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1.
Elife ; 102021 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-34605405

RESUMO

PERK is an endoplasmic reticulum (ER) transmembrane sensor that phosphorylates eIF2α to initiate the Unfolded Protein Response (UPR). eIF2α phosphorylation promotes stress-responsive gene expression most notably through the transcription factor ATF4 that contains a regulatory 5' leader. Possible PERK effectors other than ATF4 remain poorly understood. Here, we report that the bZIP transcription factor Xrp1 is required for ATF4-independent PERK signaling. Cell-type-specific gene expression profiling in Drosophila indicated that delta-family glutathione-S-transferases (gstD) are prominently induced by the UPR-activating transgene Rh1G69D. Perk was necessary and sufficient for such gstD induction, but ATF4 was not required. Instead, Perk and other regulators of eIF2α phosphorylation regulated Xrp1 protein levels to induce gstDs. The Xrp1 5' leader has a conserved upstream Open Reading Frame (uORF) analogous to those that regulate ATF4 translation. The gstD-GFP reporter induction required putative Xrp1 binding sites. These results indicate that antioxidant genes are highly induced by a previously unrecognized UPR signaling axis consisting of PERK and Xrp1.


Assuntos
Antioxidantes/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Glutationa Transferase/metabolismo , Discos Imaginais/enzimologia , eIF-2 Quinase/metabolismo , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Estresse do Retículo Endoplasmático , Fator de Iniciação 2 em Eucariotos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Glutationa Transferase/genética , Discos Imaginais/embriologia , Fases de Leitura Aberta , Fosforilação , Rodopsina/genética , Rodopsina/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Resposta a Proteínas não Dobradas , eIF-2 Quinase/genética
2.
Elife ; 102021 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-34665128

RESUMO

Enzymerhodopsins represent a recently discovered class of rhodopsins which includes histidine kinase rhodopsin, rhodopsin phosphodiesterases, and rhodopsin guanylyl cyclases (RGCs). The regulatory influence of the rhodopsin domain on the enzyme activity is only partially understood and holds the key for a deeper understanding of intra-molecular signaling pathways. Here, we present a UV-Vis and FTIR study about the light-induced dynamics of a RGC from the fungus Catenaria anguillulae, which provides insights into the catalytic process. After the spectroscopic characterization of the late rhodopsin photoproducts, we analyzed truncated variants and revealed the involvement of the cytosolic N-terminus in the structural rearrangements upon photo-activation of the protein. We tracked the catalytic reaction of RGC and the free GC domain independently by UV-light induced release of GTP from the photolabile NPE-GTP substrate. Our results show substrate binding to the dark-adapted RGC and GC alike and reveal differences between the constructs attributable to the regulatory influence of the rhodopsin on the conformation of the binding pocket. By monitoring the phosphate rearrangement during cGMP and pyrophosphate formation in light-activated RGC, we were able to confirm the M state as the active state of the protein. The described setup and experimental design enable real-time monitoring of substrate turnover in light-activated enzymes on a molecular scale, thus opening the pathway to a deeper understanding of enzyme activity and protein-protein interactions.


Assuntos
Blastocladiomycota/genética , GMP Cíclico/genética , Proteínas Fúngicas/genética , Guanilato Ciclase/genética , Rodopsina/genética , Blastocladiomycota/metabolismo , GMP Cíclico/metabolismo , Proteínas Fúngicas/metabolismo , Guanilato Ciclase/metabolismo , Rodopsina/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier
3.
Nat Commun ; 12(1): 4934, 2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34400638

RESUMO

Rhodopsin (RHO) gene mutations are a common cause of autosomal dominant retinitis pigmentosa (ADRP). The need to suppress toxic protein expression together with mutational heterogeneity pose challenges for treatment development. Mirtrons are atypical RNA interference effectors that are spliced from transcripts as short introns. Here, we develop a novel mirtron-based knockdown/replacement gene therapy for the mutation-independent treatment of RHO-related ADRP, and demonstrate efficacy in a relevant mammalian model. Splicing and potency of rhodopsin-targeting candidate mirtrons are initially determined, and a mirtron-resistant codon-modified version of the rhodopsin coding sequence is validated in vitro. These elements are then combined within a single adeno-associated virus (AAV) and delivered subretinally in a RhoP23H knock-in mouse model of ADRP. This results in significant mouse-to-human rhodopsin RNA replacement and is associated with a slowing of retinal degeneration. This provides proof of principle that synthetic mirtrons delivered by AAV are capable of reducing disease severity in vivo.


Assuntos
Terapia Genética , RNA/genética , Retinite Pigmentosa/genética , Retinite Pigmentosa/terapia , Animais , Dependovirus/genética , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Vetores Genéticos , Células HEK293 , Humanos , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , RNA/metabolismo , Interferência de RNA , Splicing de RNA , Retina , Degeneração Retiniana , Rodopsina/genética , Rodopsina/metabolismo
4.
Sci Rep ; 11(1): 16356, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381136

RESUMO

Retinitis Pigmentosa (RP) is a blinding disease that arises from loss of rods and subsequently cones. The P23H rhodopsin knock-in (P23H-KI) mouse develops retinal degeneration that mirrors RP phenotype in patients carrying the orthologous variant. Previously, we found that the P23H rhodopsin protein was degraded in P23H-KI retinas, and the Unfolded Protein Response (UPR) promoted P23H rhodopsin degradation in heterologous cells in vitro. Here, we investigated the role of a UPR regulator gene, activating transcription factor 6 (Atf6), in rhodopsin protein homeostasis in heterozygous P23H rhodopsin (Rho+/P23H) mice. Significantly increased rhodopsin protein levels were found in Atf6-/-Rho+/P23H retinas compared to Atf6+/-Rho+/P23H retinas at early ages (~ P12), while rhodopsin mRNA levels were not different. The IRE1 pathway of the UPR was hyper-activated in young Atf6-/-Rho+/P23H retinas, and photoreceptor layer thickness was unchanged at this early age in Rho+/P23H mice lacking Atf6. By contrast, older Atf6-/-Rho+/P23H mice developed significantly increased retinal degeneration in comparison to Atf6+/-Rho+/P23H mice in all retinal layers, accompanied by reduced rhodopsin protein levels. Our findings demonstrate that Atf6 is required for efficient clearance of rhodopsin protein in rod photoreceptors expressing P23H rhodopsin, and that loss of Atf6 ultimately accelerates retinal degeneration in P23H-KI mice.


Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Homeostase/fisiologia , Retina/metabolismo , Retinite Pigmentosa/metabolismo , Rodopsina/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Degeneração Retiniana/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo
5.
J Phys Chem Lett ; 12(27): 6284-6291, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34213348

RESUMO

We report a transient signature in the near-UV absorption of Krokinobacter eikastus rhodopsin 2 (KR2), which spans from the femtosecond up to the millisecond time scale. The signature rises with the all-trans to 13-cis isomerization of retinal and decays with the reisomerization to all-trans in the late photocycle, making it a promising marker band for retinal configuration. Hybrid quantum mechanics/molecular mechanics simulations show that the near-UV absorption signal corresponds to an S0 → S3 and/or an S0 → S5 transition, which is present in all photointermediates. These transitions exhibit a negligible spectral shift by the altering protein environment, in contrast to the main absorption band. This is rationalized by the extension of the transition densities that omits the Schiff base nitrogen. Further characterization and first steps into possible optogenetic applications were performed with near-UV quenching experiments of an induced photostationary state, yielding an ultrafast regeneration of the parent state of KR2.


Assuntos
Absorção Fisico-Química , Flavobacteriaceae/metabolismo , Rodopsina/química , Rodopsina/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Raios Ultravioleta , Membrana Celular/metabolismo , Flavobacteriaceae/citologia , Simulação de Dinâmica Molecular , Conformação Proteica , Análise Espectral
6.
J Chem Phys ; 154(21): 215101, 2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34240976

RESUMO

Photoisomerization in the retinal leads to a channel opening in rhodopsins that triggers translocation or pumping of ions/protons. Crystal structures of rhodopsins contain several structurally conserved water molecules. It has been suggested that water plays an active role in facilitating the ion pumping/translocation process by acting as a lubricant in these systems. In this paper, we systematically investigate the localization, structure, dynamics, and energetics of the water molecules along the channel for the resting/dark state of KR2 rhodopsin. By employing several microseconds long atomistic molecular dynamics simulation of this trans-membrane protein system, we demonstrate the presence of five distinct water containing pockets/cavities separated by gateways controlled by protein side-chains. There exists a strong hydrogen bonded network involving these buried water molecules and functionally important key residues. We present evidence of significant structural and dynamical heterogeneity in the water molecules present in these cavities, with very rare exchange between them. The exchange time scale of such buried water with the bulk has an extremely wide range, from tens of nanoseconds to >1.5 µs. The translational and rotational dynamics of buried water are found to be strongly dependent on the protein cavity size and local interactions with a classic signature of trapped diffusion and rotational anisotropy.


Assuntos
Flavobacteriaceae/química , Simulação de Dinâmica Molecular , Rodopsina/química , Sódio/química , Conformação Proteica , Rodopsina/metabolismo , Sódio/metabolismo , Água/química , Água/metabolismo
7.
Int J Mol Sci ; 22(13)2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201658

RESUMO

The death of photoreceptor cells is induced by continuous light exposure. However, it is unclear whether light damage was induced in retinal ganglion cells with photosensitivity by transduction of optogenetic genes. In this study, we evaluated the phototoxicities of continuous light exposure on retinal ganglion cells after transduction of the optogenetic gene mVChR1 using an adeno-associated virus vector. Rats were exposed to continuous light for a week, and visually evoked potentials (VEPs) were recorded. The intensities of continuous light (500, 1000, 3000, and 5000 lx) increased substantially after VEP recordings. After the final recording of VEPs, retinal ganglion cells (RGCs) were retrogradely labeled with a fluorescein tracer, FluoroGold, and the number of retinal ganglion cells was counted under a fluorescent microscope. There was no significant reduction in the amplitudes of VEPs and the number of RGCs after exposure to any light intensity. These results indicated that RGCs were photosensitive after the transduction of optogenetic genes and did not induce any phototoxicity by continuous light exposure.


Assuntos
Optogenética/métodos , Células Ganglionares da Retina/fisiologia , Rodopsina/genética , Animais , Dependovirus/genética , Potenciais Evocados Visuais , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Células HEK293 , Humanos , Luz/efeitos adversos , Técnicas de Patch-Clamp , Estimulação Luminosa , Ratos , Células Ganglionares da Retina/patologia , Rodopsina/metabolismo , Estilbamidinas/química , Estilbamidinas/metabolismo , Transdução Genética , Volvox/genética
8.
Cell Death Dis ; 12(7): 664, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34215725

RESUMO

Various retinal degenerative disorders manifest in alterations of the AKT/mTOR axis. Despite this, consensus on the therapeutic targeting of mTOR in degenerating retinas has not yet been achieved. Therefore, we investigated the role of AKT/mTOR signaling in rd16 retinas, in which we restored the AKT/mTOR axis by genetic ablation of pseudokinase TRB3, known to inhibit phosphorylation of AKT and mTOR. First, we found that TRB3 ablation resulted in preservation of photoreceptor function in degenerating retinas. Then, we learned that the mTOR downstream cellular pathways involved in the homeostasis of photoreceptors were also reprogrammed in rd16 TRB3-/- retinas. Thus, the level of inactivated translational repressor p-4E-BP1 was significantly increased in these mice along with the restoration of translational rate. Moreover, in rd16 mice manifesting decline in p-mTOR at P15, we found elevated expression of Beclin-1 and ATG5 autophagy genes. Thus, these mice showed impaired autophagy flux measured as an increase in LC3 conversion and p62 accumulation. In addition, the RFP-EGFP-LC3 transgene expression in rd16 retinas resulted in statistically fewer numbers of red puncta in photoreceptors, suggesting impaired late autophagic vacuoles. In contrast, TRIB3 ablation in these mice resulted in improved autophagy flux. The restoration of translation rate and the boost in autophagosome formation occurred concomitantly with an increase in total Ub and rhodopsin protein levels and the elevation of E3 ligase Parkin1. We propose that TRB3 may retard retinal degeneration and be a promising therapeutic target to treat various retinal degenerative disorders.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Células Fotorreceptoras de Vertebrados/enzimologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Degeneração Retiniana/enzimologia , Serina-Treonina Quinases TOR/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Autofagossomos/genética , Autofagossomos/metabolismo , Autofagossomos/patologia , Autofagia , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Proteínas de Ciclo Celular/genética , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Células Fotorreceptoras de Vertebrados/patologia , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Rodopsina/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
9.
Methods Mol Biol ; 2315: 73-97, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302671

RESUMO

With 700 members, G protein-coupled receptors (GPCRs) of the rhodopsin family (class A) form the largest membrane receptor family in humans and are the target of about 30% of presently available pharmaceutical drugs. The recent boom in GPCR structures led to the structural resolution of 57 unique receptors in different states (39 receptors in inactive state only, 2 receptors in active state only and 16 receptors in different activation states). In spite of these tremendous advances, most computational studies on GPCRs, including molecular dynamics simulations, virtual screening and drug design, rely on GPCR models obtained by homology modeling. In this protocol, we detail the different steps of homology modeling with the MODELLER software, from template selection to model evaluation. The present structure boom provides closely related templates for most receptors. If, in these templates, some of the loops are not resolved, in most cases, the numerous available structures enable to find loop templates with similar length for equivalent loops. However, simultaneously, the large number of putative templates leads to model ambiguities that may require additional information based on multiple sequence alignments or molecular dynamics simulations to be resolved. Using the modeling of the human bradykinin receptor B1 as a case study, we show how several templates are managed by MODELLER, and how the choice of template(s) and of template fragments can improve the quality of the models. We also give examples of how additional information and tools help the user to resolve ambiguities in GPCR modeling.


Assuntos
Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Sequência de Aminoácidos , Humanos , Modelos Químicos , Simulação de Dinâmica Molecular , Conformação Proteica , Receptores da Bradicinina/química , Receptores da Bradicinina/metabolismo , Rodopsina/química , Rodopsina/metabolismo , Alinhamento de Sequência/métodos , Homologia de Sequência de Aminoácidos , Software
10.
Exp Eye Res ; 210: 108700, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34245755

RESUMO

Age-related macular degeneration (AMD) is a complex retinal disease with no viable treatment strategy. The causative mechanistic pathway for this disease is not yet clear. Therefore, it is highly warranted to screen effective drugs to treat AMD. Rapamycin are known to inhibit inflammation and has been widely used in the clinic as an immunosuppressant. This study aimed to investigate the protective effect of rapamycin on the AMD retinal degeneration model. The AMD models were established by injection of 35 mg/kg sodium iodate (NaIO3) into the tail vein. Then the treated mice intraperitoneally received rapamycin (2 mg/kg) once a day. The histomorphological analysis showed that rapamycin could inhibit retinal structure damage and apoptosis. Experiments revealed that rapamycin significantly attenuated inflammatory response and oxidative stress. Our experimental results demonstrated that rapamycin has protected the retinal against degeneration induced by NaIO3. The therapeutic effect was more significant after 7 days of treatment. Therefore, our study potentially provides a powerful experimental support for the treatment of AMD.


Assuntos
Modelos Animais de Doenças , Imunossupressores/uso terapêutico , Degeneração Retiniana/prevenção & controle , Epitélio Pigmentado da Retina/efeitos dos fármacos , Sirolimo/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/metabolismo , Marcação In Situ das Extremidades Cortadas , Injeções Intraperitoneais , Iodatos/toxicidade , Masculino , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Retina/metabolismo , Retina/patologia , Degeneração Retiniana/induzido quimicamente , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Rodopsina/metabolismo , cis-trans-Isomerases/metabolismo
11.
Dev Biol ; 478: 205-211, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34265355

RESUMO

Ire1 is an endoplasmic reticulum (ER) transmembrane RNase that cleaves substrate mRNAs to help cells adapt to ER stress. Because there are cell types with physiological ER stress, loss of Ire1 results in metabolic and developmental defects in diverse organisms. In Drosophila, Ire1 mutants show developmental defects at early larval stages and in pupal eye photoreceptor differentiation. These Drosophila studies relied on a single Ire1 loss of function allele with a Piggybac insertion in the coding sequence. Here, we report that an Ire1 allele with a specific impairment in the RNase domain, H890A, unmasks previously unrecognized Ire1 phenotypes in Drosophila eye pigmentation. Specifically, we found that the adult eye pigmentation is altered, and the pigment granules are compromised in Ire1H890A homozygous mosaic eyes. Furthermore, the Ire1H890A mutant eyes had dramatically reduced Rhodopsin-1 protein levels. Drosophila eye pigment granules are most notably associated with late endosome/lysosomal defects. Our results indicate that the loss of Ire1, which would impair ER homeostasis, also results in altered adult eye pigmentation.


Assuntos
Olho Composto de Artrópodes/química , Olho Composto de Artrópodes/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , Pigmentos Biológicos/análise , Alelos , Animais , Olho Composto de Artrópodes/ultraestrutura , Drosophila melanogaster , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Cor de Olho , Mutação , Fenotiazinas/análise , Células Fotorreceptoras de Invertebrados/metabolismo , Pigmentação , Pteridinas/análise , Rodopsina/metabolismo
12.
J Neurosci ; 41(36): 7514-7531, 2021 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-34301828

RESUMO

Primary cilia exhibit a distinct complement of proteins, including G-protein-coupled receptors (GPCRs) that mediate sensory and developmental signals. The localization of GPCRs to the ciliary membrane involves ciliary localization sequences (CLSs), but it is not known how CLSs might relate to cilium type. Here, we studied the localization of two rhodopsin (RHO)-like GPCRs, somatostatin receptor (SSTR3) and RHO, in three types of cilia, from inner medullary collecting duct (IMCD3) cells, hTERT-RPE1 cells (possessing pocket cilia), and rod photoreceptors (whose cilia grow into elaborate phototransductive outer segments). SSTR3 was localized specifically to all three types of cilia, whereas RHO showed more selectivity for the photoreceptor cilium. Focusing on C-terminal CLSs, we characterized a novel CLS in the SSTR3 C terminus, which was required for the robust ciliary localization of SSTR3. Replacing the C terminus of RHO with this SSTR3 CLS-enhanced ciliary localization, compared with full-length RHO in IMCD3 and hTERT-RPE1 cells. Addition of the SSTR3 CLS to the single transmembrane protein CD8A enabled ciliary localization. In hTERT-RPE1 cells, a partial SSTR3 CLS added to CD8A effected specific localization to the periciliary (pocket) membrane, demonstrating C-terminal localization sequence targeting to this domain. Using retinas from mice, including both sexes, we show that deletion of the C terminus of RHO reduced the rod outer segment localization and that addition of the SSTR3 C-terminal CLS to the truncated RHO partly rescued this mislocalization. Overall, the study details elements of the different C termini of SSTR3 and RHO that are major effectors in determining specificity of cilium (or pericilium) localization among different types of cilia.SIGNIFICANCE STATEMENT The localization of G-protein-coupled receptors to primary cilia is key to many types of signal transduction. After characterizing a novel C-terminal CLS in SSTR3, we investigated how SSTR3 and RHO localization to the cilium relates to C-terminal CLSs and to cilium type. We found that the SSTR3 C-terminal CLS was effective in three different types of cilia, but the RHO C terminus showed a clear localization preference for the highly elaborate photoreceptor cilium. When added to CD8A, part of the SSTR3 CLS promoted specific periciliary membrane localization in hTERT-RPE1 cells, demonstrating an effective CLS for this domain. Thus, we demonstrate that elements of the C termini of SSTR3 and RHO determine different localization patterns among different types of cilia.


Assuntos
Cílios/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Somatostatina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Rodopsina/metabolismo , Animais , Linhagem Celular , Humanos , Camundongos , Retina/metabolismo , Transdução de Sinais/fisiologia
13.
Sci Rep ; 11(1): 14765, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34285294

RESUMO

Microbial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an Escherichia coli expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate Oxyrrhis marina (O. marina rhodopsin-2, OmR2) that can be expressed in E. coli cells. E. coli cells harboring the OmR2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified OmR2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-trans retinal chromophore, (2) the possession of the deprotonated counterion (pKa = 3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of OmR2 expressed in E. coli cells could build a basis for understanding and utilizing eukaryotic rhodopsins.


Assuntos
Dinoflagelados/metabolismo , Engenharia de Proteínas/métodos , Rodopsina/metabolismo , Animais , Linhagem Celular , Dinoflagelados/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Evolução Molecular , Camundongos , Processos Fotoquímicos , Bombas de Próton/genética , Bombas de Próton/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Rodopsina/genética , Bases de Schiff
14.
Molecules ; 26(10)2021 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-34069614

RESUMO

Rhodopsin is the G protein-coupled receptor of rod photoreceptor cells that mediates vertebrate vision at low light intensities. Mutations in rhodopsin cause inherited retinal degenerative diseases such as retinitis pigmentosa. Several therapeutic strategies have attempted to address and counteract the deleterious effect of rhodopsin mutations on the conformation and function of this photoreceptor protein, but none has been successful in efficiently preventing retinal degeneration in humans. These approaches include, among others, the use of small molecules, known as pharmacological chaperones, that bind to the receptor stabilizing its proper folded conformation. Valproic acid, in its sodium valproate form, has been used as an anticonvulsant in epileptic patients and in the treatment of several psychiatric disorders. More recently, this compound has been tested as a potential therapeutic agent for the treatment of retinal degeneration associated with retinitis pigmentosa caused by rhodopsin mutations. We now report on the effect of sodium valproate on the conformational stability of heterologously expressed wild-type rhodopsin and a rhodopsin mutant, I307N, which has been shown to be an appropriate model for studying retinal degeneration in mice. We found no sign of enhanced stability for the dark inactive conformation of the I307N mutant. Furthermore, the photoactivated conformation of the mutant appears to be destabilized by sodium valproate as indicated by a faster decay of its active conformation. Therefore, our results support a destabilizing effect of sodium valproate on rhodopsin I307N mutant associated with retinal degeneration. These findings, at the molecular level, agree with recent clinical studies reporting negative effects of sodium valproate on the visual function of retinitis pigmentosa patients.


Assuntos
Receptores Acoplados a Proteínas G/efeitos dos fármacos , Rodopsina/química , Ácido Valproico/farmacologia , Animais , Humanos , Mutação , Conformação Proteica , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Retinite Pigmentosa/metabolismo , Rodopsina/metabolismo
15.
Methods Mol Biol ; 2268: 43-60, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085260

RESUMO

Large-scale recombinant expression of G protein-coupled receptors (GPCRs) is required for structure and function studies where there is a need for milligram amounts of protein in pure form. Here we describe a procedure for the construction of human embryonic kidney 293S (HEK293S) stable cell lines for inducible expression of the gene encoding bovine rhodopsin. The HEK293S cell line is particularly suitable for this application because of several favorable properties as a recombinant host including: its ease of transfection, its capacity for handling large amounts of protein cargo, and its ability to perform the necessary co- and post-translational modifications required for correct folding and processing of complex membrane proteins such as GPCRs. The procedures described here will focus on the HEK293S GnTI- cell line, an HEK293S derivative that is widely used for the production of glycoproteins modified homogeneously with truncated N-glycans.


Assuntos
Processamento de Proteína Pós-Traducional , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes/metabolismo , Rodopsina/metabolismo , Animais , Bovinos , Glicosilação , Células HEK293 , Humanos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Rodopsina/genética , Rodopsina/isolamento & purificação , Transfecção
16.
Nat Commun ; 12(1): 3967, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34172734

RESUMO

Bottom-up and top-down approaches to synthetic biology each employ distinct methodologies with the common aim to harness living systems. Here, we realize a strategic merger of both approaches to convert light into proton gradients for the actuation of synthetic cellular systems. We genetically engineer E. coli to overexpress the light-driven inward-directed proton pump xenorhodopsin and encapsulate them in artificial cell-sized compartments. Exposing the compartments to light-dark cycles, we reversibly switch the pH by almost one pH unit and employ these pH gradients to trigger the attachment of DNA structures to the compartment periphery. For this purpose, a DNA triplex motif serves as a nanomechanical switch responding to the pH-trigger of the E. coli. When DNA origami plates are modified with the pH-sensitive triplex motif, the proton-pumping E. coli can trigger their attachment to giant unilamellar lipid vesicles (GUVs) upon illumination. A DNA cortex is formed upon DNA origami polymerization, which sculpts and deforms the GUVs. We foresee that the combination of bottom-up and top down approaches is an efficient way to engineer synthetic cells.


Assuntos
DNA Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Genética/métodos , Prótons , DNA Bacteriano/química , Concentração de Íons de Hidrogênio , Luz , Microrganismos Geneticamente Modificados , Bombas de Próton/genética , Bombas de Próton/metabolismo , Rodopsina/genética , Rodopsina/metabolismo
17.
Int J Mol Sci ; 22(10)2021 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-34065385

RESUMO

In retinitis pigmentosa (RP), one of many possible genetic mutations causes rod degeneration, followed by cone secondary death leading to blindness. Accumulating evidence indicates that rod death triggers multiple, non-cell-autonomous processes, which include oxidative stress and inflammation/immune responses, all contributing to cone demise. Inflammation relies on local microglia and recruitment of immune cells, reaching the retina through breakdowns of the inner blood retinal barrier (iBRB). Leakage in the inner retina vasculature suggests similarly altered outer BRB, formed by junctions between retinal pigment epithelium (RPE) cells, which are crucial for retinal homeostasis, immune response, and privilege. We investigated the RPE structural integrity in three models of RP (rd9, rd10, and Tvrm4 mice) by immunostaining for zonula occludens-1 (ZO-1), an essential regulatory component of tight junctions. Quantitative image analysis demonstrated discontinuities in ZO-1 profiles in all mutants, despite different degrees of photoreceptor loss. ZO-1 interruption zones corresponded to leakage of in vivo administered, fluorescent dextran through the choroid-RPE interface, demonstrating barrier dysfunction. Dexamethasone, administered to rd10 mice for rescuing cones, also rescued RPE structure. Thus, previously undetected, stereotyped abnormalities occur in the RPE of RP mice; pharmacological targeting of inflammation supports a feedback loop leading to simultaneous protection of cones and the RPE.


Assuntos
Retina/fisiopatologia , Epitélio Pigmentado da Retina/fisiopatologia , Retinite Pigmentosa/fisiopatologia , Animais , Dexametasona/farmacologia , Modelos Animais de Doenças , Estudos de Avaliação como Assunto , Inflamação/metabolismo , Inflamação/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Retina/efeitos dos fármacos , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Células Fotorreceptoras Retinianas Cones/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/efeitos dos fármacos , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Vasos Retinianos/efeitos dos fármacos , Vasos Retinianos/metabolismo , Retinite Pigmentosa/metabolismo , Rodopsina/metabolismo , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo
18.
PLoS Genet ; 17(6): e1009613, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34161320

RESUMO

Color vision in Drosophila melanogaster is based on the expression of five different color-sensing Rhodopsin proteins in distinct subtypes of photoreceptor neurons. Promoter regions of less than 300 base pairs are sufficient to reproduce the unique, photoreceptor subtype-specific rhodopsin expression patterns. The underlying cis-regulatory logic remains poorly understood, but it has been proposed that the rhodopsin promoters have a bipartite structure: the distal promoter region directs the highly restricted expression in a specific photoreceptor subtype, while the proximal core promoter region provides general activation in all photoreceptors. Here, we investigate whether the rhodopsin promoters exhibit a strict specialization of their distal (subtype specificity) and proximal (general activation) promoter regions, or if both promoter regions contribute to generating the photoreceptor subtype-specific expression pattern. To distinguish between these two models, we analyze the expression patterns of a set of hybrid promoters that combine the distal promoter region of one rhodopsin with the proximal core promoter region of another rhodopsin. We find that the function of the proximal core promoter regions extends beyond providing general activation: these regions play a previously underappreciated role in generating the non-overlapping expression patterns of the different rhodopsins. Therefore, cis-regulatory motifs in both the distal and the proximal core promoter regions recruit transcription factors that generate the unique rhodopsin patterns in a combinatorial manner. We compare this combinatorial regulatory logic to the regulatory logic of olfactory receptor genes and discuss potential implications for the evolution of rhodopsins.


Assuntos
Visão de Cores/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Células Fotorreceptoras de Invertebrados/metabolismo , Regiões Promotoras Genéticas , Rodopsina/genética , Fatores de Transcrição/genética , Animais , Sequência de Bases , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Evolução Molecular , Feminino , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células Fotorreceptoras de Invertebrados/classificação , Células Fotorreceptoras de Invertebrados/citologia , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Rodopsina/metabolismo , Fatores de Transcrição/metabolismo
19.
Artigo em Inglês | MEDLINE | ID: mdl-34126231

RESUMO

It is not well understood how the spectral composition (wavelength) of daylight that varies considerably during the day and seasons affects photoperiodic responses in a seasonal species. Here, we investigated the molecular underpinnings of wavelength-dependent photoperiodic induction in migratory redheaded buntings transferred to 13 h long days in neutral (white), 460 nm (blue), 500 nm (green) or 620 nm (red) wavelength that were compared with one another, and to short day controls for indices of the migratory (body fattening and weight gain, and Zugunruhe) and reproductive (testicular maturation) responses. Buntings showed wavelength-dependent photoperiodic response, with delayed Zugunruhe and slower testis maturation under 620 nm red light. Post-mortem comparison of gene expressions further revealed wavelength-dependence of the photoperiodic molecular response. Whereas there were higher retinal expressions of opn2 (rhodopsin) and opn5 (neuropsin) genes in red daylight, and of rhodopsin-like opsin (rh2) gene in green daylight, the hypothalamic opn2 mRNA levels were higher in blue daylight. Similarly, we found in birds under blue daylight an increased hypothalamic expression of genes involved in the photoperiodic induction (thyroid stimulating hormone subunit beta, tshb; eye absent 3, eya3; deiodinase type 2, dio2) and associated neural responses such as the calcium signaling (ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2, atp2a2), dopamine biosynthesis (tyrosine hydroxylase, th) and neurogenesis (brain-derived neurotrophic factor, bdnf). These results demonstrate transcriptional changes in parallel to responses associated with migration and reproduction in buntings, and suggest a role of daylight spectrum in photoperiodic induction of the vernal response in obligate spring avian migrants.


Assuntos
Migração Animal , Luz , Fotoperíodo , Rodopsina/fisiologia , Estações do Ano , Aves Canoras/fisiologia , Animais , Comportamento Animal , Encéfalo/metabolismo , Sinalização do Cálcio , Ritmo Circadiano/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Hipotálamo/metabolismo , Índia , Masculino , Opsinas , Fenótipo , Retina/metabolismo , Rodopsina/metabolismo , Testículo/crescimento & desenvolvimento
20.
J Cell Biol ; 220(8)2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34132745

RESUMO

Photoreceptors rely on distinct membrane compartments to support their specialized function. Unlike protein localization, identification of critical differences in membrane content has not yet been expanded to lipids, due to the difficulty of isolating domain-specific samples. We have overcome this by using SMA to coimmunopurify membrane proteins and their native lipids from two regions of photoreceptor ROS disks. Each sample's copurified lipids were subjected to untargeted lipidomic and fatty acid analysis. Extensive differences between center (rhodopsin) and rim (ABCA4 and PRPH2/ROM1) samples included a lower PC to PE ratio and increased LC- and VLC-PUFAs in the center relative to the rim region, which was enriched in shorter, saturated FAs. The comparatively few differences between the two rim samples likely reflect specific protein-lipid interactions. High-resolution profiling of the ROS disk lipid composition gives new insights into how intricate membrane structure and protein activity are balanced within the ROS, and provides a model for future studies of other complex cellular structures.


Assuntos
Membrana Celular/metabolismo , Proteínas do Olho/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Animais , Bovinos , Membrana Celular/ultraestrutura , Proteínas do Olho/imunologia , Lipidômica , Proteínas de Membrana/imunologia , Camundongos Endogâmicos BALB C , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Nanotecnologia , Periferinas/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/ultraestrutura , Rodopsina/metabolismo , Anticorpos de Domínio Único/imunologia , Tetraspaninas/metabolismo
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