RESUMO
Guanylate cyclase activating protein-5 (GCAP5) in zebrafish photoreceptors promotes the activation of membrane receptor retinal guanylate cyclase (GC-E). Previously, we showed the R22A mutation in GCAP5 (GCAP5R22A) abolishes dimerization of GCAP5 and activates GC-E by more than 3-fold compared to that of wild-type GCAP5 (GCAP5WT). Here, we present ITC, NMR, and functional analysis of GCAP5R22A to understand how R22A causes a decreased dimerization affinity and increased cyclase activation. ITC experiments reveal GCAP5R22A binds a total of 3 Ca2+, including two sites in the nanomolar range followed by a single micromolar site. The two nanomolar sites in GCAP5WT were not detected by ITC, suggesting that R22A may affect the binding of Ca2+ to these sites. The NMR-derived structure of GCAP5R22A is overall similar to that of GCAP5WT (RMSD = 2.3 Å), except for local differences near R22A (Q19, W20, Y21, and K23) and an altered orientation of the C-terminal helix near the N-terminal myristate. GCAP5R22A lacks an intermolecular salt bridge between R22 and D71 that may explain the weakened dimerization. We present a structural model of GCAP5 bound to GC-E in which the R22 side-chain contacts exposed hydrophobic residues in GC-E. Cyclase assays suggest that GC-E binds to GCAP5R22A with â¼25% higher affinity compared to GCAP5WT, consistent with more favorable hydrophobic contact by R22A that may help explain the increased cyclase activation.
Assuntos
Proteínas Ativadoras de Guanilato Ciclase , Guanilato Ciclase , Modelos Moleculares , Animais , Cálcio/metabolismo , Ativação Enzimática/genética , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Guanilato Ciclase/química , Proteínas Ativadoras de Guanilato Ciclase/metabolismo , Proteínas Ativadoras de Guanilato Ciclase/genética , Proteínas Ativadoras de Guanilato Ciclase/química , Mutação , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Multimerização Proteica , Retina/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/metabolismoRESUMO
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.
Assuntos
Lipossomos , Retina , Camundongos , Humanos , Animais , Distribuição Tecidual , Retina/metabolismo , Transdução de Sinal Luminoso , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ativadoras de Guanilato Ciclase/genética , Proteínas Ativadoras de Guanilato Ciclase/metabolismo , Cálcio/metabolismoRESUMO
Vertebrate photoreceptor cells are exquisite light detectors operating under very dim and bright illumination mediated by phototransduction, which is under control of the two secondary messengers cGMP and Ca2+. Feedback mechanisms enable photoreceptor cells to regain their responsiveness after light stimulation and involve neuronal Ca2+-sensor proteins, named GCAPs (guanylate cyclase-activating proteins) and recoverins. This review compares the diversity in Ca2+-related signaling mediated by GCAP and recoverin variants that exhibit differences in Ca2+-sensing, protein conformational changes, myristoyl switch mechanisms, diversity in divalent cation binding and dimer formation. In summary, both subclasses of neuronal Ca2+-sensor proteins contribute to a complex signaling network in rod and cone cells, which is perfectly suited to match the requirements for sensitive cell responses and maintaining this responsiveness in the presence of different background light intensities.
Assuntos
Cálcio , Proteínas Sensoras de Cálcio Neuronal , Proteínas Sensoras de Cálcio Neuronal/metabolismo , Cálcio/metabolismo , Retina/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Proteínas Ativadoras de Guanilato Ciclase/genética , Proteínas Ativadoras de Guanilato Ciclase/química , Recoverina/genética , Recoverina/metabolismoRESUMO
BACKGROUND: Several serious attempts to treat colorectal cancer have been made in recent decades. However, no effective treatment has yet been discovered due to the complexities of its etiology. METHODS: we used Weighted Gene Co-expression Network Analysis (WGCNA) to identify key modules, hub-genes, and mRNA-miRNA regulatory networks associated with CRC. Next, enrichment analysis of modules has been performed using Cluepedia. Next, quantitative real-time PCR (RT-qPCR) was used to validate the expression of selected hub-genes in CRC tissues. RESULTS: Based on the WGCNA results, the brown module had a significant positive correlation (r = 0.98, p-value=9e-07) with CRC. Using the survival and DEGs analyses, 22 genes were identified as hub-genes. Next, three candidate hub-genes were selected for RT-qPCR validation, and 22 pairs of cancerous and non-cancerous tissues were collected from CRC patients referred to the Gastroenterology and Liver Clinic. The RT-qPCR results revealed that the expression of GUCA2B was significantly reduced in CRC tissues, which is consistent with the results of differential expression analysis. Finally, top miRNAs correlated with GUCA2B were identified, and ROC analyses revealed that GUCA2B has a high diagnostic performance for CRC. CONCLUSIONS: The current study discovered key modules and GUCA2B as a hub-gene associated with CRC, providing references to understand the pathogenesis and be considered a novel candidate to CRC target therapy.
Assuntos
Neoplasias Colorretais/genética , Proteínas Ativadoras de Guanilato Ciclase/genética , Apoptose/fisiologia , Biomarcadores Tumorais , Regulação Neoplásica da Expressão Gênica/fisiologia , Redes Reguladoras de Genes , Humanos , Mucosa Intestinal/fisiologia , MicroRNAs/biossíntese , Peptídeos Natriuréticos/metabolismo , TranscriptomaRESUMO
Retinal guanylate cyclases (RetGCs) are regulated by a family of guanylate cyclase-activating proteins (called GCAP1-7). GCAPs form dimers that bind to Ca2+ and confer Ca2+ sensitive activation of RetGC during visual phototransduction. The GCAP5 homologue from zebrafish contains two nonconserved cysteine residues (Cys15 and Cys17) that bind to ferrous ion, which stabilizes GCAP5 dimerization and diminishes its ability to activate RetGC. Here, we present NMR and EPR-DEER structural analysis of a GCAP5 dimer in the Mg2+-bound, Ca2+-free, Fe2+-free activator state. The NMR-derived structure of GCAP5 is similar to the crystal structure of Ca2+-bound GCAP1 (root-mean-square deviation of 2.4 Å), except that the N-terminal helix of GCAP5 is extended by two residues, which allows the sulfhydryl groups of Cys15 and Cys17 to become more solvent exposed in GCAP5 to facilitate Fe2+ binding. Nitroxide spin-label probes were covalently attached to particular cysteine residues engineered in GCAP5: C15, C17, T26C, C28, N56C, C69, C105, N139C, E152C, and S159C. The intermolecular distance of each spin-label probe in dimeric GCAP5 (measured by EPR-DEER) defined restraints for calculating the dimer structure by molecular docking. The GCAP5 dimer possesses intermolecular hydrophobic contacts involving the side chain atoms of H18, Y21, M25, F72, V76, and W93, as well as an intermolecular salt bridge between R22 and D71. The structural model of the GCAP5 dimer was validated by mutations (H18E/Y21E, H18A/Y21A, R22D, R22A, M25E, D71R, F72E, and V76E) at the dimer interface that disrupt dimerization of GCAP5 and affect the activation of RetGC. We propose that GCAP5 dimerization may play a role in the Fe2+-dependent regulation of cyclase activity in zebrafish photoreceptors.
Assuntos
Proteínas Ativadoras de Guanilato Ciclase/química , Proteínas de Peixe-Zebra/química , Sequência de Aminoácidos , Animais , Cisteína/química , Espectroscopia de Ressonância de Spin Eletrônica , Proteínas Ativadoras de Guanilato Ciclase/genética , Proteínas Ativadoras de Guanilato Ciclase/metabolismo , Magnésio/química , Magnésio/metabolismo , Simulação de Acoplamento Molecular , Mutação , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
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.
Assuntos
Distrofia de Cones/patologia , GMP Cíclico/metabolismo , Proteínas Ativadoras de Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Mutação , Retina/enzimologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Adolescente , Cálcio/metabolismo , Criança , Distrofia de Cones/genética , Distrofia de Cones/metabolismo , Feminino , Humanos , Transdução de Sinal Luminoso , Masculino , Pessoa de Meia-Idade , Linhagem , Transdução de SinaisRESUMO
Different forms of photoreceptor degeneration cause blindness. Retinal degeneration-3 protein (RD3) deficiency in photoreceptors leads to recessive congenital blindness. We proposed that aberrant activation of the retinal membrane guanylyl cyclase (RetGC) by its calcium-sensor proteins (guanylyl cyclase-activating protein [GCAP]) causes this retinal degeneration and that RD3 protects photoreceptors by preventing such activation. We here present in vivo evidence that RD3 protects photoreceptors by suppressing activation of both RetGC1 and RetGC2 isozymes. We further suggested that insufficient inhibition of RetGC by RD3 could contribute to some dominant forms of retinal degeneration. The R838S substitution in RetGC1 that causes autosomal-dominant cone-rod dystrophy 6, not only impedes deceleration of RetGC1 activity by Ca2+GCAPs but also elevates this isozyme's resistance to inhibition by RD3. We found that RD3 prolongs the survival of photoreceptors in transgenic mice harboring human R838S RetGC1 (R838S+). Overexpression of GFP-tagged human RD3 did not improve the calcium sensitivity of cGMP production in R838S+ retinas but slowed the progression of retinal blindness and photoreceptor degeneration. Fluorescence of the GFP-tagged RD3 in the retina only partially overlapped with immunofluorescence of RetGC1 or GCAP1, indicating that RD3 separates from the enzyme before the RetGC1:GCAP1 complex is formed in the photoreceptor outer segment. Most importantly, our in vivo results indicate that, in addition to the abnormal Ca2+ sensitivity of R838S RetGC1 in the outer segment, the mutated RetGC1 becomes resistant to inhibition by RD3 in a different cellular compartment(s) and suggest that RD3 overexpression could be utilized to reduce the severity of cone-rod dystrophy 6 pathology.
Assuntos
Guanilato Ciclase/metabolismo , Proteínas Nucleares/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Guanilato Ciclase/genética , Proteínas Ativadoras de Guanilato Ciclase/genética , Proteínas Ativadoras de Guanilato Ciclase/metabolismo , Células HEK293 , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Camundongos Knockout , Mutação , Proteínas Nucleares/genética , Receptores de Superfície Celular/genética , Retinose Pigmentar/genética , Retinose Pigmentar/metabolismoRESUMO
Phosphodiesterase-6 (PDE6) is a key protein in the G-protein cascade converting photon information to bioelectrical signals in vertebrate photoreceptor cells. Here, we demonstrate that PDE6 is regulated by calcium, contrary to the common view that PDE1 is the unique PDE class whose activity is modulated by intracellular Ca2+. To broaden the operating range of photoreceptors, mammalian rod photoresponse recovery is accelerated mainly by two calcium sensor proteins: recoverin, modulating the lifetime of activated rhodopsin, and guanylate cyclase-activating proteins (GCAPs), regulating the cGMP synthesis. We found that decreasing rod intracellular Ca2+ concentration accelerates the flash response recovery and increases the basal PDE6 activity (ßdark) maximally by ~ 30% when recording local electroretinography across the rod outer segment layer from GCAPs-/- recoverin-/- mice. Our modeling shows that a similar elevation in ßdark can fully explain the observed acceleration of flash response recovery in low Ca2+. Additionally, a reduction of the free Ca2+ in GCAPs-/- recoverin-/- rods shifted the inhibition constants of competitive PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) against the thermally activated and light-activated forms of PDE6 to opposite directions, indicating a complex interaction between IBMX, PDE6, and calcium. The discovered regulation of PDE6 is a previously unknown mechanism in the Ca2+-mediated modulation of rod light sensitivity.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Proteínas Ativadoras de Guanilato Ciclase/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/enzimologia , Animais , GMP Cíclico/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Feminino , Proteínas Ativadoras de Guanilato Ciclase/genética , Masculino , Camundongos , Camundongos Knockout , Células Fotorreceptoras Retinianas Bastonetes/citologiaRESUMO
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.
Assuntos
Cálcio/metabolismo , Proteínas Ativadoras de Guanilato Ciclase/genética , Proteínas Ativadoras de Guanilato Ciclase/metabolismo , Magnésio/metabolismo , Mutação , Distrofias Retinianas/genética , Proteínas Ativadoras de Guanilato Ciclase/química , Humanos , Conformação Proteica , Multimerização ProteicaRESUMO
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.
Assuntos
Cálcio/metabolismo , Distrofia de Cones/genética , Distrofia de Cones/fisiopatologia , Proteínas Ativadoras de Guanilato Ciclase/genética , Mutação/genética , Células Bipolares da Retina/patologia , Células Fotorreceptoras Retinianas Bastonetes/patologia , Transmissão Sináptica , Atrofia , Cátions , Distrofia de Cones/diagnóstico por imagem , Progressão da Doença , Eletrorretinografia , Feminino , Fundo de Olho , Guanilato Ciclase/metabolismo , Proteínas Ativadoras de Guanilato Ciclase/química , Heterozigoto , Humanos , Hidrodinâmica , Interações Hidrofóbicas e Hidrofílicas , Pessoa de Meia-Idade , Simulação de Dinâmica Molecular , Fenótipo , Agregados Proteicos , Estabilidade Proteica , Estrutura Quaternária de Proteína , Células Bipolares da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Tomografia de Coerência ÓpticaRESUMO
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.
Assuntos
Cálcio/metabolismo , Distrofias de Cones e Bastonetes/genética , Proteínas Ativadoras de Guanilato Ciclase/química , Proteínas Ativadoras de Guanilato Ciclase/genética , Mutação de Sentido Incorreto/genética , Difusão Dinâmica da Luz , Proteínas Ativadoras de Guanilato Ciclase/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Simulação de Dinâmica Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação Puntual/genética , Agregados Proteicos , Multimerização Proteica , Estabilidade Proteica , Espalhamento a Baixo Ângulo , Temperatura , Difração de Raios XRESUMO
Retinal degeneration-3 (RD3) protein protects photoreceptors from degeneration by preventing retinal guanylyl cyclase (RetGC) activation via calcium-sensing guanylyl cyclase-activating proteins (GCAP), and RD3 truncation causes severe congenital blindness in humans and other animals. The three-dimensional structure of RD3 has recently been established, but the molecular mechanisms of its inhibitory binding to RetGC remain unclear. Here, we report the results of probing 133 surface-exposed residues in RD3 by single substitutions and deletions to identify side chains that are critical for the inhibitory binding of RD3 to RetGC. We tested the effects of these substitutions and deletions in vitro by reconstituting purified RD3 variants with GCAP1-activated human RetGC1. Although the vast majority of the surface-exposed residues tolerated substitutions without loss of RD3's inhibitory activity, substitutions in two distinct narrow clusters located on the opposite sides of the molecule effectively suppressed RD3 binding to the cyclase. The first surface-exposed cluster included residues adjacent to Leu63 in the loop connecting helices 1 and 2. The second cluster surrounded Arg101 on a surface of helix 3. Single substitutions in those two clusters drastically, i.e. up to 245-fold, reduced the IC50 for the cyclase inhibition. Inactivation of the two binding sites completely disabled binding of RD3 to RetGC1 in living HEK293 cells. In contrast, deletion of 49 C-terminal residues did not affect the apparent affinity of RD3 for RetGC. Our findings identify the functional interface on RD3 required for its inhibitory binding to RetGC, a process essential for protecting photoreceptors from degeneration.
Assuntos
Proteínas do Olho/metabolismo , Guanilato Ciclase/metabolismo , Receptores de Superfície Celular/metabolismo , Substituição de Aminoácidos , Animais , Bovinos , Proteínas do Olho/genética , Guanilato Ciclase/genética , Proteínas Ativadoras de Guanilato Ciclase/genética , Proteínas Ativadoras de Guanilato Ciclase/metabolismo , Células HEK293 , Humanos , Mutação de Sentido Incorreto , Ligação Proteica , Receptores de Superfície Celular/genéticaRESUMO
Primary congenital glaucoma (PCG) is a heterogeneous, inherited, and severe optical neuropathy caused by apoptotic degeneration of the retinal ganglion cell layer. Whole-exome sequencing analysis of one PCG family identified two affected siblings who carried a low-frequency homozygous nonsense GUCA1C variant (c.52G > T/p.Glu18Ter/rs143174402). This gene encodes GCAP3, a member of the guanylate cyclase activating protein family, involved in phototransduction and with a potential role in intraocular pressure regulation. Segregation analysis supported the notion that the variant was coinherited with the disease in an autosomal recessive fashion. GCAP3 was detected immunohistochemically in the adult human ocular ciliary epithelium and retina. To evaluate the ocular effect of GUCA1C loss-of-function, a guca1c knockout zebrafish line was generated by CRISPR/Cas9 genome editing. Immunohistochemistry demonstrated the presence of GCAP3 in the non-pigmented ciliary epithelium and retina of adult wild-type fishes. Knockout animals presented up-regulation of the glial fibrillary acidic protein in Müller cells and evidence of retinal ganglion cell apoptosis, indicating the existence of gliosis and glaucoma-like retinal damage. In summary, our data provide evidence for the role of GUCA1C as a candidate gene in PCG and offer new insights into the function of this gene in the ocular anterior segment and the retina.
Assuntos
Glaucoma/genética , Proteínas Ativadoras de Guanilato Ciclase/fisiologia , Retina/metabolismo , Proteínas de Peixe-Zebra/fisiologia , Adulto , Sequência de Aminoácidos , Animais , Apoptose , Sequência de Bases , Sistemas CRISPR-Cas , Feminino , Edição de Genes , Técnicas de Inativação de Genes , Glaucoma/congênito , Gliose/genética , Gliose/patologia , Proteínas Ativadoras de Guanilato Ciclase/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Retina/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Genetic heterogeneity leading to retinal disorders impairs biological processes by causing, for example, severe disorder of signal transduction in photoreceptor outer segments. A normal balance of the second messenger homeostasis in photoreceptor cells seems to be a crucial factor for healthy and normal photoreceptor function. Genes like GUCY2D coding for guanylate cyclase GC-E and GUCA1A coding for the Ca2+-sensor guanylate cyclase-activating protein GCAP1 are critical for a precisely controlled synthesis of the second messenger cGMP. Mutations in GUCA1A frequently correlate in patients with cone dystrophy and cone-rod dystrophy. Here, we report two mutations in the GUCA1A gene that were found in patients diagnosed with retinitis pigmentosa, a phenotype that was rarely detected among previous cases of GUCA1A related retinopathies. One patient was heterozygous for the missense variant c.55C > T (p.H19Y), while the other patient was heterozygous for the missense variant c.479T > G (p.V160G). Using heterologous expression and cell culture systems, we examined the functional and molecular consequences of these point mutations. Both variants showed a dysregulation of guanylate cyclase activity, either a profound shift in Ca2+-sensitivity (H19Y) or a nearly complete loss of activating potency (V160G). Functional heterogeneity became also apparent in Ca2+/Mg2+-binding properties and protein conformational dynamics. A faster progression of retinal dystrophy in the patient carrying the V160G mutation seems to correlate with the more severe impairment of this variant.
Assuntos
Proteínas Ativadoras de Guanilato Ciclase , Retinose Pigmentar , Cálcio/metabolismo , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Proteínas Ativadoras de Guanilato Ciclase/genética , Proteínas Ativadoras de Guanilato Ciclase/metabolismo , Humanos , Fenótipo , Retinose Pigmentar/genéticaRESUMO
Guanylate Cyclase activating protein 1 (GCAP1) mediates the Ca2+-dependent regulation of the retinal Guanylate Cyclase (GC) in photoreceptors, acting as a target inhibitor at high [Ca2+] and as an activator at low [Ca2+]. Recently, a novel missense mutation (G86R) was found in GUCA1A, the gene encoding for GCAP1, in patients diagnosed with cone-rod dystrophy. The G86R substitution was found to affect the flexibility of the hinge region connecting the N- and C-domains of GCAP1, resulting in decreased Ca2+-sensitivity and abnormally enhanced affinity for GC. Based on a structural model of GCAP1, here, we tested the hypothesis of a cation-π interaction between the positively charged R86 and the aromatic W94 as the main mechanism underlying the impaired activator-to-inhibitor conformational change. W94 was mutated to F or L, thus, resulting in the double mutants G86R+W94L/F. The double mutants showed minor structural and stability changes with respect to the single G86R mutant, as well as lower affinity for both Mg2+ and Ca2+, moreover, substitutions of W94 abolished "phase II" in Ca2+-titrations followed by intrinsic fluorescence. Interestingly, the presence of an aromatic residue in position 94 significantly increased the aggregation propensity of Ca2+-loaded GCAP1 variants. Finally, atomistic simulations of all GCAP1 variants in the presence of Ca2+ supported the presence of two cation-π interactions involving R86, which was found to act as a bridge between W94 and W21, thus, locking the hinge region in an activator-like conformation and resulting in the constitutive activation of the target under physiological conditions.
Assuntos
Distrofia de Cones/metabolismo , Proteínas Ativadoras de Guanilato Ciclase/química , Proteínas Ativadoras de Guanilato Ciclase/metabolismo , Guanilato Ciclase/metabolismo , Aminoácidos Aromáticos/química , Cálcio/metabolismo , Cátions/química , Dicroísmo Circular , Distrofia de Cones/genética , Difusão Dinâmica da Luz , Proteínas Ativadoras de Guanilato Ciclase/genética , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Mutação de Sentido Incorreto , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes , TermodinâmicaRESUMO
Importance: As genetic and genomic screening is becoming more widely accessed, correctly distinguishing pathogenic from nonpathogenic variants is of increasing relevance. Objective: To reevaluate a previously reported family in whom the p.(Pro50Leu) variant in the gene GUCA1A was associated with a dominant retinal dystrophy, in light of new examination findings in the proband's daughter. Design, Setting, and Participants: A genetic study relating to a family with an inherited retinal dystrophy was performed at the retinal genetics service of Moorfields Eye Hospital from October 27, 2009, to May 23, 2019, after the proband's daughter underwent fundus examination. Main Outcomes and Measures: Results of sequencing of X chromosome-linked retinitis pigmentosa genes in the proband and specific analysis of the repetitive ORF15 region of the RPGR gene. Results: A frame-shifting single-nucleotide deletion was found in the ORF15 exon of RPGR (GRCh37 [hg19] x:38145160delT; NM_001034853.1: c.3092delA p.[Glu1031Glyfs*58]), which may be associated with the loss of 121 amino acid residues at the carboxyl terminus of the protein. The p.(Pro50Leu) variant in GUCA1A was also found to be too common in a publicly available genome database to be a fully penetrant cause of a dominant retinal dystrophy. Conclusions and Relevance: The phenotype in the family is now associated with the variant in RPGR. The findings suggest that the p.(Pro50Leu) variant in GUCA1A should not be regarded as pathogenic. This report also highlights the relevance of examining relatives, of reevaluating diagnoses in light of new data, and of considering X chromosome-linked inheritance in apparently autosomal dominant pedigrees unless there is clear male-to-male transmission.
Assuntos
Proteínas Ativadoras de Guanilato Ciclase/genética , Mutação , Distrofias Retinianas/genética , Adulto , Proteínas do Olho/genética , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/genética , Humanos , MasculinoRESUMO
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.
Assuntos
GMP Cíclico/metabolismo , Proteínas Ativadoras de Guanilato Ciclase/genética , Mutação , Distrofias Retinianas/etiologia , Distrofias Retinianas/metabolismo , Transdução de Sinais , Algoritmos , Animais , Cálcio/metabolismo , Guanilato Ciclase/metabolismo , Humanos , Transdução de Sinal Luminoso , Camundongos , Modelos Teóricos , Receptores de Superfície Celular/metabolismo , Distrofias Retinianas/patologiaRESUMO
GUCA1A gene variants are associated with autosomal dominant (AD) cone dystrophy (COD) and cone-rod dystrophy (CORD). GUCA1A-associated AD-COD/CORD has never been reported in the Japanese population. The purpose of this study was to investigate clinical and genetic features of GUCA1A-associated AD-COD/CORD from a large Japanese cohort. We identified 8 variants [c.C50_80del (p.E17VfsX22), c.T124A (p.F42I), c.C204G (p.D68E), c.C238A (p.L80I), c.T295A (p.Y99N), c.A296C (p.Y99S), c.C451T (p.L151F), and c.A551G (p.Q184R)] in 14 families from our whole exome sequencing database composed of 1385 patients with inherited retinal diseases (IRDs) from 1192 families. Three variants (p.Y99N, p.Y99S, and p.L151F), which are located on/around EF-hand domains 3 and 4, were confirmed as "pathogenic", whereas the other five variants, which did not co-segregate with IRDs, were considered "non-pathogenic". Ophthalmic findings of 9 patients from 3 families with the pathogenic variants showed central visual impairment from early to middle-age onset and progressive macular atrophy. Electroretinography revealed severely decreased or non-recordable cone responses, whereas rod responses were highly variable, ranging from nearly normal to non-recordable. Our results indicate that the three pathogenic variants, two of which were novel, underlie AD-COD/CORD with progressive retinal atrophy, and the prevalence (0.25%, 3/1192 families) of GUCA1A-associated IRDs may be low among Japanese patients.
Assuntos
Distrofia de Cones/genética , Distrofias de Cones e Bastonetes/genética , Proteínas Ativadoras de Guanilato Ciclase/genética , Padrões de Herança , Polimorfismo Genético , Adolescente , Adulto , Idade de Início , Idoso , Sequência de Aminoácidos , Distrofia de Cones/diagnóstico , Distrofia de Cones/epidemiologia , Distrofia de Cones/patologia , Distrofias de Cones e Bastonetes/diagnóstico , Distrofias de Cones e Bastonetes/epidemiologia , Distrofias de Cones e Bastonetes/patologia , Análise Mutacional de DNA , Eletrorretinografia , Feminino , Angiofluoresceinografia , Expressão Gênica , Genes Dominantes , Humanos , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Linhagem , Prevalência , Retina/metabolismo , Retina/patologia , Alinhamento de SequênciaRESUMO
Signal transduction processes that are under control of changes in cytoplasmic Ca2+-concentration involve Ca2+-sensor proteins, which often undergo pronounced conformational transitions triggered by Ca2+. Consequences of conformational changes can be the structural rearrangement of single amino acids, exposition of small patches of several amino acids, or the movement of whole protein regions or domains. Furthermore, these conformational changes can lead to the exposure or movement of posttranslationally attached acyl groups. These processes could then control the function of target proteins, for example, by Ca2+-dependent protein-protein interaction. Fluorescence spectroscopy allows for mapping these Ca2+-sensitive regions but needs site-specific fluorescence labelling. We describe the application of a new group of diaminoterephthalate-derived fluorescence probes targeting either cysteines in guanylate cyclase-activating proteins, named GCAPs, or azide moieties in covalently attached acyl groups. By monitoring Ca2+-dependent changes in fluorescence emission, we identify Ca2+-sensitive protein regions in GCAPs and correlate conformational changes to protein function.
Assuntos
Cálcio/metabolismo , Proteínas Ativadoras de Guanilato Ciclase/genética , Proteínas Ativadoras de Guanilato Ciclase/metabolismo , Animais , Sítios de Ligação , Bovinos , Mutagênese Sítio-Dirigida , Ligação Proteica , Conformação Proteica , Espectrometria de Fluorescência , Coloração e RotulagemRESUMO
The guanylyl cyclase-activating protein, GCAP1, activates photoreceptor membrane guanylyl cyclase (RetGC) in the light, when free Ca2+ concentrations decline, and decelerates the cyclase in the dark, when Ca2+ concentrations rise. Here, we report a novel mutation, G86R, in the GCAP1 (GUCA1A) gene in a family with a dominant retinopathy. The G86R substitution in a "hinge" region connecting EF-hand domains 2 and 3 in GCAP1 strongly interfered with its Ca2+-dependent activator-to-inhibitor conformational transition. The G86R-GCAP1 variant activated RetGC at low Ca2+ concentrations with higher affinity than did the WT GCAP1, but failed to decelerate the cyclase at the Ca2+ concentrations characteristic of dark-adapted photoreceptors. Ca2+-dependent increase in Trp94 fluorescence, indicative of the GCAP1 transition to its RetGC inhibiting state, was suppressed and shifted to a higher Ca2+ range. Conformational changes in G86R GCAP1 detectable by isothermal titration calorimetry (ITC) also became less sensitive to Ca2+, and the dose dependence of the G86R GCAP1-RetGC1 complex inhibition by retinal degeneration 3 (RD3) protein was shifted toward higher than normal concentrations. Our results indicate that the flexibility of the hinge region between EF-hands 2 and 3 is required for placing GCAP1-regulated Ca2+ sensitivity of the cyclase within the physiological range of intracellular Ca2+ at the expense of reducing GCAP1 affinity for the target enzyme. The disease-linked mutation of the hinge Gly86, leading to abnormally high affinity for the target enzyme and reduced Ca2+ sensitivity of GCAP1, is predicted to abnormally elevate cGMP production and Ca2+ influx in photoreceptors in the dark.