alterations emerge at the protein-protein interaction level, suggesting the existence of general features shared by the arrhythmia-associated variants. All mutants showed a faster rate of dissociation from the target peptide than wild-type CaM. Integration of spectroscopic data with exhaustive molecular dynamics simulations suggests that, in the presence of Ca2+, functional recognition involves allosteric interactions initiated by the N-terminal lobe of CaM, which shows a lower affinity for Ca2+ compared to the C-terminal lobe in the isolated protein

Recombinant protein delivery enables modulation of the phototransduction cascade in mouse retina

Inherited retinal dystrophies are often associated with mutations in the genes involved in the phototransduction cascade in photoreceptors, a paradigmatic signaling pathway mediated by G protein-coupled receptors. Photoreceptor viability is strictly dependent on the levels of the second messengers cGMP and Ca2+. Here we explored the possibility of modulating the phototransduction cascade in mouse rods using direct or liposome-mediated administration of a recombinant protein crucial for regulating the interplay of the second messengers in photoreceptor outer segments. The effects of administration of the free and liposome-encapsulated human guanylate cyclase-activating protein 1 (GCAP1) were compared in biological systems of increasing complexity (in cyto, ex vivo, and in vivo). The analysis of protein biodistribution and the direct measurement of functional alteration in rod photoresponses show that the exogenous GCAP1 protein is fully incorporated into the mouse retina and photoreceptor outer segments. Furthermore, only in the presence of a point mutation associated with cone-rod dystrophy in humans p.(E111V), protein delivery induces a disease-like electrophysiological phenotype, consistent with constitutive activation of the retinal guanylate cyclase. Our study demonstrates that both direct and liposome-mediated protein delivery are powerful complementary tools for targeting signaling cascades in neuronal cells, which could be particularly important for the treatment of autosomal dominant genetic diseases

Oligomeric state, hydrodynamic properties and target recognition of human Calcium and Integrin Binding protein 2 (CIB2)

Calcium- and Integrin-Binding protein 2 (CIB2) is a small and ubiquitously expressed protein with largely unknown biological function but ascertained role in hearing physiology and disease. Recent studies found that CIB2 binds Ca2+ with moderate affinity and dimerizes under conditions mimicking the physiological ones. Here we provided new lines of evidence on CIB2 oligomeric state and the mechanism of interaction with the alpha 7B integrin target. Based on a combination of native mass spectrometry, chemical cross-linking/mass spectrometry, analytical gel filtration, dynamic light scattering and molecular dynamics simulations we conclude that CIB2 is monomeric under all tested conditions and presents uncommon hydrodynamic properties, most likely due to the high content of hydrophobic solvent accessible surface. Surface plasmon resonance shows that the interaction with alpha 7B occurs with relatively low affinity and is limited to the cytosolic region proximal to the membrane, being kinetically favored in the presence of physiological Mg2+ and in the absence of Ca2+. Although CIB2 binds to an alpha 7B peptide in a 1:1 stoichiometry, the formation of the complex might induce binding of another CIB2 molecule

Impaired Ca2+ sensitivity of a novel GCAP1 variant causes cone dystrophy and leads to abnormal synaptic transmission between photoreceptors and bipolar cells

Guanylate cyclase-activating protein 1 (GCAP1) is involved in the shutdown of the phototransduction cascade by regulating the enzymatic activity of retinal guanylate cyclase via a Ca2+/cGMP negative feedback. While the phototransduction-associated role of GCAP1 in the photoreceptor outer segment is widely established, its implication in synaptic transmission to downstream neurons remains to be clarified. Here, we present clinical and biochemical data on a novel isolate GCAP1 variant leading to a double amino acid substitution (p.N104K and p.G105R) and associated with cone dystrophy (COD) with an unusual phenotype. Severe alterations of the electroretinogram were observed under both scotopic and photopic conditions, with a negative pattern and abnormally attenuated b-wave component. The biochemical and biophysical analysis of the heterologously expressed N104K-G105R variant corroborated by molecular dynamics simulations highlighted a severely compromised Ca2+-sensitivity, accompanied by minor structural and stability alterations. Such differences reflected on the dysregulation of both guanylate cyclase isoforms (RetGC1 and RetGC2), resulting in the constitutive activation of both enzymes at physiological levels of Ca2+. As observed with other GCAP1-associated COD, perturbation of the homeostasis of Ca2+ and cGMP may lead to the toxic accumulation of second messengers, ultimately triggering cell death. However, the abnormal electroretinogram recorded in this patient also suggested that the dysregulation of the GCAP1-cyclase complex further propagates to the synaptic terminal, thereby altering the ON-pathway related to the b-wave generation. In conclusion, the pathological phenotype may rise from a combination of second messengers' accumulation and dysfunctional synaptic communication with bipolar cells, whose molecular mechanisms remain to be clarified

Luminescent and paramagnetic properties of nanoparticles shed light on their interactions with proteins

Nanoparticles have been recognized as promising tools for targeted drug-delivery and protein therapeutics. However, the mechanisms of protein-nanoparticle interaction and the dynamics underlying the binding process are poorly understood. Here, we present a general methodology for the characterization of protein-nanoparticle interaction on a molecular level. To this end we combined biophysical techniques including nuclear magnetic resonance (NMR), circular dichroism (CD), resonance energy transfer (RET) and surface plasmon resonance (SPR). Particularly, we analyzed molecular mechanisms and dynamics of the interaction of CaF2nanoparticles with the prototypical calcium sensor calmodulin (CaM). We observed the transient formation of an intermediate encounter complex involving the structural region linking the two domains. Specific interaction of CaM with CaF2NPs is driven by the N-terminal EF-hands, which seem to recognize Ca2+on the surface of the nanoparticle. We conclude that CaF2NP-CaM interaction is fully compatible with potential applications in nanomedicine. Overall, the methods presented in this work can be extended to other systems and may be useful to quantitatively characterize structural and dynamic features of protein-NP interactions with important implications for nanomedicine and nano-biotechnology

Ionic displacement of Ca2+ by Pb2+ in calmodulin is affected by arrhythmia-associated mutations

Lead is a highly toxic metal that severely perturbs physiological processes even at sub-micromolar levels, often by disrupting the Ca2+ signaling pathways. Recently, Pb2+-associated cardiac toxicity has emerged, with potential involvement of both the ubiquitous Ca2+ sensor protein calmodulin (CaM) and ryanodine receptors. In this work, we explored the hypothesis that Pb2+ contributes to the pathological phenotype of CaM variants associated with congenital arrhythmias. We performed a thorough spectroscopic and computational characterization of CaM conformational switches in the co-presence of Pb2+ and four missense mutations associated with congenital arrhythmias, namely N53I, N97S, E104A and F141L, and analyzed their effects on the recognition of a target peptide of RyR2. When bound to any of the CaM variants, Pb2+ is difficult to displace even under equimolar Ca2+ concentrations, thus locking all CaM variants in a specific conformation, which exhibits characteristics of coiled-coil assemblies. All arrhythmia-associated variants appear to be more susceptible to Pb2+ than WT CaM, as the conformational transition towards the coiled-coil conformation occurs at lower Pb2+, regardless of the presence of Ca2+, with altered cooperativity. The presence of arrhythmia-associated mutations specifically alters the cation coordination of CaM variants, in some cases involving allosteric communication between the EF-hands in the two domains. Finally, while wild type CaM increases the affinity for the RyR2 target in the presence of Pb2+, no specific pattern could be detected for all other variants, ruling out a synergistic effect of Pb2+ and mutations in the recognition process