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Auteur : Grafiati
Publié le 11 mars 2023

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1

Ames, James B. « Structural Insights into Retinal Guanylate Cyclase Activator Proteins (GCAPs) ». International Journal of Molecular Sciences 22, no 16 (13 août 2021) : 8731. http://dx.doi.org/10.3390/ijms22168731.

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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.
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2

Vinberg, Frans, Teemu T. Turunen, Hanna Heikkinen, Marja Pitkänen et Ari Koskelainen. « A novel Ca2+-feedback mechanism extends the operating range of mammalian rods to brighter light ». Journal of General Physiology 146, no 4 (28 septembre 2015) : 307–21. http://dx.doi.org/10.1085/jgp.201511412.

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Sensory cells adjust their sensitivity to incoming signals, such as odor or light, in response to changes in background stimulation, thereby extending the range over which they operate. For instance, rod photoreceptors are extremely sensitive in darkness, so that they are able to detect individual photons, but remain responsive to visual stimuli under conditions of bright ambient light, which would be expected to saturate their response given the high gain of the rod transduction cascade in darkness. These photoreceptors regulate their sensitivity to light rapidly and reversibly in response to changes in ambient illumination, thereby avoiding saturation. Calcium ions (Ca2+) play a major role in mediating the rapid, subsecond adaptation to light, and the Ca2+-binding proteins GCAP1 and GCAP2 (or guanylyl cyclase–activating proteins [GCAPs]) have been identified as important mediators of the photoreceptor response to changes in intracellular Ca2+. However, mouse rods lacking both GCAP1 and GCAP2 (GCAP−/−) still show substantial light adaptation. Here, we determined the Ca2+ dependency of this residual light adaptation and, by combining pharmacological, genetic, and electrophysiological tools, showed that an unknown Ca2+-dependent mechanism contributes to light adaptation in GCAP−/− mouse rods. We found that mimicking the light-induced decrease in intracellular [Ca2+] accelerated recovery of the response to visual stimuli and caused a fourfold decrease of sensitivity in GCAP−/− rods. About half of this Ca2+-dependent regulation of sensitivity could be attributed to the recoverin-mediated pathway, whereas half of it was caused by the unknown mechanism. Furthermore, our data demonstrate that the feedback mechanisms regulating the sensitivity of mammalian rods on the second and subsecond time scales are all Ca2+ dependent and that, unlike salamander rods, Ca2+-independent background-induced acceleration of flash response kinetics is rather weak in mouse rods.
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Avesani, Anna, Laura Bielefeld, Nicole Weisschuh, Valerio Marino, Pascale Mazzola, Katarina Stingl, Tobias B. Haack, Karl-Wilhelm Koch et Daniele Dell’Orco. « Molecular Properties of Human Guanylate Cyclase-Activating Protein 3 (GCAP3) and Its Possible Association with Retinitis Pigmentosa ». International Journal of Molecular Sciences 23, no 6 (17 mars 2022) : 3240. http://dx.doi.org/10.3390/ijms23063240.

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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.
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4

Peshenko, Igor V., Elena V. Olshevskaya et Alexander M. Dizhoor. « GUCY2D mutations in retinal guanylyl cyclase 1 provide biochemical reasons for dominant cone–rod dystrophy but not for stationary night blindness ». Journal of Biological Chemistry 295, no 52 (27 octobre 2020) : 18301–15. http://dx.doi.org/10.1074/jbc.ra120.015553.

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Mutations in the GUCY2D gene coding for the dimeric human retinal membrane guanylyl cyclase (RetGC) isozyme RetGC1 cause various forms of blindness, ranging from rod dysfunction to rod and cone degeneration. We tested how the mutations causing recessive congenital stationary night blindness (CSNB), recessive Leber's congenital amaurosis (LCA1), and dominant cone–rod dystrophy-6 (CORD6) affected RetGC1 activity and regulation by RetGC-activating proteins (GCAPs) and retinal degeneration-3 protein (RD3). CSNB mutations R666W, R761W, and L911F, as well as LCA1 mutations R768W and G982VfsX39, disabled RetGC1 activation by human GCAP1, -2, and -3. The R666W and R761W substitutions compromised binding of GCAP1 with RetGC1 in HEK293 cells. In contrast, G982VfsX39 and L911F RetGC1 retained the ability to bind GCAP1 in cyto but failed to effectively bind RD3. R768W RetGC1 did not bind either GCAP1 or RD3. The co-expression of GUCY2D allelic combinations linked to CSNB did not restore RetGC1 activity in vitro. The CORD6 mutation R838S in the RetGC1 dimerization domain strongly dominated the Ca2+ sensitivity of cyclase regulation by GCAP1 in RetGC1 heterodimer produced by co-expression of WT and the R838S subunits. It required higher Ca2+ concentrations to decelerate GCAP-activated RetGC1 heterodimer—6-fold higher than WT and 2-fold higher than the Ser838-harboring homodimer. The heterodimer was also more resistant than homodimers to inhibition by RD3. The observed biochemical changes can explain the dominant CORD6 blindness and recessive LCA1 blindness, both of which affect rods and cones, but they cannot explain the selective loss of rod function in recessive CSNB.
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5

Howes, K. A. « GCAP1 rescues rod photoreceptor response in GCAP1/GCAP2 knockout mice ». EMBO Journal 21, no 7 (1 avril 2002) : 1545–54. http://dx.doi.org/10.1093/emboj/21.7.1545.

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6

Gorczyca, Wojciech A., Marcin Kobiałka, Marianna Kuropatwa et Ewa Kurowska. « Ca2+ differently affects hydrophobic properties of guanylyl cyclase-activating proteins (GCAPs) and recoverin. » Acta Biochimica Polonica 50, no 2 (30 juin 2003) : 367–76. http://dx.doi.org/10.18388/abp.2003_3691.

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Guanylyl cyclase-activating proteins (GCAPs) and recoverin are retina-specific Ca(2+)-binding proteins involved in phototransduction. We provide here evidence that in spite of structural similarities GCAPs and recoverin differently change their overall hydrophobic properties in response to Ca(2+). Using native bovine GCAP1, GCAP2 and recoverin we show that: i) the Ca(2+)-dependent binding of recoverin to Phenyl-Sepharose is distinct from such interactions of GCAPs; ii) fluorescence intensity of 1-anilinonaphthalene-8-sulfonate (ANS) is markedly higher at high [Ca(2+)](free) (10 microM) than at low [Ca(2+)](free) (10 nM) in the presence of recoverin, while an opposing effect is observed in the presence of GCAPs; iii) fluorescence resonance energy transfer from tryptophane residues to ANS is more efficient at high [Ca(2+)](free) in recoverin and at low [Ca(2+)](free) in GCAP2. Such different changes of hydrophobicity evoked by Ca(2+) appear to be the precondition for possible mechanisms by which GCAPs and recoverin control the activities of their target enzymes.
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7

Dejda, Agnieszka, Izabela Matczak et Wojciech A. Gorczyca. « p19 detected in the rat retina and pineal gland is a guanylyl cyclase-activating protein (GCAP). » Acta Biochimica Polonica 49, no 4 (31 décembre 2002) : 899–905. http://dx.doi.org/10.18388/abp.2002_3749.

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The Ca(2+)-dependent activation of retina-specific guanylyl cyclase (retGC) is mediated by guanylyl cyclase-activating proteins (GCAPs). Here we report for the first time detection of a 19 kDa protein (p19) with GCAP properties in extracts of rat retina and pineal gland. Both extracts stimulate synthesis of cGMP in rod outer segment (ROS) membranes at low (30 nM) but not at high (1 microM) concentrations of Ca(2+). At low Ca(2+), immunoaffinity purified p19 activates guanylyl cyclase(s) in bovine ROS and rat retinal membranes. Moreover, p19 is recognized by antibodies against bovine GCAP1 and, similarly to other GCAPs, exhibits a Ca(2+)-dependent electrophoretic mobility shift.
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8

Imanishi, Yoshikazu, Lili Yang, Izabela Sokal, S?awomir Filipek, Krzysztof Palczewski et Wolfgang Baehr. « Diversity of Guanylate Cyclase-Activating Proteins (GCAPs) in Teleost Fish : Characterization of Three Novel GCAPs (GCAP4, GCAP5, GCAP7) from Zebrafish (Danio rerio) and Prediction of Eight GCAPs (GCAP1-8) in Pufferfish (Fugu rubripes) ». Journal of Molecular Evolution 59, no 2 (août 2004) : 204–17. http://dx.doi.org/10.1007/s00239-004-2614-y.

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Pennesi, M. E., K. A. Howes, W. Baehr et S. M. Wu. « Guanylate cyclase-activating protein (GCAP) 1 rescues cone recovery kinetics in GCAP1/GCAP2 knockout mice ». Proceedings of the National Academy of Sciences 100, no 11 (5 mai 2003) : 6783–88. http://dx.doi.org/10.1073/pnas.1130102100.

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10

Payne, Annette M., Susan M. Downes, David A. R. Bessant, Catherine Plant, Tony Moore, Alan C. Bird et Shomi S. Bhattacharya. « Genetic analysis of the guanylate cyclase activator 1B (GUCA1B) gene in patients with autosomal dominant retinal dystrophies : Table 1 ». Journal of Medical Genetics 36, no 9 (1 septembre 1999) : 691–93. http://dx.doi.org/10.1136/jmg.36.9.691.

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The guanylate cyclase activator proteins (GCAP1 and GCAP2) are calcium binding proteins which by activating Ret-GC1 play a key role in the recovery phase of phototransduction. Recently a mutation in theGUCA1A gene (coding for GCAP1) mapping to the 6p21.1 region was described as causing cone dystrophy in a British family. In addition mutations in Ret-GC1have been shown to cause Leber congenital amaurosis and cone-rod dystrophy. To determine whether GCAP2 is involved in dominant retinal degenerative diseases, the GCAP2 gene was screened in 400 unrelated subjects with autosomal dominant central and peripheral retinal dystrophies.A number of changes involving the intronic as well as the coding sequence were observed. In exon 1 a T to C nucleotide change was observed leaving the tyrosine residue 57 unchanged. In exon 3 a 1 bp intronic insertion, a single nucleotide substitution G to A in the intron 3′ of this exon, and a GAG to GAT change at codon 155 were observed. This latter change results in a conservative change of glutamic acid to aspartic acid. In exon 4 a 7 bp intronic insertion, a single nucleotide A to G substitution in the intron 5′ of this exon, and a single base pair change C to G in the intron 3′ of exon 4 were seen. None of these changes would be expected to affect correct splicing of this gene. All these changes were observed in controls. The results of this study do not show any evidence so far that GCAP2 is involved in the pathogenesis of autosomal dominant retinal degeneration in this group of patients. All the changes detected were found to be sequence variations or polymorphisms and not disease causing.
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11

Bonì, Francesco, Valerio Marino, Carlo Bidoia, Eloise Mastrangelo, Alberto Barbiroli, Daniele Dell’Orco et Mario Milani. « Modulation of Guanylate Cyclase Activating Protein 1 (GCAP1) Dimeric Assembly by Ca2+ or Mg2+ : Hints to Understand Protein Activity ». Biomolecules 10, no 10 (5 octobre 2020) : 1408. http://dx.doi.org/10.3390/biom10101408.

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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.
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12

Abbas, Seher, Valerio Marino, Laura Bielefeld, Karl-Wilhelm Koch et Daniele Dell’Orco. « Constitutive Activation of Guanylate Cyclase by the G86R GCAP1 Variant Is Due to “Locking” Cation-π Interactions that Impair the Activator-to-Inhibitor Structural Transition ». International Journal of Molecular Sciences 21, no 3 (23 janvier 2020) : 752. http://dx.doi.org/10.3390/ijms21030752.

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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.
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Marino, Valerio, Giuditta Dal Cortivo, Paolo Enrico Maltese, Giorgio Placidi, Elisa De Siena, Benedetto Falsini, Matteo Bertelli et Daniele Dell’Orco. « Impaired Ca2+ Sensitivity of a Novel GCAP1 Variant Causes Cone Dystrophy and Leads to Abnormal Synaptic Transmission Between Photoreceptors and Bipolar Cells ». International Journal of Molecular Sciences 22, no 8 (14 avril 2021) : 4030. http://dx.doi.org/10.3390/ijms22084030.

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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.
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Roth, Nora, Christoph Faul, Christiane Dorn, Wichard Vogel, Wolfgang A. Bethge, Lothar Kanz, Hans-Georg Rammensee et Sebastian P. Haen. « Development of New Autoimmunity Against T Cell Antigens Derived From Retinal Proteins After Allogeneic Hematopoietic Cell Transplantation. » Blood 120, no 21 (16 novembre 2012) : 3060. http://dx.doi.org/10.1182/blood.v120.21.3060.3060.

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Abstract Abstract 3060 Introduction: Graft versus host disease (GvHD) is mainly mediated by T cells recognizing major (MHC) and minor (miHAG) histocompatibility antigens (human leukocyte antigens and MHC-restricted epitopes, respectively). The clinical appearance of a GvHD affecting the central nervous system (CNS) and the retina as part of the CNS is rare and evidence is limited to single case reports. Some publications describe the development of new autoimmunity after hematopoietic cell transplantation (HCT) manifested as hemolytic anemia (AIHA), immune thrombocytopenia (ITP) or myasthenia gravis. Of note, new autoinflammatory diseases affecting the retina have not been reported. In this study we investigated the GvHD of the retina and examined the development of new autoimmune T cell responses against epitopes derived from proteins exclusively expressed in the retina. Patients and Methods: We analyzed T cells from 8 women and 12 men with a median age of 55 years (range 29 – 69 years) that had underwent HCT. Underlying diseases were acute lymphoblastic leukemia (n = 1), acute myeloid leukemia, (n = 6), chronic myeloid leukemia (n = 1), myelodysplastic syndrome (n = 3), myeloproliferative syndromes (primary myelofibrosis, n = 2; essential thrombocytemia with secondary myelofibrosis, n = 2; polycythemia vera with secondary myelofibrosis, n = 1), B-cell non-Hodgkin lymphoma (gray zone lymphoma, n = 1; follicular lymphoma, n = 1; peripheral T cell lymphoma, n = 1) and Hodgkin's lymphoma (n = 1). Potential T cell epitopes from four unique highly polymorphic retinal proteins (membrane-bound retinal guanylate cyclase 1 protein (retGC), the guanylate cyclase activating proteins 1 and 2 (GCAP1 and GCAP2) and the retinoid binding protein 3 (RBP3)) were identified using 2 approaches. First, genomic DNA derived from both donor and recipient coding for these proteins was sequenced by Sanger sequencing in search of single nucleotide polymorphisms (SNP). Second, alternate peptide expression based on known SNP was predicted using internet based databases (EpiToolKit). The predicted epitopes were synthesized and used in T cell assays. Peripheral blood mononuclear cells (PBMCs) from patients after hematopoietic regeneration (neutrophils > 500/μl) were stimulated with SNP peptide pairs (peptides pairs differing in one amino acid) and analyzed by IFNg-ELISPOT and flow cytometry. Results: In 5 out of 20 patients (25%), strong T cell responses against peptides derived from retGC as well as from GCAP1 and GCAP2 were observed which were not detectable before HCT and not reflected by a difference in the DNA sequence between donor and recipient. Two patients of the cohort presented with visual loss which was due to cone dystrophy (n = 1) and retrobulbar optic neuritis (n = 1). In the patient with cone dystrophy, we observed circulating antigen specific T cells against peptides derived from retGC. The patient with retrobulbar optic neuritis did not have antigen specific T cell responses. In 2 clinically silent patients, we found IFNg producing CD4+ T cells that recognized a predicted GCAP1-derived self-peptide. One patient also had a strong T cell response against a GCAP2-derived self-peptide. The T cells specifically recognized the peptide represented in the autologous DNA sequence; no reactivity was seen after stimulation with the SNP peptide. Furthermore, the T cell reactions persisted over time and were still detectable one year after HCT. In another patient, T cell responses against the pair of GCAP2 peptides were detected. Here, the reactivity against one peptide could not be discriminated due to limited availability of patient T cells. One further patient displayed T cell responses against GCAP and retGC peptides, which were directed against both self- and SNP peptides. As controls we stimulated T cells from 5 HLA-matched healthy individuals with all respective peptides and observed no T cell reaction. Conclusions: 25% of the patients revealed strong T cell responses against retinal autoantigens after HCT. T cell responses detected late after HCT as observed in 3 patients might indicate a chronic antigen exposure. Clinical manifestations were cone dystrophy (here, antigen-specific T cells against cone protein-derived peptides could be detected) and retrobulbar optic neuritis. To our knowledge, this is the first report on antigen-specificity of neoautoinflammatory cells after allogeneic HCT. Disclosures: No relevant conflicts of interest to declare.
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Marino, Valerio, Alberto Borsatto, Farina Vocke, Karl-Wilhelm Koch et Daniele Dell'Orco. « CaF2nanoparticles as surface carriers of GCAP1, a calcium sensor protein involved in retinal dystrophies ». Nanoscale 9, no 32 (2017) : 11773–84. http://dx.doi.org/10.1039/c7nr03288a.

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Biasi, Amedeo, Valerio Marino, Giuditta Dal Cortivo, Paolo Enrico Maltese, Antonio Mattia Modarelli, Matteo Bertelli, Leonardo Colombo et Daniele Dell’Orco. « A Novel GUCA1A Variant Associated with Cone Dystrophy Alters cGMP Signaling in Photoreceptors by Strongly Interacting with and Hyperactivating Retinal Guanylate Cyclase ». International Journal of Molecular Sciences 22, no 19 (6 octobre 2021) : 10809. http://dx.doi.org/10.3390/ijms221910809.

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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.
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Wilkie, Susan E., Inez Stinton, Phillippa Cottrill, Evelyne Deery, Richard Newbold, Martin J. Warren, Shomi S. Bhattacharya et David M. Hunt. « Characterisation of two genes for guanylate cyclase activator protein (GCAP1 and GCAP2) in the Japanese pufferfish, Fugu rubripes ». Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression 1577, no 1 (août 2002) : 73–80. http://dx.doi.org/10.1016/s0167-4781(02)00413-x.

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Vladimirov, Vasiliy I., Viktoriia E. Baksheeva, Irina V. Mikhailova, Ramis G. Ismailov, Ekaterina A. Litus, Natalia K. Tikhomirova, Aliya A. Nazipova, Sergei E. Permyakov, Evgeni Yu Zernii et Dmitry V. Zinchenko. « A Novel Approach to Bacterial Expression and Purification of Myristoylated Forms of Neuronal Calcium Sensor Proteins ». Biomolecules 10, no 7 (10 juillet 2020) : 1025. http://dx.doi.org/10.3390/biom10071025.

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N-terminal myristoylation is a common co-and post-translational modification of numerous eukaryotic and viral proteins, which affects their interaction with lipids and partner proteins, thereby modulating various cellular processes. Among those are neuronal calcium sensor (NCS) proteins, mediating transduction of calcium signals in a wide range of regulatory cascades, including reception, neurotransmission, neuronal growth and survival. The details of NCSs functioning are of special interest due to their involvement in the progression of ophthalmological and neurodegenerative diseases and their role in cancer. The well-established procedures for preparation of native-like myristoylated forms of recombinant NCSs via their bacterial co-expression with N-myristoyl transferase from Saccharomyces cerevisiae often yield a mixture of the myristoylated and non-myristoylated forms. Here, we report a novel approach to preparation of several NCSs, including recoverin, GCAP1, GCAP2, neurocalcin δ and NCS-1, ensuring their nearly complete N-myristoylation. The optimized bacterial expression and myristoylation of the NCSs is followed by a set of procedures for separation of their myristoylated and non-myristoylated forms using a combination of hydrophobic interaction chromatography steps. We demonstrate that the refolded and further purified myristoylated NCS-1 maintains its Ca2+-binding ability and stability of tertiary structure. The developed approach is generally suited for preparation of other myristoylated proteins.
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Newbold, R. J., E. C. Raux, C. E. Walker, S. E. Wilkie, D. M. Hunt, S. S. Bhattacharya et M. J. Warren. « Why a new mutation in human GCAP1 causes retinal degeneration ». Biochemical Society Transactions 28, no 5 (1 octobre 2000) : A380. http://dx.doi.org/10.1042/bst028a380a.

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Lim, Sunghyuk, Igor V. Peshenko, Alexander M. Dizhoor et James B. Ames. « Structural Insights for Activation of Retinal Guanylate Cyclase by GCAP1 ». PLoS ONE 8, no 11 (13 novembre 2013) : e81822. http://dx.doi.org/10.1371/journal.pone.0081822.

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Surguchov, Andrei, J. Darin Bronson, Poulabi Banerjee, James A. Knowles, Claudia Ruiz, Iswari Subbaraya, Krzysztof Palczewski et Wolfgang Baehr. « The Human GCAP1 and GCAP2 Genes Are Arranged in a Tail-to-Tail Array on the Short Arm of Chromosome 6 (p21.1) ». Genomics 39, no 3 (février 1997) : 312–22. http://dx.doi.org/10.1006/geno.1996.4513.

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Sokal, Izabela, Ning Li, Irina Surgucheva, Martin J. Warren, Annette M. Payne, Shomi S. Bhattacharya, Wolfgang Baehr et Krzysztof Palczewski. « GCAP1(Y99C) Mutant Is Constitutively Active in Autosomal Dominant Cone Dystrophy ». Molecular Cell 2, no 1 (juillet 1998) : 129–33. http://dx.doi.org/10.1016/s1097-2765(00)80121-5.

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Peshenko, Igor V., et Alexander M. Dizhoor. « Two clusters of surface-exposed amino acid residues enable high-affinity binding of retinal degeneration-3 (RD3) protein to retinal guanylyl cyclase ». Journal of Biological Chemistry 295, no 31 (3 juin 2020) : 10781–93. http://dx.doi.org/10.1074/jbc.ra120.013789.

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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.
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Jiang, Li, Tansy Z. Li, Shannon E. Boye, William W. Hauswirth, Jeanne M. Frederick et Wolfgang Baehr. « RNAi-Mediated Gene Suppression in a GCAP1(L151F) Cone-Rod Dystrophy Mouse Model ». PLoS ONE 8, no 3 (5 mars 2013) : e57676. http://dx.doi.org/10.1371/journal.pone.0057676.

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Peshenko, Igor V., Elena V. Olshevskaya et Alexander M. Dizhoor. « Binding of Guanylyl Cyclase Activating Protein 1 (GCAP1) to Retinal Guanylyl Cyclase (RetGC1) ». Journal of Biological Chemistry 283, no 31 (9 juin 2008) : 21747–57. http://dx.doi.org/10.1074/jbc.m801899200.

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Peshenko, Igor V., Elena V. Olshevskaya, Sunghyuk Lim, James B. Ames et Alexander M. Dizhoor. « Identification of Target Binding Site in Photoreceptor Guanylyl Cyclase-activating Protein 1 (GCAP1) ». Journal of Biological Chemistry 289, no 14 (24 février 2014) : 10140–54. http://dx.doi.org/10.1074/jbc.m113.540716.

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Dell’Orco, Daniele, Stefan Sulmann, Patrick Zägel, Valerio Marino et Karl-Wilhelm Koch. « Impact of cone dystrophy-related mutations in GCAP1 on a kinetic model of phototransduction ». Cellular and Molecular Life Sciences 71, no 19 (25 février 2014) : 3829–40. http://dx.doi.org/10.1007/s00018-014-1593-4.

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Sokal, Izabela, Ning Li, Candice S. Klug, SBawomir Filipek, Wayne L. Hubbell, Wolfgang Baehr et Krzysztof Palczewski. « Calcium-sensitive Regions of GCAP1 as Observed by Chemical Modifications, Fluorescence, and EPR Spectroscopies ». Journal of Biological Chemistry 276, no 46 (27 août 2001) : 43361–73. http://dx.doi.org/10.1074/jbc.m103614200.

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Yang, Sufang, Alexander Dizhoor, David J. Wilson et Grazyna Adamus. « GCAP1, Rab6, and HSP27 : Novel Autoantibody Targets in Cancer-Associated Retinopathy and Autoimmune Retinopathy ». Translational Vision Science & ; Technology 5, no 3 (2 mai 2016) : 1. http://dx.doi.org/10.1167/tvst.5.3.1.

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Lim, Sunghyuk, Igor V. Peshenko, Alexander M. Dizhoor et James B. Ames. « Backbone 1H, 13C, and 15N resonance assignments of guanylyl cyclase activating protein-1, GCAP1 ». Biomolecular NMR Assignments 7, no 1 (6 mars 2012) : 39–42. http://dx.doi.org/10.1007/s12104-012-9373-2.

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Pertzev, Alexandre, Teresa Duda et Rameshwar K. Sharma. « Ca2+Sensor GCAP1 : A Constitutive Element of the ONE-GC-Modulated Odorant Signal Transduction Pathway ». Biochemistry 49, no 34 (31 août 2010) : 7303–13. http://dx.doi.org/10.1021/bi101001v.

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Coleman, Jason E., Yan Zhang, Gary A. J. Brown et Susan L. Semple-Rowland. « Cone Cell Survival and Downregulation of GCAP1 Protein in the Retinas of GC1 Knockout Mice ». Investigative Opthalmology & ; Visual Science 45, no 10 (1 octobre 2004) : 3397. http://dx.doi.org/10.1167/iovs.04-0392.

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Buch, Prateek K., Marija Mihelec, Phillippa Cottrill, Susan E. Wilkie, Rachael A. Pearson, Yanai Duran, Emma L. West, Michel Michaelides, Robin R. Ali et David M. Hunt. « Dominant Cone-Rod Dystrophy : A Mouse Model Generated by Gene Targeting of the GCAP1/Guca1a Gene ». PLoS ONE 6, no 3 (28 mars 2011) : e18089. http://dx.doi.org/10.1371/journal.pone.0018089.

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Jiang, Li, Dianna Wheaton, Grzegorz Bereta, Kang Zhang, Krzysztof Palczewski, David G. Birch et Wolfgang Baehr. « A novel GCAP1(N104K) mutation in EF-hand 3 (EF3) linked to autosomal dominant cone dystrophy ». Vision Research 48, no 23-24 (octobre 2008) : 2425–32. http://dx.doi.org/10.1016/j.visres.2008.07.016.

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Dell’Orco, Daniele, Petra Behnen, Sara Linse et Karl-Wilhelm Koch. « Calcium binding, structural stability and guanylate cyclase activation in GCAP1 variants associated with human cone dystrophy ». Cellular and Molecular Life Sciences 67, no 6 (7 janvier 2010) : 973–84. http://dx.doi.org/10.1007/s00018-009-0243-8.

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Newbold, R. J. « The destabilization of human GCAP1 by a proline to leucine mutation might cause cone-rod dystrophy ». Human Molecular Genetics 10, no 1 (1 janvier 2001) : 47–54. http://dx.doi.org/10.1093/hmg/10.1.47.

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Abbas, Seher, Valerio Marino, Nicole Weisschuh, Sinja Kieninger, Maria Solaki, Daniele Dell’Orco et Karl-Wilhelm Koch. « Neuronal Calcium Sensor GCAP1 Encoded by GUCA1A Exhibits Heterogeneous Functional Properties in Two Cases of Retinitis Pigmentosa ». ACS Chemical Neuroscience 11, no 10 (16 avril 2020) : 1458–70. http://dx.doi.org/10.1021/acschemneuro.0c00111.

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38

Nishiguchi, Koji M., Izabela Sokal, Lili Yang, Nirmalya Roychowdhury, Krzysztof Palczewski, Eliot L. Berson, Thaddeus P. Dryja et Wolfgang Baehr. « A Novel Mutation (I143NT) in Guanylate Cyclase-Activating Protein 1 (GCAP1) Associated with Autosomal Dominant Cone Degeneration ». Investigative Opthalmology & ; Visual Science 45, no 11 (1 novembre 2004) : 3863. http://dx.doi.org/10.1167/iovs.04-0590.

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39

Sokal, Izabela, William J. Dupps, Michael A. Grassi, Jeremiah Brown, Louisa M. Affatigato, Nirmalya Roychowdhury, Lili Yang et al. « A Novel GCAP1 Missense Mutation (L151F) in a Large Family with Autosomal Dominant Cone-Rod Dystrophy (adCORD) ». Investigative Opthalmology & ; Visual Science 46, no 4 (1 avril 2005) : 1124. http://dx.doi.org/10.1167/iovs.04-1431.

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40

Peshenko, Igor V., Elena V. Olshevskaya, Suxia Yao, Hany H. Ezzeldin, Steven J. Pittler et Alexander M. Dizhoor. « Activation of Retinal Guanylyl Cyclase RetGC1 by GCAP1 : Stoichiometry of Binding and Effect of New LCA-Related Mutations ». Biochemistry 49, no 4 (2 février 2010) : 709–17. http://dx.doi.org/10.1021/bi901495y.

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Lim, Sunghyuk, Igor V. Peshenko, Elena V. Olshevskaya, Alexander M. Dizhoor et James B. Ames. « Structure of Guanylyl Cyclase Activator Protein 1 (GCAP1) Mutant V77E in a Ca2+-free/Mg2+-bound Activator State ». Journal of Biological Chemistry 291, no 9 (24 décembre 2015) : 4429–41. http://dx.doi.org/10.1074/jbc.m115.696161.

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42

Sokal, Izabela, Annie E. Otto-Bruc, Irina Surgucheva, Christophe L. M. J. Verlinde, Chien-Kao Wang, Wolfgang Baehr et Krzysztof Palczewski. « Conformational Changes in Guanylyl Cyclase-activating Protein 1 (GCAP1) and Its Tryptophan Mutants as a Function of Calcium Concentration ». Journal of Biological Chemistry 274, no 28 (9 juillet 1999) : 19829–37. http://dx.doi.org/10.1074/jbc.274.28.19829.

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43

Dal Cortivo, Giuditta, Valerio Marino, Francesco Bonì, Mario Milani et Daniele Dell'Orco. « Missense mutations affecting Ca2+-coordination in GCAP1 lead to cone-rod dystrophies by altering protein structural and functional properties ». Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1867, no 10 (octobre 2020) : 118794. http://dx.doi.org/10.1016/j.bbamcr.2020.118794.

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Wilkie, Susan E., Yang Li, Evelyne C. Deery, Richard J. Newbold, Daniel Garibaldi, J. Bronwyn Bateman, Heidi Zhang et al. « Identification and Functional Consequences of a New Mutation (E155G) in the Gene for GCAP1 That Causes Autosomal Dominant Cone Dystrophy ». American Journal of Human Genetics 69, no 3 (septembre 2001) : 471–80. http://dx.doi.org/10.1086/323265.

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Olshevskaya, E. V., I. V. Peshenko, A. B. Savchenko et A. M. Dizhoor. « Retinal Guanylyl Cyclase Isozyme 1 Is the Preferential In Vivo Target for Constitutively Active GCAP1 Mutants Causing Congenital Degeneration of Photoreceptors ». Journal of Neuroscience 32, no 21 (23 mai 2012) : 7208–17. http://dx.doi.org/10.1523/jneurosci.0976-12.2012.

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Peshenko, Igor V., Artur V. Cideciyan, Alexander Sumaroka, Elena V. Olshevskaya, Alexander Scholten, Seher Abbas, Karl-Wilhelm Koch, Samuel G. Jacobson et Alexander M. Dizhoor. « A G86R mutation in the calcium-sensor protein GCAP1 alters regulation of retinal guanylyl cyclase and causes dominant cone-rod degeneration ». Journal of Biological Chemistry 294, no 10 (8 janvier 2019) : 3476–88. http://dx.doi.org/10.1074/jbc.ra118.006180.

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47

Stephen, Ricardo, Krzysztof Palczewski et Marcelo C. Sousa. « The Crystal Structure of GCAP3 Suggests Molecular Mechanism of GCAP-linked Cone Dystrophies ». Journal of Molecular Biology 359, no 2 (juin 2006) : 266–75. http://dx.doi.org/10.1016/j.jmb.2006.03.042.

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48

Semple-Rowland, Susan L., Wojciech A. Gorczyca, Janina Buczylko, Bharati S. Helekar, Claudia C. Ruiz, Iswari Subbaraya, Krzysztof Palczewski et Wolfgang Baehr. « Expression of GCAP 1 and GCAP2 in the retinal degeneration (rd ) mutant chicken retina ». FEBS Letters 385, no 1-2 (29 avril 1996) : 47–52. http://dx.doi.org/10.1016/0014-5793(96)00345-6.

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49

Solessio, Eduardo, Shobana S. Mani, Nicolas Cuenca, Gustav A. Engbretson, Robert B. Barlow et Barry E. Knox. « Developmental regulation of calcium-dependent feedback in Xenopus rods ». Journal of General Physiology 124, no 5 (25 octobre 2004) : 569–85. http://dx.doi.org/10.1085/jgp.200409162.

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The kinetics of activation and inactivation in the phototransduction pathway of developing Xenopus rods were studied. The gain of the activation steps in transduction (amplification) increased and photoresponses became more rapid as the rods matured from the larval to the adult stage. The time to peak was significantly shorter in adults (1.3 s) than tadpoles (2 s). Moreover, adult rods recovered twice as fast from saturating flashes than did larval rods without changes of the dominant time constant (2.5 s). Guanylate cyclase (GC) activity, determined using IBMX steps, increased in adult rods from ∼1.1 s−1 to 3.7 s−1 5 s after a saturating flash delivering 6,000 photoisomerizations. In larval rods, it increased from 1.8 s−1 to 4.0 s−1 9 s after an equivalent flash. However, the ratio of amplification to the measured dark phosphodiesterase activity was constant. Guanylate cyclase–activating protein (GCAP1) levels and normalized Na+/Ca2+, K+ exchanger currents were increased in adults compared with tadpoles. Together, these results are consistent with the acceleration of the recovery phase in adult rods via developmental regulation of calcium homeostasis. Despite these large changes, the single photon response amplitude was ∼0.6 pA throughout development. Reduction of calcium feedback with BAPTA increased adult single photon response amplitudes threefold and reduced its cutoff frequency to that observed with tadpole rods. Linear mathematical modeling suggests that calcium-dependent feedback can account for the observed differences in the power spectra of larval and adult rods. We conclude that larval Xenopus maximize sensitivity at the expense of slower response kinetics while adults maximize response kinetics at the expense of sensitivity.
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Hoylaerts, M. F., T. Manes et J. L. Millán. « Allelic Amino Acid Substitutions Affect the Conformation and Immunoreactivity of Germ-Cell Alkaline Phosphatase Phenotypes ». Clinical Chemistry 38, no 12 (1 décembre 1992) : 2493–500. http://dx.doi.org/10.1093/clinchem/38.12.2493.

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Abstract The gene encoding placental alkaline phosphatase (PLAP) displays a well-documented allelic polymorphism. Likewise, different phenotypes exist for the PLAP-related germ-cell alkaline phosphatase (GCAP). We investigated the extent to which various allelic GCAP positions are critical in determining the enzymatic, structural, and immunological properties of GCAP phenotypes. Three homozygous GCAP phenotypes [JEG3, BeWo, and wild-type (wt) GCAP] were analyzed and compared with a "core" GCAP mutant that contains the seven amino acid substitutions that are consistently different between PLAP and GCAP but are common to the three known allelic GCAP genotypes. Although some substitutions could influence the electrophoretic behavior of the phenotypes, the allelic differences did not affect the kinetic properties of GCAP. However, they did affect the immunoreactivity and conformation of the variants as detected with a panel of 18 epitope-mapped monoclonal antibodies (MAbs) to PLAP. The selective immunoreactivity of the PLAP/GCAP-discriminating MAb C2 was critically dependent on the nature of the allelic residues 133 and 361 in GCAP. Residue 133 was also important for the general stability of the molecule because BeWo and wt GCAP, which have Asn133 and Val133, respectively, instead of Met133, showed a consistently reduced heat stability compared to core GCAP and JEG3. Because the core GCAP mutant consistently shows the characteristics of wt GCAP, its use as an antigen should allow the generation of monoclonal antibodies to GCAP that will not cross-react with PLAP and whose immunoreactivity will only marginally be influenced by allelic GCAP variation.
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