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1.
J Biol Chem ; 294(10): 3476-3488, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30622141

RESUMO

The guanylyl cyclase-activating protein, GCAP1, activates photoreceptor membrane guanylyl cyclase (RetGC) in the light, when free Ca2+ concentrations decline, and decelerates the cyclase in the dark, when Ca2+ concentrations rise. Here, we report a novel mutation, G86R, in the GCAP1 (GUCA1A) gene in a family with a dominant retinopathy. The G86R substitution in a "hinge" region connecting EF-hand domains 2 and 3 in GCAP1 strongly interfered with its Ca2+-dependent activator-to-inhibitor conformational transition. The G86R-GCAP1 variant activated RetGC at low Ca2+ concentrations with higher affinity than did the WT GCAP1, but failed to decelerate the cyclase at the Ca2+ concentrations characteristic of dark-adapted photoreceptors. Ca2+-dependent increase in Trp94 fluorescence, indicative of the GCAP1 transition to its RetGC inhibiting state, was suppressed and shifted to a higher Ca2+ range. Conformational changes in G86R GCAP1 detectable by isothermal titration calorimetry (ITC) also became less sensitive to Ca2+, and the dose dependence of the G86R GCAP1-RetGC1 complex inhibition by retinal degeneration 3 (RD3) protein was shifted toward higher than normal concentrations. Our results indicate that the flexibility of the hinge region between EF-hands 2 and 3 is required for placing GCAP1-regulated Ca2+ sensitivity of the cyclase within the physiological range of intracellular Ca2+ at the expense of reducing GCAP1 affinity for the target enzyme. The disease-linked mutation of the hinge Gly86, leading to abnormally high affinity for the target enzyme and reduced Ca2+ sensitivity of GCAP1, is predicted to abnormally elevate cGMP production and Ca2+ influx in photoreceptors in the dark.


Assuntos
Cálcio/metabolismo , Distrofias de Cones e Bastonetes/genética , Proteínas Ativadoras de Guanilato Ciclase/genética , Proteínas Ativadoras de Guanilato Ciclase/metabolismo , Guanilato Ciclase/metabolismo , Mutação , Retina/enzimologia , Morte Celular/genética , Distrofias de Cones e Bastonetes/enzimologia , Distrofias de Cones e Bastonetes/metabolismo , Distrofias de Cones e Bastonetes/patologia , Proteínas Ativadoras de Guanilato Ciclase/química , Humanos , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Retina/patologia , Células Fotorreceptoras Retinianas Cones/patologia , Células Fotorreceptoras Retinianas Bastonetes/patologia
2.
J Biol Chem ; 293(45): 17546-17558, 2018 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-30228185

RESUMO

RAB28, a member of the RAS oncogene family, is a ubiquitous, farnesylated, small GTPase of unknown function present in photoreceptors and the retinal pigmented epithelium (RPE). Nonsense mutations of the human RAB28 gene cause recessive cone-rod dystrophy 18 (CRD18), characterized by macular hyperpigmentation, progressive loss of visual acuity, RPE atrophy, and severely attenuated cone and rod electroretinography (ERG) responses. In an attempt to elucidate the disease-causing mechanism, we generated Rab28-/- mice by deleting exon 3 and truncating RAB28 after exon 2. We found that Rab28-/- mice recapitulate features of the human dystrophy (i.e. they exhibited reduced cone and rod ERG responses and progressive retina degeneration). Cones of Rab28-/- mice extended their outer segments (OSs) to the RPE apical processes and formed enlarged, balloon-like distal tips before undergoing degeneration. The visual pigment content of WT and Rab28-/- cones was comparable before the onset of degeneration. Cone phagosomes were almost absent in Rab28-/- mice, whereas rod phagosomes displayed normal levels. A protein-protein interaction screen identified several RAB28-interacting proteins, including the prenyl-binding protein phosphodiesterase 6 δ-subunit (PDE6D) and voltage-gated potassium channel subfamily J member 13 (KCNJ13) present in the RPE apical processes. Of note, the loss of PDE6D prevented delivery of RAB28 to OSs. Taken together, these findings reveal that RAB28 is required for shedding and phagocytosis of cone OS discs.


Assuntos
Fagocitose , Células Fotorreceptoras Retinianas Cones/enzimologia , Epitélio Pigmentado da Retina/enzimologia , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Distrofias de Cones e Bastonetes/enzimologia , Distrofias de Cones e Bastonetes/genética , Distrofias de Cones e Bastonetes/patologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Camundongos , Camundongos Knockout , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Epitélio Pigmentado da Retina/patologia , Células Fotorreceptoras Retinianas Bastonetes/enzimologia , Células Fotorreceptoras Retinianas Bastonetes/patologia , Proteínas rab de Ligação ao GTP/genética
3.
Adv Exp Med Biol ; 1074: 367-373, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29721965

RESUMO

Photoreceptor cell death in inherited retinal degeneration is accompanied by over-activation of histone deacetylases (HDAC). Excessive HDAC activity is found both in primary rod degeneration (such as in the rd10 mouse) and in primary cone death, including the cone photoreceptor function loss 1 (cpfl1) mouse. We evaluated the potential of pharmacological HDAC inhibition to prevent photoreceptor degeneration in primary rod and cone degeneration. We show that a single in vivo treatment of cpfl1 mice with the HDAC inhibitor trichostatin A (TSA) resulted in a significant protection of cpfl1 mutant cones. Similarly, HDAC inhibition with the clinically approved HDAC inhibitor vorinostat (SAHA) resulted in a significant improvement of rod survival in rd10 retinal explant cultures. Altogether, these results highlight the feasibility of targeted neuroprotection in vivo and create hope to maintain vision in patients suffering from both rod and cone dystrophies.


Assuntos
Distrofias de Cones e Bastonetes/prevenção & controle , Inibidores de Histona Desacetilases/uso terapêutico , Ácidos Hidroxâmicos/uso terapêutico , Vorinostat/uso terapêutico , Animais , Animais Congênicos , Morte Celular , Distrofias de Cones e Bastonetes/tratamento farmacológico , Distrofias de Cones e Bastonetes/enzimologia , Distrofias de Cones e Bastonetes/genética , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Inibidores de Histona Desacetilases/administração & dosagem , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/administração & dosagem , Ácidos Hidroxâmicos/farmacologia , Injeções Intravítreas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Técnicas de Cultura de Órgãos , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Células Fotorreceptoras Retinianas Cones/patologia , Células Fotorreceptoras Retinianas Bastonetes/efeitos dos fármacos , Células Fotorreceptoras Retinianas Bastonetes/patologia , Vorinostat/administração & dosagem , Vorinostat/farmacologia
4.
Mol Cell Biochem ; 448(1-2): 91-105, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29427171

RESUMO

This study with recombinant reconstituted system mimicking the cellular conditions of the native cones documents that photoreceptor ROS-GC1 is modulated by gaseous CO2. Mechanistically, CO2 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 Ca2+-modulated phototransduction, the CO2 pathway is Ca2+-independent, yet is linked with it and synergizes it. It, through R787C 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 Ca2+ 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.


Assuntos
Dióxido de Carbono/metabolismo , Anidrase Carbônica II/metabolismo , Distrofias de Cones e Bastonetes/enzimologia , Guanilato Ciclase/metabolismo , Receptores de Superfície Celular/metabolismo , Células Fotorreceptoras Retinianas Cones/enzimologia , Células Fotorreceptoras Retinianas Bastonetes/enzimologia , Animais , Células COS , Anidrase Carbônica II/genética , Catálise , Bovinos , Chlorocebus aethiops , Distrofias de Cones e Bastonetes/genética , Distrofias de Cones e Bastonetes/patologia , GMP Cíclico/genética , GMP Cíclico/metabolismo , Guanilato Ciclase/genética , Proteínas Ativadoras de Guanilato Ciclase/genética , Proteínas Ativadoras de Guanilato Ciclase/metabolismo , Receptores de Superfície Celular/genética , Células Fotorreceptoras Retinianas Cones/patologia , Células Fotorreceptoras Retinianas Bastonetes/patologia
5.
Hum Mol Genet ; 25(20): 4546-4555, 2016 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-28173158

RESUMO

Hereditary retinal degenerations encompass a group of genetic diseases characterized by extreme clinical variability. Following next-generation sequencing and autozygome-based screening of patients presenting with a peculiar, recessive form of cone-dominated retinopathy, we identified five homozygous variants [p.(Asp594fs), p.(Gln117*), p.(Met712fs), p.(Ile756Phe), and p.(Glu543Lys)] in the polyglutamylase-encoding gene TTLL5, in eight patients from six families. The two male patients carrying truncating TTLL5 variants also displayed a substantial reduction in sperm motility and infertility, whereas those carrying missense changes were fertile. Defects in this polyglutamylase in humans have recently been associated with cone photoreceptor dystrophy, while mouse models carrying truncating mutations in the same gene also display reduced fertility in male animals. We examined the expression levels of TTLL5 in various human tissues and determined that this gene has multiple viable isoforms, being highly expressed in testis and retina. In addition, antibodies against TTLL5 stained the basal body of photoreceptor cells in rat and the centrosome of the spermatozoon flagellum in humans, suggesting a common mechanism of action in these two cell types. Taken together, our data indicate that mutations in TTLL5 delineate a novel, allele-specific syndrome causing defects in two as yet pathogenically unrelated functions, reproduction and vision.


Assuntos
Proteínas de Transporte/genética , Distrofias de Cones e Bastonetes/enzimologia , Expressão Gênica , Infertilidade Masculina/enzimologia , Mutação , Adolescente , Adulto , Idoso , Animais , Distrofias de Cones e Bastonetes/genética , Análise Mutacional de DNA , Modelos Animais de Doenças , Proteínas do Olho/genética , Feminino , Homozigoto , Humanos , Infertilidade Masculina/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Especificidade de Órgãos , Linhagem , Células Fotorreceptoras de Vertebrados/enzimologia , Ratos , Motilidade dos Espermatozoides , Espermatozoides/enzimologia , Testículo/enzimologia
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