Assessing the predicted impact of single amino acid substitutions in calmodulin for CAGI6 challenges.

Recent thermodynamic and functional studies have been conducted to evaluate the impact of amino acid substitutions on Calmodulin (CaM). The Critical Assessment of Genome Interpretation (CAGI) data provider at University of Verona (Italy) measured the melting temperature (T) and the percentage of unfolding (%unfold) of a set of CaM variants (CaM challenge dataset). Thermodynamic measurements for the equilibrium unfolding of CaM were obtained by monitoring far-UV Circular Dichroism as a function of temperature.

Structural dynamics of calcium and integrin-binding protein 2 (CIB2) reveal uncommon flexibility and heterogeneous calcium and magnesium loading.

Calcium- and Integrin-Binding protein 2 (CIB2) is a widely expressed protein with an uncertain biological role. Two of its four EF-hand motifs bind Mg(II) and/or Ca(II), thus triggering conformational changes. Although previous studies suggested that CIB2 preferentially binds Mg(II) over Ca(II) under physiological conditions, an atomic level characterization of CIB2 in the presence of both cations was lacking.

Supramolecular complexes of GCAP1: implications for inherited retinal dystrophies.

Guanylate Cyclase Activating Protein 1 (GCAP1) is a calcium sensor that regulates the enzymatic activity of retinal Guanylate Cyclase 1 (GC1) in photoreceptors in a Ca/Mg dependent manner. While point mutations in GCAP1 have been associated with inherited retinal dystrophies (IRDs), their impact on protein dimerization or on the possible interaction with the potent GC1 inhibitor RD3 (retinal degeneration protein 3) has never been investigated. Here, we integrate exhaustive in silico investigations with biochemical assays to evaluate the effects of the p.

Comprehensive analysis of two hotspot codons in the TUBB4B gene and associated phenotypes.

Our purpose was to elucidate the genotype and ophthalmological and audiological phenotype in TUBB4B-associated inherited retinal dystrophy (IRD) and sensorineural hearing loss (SNHL), and to model the effects of all possible amino acid substitutions at the hotspot codons Arg390 and Arg391. Six patients from five families with heterozygous missense variants in TUBB4B were included in this observational study. Ophthalmological testing included best-corrected visual acuity, fundus examination, optical coherence tomography, fundus autofluorescence imaging, and full-field electroretinography (ERG).

Disrupted protein interaction dynamics in a genetic neurodevelopmental disorder revealed by structural bioinformatics and genetic code expansion.

Deciphering the structural effects of gene variants is essential for understanding the pathophysiological mechanisms of genetic diseases. Using a neurodevelopmental disorder called Bosch-Boonstra-Schaaf Optic Atrophy Syndrome (BBSOAS) as a genetic disease model, we applied structural bioinformatics and Genetic Code Expansion (GCE) strategies to assess the pathogenic impact of human NR2F1 variants and their binding with known and novel partners. While the computational analyses of the NR2F1 structure delineated the molecular basis of the impact of several variants on the isolated and complexed structures, the GCE enabled covalent and site-specific capture of transient supramolecular interactions in living cells.

Impact of calmodulin missense variants associated with congenital arrhythmia on the thermal stability and the degree of unfolding.

Thermal denaturation profiles of proteins that bind several ligands may deviate from the single transition, making their thermodynamic description challenging. We report an empirical method that estimates melting temperatures (T) from multi-transition thermal denaturation profiles of 16 variants of calmodulin (CaM) associated with congenital arrhythmia. Differences in T estimated by empirical fitting correlate (for apo CaM variants) with those obtained by thermodynamic models.

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 Ca. 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.

Ionic displacement of Ca by Pb 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 Ca signaling pathways. Recently, Pb-associated cardiac toxicity has emerged, with potential involvement of both the ubiquitous Ca sensor protein calmodulin (CaM) and ryanodine receptors. In this work, we explored the hypothesis that Pb contributes to the pathological phenotype of CaM variants associated with congenital arrhythmias.

Biallelic Variants in TULP1 Are Associated with Heterogeneous Phenotypes of Retinal Dystrophy.

Biallelic pathogenic variants in are mostly associated with severe rod-driven inherited retinal degeneration. In this study, we analyzed clinical heterogeneity in 17 patients and characterized the underlying biallelic variants in . All patients underwent thorough ophthalmological examinations.

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.

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 Mg and Ca 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.

Molecular Properties of Human Guanylate Cyclase-Activating Protein 3 (GCAP3) and Its Possible Association with Retinitis Pigmentosa.

The cone-specific guanylate cyclase-activating protein 3 (GCAP3), encoded by the GUCA1C gene, has been shown to regulate the enzymatic activity of membrane-bound guanylate cyclases (GCs) in bovine and teleost fish photoreceptors, to an extent comparable to that of the paralog protein GCAP1. To date, the molecular mechanisms underlying GCAP3 function remain largely unexplored. In this work, we report a thorough characterization of the biochemical and biophysical properties of human GCAP3, moreover, we identified an isolated case of retinitis pigmentosa, in which a patient carried the c.

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

Calmodulin (CaM), a ubiquitous and highly conserved Ca-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 Ca-binding motif and of the D129V and D131H/E variants in the EF4 motif, and probed their interaction with RyR2.

A Novel Variant Associated with Cone Dystrophy Alters cGMP Signaling in Photoreceptors by Strongly Interacting with and Hyperactivating Retinal Guanylate Cyclase.

Guanylate cyclase-activating protein 1 (GCAP1), encoded by the gene, is a neuronal calcium sensor protein involved in shaping the photoresponse kinetics in cones and rods. GCAP1 accelerates or slows the cGMP synthesis operated by retinal guanylate cyclase (GC) based on the light-dependent levels of intracellular Ca, thereby ensuring a timely regulation of the phototransduction cascade. We found a novel variant of in a patient affected by autosomal dominant cone dystrophy (adCOD), leading to the Asn104His (N104H) amino acid substitution at the protein level.

Mutations at a split codon in the GTPase-encoding domain of OPA1 cause dominant optic atrophy through different molecular mechanisms.

Exonic (i.e. coding) variants in genes associated with disease can exert pathogenic effects both at the protein and mRNA level, either by altering the amino acid sequence or by affecting pre-mRNA splicing.

A hybrid stochastic/deterministic model of single photon response and light adaptation in mouse rods.

The phototransduction cascade is paradigmatic for signaling pathways initiated by G protein-coupled receptors and is characterized by a fine regulation of photoreceptor sensitivity and electrical response to a broad range of light stimuli. Here, we present a biochemically comprehensive model of phototransduction in mouse rods based on a hybrid stochastic and deterministic mathematical framework, and a quantitatively accurate description of the rod impedance in the dark. The latter, combined with novel patch clamp recordings from rod outer segments, enables the interconversion of dim flash responses between photovoltage and photocurrent and thus direct comparison with the simulations.

Impaired Ca 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 Ca/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.

Autosomal Dominant Gyrate Atrophy-Like Choroidal Dystrophy Revisited: 45 Years Follow-Up and Association with a Novel Missense Variant.

We present a long-term follow-up in autosomal dominant gyrate atrophy-like choroidal dystrophy (adGALCD) and propose a possible genotype/phenotype correlation. Ophthalmic examination of six patients from two families revealed confluent areas of choroidal atrophy resembling gyrate atrophy, starting in the second decade of life. Progression continued centrally, reaching the fovea at about 60 years of age.

Simultaneous detection of reciprocal interactions between calmodulin, Ca2+ and molecular targets: a focus on the calmodulin-RyR2 complex.

In a recent issue of Biochemical Journal, Brohus et al. (Biochem. J.

Bringing the Ca sensitivity of myristoylated recoverin into the physiological range.

The prototypical Ca-sensor protein recoverin (Rec) is thought to regulate the activity of rhodopsin kinase (GRK1) in photoreceptors by switching from a relaxed (R) disc membrane-bound conformation in the dark to a more compact, cytosol-diffusing tense (T) conformation upon cell illumination. However, the apparent affinity for Ca of its physiologically relevant form (myristoylated recoverin) is almost two orders of magnitude too low to support this mechanism . In this work, we compared the individual and synergistic roles of the myristic moiety, the GRK1 target and the disc membrane in modulating the calcium sensitivity of Rec.