RESUMEN
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 Ca2+/Mg2+ 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.(E111V) substitution, associated with a severe form of IRD, on GCAP1 homo- and hetero-dimerization, and demonstrate that WT-GCAP1 directly interacts with RD3. Although inducing constitutive activation in GC1, the E111V substitution only slightly affects the dimerization of GCAP1. Both wild type and E111V-GCAP1 are predominantly monomeric in the absence of the GC1 target, however E111V-GCAP1 shows a stronger tendency to be monomeric in the Ca2+-bound form, corresponding to GC1 inhibiting state. Reconstitution experiments performed in the co-presence of wild type GCAP1, E111V-GCAP1 and RD3 restored nearly physiological regulation of the GC1 enzymatic activity in terms of cGMP synthesis and Ca2+-sensitivity, suggesting new scenarios for biologics-mediated treatment of GCAP1-associated IRDs.
RESUMEN
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). Audiological examination included pure-tone and speech audiometry in adult patients and auditory brainstem response testing in a child. Genetic testing was performed by disease gene panel analysis based on genome sequencing. The molecular consequences of the substitutions of residues 390 and 391 on TUBB4B and its interaction with α-tubulin were predicted in silico on its three-dimensional structure obtained by homology modelling. Two independent patients had amino acid exchanges at position 391 (p.(Arg391His) or p.(Arg391Cys)) of the TUBB4B protein. Both had a distinct IRD phenotype with peripheral round yellowish lesions with pigmented spots and mild or moderate SNHL, respectively. Yet the phenotype was milder with a sectorial pattern of bone spicules in one patient, likely due to a genetically confirmed mosaicism for p.(Arg391His). Three patients were heterozygous for an amino acid exchange at position 390 (p.(Arg390Gln) or p.(Arg390Trp)) and presented with another distinct retinal phenotype with well demarcated pericentral retinitis pigmentosa. All showed SNHL ranging from mild to severe. One additional patient showed a variant distinct from codon 390 or 391 (p.(Tyr310His)), and presented with congenital profound hearing loss and reduced responses in ERG. Variants at codon positions 390 and 391 were predicted to decrease the structural stability of TUBB4B and its complex with α-tubulin, as well as the complex affinity. In conclusion, the twofold larger reduction in heterodimer affinity exhibited by Arg391 substitutions suggested an association with the more severe retinal phenotype, compared to the substitution at Arg390.
Asunto(s)
Codón , Pérdida Auditiva Sensorineural , Fenotipo , Tubulina (Proteína) , Humanos , Femenino , Tubulina (Proteína)/genética , Tubulina (Proteína)/química , Masculino , Adulto , Pérdida Auditiva Sensorineural/genética , Codón/genética , Persona de Mediana Edad , Mutación Missense , Niño , Linaje , Adolescente , Sustitución de Aminoácidos , Adulto Joven , Retinitis Pigmentosa/genéticaRESUMEN
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. This revealed the variable quaternary conformations of NR2F1 variants and highlighted the disrupted interplay with dimeric partners and the newly identified co-factor, CRABP2. The disclosed consequence of the pathogenic mutations on the conformation, supramolecular interplay, and alterations in the cell cycle, viability, and sub-cellular localization of the different variants reflect the heterogeneous disease spectrum of BBSOAS and set up novel foundation for unveiling the complexity of neurodevelopmental diseases.
Asunto(s)
Discapacidad Intelectual , Humanos , Mutación , Discapacidad Intelectual/genética , Código GenéticoRESUMEN
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 (Tm) from multi-transition thermal denaturation profiles of 16 variants of calmodulin (CaM) associated with congenital arrhythmia. Differences in Tm estimated by empirical fitting correlate (for apo CaM variants) with those obtained by thermodynamic models. Most CaM variants were more stable than the wild type (WT) in the absence of Ca2+, but less stable in the presence of Ca2+, and displayed either WT-like or higher unfolding percentages in their apo-form, as evaluated by circular dichroism spectroscopy.
RESUMEN
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.
Asunto(s)
Liposomas , Retina , Ratones , Humanos , Animales , Distribución Tisular , Retina/metabolismo , Fototransducción , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Activadoras de la Guanilato-Ciclasa/genética , Proteínas Activadoras de la Guanilato-Ciclasa/metabolismo , Calcio/metabolismoRESUMEN
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 wild type (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 WT 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.
Asunto(s)
Calmodulina , Plomo , Humanos , Calmodulina/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Arritmias Cardíacas/genética , MutaciónRESUMEN
Biallelic pathogenic variants in TULP1 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 TULP1. All patients underwent thorough ophthalmological examinations. Minigene assays and structural analyses were performed to assess the consequences of splice variants and missense variants. Three patients were diagnosed with Leber congenital amaurosis, nine with early onset retinitis pigmentosa, two with retinitis pigmentosa with an onset in adulthood, one with cone dystrophy, and two with cone-rod dystrophy. Seventeen different alleles were identified, namely eight missense variants, six nonsense variants, one in-frame deletion variant, and two splice site variants. For the latter two, minigene assays revealed aberrant transcripts containing frameshifts and premature termination codons. Structural analysis and molecular modeling suggested different degrees of structural destabilization for the missense variants. In conclusion, we report the largest cohort of patients with TULP1-associated IRD published to date. Most of the patients exhibited rod-driven disease, yet a fraction of the patients exhibited cone-driven disease. Our data support the hypothesis that TULP1 variants do not fold properly and thus trigger unfolded protein response, resulting in photoreceptor death.
Asunto(s)
Distrofias Retinianas , Retinitis Pigmentosa , Humanos , Distrofias Retinianas/genética , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/diagnóstico , Fenotipo , Células Fotorreceptoras Retinianas Conos/metabolismo , Mutación Missense , Mutación , Linaje , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismoRESUMEN
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.301G>C mutation in GUCA1C, resulting in the substitution of a highly conserved aspartate residue by a histidine (p.(D101H)). We found that myristoylated GCAP3 can activate GC1 with a similar Ca2+-dependent profile, but significantly less efficiently than GCAP1. The non-myristoylated form did not induce appreciable regulation of GC1, nor did the p.D101H variant. GCAP3 forms dimers under physiological conditions, but at odds with its paralogs, it tends to form temperature-dependent aggregates driven by hydrophobic interactions. The peculiar properties of GCAP3 were confirmed by 2 ms molecular dynamics simulations, which for the p.D101H variant highlighted a very high structural flexibility and a clear tendency to lose the binding of a Ca2+ ion to EF3. Overall, our data show that GCAP3 has unusual biochemical properties, which make the protein significantly different from GCAP1 and GCAP2. Moreover, the newly identified point mutation resulting in a substantially unfunctional protein could trigger retinitis pigmentosa through a currently unknown mechanism.
Asunto(s)
Proteínas Activadoras de la Guanilato-Ciclasa/metabolismo , Retinitis Pigmentosa , Animales , Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Bovinos , Guanilato Ciclasa/genética , Guanilato Ciclasa/metabolismo , Proteínas Activadoras de la Guanilato-Ciclasa/química , Humanos , Células Fotorreceptoras Retinianas Conos/metabolismo , Retinitis Pigmentosa/genéticaRESUMEN
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.
Asunto(s)
Arritmias Cardíacas , Calmodulina , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Arritmias Cardíacas/congénito , Arritmias Cardíacas/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Humanos , Unión Proteica , Conformación Proteica , Dominios ProteicosRESUMEN
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. The latter is often neglected due to the lack of RNA analyses in genetic diagnostic testing. In this study we considered both pathomechanisms and performed a comprehensive analysis of nine exonic nucleotide changes in OPA1, which is the major gene underlying autosomal dominant optic atrophy (DOA) and is characterized by pronounced allelic heterogeneity. We focused on the GTPase-encoding domain of OPA1, which harbors most of the missense variants associated with DOA. Given that the consensus splice sites extend into the exons, we chose a split codon, namely codon 438, for our analyses. Variants at this codon are the second most common cause of disease in our large cohort of DOA patients harboring disease-causing variants in OPA1. In silico splice predictions, heterologous splice assays, analysis of patient's RNA when available, and protein modeling revealed different molecular outcomes for variants at codon 438. The wildtype aspartate residue at amino acid position 438 is directly involved in the dimerization of OPA1 monomers. We found that six amino acid substitutions at codon 438 (i.e. all substitutions of the first and second nucleotide of the codon) destabilized dimerization while only substitutions of the first nucleotide of the codon caused exon skipping. Our study highlights the value of combining RNA analysis and protein modeling approaches to accurately assign patients to future precision therapies.
Asunto(s)
Atrofia Óptica Autosómica Dominante , Codón/genética , Análisis Mutacional de ADN , GTP Fosfohidrolasas/genética , Humanos , Mutación , Nucleótidos , Atrofia Óptica Autosómica Dominante/genética , Atrofia Óptica Autosómica Dominante/patología , LinajeRESUMEN
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.
RESUMEN
Guanylate cyclase-activating protein 1 (GCAP1), encoded by the GUCA1A 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 Ca2+, thereby ensuring a timely regulation of the phototransduction cascade. We found a novel variant of GUCA1A in a patient affected by autosomal dominant cone dystrophy (adCOD), leading to the Asn104His (N104H) amino acid substitution at the protein level. While biochemical analysis of the recombinant protein showed impaired Ca2+ sensitivity of the variant, structural properties investigated by circular dichroism and limited proteolysis excluded major structural rearrangements induced by the mutation. Analytical gel filtration profiles and dynamic light scattering were compatible with a dimeric protein both in the presence of Mg2+ alone and Mg2+ and Ca2+. Enzymatic assays showed that N104H-GCAP1 strongly interacts with the GC, with an affinity that doubles that of the WT. The doubled IC50 value of the novel variant (520 nM for N104H vs. 260 nM for the WT) is compatible with a constitutive activity of GC at physiological levels of Ca2+. The structural region at the interface with the GC may acquire enhanced flexibility under high Ca2+ conditions, as suggested by 2 µs molecular dynamics simulations. The altered interaction with GC would cause hyper-activity of the enzyme at both low and high Ca2+ levels, which would ultimately lead to toxic accumulation of cGMP and Ca2+ in the photoreceptor outer segment, thus triggering cell death.
Asunto(s)
Distrofia del Cono/patología , GMP Cíclico/metabolismo , Proteínas Activadoras de la Guanilato-Ciclasa/genética , Guanilato Ciclasa/metabolismo , Mutación , Retina/enzimología , Células Fotorreceptoras Retinianas Bastones/metabolismo , Adolescente , Calcio/metabolismo , Niño , Distrofia del Cono/genética , Distrofia del Cono/metabolismo , Femenino , Humanos , Fototransducción , Masculino , Persona de Mediana Edad , Linaje , Transducción de SeñalRESUMEN
In murine and bovine photoreceptors, guanylate cyclase-activating protein 2 (GCAP2) activates retinal guanylate cyclases (GCs) at low Ca2+ levels, thus contributing to the Ca2+/cGMP negative feedback on the cyclase together with its paralog guanylate cyclase-activating protein 1, which has the same function but different Ca2+ sensitivity. In humans, a GCAP2 missense mutation (G157R) has been associated with inherited retinal degeneration (IRD) via an unknown molecular mechanism. Here, we characterized the biochemical properties of human GCAP2 and the G157R variant, focusing on its dimerization and the Ca2+/Mg2+-binding processes in the presence or absence of N-terminal myristoylation. We found that human GCAP2 and its bovine/murine orthologs significantly differ in terms of oligomeric properties, cation binding, and GC regulation. Myristoylated GCAP2 endothermically binds up to 3 Mg2+ with high affinity and forms a compact dimer that may reversibly dissociate in the presence of Ca2+. Conversely, nonmyristoylated GCAP2 does not bind Mg2+ over the physiological range and remains as a monomer in the absence of Ca2+. Both myristoylated and nonmyristoylated GCAP2 bind Ca2+ with high affinity. At odds with guanylate cyclase-activating protein 1 and independently of myristoylation, human GCAP2 does not significantly activate retinal GC1 in a Ca2+-dependent fashion. The IRD-associated G157R variant is characterized by a partly misfolded, molten globule-like conformation with reduced affinity for cations and prone to form aggregates, likely mediated by hydrophobic interactions. Our findings suggest that GCAP2 might be mostly implicated in processes other than phototransduction in human photoreceptors and suggest a possible molecular mechanism for G157R-associated IRD.
Asunto(s)
Calcio/metabolismo , Proteínas Activadoras de la Guanilato-Ciclasa/genética , Proteínas Activadoras de la Guanilato-Ciclasa/metabolismo , Magnesio/metabolismo , Mutación , Distrofias Retinianas/genética , Proteínas Activadoras de la Guanilato-Ciclasa/química , Humanos , Conformación Proteica , Multimerización de ProteínaRESUMEN
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.
Asunto(s)
Calcio/metabolismo , Distrofia del Cono/genética , Distrofia del Cono/fisiopatología , Proteínas Activadoras de la Guanilato-Ciclasa/genética , Mutación/genética , Células Bipolares de la Retina/patología , Células Fotorreceptoras Retinianas Bastones/patología , Transmisión Sináptica , Atrofia , Cationes , Distrofia del Cono/diagnóstico por imagen , Progresión de la Enfermedad , Electrorretinografía , Femenino , Fondo de Ojo , Guanilato Ciclasa/metabolismo , Proteínas Activadoras de la Guanilato-Ciclasa/química , Heterocigoto , Humanos , Hidrodinámica , Interacciones Hidrofóbicas e Hidrofílicas , Persona de Mediana Edad , Simulación de Dinámica Molecular , Fenotipo , Agregado de Proteínas , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Células Bipolares de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Células Fotorreceptoras Retinianas Bastones/metabolismo , Tomografía de Coherencia ÓpticaRESUMEN
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. Subretinal deposits, retinal pigmentation or choroidal neovascularization as seen in late-onset retinal degeneration (LORD) were not observed. Whole genome sequencing revealed a novel missense variant in the C1QTNF5 gene (p.(Q180E)) which was found in heterozygous state in all affected subjects. Haplotype analysis showed that this variant found in both families is identical by descent. Three-dimensional modeling of the possible supramolecular assemblies of C1QTNF5 revealed that the p.(Q180E) variant led to the destabilization of protein tertiary and quaternary structures, affecting both the stability of the single protomer and the entire globular head, thus exerting detrimental effects on the formation of C1QTNF5 trimeric globular domains and their interaction. In conclusion, we propose that the p.(Q180E) variant causes a specific phenotype, adGALCD, that differs in multiple clinical aspects from LORD. Disruption of optimal cell-adhesion mechanisms is expected when analyzing the effects of the point mutation at the protein level.
Asunto(s)
Coroides/patología , Colágeno/genética , Genes Dominantes , Atrofia Girata/genética , Adolescente , Adulto , Edad de Inicio , Colágeno/química , Progresión de la Enfermedad , Femenino , Estudios de Seguimiento , Fondo de Ojo , Humanos , Masculino , Persona de Mediana Edad , Modelos Moleculares , Mutación Missense , Linaje , Fenotipo , Dominios Proteicos , Electricidad Estática , Tomografía de Coherencia Óptica , Campos VisualesRESUMEN
The prototypical Ca2+-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 Ca2+ of its physiologically relevant form (myristoylated recoverin) is almost two orders of magnitude too low to support this mechanism in vivo. 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. We show that the sole presence of the target or the disc membrane alone are not sufficient to achieve a physiological response to changes in intracellular [Ca2+]. Instead, the simultaneous presence of GRK1 and membrane allows the T to R transition to occur in a physiological range of [Ca2+] with high cooperativity via a conformational selection mechanism that drives the structural transitions of Rec in the presence of multiple ligands. Our conclusions may apply to other sensory transduction systems involving protein complexes and biological membranes.
Asunto(s)
Calcio/metabolismo , Recoverina/metabolismo , Animales , Apoproteínas/química , Apoproteínas/metabolismo , Dicroismo Circular , Ácido Egtácico/análogos & derivados , Ácido Egtácico/química , Transferencia Resonante de Energía de Fluorescencia , Quinasa 1 del Receptor Acoplado a Proteína-G/química , Quinasa 1 del Receptor Acoplado a Proteína-G/metabolismo , Iones/química , Péptidos/química , Péptidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Recoverina/química , Recoverina/genéticaRESUMEN
The guanylyl cyclase-activating protein 1, GCAP1, activates or inhibits retinal guanylyl cyclase (retGC) depending on cellular Ca2+ concentrations. Several point mutations of GCAP1 have been associated with impaired calcium sensitivity that eventually triggers progressive retinal degeneration. In this work, we demonstrate that the recombinant human protein presents a highly dynamic monomer-dimer equilibrium, whose dissociation constant is influenced by salt concentration and, more importantly, by protein binding to Ca2+ or Mg2+. Based on small-angle X-ray scattering data, protein-protein docking, and molecular dynamics simulations we propose two novel three-dimensional models of Ca2+-bound GCAP1 dimer. The different propensity of human GCAP1 to dimerize suggests structural differences induced by cation binding potentially involved in the regulation of retGC activity.
Asunto(s)
Calcio/química , Proteínas Activadoras de la Guanilato-Ciclasa/química , Magnesio/química , Simulación de Dinámica Molecular , Multimerización de Proteína , Calcio/metabolismo , Proteínas Activadoras de la Guanilato-Ciclasa/metabolismo , Humanos , Magnesio/metabolismoRESUMEN
Lipedema is an often underdiagnosed chronic disorder that affects subcutaneous adipose tissue almost exclusively in women, which leads to disproportionate fat accumulation in the lower and upper body extremities. Common comorbidities include anxiety, depression, and pain. The correlation between mood disorder and subcutaneous fat deposition suggests the involvement of steroids metabolism and neurohormones signaling, however no clear association has been established so far. In this study, we report on a family with three patients affected by sex-limited autosomal dominant nonsyndromic lipedema. They had been screened by whole exome sequencing (WES) which led to the discovery of a missense variant p.(Leu213Gln) in AKR1C1, the gene encoding for an aldo-keto reductase catalyzing the reduction of progesterone to its inactive form, 20-α-hydroxyprogesterone. Comparative molecular dynamics simulations of the wild-type vs. variant enzyme, corroborated by a thorough structural and functional bioinformatic analysis, suggest a partial loss-of-function of the variant. This would result in a slower and less efficient reduction of progesterone to hydroxyprogesterone and an increased subcutaneous fat deposition in variant carriers. Overall, our results suggest that AKR1C1 is the first candidate gene associated with nonsyndromic lipedema.
Asunto(s)
20-Hidroxiesteroide Deshidrogenasas/genética , Secuenciación del Exoma/métodos , Lipedema/genética , Mutación Missense , 20-Hidroxiesteroide Deshidrogenasas/química , 20-Hidroxiesteroide Deshidrogenasas/metabolismo , 20-alfa-Dihidroprogesterona/metabolismo , Adulto , Anciano , Femenino , Humanos , Lipedema/metabolismo , Mutación con Pérdida de Función , Persona de Mediana Edad , Modelos Moleculares , Simulación de Dinámica Molecular , Linaje , Progesterona/metabolismo , Conformación ProteicaRESUMEN
Guanylate cyclase activating protein 1 (GCAP1) is a neuronal calcium sensor (NCS) involved in the early biochemical steps underlying the phototransduction cascade. By switching from a Ca2+-bound form in the dark to a Mg2+-bound state following light activation of the cascade, GCAP1 triggers the activation of the retinal guanylate cyclase (GC), thus replenishing the levels of 3',5'-cyclic monophosphate (cGMP) necessary to re-open CNG channels. Here, we investigated the structural and functional effects of three missense mutations in GCAP1 associated with cone-rod dystrophy, which severely perturb the homeostasis of cGMP and Ca2+. Substitutions affect residues directly involved in Ca2+ coordination in either EF3 (D100G) or EF4 (E155A and E155G) Ca2+ binding motifs. We found that all GCAP1 variants form relatively stable dimers showing decreased apparent affinity for Ca2+ and blocking the enzyme in a constitutively active state at physiological levels of Ca2+. Interestingly, by corroborating spectroscopic experiments with molecular dynamics simulations we show that beside local structural effects, mutation of the bidentate glutamate in an EF-hand calcium binding motif can profoundly perturb the flexibility of the adjacent EF-hand as well, ultimately destabilizing the whole domain. Therefore, while Ca2+-binding to GCAP1 per se occurs sequentially, allosteric effects may connect EF hand motifs, which appear to be essential for the integrity of the structural switch mechanism in GCAP1, and perhaps in other NCS proteins.