RESUMEN
In the vertebrate retina, phosphorylation of photoactivated visual pigments in rods and cones by G protein-coupled receptor kinases (GRKs) is essential for sustained visual function. Previous in vitro analysis demonstrated that GRK1 and GRK7 are phosphorylated by PKA, resulting in a reduced capacity to phosphorylate rhodopsin. In vivo observations revealed that GRK phosphorylation occurs in the dark and is cAMP dependent. In many vertebrates, including humans and zebrafish, GRK1 is expressed in both rods and cones while GRK7 is expressed only in cones. However, mice express only GRK1 in both rods and cones and lack GRK7. We recently generated a mutation in Grk1 that deletes the phosphorylation site, Ser21. This mutant demonstrated delayed dark adaptation in mouse rods but not in cones in vivo, suggesting GRK1 may serve a different role depending upon the photoreceptor cell type in which it is expressed. Here, zebrafish were selected to evaluate the role of cAMP-dependent GRK phosphorylation in cone photoreceptor recovery. Electroretinogram analyses of larvae treated with forskolin show that elevated intracellular cAMP significantly decreases recovery of the cone photoresponse, which is mediated by Grk7a rather than Grk1b. Using a cone-specific dominant negative PKA transgene, we show for the first time that PKA is required for Grk7a phosphorylation in vivo. Lastly, immunoblot analyses of rod grk1a-/- and cone grk1b-/- zebrafish and Nrl-/- mouse show that cone-expressed Grk1 does not undergo cAMP-dependent phosphorylation in vivo. These results provide a better understanding of the function of Grk phosphorylation relative to cone adaptation and recovery.
Asunto(s)
Quinasas de Receptores Acoplados a Proteína-G , Células Fotorreceptoras Retinianas Conos , Proteínas de Pez Cebra , Pez Cebra , Animales , Quinasa 1 del Receptor Acoplado a Proteína-G/genética , Quinasa 1 del Receptor Acoplado a Proteína-G/metabolismo , Quinasas de Receptores Acoplados a Proteína-G/genética , Quinasas de Receptores Acoplados a Proteína-G/metabolismo , Fosforilación , Células Fotorreceptoras Retinianas Conos/metabolismo , Rodopsina/genética , Rodopsina/metabolismo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Purpose: Light detection in retinal rod photoreceptors is initiated by activation of the visual pigment rhodopsin. A critical, yet often-overlooked, step enabling efficient perception of light is rhodopsin dephosphorylation mediated by protein phosphatase 2A (PP2A). PP2A deficiency has been reported to impair rhodopsin regeneration after phosphorylation by G protein receptor kinase 1 (GRK1) and binding of arrestin (Arr1), thereby delaying rod dark adaptation. However, its effects on the viability of photoreceptors in the absence of GRK1 and Arr1 remain unclear. Here, we investigated the effects of PP2A deficiency in the absence of GRK1 or Arr1, both of which have been implicated in Oguchi disease, a form of night blindness. Methods: Rod-specific mice lacking the predominant catalytic Cα-subunit of PP2A were crossed with the Grk1-/- or Arr1-/- strains to obtain double knockout lines. Rod photoreceptor viability was analyzed in histological cross-sections of the retina stained with hematoxylin and eosin, and rod function was evaluated by ex vivo electroretinography. Results: PP2A deficiency alone did not impair photoreceptor viability up to 12 months of age. Retinal degeneration was more pronounced in rods lacking GRK1 compared to rods lacking Arr1, and degeneration was accelerated in both Grk1-/- or Arr1-/- strains where PP2A was also deleted. In Arr1-/- mice, rod maximal photoresponse amplitudes were reduced by 80% at 3 months, and this diminution was enhanced further with concomitant PP2A deficiency. Conclusions: These results suggest that although PP2A is not required for the survival of rods, its deletion accelerates the degeneration induced by the absence of either GRK1 or Arr1.
Asunto(s)
Arrestina , Degeneración Retiniana , Animales , Arrestina/metabolismo , Quinasa 1 del Receptor Acoplado a Proteína-G/genética , Ratones , Ratones Noqueados , Proteína Fosfatasa 2 , Degeneración Retiniana/genética , Degeneración Retiniana/metabolismo , Células Fotorreceptoras Retinianas Bastones/fisiología , Rodopsina/metabolismoRESUMEN
BACKGROUND: Oguchi disease is a rare autosomal recessive form of congenital quiescent night blindness. Oguchi disease has been found to be associated with gene mutations in SAG and GRK1, which are vital factors in the recovery phase of phototransduction after light stimuli. We report a case of Oguchi disease with novel heterozygous mutations in SAG. CASE PRESENTATION: A 7-year-old girl with a history of night blindness since childhood, was referred to our hospital. Ophthalmologic examinations included visual acuity, fundus examinations, fundus photography, spectral-domain optical coherence tomography, electroretinographic (ERG). Mutation screening of the SAG and GRK1 genes was performed. This patient exhibited typical clinical characteristics of Oguchi disease, including night blindness, golden fundus with the Mizuo-Nakamura phenomenon, packed structure of the parafovea in optical coherence tomography and reduced a-waves and b-waves in scotopic 3.0 ERG. Genetic testing revealed a heterozygous change in nucleotide c.72_75+15delATCGGTGAGTGGTGCACAA in exon 2 of the SAG gene in this patient, her unaffected mother and younger brother. A splicing alteration of nucleotide c.376-2A>C was identified in exon 6 of the SAG gene with heterozygous status in this patient and her unaffected father. CONCLUSIONS: Compound heterozygosity of a nonsense p.S25X mutation in exon 2 and a splicing alteration in exon 6 of the SAG gene is the cause of this patient with Oguchi type 1 disease in China.
Asunto(s)
Enfermedades Hereditarias del Ojo , Ceguera Nocturna , Arrestinas/genética , Niño , Electrorretinografía , Enfermedades Hereditarias del Ojo/diagnóstico , Enfermedades Hereditarias del Ojo/genética , Femenino , Quinasa 1 del Receptor Acoplado a Proteína-G/genética , Humanos , Mutación , Ceguera Nocturna/diagnóstico , Ceguera Nocturna/genética , LinajeRESUMEN
BACKGROUND: Oguchi disease is a rare autosomal recessive retinal dystrophy, characterized by congenital stationary blindness and caused by pathogenic variants in SAG and GRK1 genes. The present study aimed to report an Italian patient affected by Oguchi disease, evaluated by means of a multimodal retinal imaging study and harboring two novel heterozygous pathogenic variants in the SAG gene. MATERIALS AND METHODS: A 60-year-old female complaining congenital stationary night blindness was investigated through fundus photograph, optical coherence tomography (OCT), electroretinography (ERG), and genetic testing. RESULTS: Fundus examination showed a golden-grayish fundus aspect. The rod response of the scotopic ERG was undetectable and mixed rod-cone response was electronegative. Fundus photographs obtained in light and in prolonged dark-adapted conditions allowed to detect the Mizuo-Nakamura phenomenon. Light condition OCT over the abnormal retinal regions showed high-intensity areas in the outer photoreceptor segment layer, that reduced with prolonged dark adaption. Genetic testing identified two rare heterozygous sequence variants in the SAG gene: NM_000541.5:c.807delA p.(Glu270Lysfs*9) and NM_000541.5:c.1047-1G>C confirming the diagnosis of Oguchi disease. CONCLUSIONS: We identified the first Italian compound heterozygous patient harboring two novel alterations in the SAG gene (a frameshift deletion and a splicing variant). The involvement of the SAG gene in Oguchi disease is a common finding in Japanese population, but rarely identified in Caucasians. Clinical suspicion should prompt the molecular analysis of genes associated with this condition.
Asunto(s)
Enfermedades Hereditarias del Ojo , Ceguera Nocturna , Electrorretinografía , Enfermedades Hereditarias del Ojo/diagnóstico , Enfermedades Hereditarias del Ojo/genética , Femenino , Quinasa 1 del Receptor Acoplado a Proteína-G/genética , Humanos , Persona de Mediana Edad , Mutación , Ceguera Nocturna/diagnóstico , Ceguera Nocturna/genética , Trastornos de la VisiónRESUMEN
Neuronal calcium sensor-1 (NCS-1) is a four-EF-hand ubiquitous signaling protein modulating neuronal function and survival, which participates in neurodegeneration and carcinogenesis. NCS-1 recognizes specific sites on cellular membranes and regulates numerous targets, including G-protein coupled receptors and their kinases (GRKs). Here, with the use of cellular models and various biophysical and computational techniques, we demonstrate that NCS-1 is a redox-sensitive protein, which responds to oxidizing conditions by the formation of disulfide dimer (dNCS-1), involving its single, highly conservative cysteine C38. The dimer content is unaffected by the elevation of intracellular calcium levels but increases to 10-30% at high free zinc concentrations (characteristic of oxidative stress), which is accompanied by accumulation of the protein in punctual clusters in the perinuclear area. The formation of dNCS-1 represents a specific Zn2+-promoted process, requiring proper folding of the protein and occurring at redox potential values approaching apoptotic levels. The dimer binds Ca2+ only in one EF-hand per monomer, thereby representing a unique state, with decreased α-helicity and thermal stability, increased surface hydrophobicity, and markedly improved inhibitory activity against GRK1 due to 20-fold higher affinity towards the enzyme. Furthermore, dNCS-1 can coordinate zinc and, according to molecular modeling, has an asymmetrical structure and increased conformational flexibility of the subunits, which may underlie their enhanced target-binding properties. In HEK293 cells, dNCS-1 can be reduced by the thioredoxin system, otherwise accumulating as protein aggregates, which are degraded by the proteasome. Interestingly, NCS-1 silencing diminishes the susceptibility of Y79 cancer cells to oxidative stress-induced apoptosis, suggesting that NCS-1 may mediate redox-regulated pathways governing cell death/survival in response to oxidative conditions.
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Señalización del Calcio/genética , Quinasa 1 del Receptor Acoplado a Proteína-G/genética , Neoplasias/genética , Proteínas Sensoras del Calcio Neuronal/genética , Neuronas/metabolismo , Neuropéptidos/genética , Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Línea Celular Tumoral , Dimerización , Disulfuros/química , Motivos EF Hand/genética , Células HEK293 , Humanos , Cinética , Neoplasias/patología , Proteínas Sensoras del Calcio Neuronal/antagonistas & inhibidores , Neuronas/química , Neuropéptidos/antagonistas & inhibidores , Oxidación-Reducción , Receptores Acoplados a Proteínas G/genética , Transducción de Señal/genética , Zinc/metabolismoRESUMEN
Eukaryotes generally display a circadian rhythm as an adaption to the reoccurring day/night cycle. This is particularly true for visual physiology that is directly affected by changing light conditions. Here we investigate the influence of the circadian rhythm on the expression and function of visual transduction cascade regulators in diurnal zebrafish and nocturnal mice. We focused on regulators of shut-off kinetics such as Recoverins, Arrestins, Opsin kinases, and Regulator of G-protein signaling that have direct effects on temporal vision. Transcript as well as protein levels of most analyzed genes show a robust circadian rhythm-dependent regulation, which correlates with changes in photoresponse kinetics. Electroretinography demonstrates that photoresponse recovery in zebrafish is delayed in the evening and accelerated in the morning. Functional rhythmicity persists in continuous darkness, and it is reversed by an inverted light cycle and disrupted by constant light. This is in line with our finding that orthologous gene transcripts from diurnal zebrafish and nocturnal mice are often expressed in an anti-phasic daily rhythm.
Asunto(s)
Ritmo Circadiano/efectos de la radiación , Células Fotorreceptoras de Vertebrados/efectos de la radiación , Células Fotorreceptoras Retinianas Conos/efectos de la radiación , Animales , Arrestinas/genética , Arrestinas/metabolismo , Oscuridad , Electrorretinografía , Femenino , Quinasa 1 del Receptor Acoplado a Proteína-G/genética , Quinasa 1 del Receptor Acoplado a Proteína-G/metabolismo , Luz , Fototransducción , Masculino , Ratones , Modelos Animales , Células Fotorreceptoras de Vertebrados/metabolismo , Proteínas RGS/genética , Proteínas RGS/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismo , Visión Ocular/efectos de la radiación , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
Adaptive evolution to cellular stress is a process implicated in a wide range of biological and clinical phenomena. Two major routes of adaptation have been identified: non-genetic changes, which allow expression of different phenotypes in novel environments, and genetic variation achieved by selection of fitter phenotypes. While these processes are broadly accepted, their temporal and epistatic features in the context of cellular evolution and emerging drug resistance are contentious. In this manuscript, we generated hypomorphic alleles of the essential nuclear pore complex (NPC) gene NUP58. By dissecting early and long-term mechanisms of adaptation in independent clones, we observed that early physiological adaptation correlated with transcriptome rewiring and upregulation of genes known to interact with the NPC; long-term adaptation and fitness recovery instead occurred via focal amplification of NUP58 and restoration of mutant protein expression. These data support the concept that early phenotypic plasticity allows later acquisition of genetic adaptations to a specific impairment. We propose this approach as a genetic model to mimic targeted drug therapy in human cells and to dissect mechanisms of adaptation.
Asunto(s)
Adaptación Fisiológica/genética , Alelos , Quinasa 1 del Receptor Acoplado a Proteína-G/genética , Aptitud Genética , N-Glicosil Hidrolasas/genética , Proteínas de Complejo Poro Nuclear/genética , Sistemas CRISPR-Cas , Línea Celular Tumoral , Quinasa 1 del Receptor Acoplado a Proteína-G/metabolismo , Edición Génica , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HCT116 , Células HEK293 , Haploidia , Humanos , Carioferinas/genética , Carioferinas/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Mutación , Células Mieloides/metabolismo , Células Mieloides/patología , N-Glicosil Hidrolasas/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Transducción de Señal , Transcriptoma , Proteína Fluorescente RojaRESUMEN
Biallelic mutations in G-Protein coupled receptor kinase 1 (GRK1) cause Oguchi disease, a rare subtype of congenital stationary night blindness (CSNB). The purpose of this study was to identify disease causing GRK1 variants and use in-depth bioinformatic analyses to evaluate how their impact on protein structure could lead to pathogenicity. Patients' genomic DNA was sequenced by whole genome, whole exome or focused exome sequencing. Disease associated variants, published and novel, were compared to nondisease associated missense variants. The impact of GRK1 missense variants at the protein level were then predicted using a series of computational tools. We identified twelve previously unpublished cases with biallelic disease associated GRK1 variants, including eight novel variants, and reviewed all GRK1 disease associated variants. Further structure-based scoring revealed a hotspot for missense variants in the kinase domain. In addition, to aid future clinical interpretation, we identified the bioinformatics tools best able to differentiate disease associated from nondisease associated variants. We identified GRK1 variants in Oguchi disease patients and investigated how disease-causing variants may impede protein function in-silico.
Asunto(s)
Enfermedades Hereditarias del Ojo , Quinasa 1 del Receptor Acoplado a Proteína-G , Ceguera Nocturna , Enfermedades Hereditarias del Ojo/genética , Quinasa 1 del Receptor Acoplado a Proteína-G/genética , Humanos , Ceguera Nocturna/genéticaRESUMEN
PURPOSE: The available literature regarding Oguchi disease is limited, with around 50 cases described to date. Caused by mutations to either the SAG gene coding for arrestin (Hayashi et al. in Ophthalmic Res 46:175-180, 2011) or the GRK1 gene coding for rhodopsin kinase (Yamamoto et al. in Nat Genet 15:175-178. https://doi.org/10.1038/ng0297-175 , 1997), Oguchi disease is an autosomal recessive condition with a good visual prognosis. The clinical diagnosis of the condition is based on the presence of night blindness (nyctalopia), as well as fundoscopic observation of the Mizuo-Nakamura phenomenon. The Mizuo-Nakamura phenomenon refers to a fundus discolouration described as a golden-brown colour with a yellow-grey metallic sheen most prominent in the peripheral retina; after prolonged dark adaptation, the fundus appears normal. The prevalence of Oguchi disease is highest in Japan, particularly with SAG mutations (Nakazawa et al. in Retina 17:17-22, 1997), although patients from Europe, Pakistan and India have also been described. Formal diagnosis requires genetic testing. METHODS: Wide-field fundus images were obtained in both dark-adapted and light-adapted retina. Optical coherence tomography and dark-adapted electroretinography responses were used to further characterize the clinical phenotype. RESULTS: Existing descriptions of Oguchi disease have been limited by available technology. The flashes required for 45°-montage photographs in a dark-adapted eye quickly cause light adaptation. Recent advances in technology enable the capture of larger retinal areas in a single image. Wide-field 133° images were obtained of the native and dark-adapted fundus in natural colour. To our knowledge, these represent the first reported single-wide-field images of Oguchi disease, showing the characteristic Mizuo-Nakamura phenomenon in true colour. Genetic testing revealed a novel homozygous mutation in GRK1. CONCLUSIONS: Here, we demonstrate how characterizing this condition with single-shot true-colour wide-field imaging has distinct advantages over scanning laser technology, which applies artificial colouration, or stitched true-colour images. Images captured with wide-field systems create a much better representation of the native and dark-adapted fundus than can be observed by the ophthalmologist using direct fundoscopy and are essential in the clinical characterization of new mutations.
Asunto(s)
Enfermedades Hereditarias del Ojo/genética , Enfermedades Hereditarias del Ojo/fisiopatología , Quinasa 1 del Receptor Acoplado a Proteína-G/genética , Mutación , Ceguera Nocturna/genética , Ceguera Nocturna/fisiopatología , Retina/fisiopatología , Adaptación a la Oscuridad , Electrorretinografía , Enfermedades Hereditarias del Ojo/diagnóstico por imagen , Humanos , Masculino , Persona de Mediana Edad , Ceguera Nocturna/diagnóstico por imagen , Oftalmoscopía , Estimulación Luminosa , Retina/diagnóstico por imagen , Tomografía de Coherencia ÓpticaRESUMEN
Timely recovery of the light response in photoreceptors requires efficient inactivation of photoactivated rhodopsin. This process is initiated by phosphorylation of its carboxyl terminus by G protein-coupled receptor kinase 1 (GRK1). Previously, we showed that GRK1 is phosphorylated in the dark at Ser21 in a cAMP-dependent manner and dephosphorylated in the light. Results in vitro indicate that dephosphorylation of Ser21 increases GRK1 activity, leading to increased phosphorylation of rhodopsin. This creates the possibility of light-dependent regulation of GRK1 activity and its efficiency in inactivating the visual pigment. To address the functional role of GRK1 phosphorylation in rods and cones in vivo, we generated mutant mice in which Ser21 is substituted with alanine (GRK1-S21A), preventing dark-dependent phosphorylation of GRK1. GRK1-S21A mice had normal retinal morphology, without evidence of degeneration. The function of dark-adapted GRK1-S21A rods and cones was also unaffected, as demonstrated by the normal amplitude and kinetics of their responses obtained by ex vivo and in vivo ERG recordings. In contrast, rod dark adaptation following exposure to bright bleaching light was significantly delayed in GRK1-S21A mice, suggesting that the higher activity of this kinase results in enhanced rhodopsin phosphorylation and therefore delays its regeneration. In contrast, dark adaptation of cones was unaffected by the S21A mutation. Taken together, these data suggest that rhodopsin phosphorylation/dephosphorylation modulates the recovery of rhodopsin to the ground state and rod dark adaptation. They also reveal a novel role for cAMP-dependent phosphorylation of GRK1 in regulating the dark adaptation of rod but not cone photoreceptors.
Asunto(s)
Adaptación a la Oscuridad/fisiología , Quinasa 1 del Receptor Acoplado a Proteína-G/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismo , Serina/metabolismo , Animales , Quinasa 1 del Receptor Acoplado a Proteína-G/genética , Cinética , Ratones Transgénicos , Fosforilación , Retina/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Rodopsina/metabolismoRESUMEN
"Universal" synthetic antibody (sAB)-based fiducial marks have been generated by customized phage display selections to facilitate the rapid structure determination of G protein-coupled receptor (GPCR) signaling complexes by single-particle cryo-electron microscopy (SP cryo-EM). sABs were generated to the two major G protein subclasses: trimeric Gi and Gs, as well as mini-Gs, and were tested to ensure binding in the context of their cognate GPCRs. Epitope binning revealed that multiple distinct epitopes exist for each G(αßγ) protein. Several Gßγ-specific sABs, cross-reactive between trimeric Gi and Gs, were identified suggesting they could be used across all subclasses in a "plug and play" fashion. sABs were also generated to a representative of another class of GPCR signaling partner, G protein receptor kinase 1 (GRK1) and evaluated further, supporting the generalizability of the approach. EM data suggested that the subclass-specific sABs provide effective single and dual fiducials for multiple GPCR signaling complexes.
Asunto(s)
Anticuerpos/química , Quinasa 1 del Receptor Acoplado a Proteína-G/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gs/química , Biblioteca de Péptidos , Secuencia de Aminoácidos , Anticuerpos/genética , Anticuerpos/metabolismo , Especificidad de Anticuerpos , Sitios de Unión , Clonación Molecular , Microscopía por Crioelectrón , Escherichia coli/genética , Escherichia coli/metabolismo , Quinasa 1 del Receptor Acoplado a Proteína-G/genética , Quinasa 1 del Receptor Acoplado a Proteína-G/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Cinética , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TermodinámicaRESUMEN
G protein-coupled receptor kinase 1 (GRK1) or rhodopsin kinase is under specific control of the neuronal Ca2+-sensor protein recoverin, which is a critical feedback mechanism responsible for the modulation of the shape and sensitivity of the rod cell photoresponse. This process requires the precise matching of interacting protein surfaces and the dynamic changes in protein conformations. Here we study the molecular recognition process of recoverin and GRK1 by testing the hypothesis of a cation-π interaction pair in the recoverin-GRK1 complex. The critical role of residue K192 in recoverin was investigated by site-directed mutagenesis and subsequent structural and functional analysis. The following methods were used: isothermal titration calorimetry, fluorescence and circular dichroism spectroscopy, Ca2+-dependent membrane binding, and protein-protein interaction analysis by back scattering interferometry and surface plasmon resonance. While neutralizing the charge at K in the mutant K192L did not prevent binding of recoverin to GRK1, reversing the charge from K to E led to more distortions in the interaction process, but both mutations increased the stability of the protein conformation. Molecular dynamics simulations provided an explanation for these findings as they let us suggest that residue 192 per se is not a major stabilizer of the interaction between recoverin and its target but rather that the native K is involved in a network of switching electrostatic interactions in wild-type recoverin.
Asunto(s)
Quinasa 1 del Receptor Acoplado a Proteína-G/metabolismo , Recoverina/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Calcio/metabolismo , Bovinos , Escherichia coli/genética , Quinasa 1 del Receptor Acoplado a Proteína-G/química , Quinasa 1 del Receptor Acoplado a Proteína-G/genética , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Mutación Puntual , Unión Proteica , Conformación Proteica , Recoverina/química , Recoverina/genética , Electricidad EstáticaRESUMEN
The tiny picoalga, Ostreococcus tauri, originating from the Thau Lagoon is a member of the marine phytoplankton. Because of its highly reduced genome and small cell size, while retaining the fundamental requirements of a eukaryotic photosynthetic cell, it became a popular model organism for studying photosynthesis or circadian clock-related processes. We analyzed the spectroscopic properties of the photoreceptor domain of the histidine kinase rhodopsin Ot-HKR that is suggested to be involved in the light-induced entrainment of the Ostreococcus circadian clock. We found that the rhodopsin, Ot-Rh, dark state absorbs maximally at 505 nm. Exposure to green-orange light led to the accumulation of a blue-shifted M-state-like absorbance form with a deprotonated Schiff base. This Ot-Rh P400 state had an unusually long lifetime of several minutes. A second long-living photoproduct with a red-shifted absorbance, P560, accumulated upon illumination with blue/UVA light. The resulting photochromicity of the rhodopsin is expected to be advantageous to its function as a molecular control element of the signal transducing HKR domains. The light intensity and the ratio of blue vs green light are reflected by the ratio of rhodopsin molecules in the long-living absorbance forms. Furthermore, dark-state absorbance and the photocycle kinetics vary with the salt content of the environment substantially. This observation is attributed to anion binding in the dark state and a transient anion release during the photocycle, indicating that the salinity affects the photoinduced processes.
Asunto(s)
Proteínas Algáceas/metabolismo , Chlorophyta/metabolismo , Quinasa 1 del Receptor Acoplado a Proteína-G/metabolismo , Histidina Quinasa/metabolismo , Rodopsina/metabolismo , Agua de Mar/microbiología , Proteínas Algáceas/genética , Secuencia de Aminoácidos , Chlorophyta/genética , Chlorophyta/efectos de la radiación , Relojes Circadianos/efectos de la radiación , Ritmo Circadiano/efectos de la radiación , Quinasa 1 del Receptor Acoplado a Proteína-G/genética , Histidina Quinasa/genética , Cinética , Luz , Rodopsina/genética , Salinidad , Agua de Mar/química , Homología de Secuencia de Aminoácido , Transducción de Señal/efectos de la radiación , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Applied Genetic Technologies Corporation (AGTC) is developing a recombinant adeno-associated virus (rAAV) vector AGTC-501, also designated AAV2tYF-GRK1-RPGRco, to treat retinitis pigmentosa (RP) in patients with mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. The vector contains a codon-optimized human RPGR cDNA (RPGRco) driven by a photoreceptor-specific promoter (G protein-coupled receptor kinase 1, GRK1) and is packaged in an AAV2 capsid with three surface tyrosine residues changed to phenylalanine (AAV2tYF). We conducted a safety and potency study of this vector administered by subretinal a injection in the naturally occurring RPGR-deficient Rd9 mouse model. Sixty Rd9 mice (20 per group) received a subretinal injection in the right eye of vehicle (control) or AAV2tYF-GRK1-RPGRco at one of two dose levels (4 × 108 or 4 × 109 vg/eye) and were followed for 12 weeks after injection. Vector injections were well tolerated, with no systemic toxicity. There was a trend towards reduced electroretinography b-wave amplitudes in the high vector dose group that was not statistically significant. There were no clinically important changes in hematology or clinical chemistry parameters and no vector-related ocular changes in life or by histological examination. Dose-dependent RPGR protein expression, mainly in the inner segment of photoreceptors and the adjacent connecting cilium region, was observed in all vector-treated eyes examined. Sequence integrity of the codon-optimized RPGR was confirmed by sequencing of PCR-amplified DNA, or cDNA reverse transcribed from total RNA extracted from vector-treated retinal tissues, and by sequencing of RPGR protein obtained from transfected HEK 293 cells. These results support the use of rAAV2tYF-GRK1-RPGRco in clinical studies in patients with XLRP caused by RPGR mutations.
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Proteínas Portadoras/genética , Dependovirus/genética , Proteínas del Ojo/genética , Quinasa 1 del Receptor Acoplado a Proteína-G/genética , Terapia Genética/métodos , Retinitis Pigmentosa/terapia , Animales , Proteínas Portadoras/metabolismo , Codón/genética , Codón/metabolismo , Dependovirus/metabolismo , Proteínas del Ojo/metabolismo , Quinasa 1 del Receptor Acoplado a Proteína-G/metabolismo , Terapia Genética/efectos adversos , Ratones , Retinitis Pigmentosa/genéticaRESUMEN
The visual systems of snakes are heavily modified relative to other squamates, a condition often thought to reflect their fossorial origins. Further modifications are seen in caenophidian snakes, where evolutionary transitions between rod and cone photoreceptors, termed photoreceptor transmutations, have occurred in many lineages. Little previous work, however, has focused on the molecular evolutionary underpinnings of these morphological changes. To address this, we sequenced seven snake eye transcriptomes and utilized new whole-genome and targeted capture sequencing data. We used these data to analyze gene loss and shifts in selection pressures in phototransduction genes that may be associated with snake evolutionary origins and photoreceptor transmutation. We identified the surprising loss of rhodopsin kinase (GRK1), despite a low degree of gene loss overall and a lack of relaxed selection early during snake evolution. These results provide some of the first evolutionary genomic corroboration for a dim-light ancestor that lacks strong fossorial adaptations. Our results also indicate that snakes with photoreceptor transmutation experienced significantly different selection pressures from other reptiles. Significant positive selection was found primarily in cone-specific genes, but not rod-specific genes, contrary to our expectations. These results reveal potential molecular adaptations associated with photoreceptor transmutation and also highlight unappreciated functional differences between rod- and cone-specific phototransduction proteins. This intriguing example of snake visual system evolution illustrates how the underlying molecular components of a complex system can be reshaped in response to changing selection pressures.
Asunto(s)
Colubridae/genética , Evolución Molecular , Quinasa 1 del Receptor Acoplado a Proteína-G/genética , Selección Genética , Visión Ocular/genética , AnimalesRESUMEN
Purpose: Recessive mutations in the human IQCB1/NPHP5 gene are associated with Senior-Løken syndrome (SLS), a ciliopathy presenting with nephronophthisis and Leber congenital amaurosis (LCA). Nphp5-knockout mice develop LCA without nephronophthisis. Mutant rods rapidly degenerate while mutant cones survive for months. The purpose of this study was to reinitiate cone ciliogenesis in a Nphp5 -/-; Nrl -/- mouse with viral expression of full-length NPHP5 and rescue function. Methods: Nphp5 -/- mice were mated with Nrl -/- mice to generate Nphp5-/-; Nrl-/- double-knockouts. Nphp5-/-; Nrl-/- mice and Nphp5+/-; Nrl-/- controls were phenotyped with confocal microscopy from postnatal day 10 (P10) until 6 months of age. Nphp5-/-; Nrl-/- mice and Nphp5+/-; Nrl-/- controls were injected at P15 with self-complementary adenoassociated virus 8 (Y733F) (AAV8(Y733F)) expressing GRK1-FL-cNPHP5. Expression of mutant NPHP5 was verified with confocal microscopy and electroretinography (ERG). Results: In the Nphp5 -/- and cone-only Nphp5 -/-; Nrl -/- mice, cone outer segments did not form, but mutant cones continued to express cone pigments in the inner segments without obvious signs of cone cell death. The mutant cone outer nuclear layer (ONL) and the inner segments were stable for more than 6 months in the cone-only Nphp5 -/-; Nrl -/- retinas. Viral expression of NPHP5 initiated after eye opening showed that connecting cilia and RP1-positive axonemes were formed. Furthermore, cone pigments and other cone outer segment proteins (cone transducin and cone PDE6) were present in the nascent mutant cone outer segments, and rescued mutant cones exhibited a significant photopic b-wave (30% of Nphp5 +/-; Nrl -/- controls). Conclusions: Nphp5-/-; Nrl-/- cones persistently express cone pigments in the inner segments without obvious degeneration, providing an extended duration interval for viral gene expression. Viral expression of full-length NPHP5 initiates ciliogenesis between P15 and P60, and mutant cones are, in part, functional, encouraging future retina gene replacement therapy.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Proteínas de Unión a Calmodulina/genética , Proteínas del Ojo/genética , Amaurosis Congénita de Leber/genética , Amaurosis Congénita de Leber/terapia , Células Fotorreceptoras Retinianas Conos/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Secuencia de Aminoácidos , Animales , Axonema/metabolismo , Axonema/ultraestructura , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/deficiencia , Proteínas de Unión a Calmodulina/deficiencia , Cilios/metabolismo , Cilios/ultraestructura , Cruzamientos Genéticos , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Modelos Animales de Enfermedad , Proteínas del Ojo/metabolismo , Femenino , Quinasa 1 del Receptor Acoplado a Proteína-G/genética , Quinasa 1 del Receptor Acoplado a Proteína-G/metabolismo , Subunidades alfa de la Proteína de Unión al GTP/genética , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Regulación de la Expresión Génica , Terapia Genética/métodos , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Amaurosis Congénita de Leber/metabolismo , Amaurosis Congénita de Leber/patología , Masculino , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Fenotipo , Células Fotorreceptoras Retinianas Conos/patología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducina/genética , Transducina/metabolismoAsunto(s)
Quinasa 1 del Receptor Acoplado a Proteína-G/genética , Mutación Missense , Ceguera Nocturna/genética , Adulto , Electrorretinografía , Enfermedades Hereditarias del Ojo , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Italia , Ceguera Nocturna/diagnóstico , Ceguera Nocturna/fisiopatología , Retina/fisiopatología , Tomografía de Coherencia Óptica , Campos Visuales/fisiologíaRESUMEN
X-linked retinitis pigmentosa (XLRP) caused by mutations in the RPGR gene is an early onset and severe cause of blindness. Successful proof-of-concept studies in a canine model have recently shown that development of a corrective gene therapy for RPGR-XLRP may now be an attainable goal. In preparation for a future clinical trial, we have here optimized the therapeutic AAV vector construct by showing that GRK1 (rather than IRBP) is a more efficient promoter for targeting gene expression to both rods and cones in non-human primates. Two transgenes were used in RPGR mutant (XLPRA2) dogs under the control of the GRK1 promoter. First was the previously developed stabilized human RPGR (hRPGRstb). Second was a new full-length stabilized and codon-optimized human RPGR (hRPGRco). Long-term (>2 years) studies with an AAV2/5 vector carrying hRPGRstb under control of the GRK1 promoter showed rescue of rods and cones from degeneration and retention of vision. Shorter term (3 months) studies demonstrated comparable preservation of photoreceptors in canine eyes treated with an AAV2/5 vector carrying either transgene under the control of the GRK1 promoter. These results provide the critical molecular components (GRK1 promoter, hRPGRco transgene) to now construct a therapeutic viral vector optimized for RPGR-XLRP patients.
Asunto(s)
Proteínas Portadoras/genética , Proteínas del Ojo/genética , Genes Ligados a X , Terapia Genética , Mutación , Retina/metabolismo , Retinitis Pigmentosa/genética , Animales , Dependovirus/genética , Modelos Animales de Enfermedad , Perros , Quinasa 1 del Receptor Acoplado a Proteína-G/genética , Expresión Génica , Orden Génico , Genes Reporteros , Vectores Genéticos/genética , Humanos , Fenotipo , Células Fotorreceptoras de Vertebrados/metabolismo , Primates , Regiones Promotoras Genéticas , Retinitis Pigmentosa/diagnóstico , Retinitis Pigmentosa/terapia , Transducción Genética , Transgenes , Pruebas de VisiónRESUMEN
Shroom-mediated remodeling of the actomyosin cytoskeleton is a critical driver of cellular shape and tissue morphology that underlies the development of many tissues including the neural tube, eye, intestines, and vasculature. Shroom uses a conserved SD2 domain to direct the subcellular localization of Rho-associated kinase (Rock), which in turn drives changes in the cytoskeleton and cellular morphology through its ability to phosphorylate and activate non-muscle myosin II. Here, we present the structure of the human Shroom-Rock binding module, revealing an unexpected stoichiometry for Shroom in which two Shroom SD2 domains bind independent surfaces on Rock. Mutation of interfacial residues impaired Shroom-Rock binding in vitro and resulted in altered remodeling of the cytoskeleton and loss of Shroom-mediated changes in cellular morphology. Additionally, we provide the first direct evidence that Shroom can function as a Rock activator. These data provide molecular insight into the Shroom-Rock interface and demonstrate that Shroom directly participates in regulating cytoskeletal dynamics, adding to its known role in Rock localization.
Asunto(s)
Quinasa 1 del Receptor Acoplado a Proteína-G/química , Proteínas de la Membrana/química , Proteínas de Microfilamentos/química , Complejos Multiproteicos/química , Quinasa 1 del Receptor Acoplado a Proteína-G/genética , Quinasa 1 del Receptor Acoplado a Proteína-G/metabolismo , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Miosina Tipo II/química , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Dominios Proteicos , Estructura Cuaternaria de Proteína , Relación Estructura-ActividadRESUMEN
Oguchi disease is a rare form of congenital stationary night blindness with an autosomal recessive inheritance pattern. The presence of Santigen (SAG) and Gproteindependent receptor kinase 1 (GRK1) mutations were investigated in the family members with Oguchi disease. All exons of the SAG and GRK1 genes were amplified by polymerase chain reaction and sequenced. The patients were shown to have characteristic clinical features of Oguchi disease. Gene analysis determined a novel GRK1 mutation c.923T>C, which caused Oguchi disease in all siblings. This mutation, was demonstrated by amino acid alignment analysis to be in a phylogenetically conserved region and resulted in an amino acid change from leucine to proline at position 308. Thus, the present study reports a novel missense mutation of GRK1 in the affected members of a consanguineous Turkish family. Homozygosity at position 308, which resides in the catalytic domain of the GRK1 gene, is the cause of Oguchi disease in this Turkish family.