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Graphical abstract, Highlights • A hybrid stochastic/deterministic model of mouse rod phototransduction is presented. • Rod photocurrent to photovoltage conversion in darkness is accurately characterized. • Photoresponses to dim and bright stimuli and in various mutants are well reproduced. • Recently debated molecular mechanisms of the phototransduction cascade are examined., 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. The model reproduces the salient features of the experimental photoresponses at very dim and bright stimuli, for both normal photoreceptors and those with genetically modified cascade components. Our modelling approach recapitulates a number of recent findings in vertebrate phototransduction. First, our results are in line with the recently established requirement of dimeric activation of PDE6 by transducin and further show that such conditions can be fulfilled at the expense of a significant excess of G protein activated by rhodopsin. Secondly, simulations suggest a crucial role of the recoverin-mediated Ca2+-feedback on rhodopsin kinase in accelerating the shutoff, when light flashes are delivered in the presence of a light background. Finally, stochastic simulations suggest that transient complexes between dark rhodopsin and transducin formed prior to light stimulation increase the reproducibility of single photon responses. Current limitations of the model are likely associated with the yet unknown mechanisms governing the shutoff of the cascade.

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.

Membrane-bound guanylate cyclases (GCs), which synthesize the second messenger guanosine-3', 5'-cyclic monophosphate, differ in their activation modes to reach the active state. Hormone peptides bind to the extracellular domain in hormone-receptor-type GCs and trigger a conformational change in the intracellular, cytoplasmic part of the enzyme. Sensory GCs that are present in rod and cone photoreceptor cells have intracellular binding sites for regulatory Ca

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.

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.

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.

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.

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.

Orientador: Fabíola Taufic Monica Iglesias Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas Resumo: Duas classes de medicamentos denominadas estimuladores e ativadores da guanilato ciclase solúvel (GCs) foram desenvolvidas para uso terapêutico em situações patológicas onde há menor formação ou biodisponibilidade NO ou tolerância farmacológica. A GCs é uma enzima heterodímera, composta pelas subunidades alfa (?) e beta (?), nas quais há a presença do grupo prostético heme e que catalisa a conversão da guanosina trifosfato (GTP) em guanosina monofosfato cíclico (GMPc) pela ação do NO. Em situações patológicas o átomo de ferro pode encontrar-se na sua forma oxidada (Fe3+), diminuindo assim a resposta máxima do óxido nítrico (NO). A principal diferença entre os moduladores da GCs é que os ativadores (BAY 58-2667, HMR 1766, BAY 60-2770) atuam de maneira mais eficaz mesmo quando a enzima encontra-se no estado oxidado. O objetivo do presente trabalho foi caracterizar funcionalmente o relaxamento induzido pelo BAY 60-2770 em artéria pulmonar isolada de coelho. O BAY 60-2770 (0,0001-100 ?M) relaxou de maneira potente (10,1 ± 0.04) e eficaz (105 ± 0,9 %) a artéria pulmonar, sendo este efeito significativamente potencializado na presença dos inibidores da GCs (ODQ, 10 ?M, 4,9 vezes), da fosfodiesterase tipo 5 (tadalafil, 100 ?M, 5,6 vezes) ou da sintase de óxido nítrico (LNAME, 100 ?M, 3,0 vezes). A presença do sequestrador de NO, do doador de NO, da indometacina, do bloqueador do canal de potássio ou a remoção endotelial não interferiram no relaxamento induzido pelo BAY 60-2770. A fenilefrina (0,00001-3 mM) e a estimulação elétrica (4-16 Hz) produziram contração dependente da concentração e frequência, respectivamente. Na presença de tetrodotoxina (TTX, 1 ?M) e fentolamina (1 ?M) houve abolição da resposta contrátil a estimulação elétrica, mostrando a liberação neurogênica de catecolamina. Na presença de BAY 60-2770 co-incubado com ODQ uma redução significativa na contração induzida pela estimulação elétrica foi observada. Apesar desta mesma redução ter sido observada na presença do L-NAME, a mesma não foi estatisticamente significante em comparação aos anéis incubados somente com BAY 60-2770 (1 ?M). Nossos resultados mostraram que a oxidação do grupamento heme, a inibição da fosfodiesterase e a ausência do NO favoreceram a resposta relaxante do BAY 60-2770 Abstract: Soluble guanylate cyclase (sGC) stimulators and activators have been developed for use in pathophysiological condition when NO formation or bioavailability are impaired or when NO tolerance gas developed. Soluble guanylate cyclase is a heterodimer enzyme composed by alpha (?) and beta (?) subunits and a prostetic heme group. Soluble guanylate cyclase converts guanosine triphosphate (GTP) into cyclic guanosine monophosphate (GMPc) after nitric oxide (NO) activaton. Under pathophysiological conditions heme can be oxidized (Fe3+), thus reduzing NO efficacy. The main difference between stimulators and activators (BAY 58-2667, BAY 60-2770 and HMR 1766) is that the latter class of drugs is more efficacious when heme is oxidized. The aim of the present study is to characterize the relaxation induced by BAY 60-2770 in isolated pulmonary artery from rabbit. BAY 60-2770 (0.0001-100 ?M) produced concentration dependent relaxation with potency and maxima response values of 10,1 ± 0.04 and 105 ± 0.9%, respectively. The inhibition of sGC (ODQ, 10 ?M) or phosphodiesterase type 5 (tadalafil, 100 ?M) or the nitric oxide synthase (L-NAME, 100 ?M) produced significantly leftward shifts by, approximately, 4.9, 5.4 and 3.0, respectively. The NO-scavenger, the NO-donor, the cyclooxygenase inhibition, the potassium channel blocker or endothelial removal did not interfere on the pharmacological parameters of BAY 60-2770. Phenylephrine (PE, 0.0001- 3 mM) and electrical field stimulation (EFS, 4-16 Hz) induced concentration and frequency dependent-contraction, respectively. Phentolamine (1 ?M) and tetrodotoxin (TTX, 1 ??) practically abolished EFS-induced contraction, showing the neurogenic source of catecholamines. Co-treatment with BAY 60-2770 with ODQ reduced significantly the EFS-induced contraction in comparison with BAY 60- 2770 (1 ?M) alone. Although we have observed a tendency of reduction in the amplitude of contraction when BAY 60-2770 was co-incubated with L-NAME, it was not statistically significant. Therefore, our results showed that the oxidation of heme group, the inhibition of phosphodiesterase and lower levels of NO favoured the relaxing response of BAY 60- 2770 in isolated rabbit pulmonary artery Mestrado Farmacologia Mestre em Farmacologia

Retinal guanylate cyclases (RetGCs) promote the Ca2+-dependent synthesis of cGMP that coordinates the recovery phase of visual phototransduction in retinal rods and cones. The Ca2+-sensitive activation of RetGCs is controlled by a family of photoreceptor Ca2+ binding proteins known as guanylate cyclase activator proteins (GCAPs). The Mg2+-bound/Ca2+-free GCAPs bind to RetGCs and activate cGMP synthesis (cyclase activity) at low cytosolic Ca2+ levels in light-activated photoreceptors. By contrast, Ca2+-bound GCAPs bind to RetGCs and inactivate cyclase activity at high cytosolic Ca2+ levels found in dark-adapted photoreceptors. Mutations in both RetGCs and GCAPs that disrupt the Ca2+-dependent cyclase activity are genetically linked to various retinal diseases known as cone-rod dystrophies. In this review, I will provide an overview of the known atomic-level structures of various GCAP proteins to understand how protein dimerization and Ca2+-dependent conformational changes in GCAPs control the cyclase activity of RetGCs. This review will also summarize recent structural studies on a GCAP homolog from zebrafish (GCAP5) that binds to Fe2+ and may serve as a Fe2+ sensor in photoreceptors. The GCAP structures reveal an exposed hydrophobic surface that controls both GCAP1 dimerization and RetGC binding. This exposed site could be targeted by therapeutics designed to inhibit the GCAP1 disease mutants, which may serve to mitigate the onset of retinal cone-rod dystrophies.

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.

Copyright: This record is sourced from MEDLINE/PubMed, a database of the U.S. National Library of Medicine 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 (βdark) 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 βdark 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.

Retinal degeneration-3 protein (RD3) deficiency causes photoreceptor dysfunction and rapid degeneration in the rd3 mouse strain and in human Leber's congenital amaurosis, a congenital retinal dystrophy that results in early vision loss. However, the mechanisms responsible for photoreceptor death remain unclear. Here, we tested two hypothesized biochemical events that may underlie photoreceptor death: (i) the failure to prevent aberrant activation of retinal guanylyl cyclase (RetGC) by calcium-sensor proteins (GCAPs) versus (ii) the reduction of GMP phosphorylation rate, preventing its recycling to GDP/GTP. We found that GMP converts to GDP/GTP in the photoreceptor fraction of the retina ∼24-fold faster in WT mice and ∼400-fold faster in rd3 mice than GTP conversion to cGMP by RetGC. Adding purified RD3 to the retinal extracts inhibited RetGC 4-fold but did not affect GMP phosphorylation in wildtype or rd3 retinas. RD3-deficient photoreceptors rapidly degenerated in rd3 mice that were reared in constant darkness to prevent light-activated GTP consumption via RetGC and phosphodiesterase 6. In contrast, rd3 degeneration was alleviated by deletion of GCAPs. After 2.5 months, only ∼40% of photoreceptors remained in rd3/rd3 retinas. Deletion of GCAP1 or GCAP2 alone preserved 68% and 57% of photoreceptors, respectively, whereas deletion of GCAP1 and GCAP2 together preserved 86%. Taken together, our in vitro and in vivo results support the hypothesis that RD3 prevents photoreceptor death primarily by suppressing activation of RetGC by both GCAP1 and GCAP2 but do not support the hypothesis that RD3 plays a significant role in GMP recycling.

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 Ca

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 &gt, 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 M&uuml, ller 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.

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

Loss-of-function mutations in the retinal degeneration 3 (RD3) gene cause inherited retinopathy with impaired rod and cone function and fast retinal degeneration in patients and in the natural strain of rd3 mice. The underlying physiopathology mechanisms are not well understood. We previously proposed that guanylate cyclase-activating proteins (GCAPs) might be key Ca2+-sensors mediating the physiopathology of this disorder, based on the demonstrated toxicity of GCAP2 when blocked in its Ca2+-free form at photoreceptor inner segments. We here show that the retinal degeneration in rd3 mice is substantially delayed by GCAPs ablation. While the number of retinal photoreceptor cells is halved in 6 weeks in rd3 mice, it takes 8 months to halve in rd3/rd3 GCAPs−/− mice. Although this substantial morphological rescue does not correlate with recovery of visual function due to very diminished guanylate cyclase activity in rd3 mice, it is very informative of the mechanisms underlying photoreceptor cell death. By showing that GCAP2 is mostly in its Ca2+-free-phosphorylated state in rd3 mice, we infer that the [Ca2+]i at rod inner segments is permanently low. GCAPs are therefore retained at the inner segment in their Ca2+-free, guanylate cyclase activator state. We show that in this conformational state GCAPs induce endoplasmic reticulum (ER) stress, mitochondrial swelling, and cell death. ER stress and mitochondrial swelling are early hallmarks of rd3 retinas preceding photoreceptor cell death, that are substantially rescued by GCAPs ablation. By revealing the involvement of GCAPs-induced ER stress in the physiopathology of Leber’s congenital amaurosis 12 (LCA12), this work will aid to guide novel therapies to preserve retinal integrity in LCA12 patients to expand the window for gene therapy intervention to restore vision.

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-&pi, 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 &ldquo, phase II&rdquo, 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-&pi, 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.

Light adaptation of photoreceptor cells is mediated by Ca(2+)-dependent mechanisms. In darkness, Ca(2+) influx through cGMP-gated channels into the outer segment of photoreceptors is balanced by Ca(2+) extrusion via Na(+)/Ca(2+), K(+) exchangers (NCKXs). Light activates a G protein signaling cascade, which closes cGMP-gated channels and decreases Ca(2+) levels in photoreceptor outer segment because of continuing Ca(2+) extrusion by NCKXs. Guanylate cyclase–activating proteins (GCAPs) then up-regulate cGMP synthesis by activating retinal membrane guanylate cyclases (RetGCs) in low Ca(2+). This activation of RetGC accelerates photoresponse recovery and critically contributes to light adaptation of the nighttime rod and daytime cone photoreceptors. In mouse rod photoreceptors, GCAP1 and GCAP2 both contribute to the Ca(2+)-feedback mechanism. In contrast, only GCAP1 appears to modulate RetGC activity in mouse cones because evidence of GCAP2 expression in cones is lacking. Surprisingly, we found that GCAP2 is expressed in cones and can regulate light sensitivity and response kinetics as well as light adaptation of GCAP1-deficient mouse cones. Furthermore, we show that GCAP2 promotes cGMP synthesis and cGMP-gated channel opening in mouse cones exposed to low Ca(2+). Our biochemical model and experiments indicate that GCAP2 significantly contributes to the activation of RetGC1 at low Ca(2+) when GCAP1 is not present. Of note, in WT mouse cones, GCAP1 dominates the regulation of cGMP synthesis. We conclude that, under normal physiological conditions, GCAP1 dominates the regulation of cGMP synthesis in mouse cones, but if its function becomes compromised, GCAP2 contributes to the regulation of phototransduction and light adaptation of cones.

The Arg838Ser mutation in retinal membrane guanylyl cyclase 1 (RetGC1) has been linked to autosomal dominant cone–rod dystrophy type 6 (CORD6). It is believed that photoreceptor degeneration is caused by the altered sensitivity of RetGC1 to calcium regulation via guanylyl cyclase activating proteins (GCAPs). To determine the mechanism by which this mutation leads to degeneration, we investigated the structure and function of rod photoreceptors in two transgenic mouse lines, 362 and 379, expressing R838S RetGC1. In both lines, rod outer segments became shorter than in their nontransgenic siblings by 3–4 weeks of age, before the eventual photoreceptor degeneration. Despite the shortening of their outer segments, the dark current of transgenic rods was 1.5–2.2-fold higher than in nontransgenic controls. Similarly, the dim flash response amplitude inR838S+rods was larger, time to peak was delayed, and flash sensitivity was increased, all suggesting elevated dark-adapted free cGMP in transgenic rods. In rods expressing R838S RetGC1, dark-current noise increased and the exchange current, detected after a saturating flash, became more pronounced. These results suggest disrupted Ca2+phototransduction feedback and abnormally high free-Ca2+concentration in the outer segments. Notably, photoreceptor degeneration, which typically occurred after 3 months of age in R838S RetGC1 transgenic mice inGCAP1,2+/+orGCAP1,2+/−backgrounds, was prevented inGCAP1,2−/−mice lacking Ca2+feedback to guanylyl cyclase. In summary, the dysregulation of guanylyl cyclase in RetGC1-linked CORD6 is a “phototransduction disease,” which means it is associated with increased free-cGMP and Ca2+levels in photoreceptors.SIGNIFICANCE STATEMENTIn a mouse model expressing human membrane guanylyl cyclase 1 (RetGC1,GUCY2D), a mutation associated with early progressing congenital blindness, cone–rod dystrophy type 6 (CORD6), deregulates calcium-sensitive feedback of phototransduction to the cyclase mediated by guanylyl cyclase activating proteins (GCAPs), which are calcium-sensor proteins. The abnormal calcium sensitivity of the cyclase increases cGMP-gated dark current in the rod outer segments, reshapes rod photoresponses, and triggers photoreceptor death. This work is the first to demonstrate a direct physiological effect ofGUCY2DCORD6-linked mutation on photoreceptor physiologyin vivo. It also identifies the abnormal regulation of the cyclase by calcium-sensor proteins as the main trigger for the photoreceptor death.

This study with recombinant reconstituted system mimicking the cellular conditions of the native cones, documents that photoreceptor ROS-GC1 is modulated by gaseous CO(2). Mechanistically, CO(2) is sensed by carbonic anhydrase (CAII), generates bicarbonate that, in turn, directly targets the core catalytic domain of ROS-GC1 and activates it to increased synthesis of cyclic GMP. This, then, functions as a second messenger for the cone phototransduction. The study demonstrates that, in contrast to the Ca(2+)−modulated phototransduction, the CO(2) pathway is Ca(2+)−independent, yet is linked with it and synergizes it. It, through R(787)C mutation in the third heptad of the signal helix domain of ROS-GC1, affects cone rod dystrophy, CORD6. CORD6 is caused firstly by lowered basal and GCAP1-dependent ROS-GC1 activity and secondly, by a shift in Ca(2+) sensitivity of the ROS-GC1/GCAP1 complex that remains active in darkness. Remarkably, the first but not the second defect disappears with bicarbonate thus explaining the basis for CORD6 pathological severity. Because cones, but not rods, express CAII, the excessive synthesis of cyclic GMP would be most acute in cones.

Purpose Sixteen different mutations in the guanylate cyclase activator 1A gene (GUCA1A), have been previously identified to cause autosomal dominant cone dystrophy (adCOD), cone–rod dystrophy (adCORD), macular dystrophy (adMD), and in an isolated patient, retinitis pigmentosa (RP). The purpose of this study is to report on two novel mutations and the patients’ clinical features. Methods Clinical investigations included visual acuity and visual field testing, fundus examination, high-resolution spectral-domain optical coherence tomography (OCT), fundus autofluorescence imaging, and full-field and multifocal electroretinogram (ERG) recordings. GUCA1A was screened by Sanger sequencing in a cohort of 12 French families with adCOD, adCORD, and adMD. Results We found two novel GUCA1A mutations—one amino acid deletion, c.302_304delTAG (p.Val101del), and one missense mutation, c.444T>A (p.Asp148Glu)—each of which was found in one family. The p.Asp148Glu mutation affected one of the Ca2+-binding amino acids of the EF4 hand, while the p.Val101del mutation resulted in the in-frame deletion of Valine-101, localized between two Ca2+-binding aspartic acid residues at positions 100 and 102 of the EF3 hand. Both families complained of visual acuity loss worsening with age. However, the p.Asp148Glu mutation was present in one family with adCOD involving abnormal cone function and an absence of macular atrophy, whereas p.Val101del mutation was encountered in another family with adMD without a generalized cone defect. Conclusions The two novel mutations described in this study are associated with distinct phenotypes, MD for p.Val101del and COD for p.Asp148Glu, with no intrafamilial phenotypic heterogeneity.

Purpose: The aim of this study was to investigate the genetic basis and pathogenic mechanism of variable maculopathies, ranging from mild photoreceptor degeneration to central areolar choroidal dystrophy, in a five-generation family. Methods: Clinical characterizations, whole-exome sequencing, and genome-wide linkage analysis were carried out on the family. Zebrafish models were used to investigate the pathogenesis of GUCA1A mutations. Results: A novel mutation, GUCA1A p.R120L, was identified in the family and predicted to alter the tertiary structure of guanylyl cyclase-activating protein 1, a photoreceptor-expressed protein encoded by the GUCA1A gene. The mutation was shown in zebrafish to cause significant disruptions in photoreceptors and retinal pigment epithelium, together with atrophies of retinal vessels and choriocapillaris. Those phenotypes could not be fully rescued by exogenous wild-type GUCA1A, suggesting a likely gain-of-function mechanism for p.R120L. GUCA1A p.D100E, another mutation previously implicated in cone dystrophy, also impaired the retinal pigment epithelium and photoreceptors in zebrafish, but probably via a dominant negative effect. Conclusion: We conclude that GUCA1A mutations could cause significant variability in maculopathies, including central areolar choroidal dystrophy, which represents a severe pattern of maculopathy. The diverse pathogenic modes of GUCA1A mutations may explain the phenotypic diversities. Genet Med advance online publication 26 January 2017

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.

Deficiency of RD3 (retinal degeneration 3) protein causes recessive blindness and photoreceptor degeneration in humans and in the rd3 mouse strain, but the disease mechanism is unclear. Here, we present evidence that RD3 protects photoreceptors from degeneration by competing with guanylyl cyclase-activating proteins (GCAPs), which are calcium sensor proteins for retinal membrane guanylyl cyclase (RetGC). RetGC activity in rd3/rd3 retinas was drastically reduced but stimulated by the endogenous GCAPs at low Ca(2+) concentrations. RetGC activity completely failed to accelerate in rd3/rd3GCAPs(−/−) hybrid photoreceptors, whose photoresponses remained drastically suppressed compared with the WT. However, ∼70% of the hybrid rd3/rd3GCAPs(−/−) photoreceptors survived past 6 months, in stark contrast to

Retinal membrane guanylyl cyclase (RetGC) in photoreceptor rod and cone cells is regulated by a family of guanylyl cyclase activating proteins (GCAP1-7). GCAP5 is expressed in zebrafish photoreceptors and promotes Ca(2+)-dependent regulation of RetGC enzymatic activity that regulates visual phototransduction. We report NMR chemical shift assignments of the Ca(2+)-free activator form of GCAP5 (BMRB no. 27705).

Signal transduction processes that are under control of changes in cytoplasmic Ca

The guanylyl cyclase-activating protein, GCAP1, activates photoreceptor membrane guanylyl cyclase (RetGC) in the light, when free Ca(2+) concentrations decline, and decelerates the cyclase in the dark, when Ca(2+) 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 Ca(2+)-dependent activator-to-inhibitor conformational transition. The G86R-GCAP1 variant activated RetGC at low Ca(2+) concentrations with higher affinity than did the WT GCAP1, but failed to decelerate the cyclase at the Ca(2+) concentrations characteristic of dark-adapted photoreceptors. Ca(2+)-dependent increase in Trp(94) fluorescence, indicative of the GCAP1 transition to its RetGC inhibiting state, was suppressed and shifted to a higher Ca(2+) range. Conformational changes in G86R GCAP1 detectable by isothermal titration calorimetry (ITC) also became less sensitive to Ca(2+), 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 Ca(2+) sensitivity of the cyclase within the physiological range of intracellular Ca(2+) at the expense of reducing GCAP1 affinity for the target enzyme. The disease-linked mutation of the hinge Gly(86), leading to abnormally high affinity for the target enzyme and reduced Ca(2+) sensitivity of GCAP1, is predicted to abnormally elevate cGMP production and Ca(2+) influx in photoreceptors in the dark.

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.

Myristoylation of most neuronal calcium sensor proteins, a group of EF-hand calcium-binding proteins mainly expressed in neuronal tissue, can have a strong impact on protein dynamics and functional properties. Intracellular oscillations of the free Ca(2+) concentration can trigger conformational changes in Ca(2+) sensors. The position and possible movements of the myristoyl group in the photoreceptor cell-specific Ca(2+) sensor GCAP2 are not well-defined but appear to be different from those of the highly homologous cognate GCAP1. We designed and applied a new group of diaminoterephthalate-derived fluorescent probes to label GCAP2 at a covalently attached 12-azido-dodecanoic acid (a myristoyl substitute) and at cysteine residues in critical positions. Fluorescence emission of dye-labeled GCAP2 decreased when going from low (10(-9) M) to high [Ca(2+)] (10(-3) M), reaching a half-maximal effect of fluorescence emission at 0.44 ± 0.07 μM. The modified acyl group can therefore monitor changes in the protein conformation during binding and dissociation of Ca(2+) in the physiological range of free [Ca(2+)]. However, fluorescence quenching studies showed that the dye-acyl chain was shielded from the quencher by an adjacent polypeptide region. Further probing three cysteine positions (C35, C111, and C131) by dye labeling revealed that all positions were also sensitive to a change in [Ca(2+)], but only one (C131) was sensitive to a change in [Mg(2+)]. We suggest a scenario during illumination of the photoreceptor cell in which Ca(2+) dissociates first from low and medium affinity binding sites. These steps are sensed by dyes in cysteines at positions 35 and 111. Release of Ca(2+) from high affinity sites is sensed by regions adjacent to the dye-labeled fatty acid and involves the critical conformational change leading to activating guanylate cyclase.

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.

Guanylate Cyclase-Activating Protein 1 (GCAP1) regulates the enzymatic activity of the photoreceptor guanylate cyclases (GC), leading to inhibition or activation of the cyclic guanosine monophosphate (cGMP) synthesis depending on its Ca2+- or Mg2+-loaded state. By genetically screening a family of patients diagnosed with cone-rod dystrophy, we identified a novel missense mutation with autosomal dominant inheritance pattern (c.332A>T; p.(Glu111Val); E111V from now on) in the GUCA1A gene coding for GCAP1. We performed a thorough biochemical and biophysical investigation of wild type (WT) and E111V human GCAP1 by heterologous expression and purification of the recombinant proteins. The E111V substitution disrupts the coordination of the Ca2+ ion in the high-affinity site (EF-hand 3, EF3), thus significantly decreasing the ability of GCAP1 to sense Ca2+ (∼80-fold higher Kdapp compared to WT). Both WT and E111V GCAP1 form dimers independently on the presence of cations, but the E111V Mg2+-bound form is prone to severe aggregation over time. Molecular dynamics simulations suggest a significantly increased flexibility of both the EF3 and EF4 cation binding loops for the Ca2+-bound form of E111V GCAP1, in line with the decreased affinity for Ca2+. In contrast, a more rigid backbone conformation is observed in the Mg2+-bound state compared to the WT, which results in higher thermal stability. Functional assays confirm that E111V GCAP1 interacts with the target GC with a similar apparent affinity (EC50); however, the mutant shifts the GC inhibition out of the physiological [Ca2+] (IC50E111V ∼10 μM), thereby leading to the aberrant constitutive synthesis of cGMP under conditions of dark-adapted photoreceptors.