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1.
Nat Commun ; 15(1): 3119, 2024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38600129

RESUMO

Light-driven sodium pumps (NaRs) are unique ion-transporting microbial rhodopsins. The major group of NaRs is characterized by an NDQ motif and has two aspartic acid residues in the central region essential for sodium transport. Here we identify a subgroup of the NDQ rhodopsins bearing an additional glutamic acid residue in the close vicinity to the retinal Schiff base. We thoroughly characterize a member of this subgroup, namely the protein ErNaR from Erythrobacter sp. HL-111 and show that the additional glutamic acid results in almost complete loss of pH sensitivity for sodium-pumping activity, which is in contrast to previously studied NaRs. ErNaR is capable of transporting sodium efficiently even at acidic pH levels. X-ray crystallography and single particle cryo-electron microscopy reveal that the additional glutamic acid residue mediates the connection between the other two Schiff base counterions and strongly interacts with the aspartic acid of the characteristic NDQ motif. Hence, it reduces its pKa. Our findings shed light on a subgroup of NaRs and might serve as a basis for their rational optimization for optogenetics.


Assuntos
Bases de Schiff , ATPase Trocadora de Sódio-Potássio , ATPase Trocadora de Sódio-Potássio/metabolismo , Bases de Schiff/química , Ácido Aspártico , Microscopia Crioeletrônica , Ácido Glutâmico , Rodopsinas Microbianas/metabolismo , Sódio/metabolismo , Rodopsina/química
2.
Curr Biol ; 34(7): 1492-1505.e6, 2024 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-38508186

RESUMO

Vision under dim light relies on primary cilia elaborated by rod photoreceptors in the retina. This specialized sensory structure, called the rod outer segment (ROS), comprises hundreds of stacked, membranous discs containing the light-sensitive protein rhodopsin, and the incorporation of new discs into the ROS is essential for maintaining the rod's health and function. ROS renewal appears to be primarily regulated by extrinsic factors (light); however, results vary depending on different model organisms. We generated two independent transgenic mouse lines where rhodopsin's fate is tracked by a fluorescently labeled rhodopsin fusion protein (Rho-Timer) and show that rhodopsin incorporation into nascent ROS discs appears to be regulated by both external lighting cues and autonomous retinal clocks. Live-cell imaging of the ROS isolated from mice exposed to six unique lighting conditions demonstrates that ROS formation occurs in a periodic manner in cyclic light, constant darkness, and artificial light/dark cycles. This alternating bright/weak banding of Rho-Timer along the length of the ROS relates to inhomogeneities in rhodopsin density and potential points of structural weakness. In addition, we reveal that prolonged dim ambient light exposure impacts not only the rhodopsin content of new discs but also that of older discs, suggesting a dynamic interchange of material between new and old discs. Furthermore, we show that rhodopsin incorporation into the ROS is greatly altered in two autosomal recessive retinitis pigmentosa mouse models, potentially contributing to the pathogenesis. Our findings provide insights into how extrinsic (light) and intrinsic (retinal clocks and genetic mutation) factors dynamically regulate mammalian ROS renewal.


Assuntos
Células Fotorreceptoras Retinianas Bastonetes , Rodopsina , Animais , Camundongos , Luz , Camundongos Transgênicos , Espécies Reativas de Oxigênio/metabolismo , Rodopsina/genética , Rodopsina/metabolismo , Segmento Externo da Célula Bastonete/metabolismo
3.
Exp Eye Res ; 241: 109856, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38479725

RESUMO

Rhodopsin-mediated autosomal dominant retinitis pigmentosa (RHO-adRP) causes progressive vision loss and is potentially incurable, accounting for 25% of adRP cases. Studies on RHO-adRP mechanism were at large based on the biochemical and cellular properties, especially class-3. Nonetheless, the absence of an appropriate model for class-3 RHO-adRP has impeded comprehensive exploration. Here, induced pluripotent stem cells (iPSCs) were generated from a healthy control and two sibling RP patients with the same point mutation, c.403C>T (p.R135W). The first three-dimensional (3D) retinal organoid model of a class-3 RHO point mutation from patient-derived iPSCs was generated. Significant defects were observed in rod photoreceptors in terms of localization, morphology, transcriptional profiling and single cell resolution, to better understand the human disease resulting from RHO mutations from a developmental perspective. This first human model of class-3 RHO-adRP provides a representation of patient's retina in vitro and displays features of RHO-adRP retinal organoids relevant for therapeutic development.


Assuntos
Retina , Retinite Pigmentosa , Humanos , Retinite Pigmentosa/genética , Mutação , Rodopsina/genética , Organoides
4.
Cell Signal ; 118: 111149, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38522808

RESUMO

G protein-coupled receptors (GPCR) and glycosaminoglycans (GAGs) are two essential components of the cell surface that regulate physiological processes in the body. GPCRs are the most extensive family of transmembrane receptors that control cellular responses to extracellular stimuli, while GAGs are polysaccharides that contribute to the function of the extracellular matrix (ECM). Due to their proximity to the plasma membrane, GAGs participate in signal transduction by interacting with various extracellular molecules and cell surface receptors. GAGs can directly interact with certain GPCRs or their ligands (chemokines, peptide hormones and neuropeptides, structural proteins, and enzymes) from the glutamate receptor family, the rhodopsin receptor family, the adhesion receptor family, and the secretin receptor family. These interactions have recently become an emerging topic, providing a new avenue for understanding how GPCR signaling is regulated. This review discusses our current state of knowledge about the role of GAGs in GPCR signaling and function.


Assuntos
Glicosaminoglicanos , Receptores Acoplados a Proteínas G , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia , Membrana Celular/metabolismo , Rodopsina/metabolismo
5.
Genome Biol Evol ; 16(4)2024 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-38451738

RESUMO

Evolutionary convergences are observed at all levels, from phenotype to DNA and protein sequences, and changes at these different levels tend to be correlated. Notably, convergent mutations can lead to convergent changes in phenotype, such as changes in metabolism, drug resistance, and other adaptations to changing environments. We propose a two-component approach to detect mutations subject to convergent evolution in protein alignments. The "Emergence" component selects mutations that emerge more often than expected, while the "Correlation" component selects mutations that correlate with the convergent phenotype under study. With regard to Emergence, a phylogeny deduced from the alignment is provided by the user and is used to simulate the evolution of each alignment position. These simulations allow us to estimate the expected number of mutations in a neutral model, which is compared to the observed number of mutations in the data studied. In Correlation, a comparative phylogenetic approach, is used to measure whether the presence of each of the observed mutations is correlated with the convergent phenotype. Each component can be used on its own, for example Emergence when no phenotype is available. Our method is implemented in a standalone workflow and a webserver, called ConDor. We evaluate the properties of ConDor using simulated data, and we apply it to three real datasets: sedge PEPC proteins, HIV reverse transcriptase, and fish rhodopsin. The results show that the two components of ConDor complement each other, with an overall accuracy that compares favorably to other available tools, especially on large datasets.


Assuntos
Evolução Molecular , Peixes , Animais , Filogenia , Peixes/genética , Rodopsina/genética , Mutação
6.
Biochemistry ; 63(7): 843-854, 2024 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-38458614

RESUMO

Ligand-triggered activation of G protein-coupled receptors (GPCRs) relies on the phenomenon of loose allosteric coupling, which involves conformational alterations spanning from the extracellular ligand-binding domain to the cytoplasmic region, where interactions with G proteins occur. During the GPCR activation process, several intermediate and equilibrium states orchestrate the movement of the flexible and rigid transmembrane (TM) segments of the GPCR. Monitoring early conformational changes is important in unraveling the structural intricacies of the loose allosteric coupling. Here, we focus on the lumi intermediate formed by thermal relaxation from the initial photointermediate, batho in primate green cone pigment (MG), a light-sensitive GPCR responsible for color vision. Our findings from light-induced Fourier transform infrared difference spectroscopy reveal its similarity with rhodopsin, which mediates twilight vision, specifically involving the flip motion of the ß-ionone ring, the relaxation of the torsional structure of the retinal, and local perturbations in the α-helix upon lumi intermediate formation. Conversely, we observe a hydrogen bond modification specific to MG's protonated carboxylic acid, identifying its origin as Glu1022.53 situated in TM2. The weakening of the hydrogen bond strength at Glu1022.53 during the transition from the batho to the lumi intermediates corresponds to a slight outward movement of TM2. Additionally, within the X-ray crystal structure of the rhodopsin lumi intermediate, we note the relocation of the Met862.53 side chain in TM2, expanding the volume of the retinal binding pocket. Consequently, the position of 2.53 emerges as the early step in the conformational shift toward light-induced activation. Moreover, given the prevalence of IR-insensitive hydrophobic amino acids at position 2.53 in many rhodopsin-like GPCRs, including rhodopsin, the hydrogen bond alteration in the C═O stretching band at Glu1022.53 of MG can be used as a probe for tracing conformational changes during the GPCR activation process.


Assuntos
Receptores Acoplados a Proteínas G , Rodopsina , Animais , Rodopsina/química , Ligantes , Espectroscopia de Infravermelho com Transformada de Fourier
7.
Phys Chem Chem Phys ; 26(13): 10343-10356, 2024 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-38501246

RESUMO

Rhodopsins are light-responsive proteins forming two vast and evolutionary distinct superfamilies whose functions are invariably triggered by the photoisomerization of a single retinal chromophore. In 2018 a third widespread superfamily of rhodopsins called heliorhodopsins was discovered using functional metagenomics. Heliorhodopsins, with their markedly different structural features with respect to the animal and microbial superfamilies, offer an opportunity to study how evolution has manipulated the chromophore photoisomerization to achieve adaptation. One question is related to the mechanism of such a reaction and how it differs from that of animal and microbial rhodopsins. To address this question, we use hundreds of quantum-classical trajectories to simulate the spectroscopically documented picosecond light-induced dynamics of a heliorhodopsin from the archaea thermoplasmatales archaeon (TaHeR). We show that, consistently with the observations, the trajectories reveal two excited state decay channels. However, inconsistently with previous hypotheses, only one channel is associated with the -C13C14- rotation of microbial rhodopsins while the second channel is characterized by the -C11C12- rotation typical of animal rhodopsins. The fact that such -C11C12- rotation is aborted upon decay and ground state relaxation, explains why illumination of TaHeR only produces the 13-cis isomer with a low quantum efficiency. We argue that the documented lack of regioselectivity in double-bond excited state twisting motion is the result of an "adaptation" that could be completely lost via specific residue substitutions modulating the steric hindrance experienced along the isomerization motion.


Assuntos
Rodopsina , Rodopsinas Microbianas , Animais , Isomerismo , Rodopsinas Microbianas/química , Rodopsina/química , Rotação
8.
Proc Natl Acad Sci U S A ; 121(12): e2318996121, 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38478688

RESUMO

Bestrhodopsins constitute a class of light-regulated pentameric ion channels that consist of one or two rhodopsins in tandem fused with bestrophin ion channel domains. Here, we report on the isomerization dynamics in the rhodopsin tandem domains of Phaeocystis antarctica bestrhodopsin, which binds all-trans retinal Schiff-base (RSB) absorbing at 661 nm and, upon illumination, converts to the meta-stable P540 state with an unusual 11-cis RSB. The primary photoproduct P682 corresponds to a mixture of highly distorted 11-cis and 13-cis RSB directly formed from the excited state in 1.4 ps. P673 evolves from P682 in 500 ps and contains highly distorted 13-cis RSB, indicating that the 11-cis fraction in P682 converts to 13-cis. Next, P673 establishes an equilibrium with P595 in 1.2 µs, during which RSB converts to 11-cis and then further proceeds to P560 in 48 µs and P540 in 1.0 ms while remaining 11-cis. Hence, extensive isomeric switching occurs on the early ground state potential energy surface (PES) on the hundreds of ps to µs timescale before finally settling on a metastable 11-cis photoproduct. We propose that P682 and P673 are trapped high up on the ground-state PES after passing through either of two closely located conical intersections that result in 11-cis and 13-cis RSB. Co-rotation of C11=C12 and C13=C14 bonds results in a constricted conformational landscape that allows thermal switching between 11-cis and 13-cis species of highly strained RSB chromophores. Protein relaxation may release RSB strain, allowing it to evolve to a stable 11-cis isomeric configuration in microseconds.


Assuntos
Diterpenos , Retinaldeído , Rodopsina , Isomerismo , Conformação Proteica , Rodopsina/metabolismo , Retinaldeído/química
9.
Sci Rep ; 14(1): 6940, 2024 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-38521799

RESUMO

Whole-body physical exercise has been shown to promote retinal structure and function preservation in animal models of retinal degeneration. It is currently unknown how exercise modulates retinal inflammatory responses. In this study, we investigated cytokine alterations associated with retinal neuroprotection induced by voluntary running wheel exercise in a retinal degeneration mouse model of class B1 autosomal dominant retinitis pigmentosa, I307N Rho. I307N Rho mice undergo rod photoreceptor degeneration when exposed to bright light (induced). Our data show, active induced mice exhibited significant preservation of retinal and visual function compared to inactive induced mice after 4 weeks of exercise. Retinal cytokine expression revealed significant reductions of proinflammatory chemokines, keratinocyte-derived chemokine (KC) and interferon gamma inducible protein-10 (IP-10) expression in active groups compared to inactive groups. Through immunofluorescence, we found KC and IP-10 labeling localized to retinal vasculature marker, collagen IV. These data show that whole-body exercise lowers specific retinal cytokine expression associated with retinal vasculature. Future studies should determine whether suppression of inflammatory responses is requisite for exercise-induced retinal protection.


Assuntos
Degeneração Retiniana , Retinite Pigmentosa , Camundongos , Animais , Degeneração Retiniana/metabolismo , Quimiocina CXCL10 , Rodopsina/metabolismo , Retinite Pigmentosa/metabolismo , Modelos Animais de Doenças
10.
J Phys Chem B ; 128(12): 2864-2873, 2024 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-38489248

RESUMO

Bovine rhodopsin is among the most studied proteins in the rhodopsin family. Its primary activation mechanism is the photoisomerization of 11-cis retinal, triggered by the absorption of a UV-visible photon. Different mutants of the same rhodopsin show different absorption wavelengths due to the influence of the specific amino acid residues forming the cavity in which the retinal chromophore is embedded, and rhodopsins activated at different wavelengths are, for example, exploited in the field of optogenetics. In this letter, we present a procedure for systematically investigating color tuning in models of bovine rhodopsin and a set of its mutants embedded in a membrane bilayer. Vertical excitation energy calculations were carried out with the polarizable embedding potential for describing the environment surrounding the chromophore. We show that polarizable embedding outperformed regular electrostatic embedding in determining both the vertical excitation energies and associated oscillator strengths of the systems studied.


Assuntos
Retina , Rodopsina , Animais , Bovinos , Rodopsina/química , Retinaldeído , Fótons
11.
J Phys Chem B ; 128(10): 2389-2397, 2024 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-38433395

RESUMO

The properties of a prosthetic group are broadened by interactions with its neighboring residues in proteins. The retinal chromophore in rhodopsins absorbs light, undergoes structural changes, and drives functionally important structural changes in proteins during the photocycle. It is therefore crucial to understand how chromophore-protein interactions regulate the molecular structure and electronic state of chromophores in rhodopsins. Schizorhodopsin is a newly discovered subfamily of rhodopsins found in the genomes of Asgard archaea, which are extant prokaryotes closest to the last common ancestor of eukaryotes and of other microbial species. Here, we report the effects of a hydrogen bond between a retinal Schiff base and its counterion on the twist of the polyene chain and the color of the retinal chromophore. Correlations between spectral features revealed the unexpected fact that the twist of the polyene chain is reduced as the hydrogen bond becomes stronger, suggesting that the twist is caused by tight atomic contacts between the chromophore and nearby residues. In addition, the strength of the hydrogen bond is the primary factor affecting the color-tuning of the retinal chromophore in schizorhodopsins. The findings of this study are valuable for manipulating the molecular structure and electronic state of the chromophore by controlling chromophore-protein interactions.


Assuntos
Retinaldeído , Rodopsina , Retinaldeído/química , Estrutura Molecular , Polienos , Bases de Schiff/química
12.
Int J Mol Sci ; 25(3)2024 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-38339118

RESUMO

Variants within the Retinitis Pigmentosa GTPase regulator (RPGR) gene are the predominant cause of X-Linked Retinitis Pigmentosa (XLRP), a common and severe form of inherited retinal disease. XLRP is characterised by the progressive degeneration and loss of photoreceptors, leading to visual loss and, ultimately, bilateral blindness. Unfortunately, there are no effective approved treatments for RPGR-associated XLRP. We sought to investigate the efficacy of RPGRORF15 gene supplementation using a clinically relevant construct in human RPGR-deficient retinal organoids (ROs). Isogenic RPGR knockout (KO)-induced pluripotent stem cells (IPSCs) were generated using established CRISPR/Cas9 gene editing methods targeting RPGR. RPGR-KO and isogenic wild-type IPSCs were differentiated into ROs and utilised to test the adeno associated virus (AAV) RPGR (AAV-RPGR) clinical vector construct. The transduction of RPGR-KO ROs using AAV-RPGR successfully restored RPGR mRNA and protein expression and localisation to the photoreceptor connecting cilium in rod and cone photoreceptors. Vector-derived RPGR demonstrated equivalent levels of glutamylation to WT ROs. In addition, treatment with AAV-RPGR restored rhodopsin localisation within RPGR-KO ROs, reducing mislocalisation to the photoreceptor outer nuclear layer. These data provide mechanistic insights into RPGRORF15 gene supplementation functional potency in human photoreceptor cells and support the previously reported Phase I/II trial positive results using this vector construct in patients with RPGR-associated XLRP, which is currently being tested in a Phase III clinical trial.


Assuntos
Opsinas , Retinite Pigmentosa , Humanos , Opsinas/genética , Dependovirus/genética , Dependovirus/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Transporte/metabolismo , Retinite Pigmentosa/genética , Retinite Pigmentosa/terapia , Retinite Pigmentosa/metabolismo , Rodopsina/genética , Terapia Genética/métodos , Mutação
13.
J Mol Evol ; 92(1): 61-71, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38324225

RESUMO

Eukaryotic cells use G protein-coupled receptors (GPCRs) to convert external stimuli into internal signals to elicit cellular responses. However, how mutations in GPCR-coding genes affect GPCR activation and downstream signaling pathways remain poorly understood. Approaches such as deep mutational scanning show promise in investigations of GPCRs, but a high-throughput method to measure rhodopsin activation has yet to be achieved. Here, we scale up a fluorescent reporter assay in budding yeast that we engineered to study rhodopsin's light-activated signal transduction. Using this approach, we measured the mutational effects of over 1200 individual human rhodopsin mutants, generated by low-frequency random mutagenesis of the GPCR rhodopsin (RHO) gene. Analysis of the data in the context of rhodopsin's three-dimensional structure reveals that transmembrane helices are generally less tolerant to mutations compared to flanking helices that face the lipid bilayer, which suggest that mutational tolerance is contingent on both the local environment surrounding specific residues and the specific position of these residues in the protein structure. Comparison of functional scores from our screen to clinically identified rhodopsin disease variants found many pathogenic mutants to be loss of function. Lastly, functional scores from our assay were consistent with a complex counterion mechanism involved in ligand-binding and rhodopsin activation. Our results demonstrate that deep mutational scanning is possible for rhodopsin activation and can be an effective method for revealing properties of mutational tolerance that may be generalizable to other transmembrane proteins.


Assuntos
Receptores Acoplados a Proteínas G , Rodopsina , Humanos , Rodopsina/genética , Rodopsina/química , Rodopsina/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/química , Transdução de Sinais , Estrutura Secundária de Proteína , Mutação
14.
J Phys Chem Lett ; 15(7): 1993-1998, 2024 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-38349321

RESUMO

Microbial rhodopsins are photoreceptors containing the retinal Schiff base chromophore and are ubiquitous among microorganisms. The Schiff base configuration of the chromophore, 15-anti (C═N trans) or 15-syn (C═N cis), is structurally important for their functions, such as membrane ion transport, because this configuration dictates the orientation of the positively charged NH group that interacts with substrate ions. The 15-anti/syn configuration is thus essential for elucidating the ion-transport mechanisms in microbial rhodopsins. Here, we identified the Schiff base configuration during the photoreaction of a sodium pumping rhodopsin from Indibacter alkaliphilus using Raman spectroscopy. We found that the unique configurational change from the 13-cis, 15-anti to all-trans, 15-syn form occurs between the photointermediates termed O1 and O2, which accomplish the Na+ uptake and release, respectively. This isomerization is considered to give rise to the highly irreversible O1 → O2 step that is crucial for unidirectional Na+ transport.


Assuntos
Rodopsina , Bases de Schiff , Rodopsina/química , Bases de Schiff/química , Íons , Transporte de Íons , Rodopsinas Microbianas , Sódio/química
15.
Exp Eye Res ; 240: 109826, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38340947

RESUMO

Retinitis pigmentosa (RP) is an inherited retinal disorder characterized by the degeneration of photoreceptors. RhoP23H/+ mice, which carry a Pro23His mutation in the RHODOPSIN (Rho) gene, are one of the most studied animal models for RP. However, except for the photoreceptors, other retinal neural cells have not been fully investigated in this model. Here, we record the temporal changes of the retina by optical coherence tomography (OCT) imaging of the RhoP23H/+ mice, from early to mid-phase of retinal degeneration. Based on thickness analysis, we identified a natural retinal thickness adaption in wild-type mice during early adulthood and observed morphological compensation of the inner retina layer to photoreceptor degeneration in the RhoP23H/+ mice, primarily on the inner nuclear layer (INL). RhoP23H/+ mice findings were further validated via: histology showing the negative correlation of INL and ONL thicknesses; as well as electroretinogram (ERG) showing an increased b-wave to a-wave ratio. These results unravel the sequential morphologic events in this model and suggest a better understanding of retinal degeneration of RP for future studies.


Assuntos
Degeneração Retiniana , Retinite Pigmentosa , Camundongos , Animais , Degeneração Retiniana/diagnóstico , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Rodopsina/genética , Retina/patologia , Retinite Pigmentosa/genética , Retinite Pigmentosa/patologia , Eletrorretinografia , Modelos Animais de Doenças
16.
Hum Gene Ther ; 35(5-6): 151-162, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38368562

RESUMO

Mutations in the rhodopsin (RHO) gene are the predominant causes of autosomal dominant retinitis pigmentosa (adRP). Given the diverse gain-of-function mutations, therapeutic strategies targeting specific sequences face significant challenges. Here, we provide a universal approach to conquer this problem: we have devised a CRISPR-Cas12i-based, mutation-independent gene knockout and replacement compound therapy carried by a dual AAV2/8 system. In this study, we successfully delayed the progression of retinal degeneration in the classic mouse disease model RhoP23H, and also RhoP347S, a new native mouse mutation model we developed. Our research expands the horizon of potential options for future treatments of RHO-mediated adRP.


Assuntos
Degeneração Retiniana , Retinite Pigmentosa , Camundongos , Animais , Rodopsina/genética , Camundongos Knockout , Retinite Pigmentosa/genética , Retinite Pigmentosa/terapia , Degeneração Retiniana/genética , Degeneração Retiniana/terapia , Mutação , Genes Dominantes
17.
Mol Biol Evol ; 41(2)2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38314890

RESUMO

Intraspecific functional variation is critical for adaptation to rapidly changing environments. For visual opsins, functional variation can be characterized in vitro and often reflects a species' ecological niche but is rarely considered in the context of intraspecific variation or the impact of recent environmental changes on species of cultural or commercial significance. Investigation of adaptation in postglacial lakes can provide key insight into how rapid environmental changes impact functional evolution. Here, we report evidence for molecular adaptation in vision in 2 lineages of Nearctic fishes that are deep lake specialists: ciscoes and deepwater sculpin. We found depth-related variation in the dim-light visual pigment rhodopsin that evolved convergently in these 2 lineages. In vitro characterization of spectral sensitivity of the convergent deepwater rhodopsin alleles revealed blue-shifts compared with other more widely distributed alleles. These blue-shifted rhodopsin alleles were only observed in deep clear postglacial lakes with underwater visual environments enriched in blue light. This provides evidence of remarkably rapid and convergent visual adaptation and intraspecific functional variation in rhodopsin. Intraspecific functional variation has important implications for conservation, and these fishes are of conservation concern and great cultural, commercial, and nutritional importance to Indigenous communities. We collaborated with the Saugeen Ojibway Nation to develop and test a metabarcoding approach that we show is efficient and accurate in recovering the ecological distribution of functionally relevant variation in rhodopsin. Our approach bridges experimental analyses of protein function and genetics-based tools used in large-scale surveys to better understand the ecological extent of adaptive functional variation.


Assuntos
Evolução Molecular , Rodopsina , Animais , Rodopsina/genética , Rodopsina/metabolismo , Peixes/genética , Peixes/metabolismo , Visão Ocular , Ecossistema
18.
Nat Commun ; 15(1): 1451, 2024 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-38365903

RESUMO

Mutations in rhodopsin can cause it to misfold and lead to retinal degeneration. A distinguishing feature of these mutants in vitro is that they mislocalize and aggregate. It is unclear whether or not these features contribute to retinal degeneration observed in vivo. The effect of P23H and G188R misfolding mutations were examined in a heterologous expression system and knockin mouse models, including a mouse model generated here expressing the G188R rhodopsin mutant. In vitro characterizations demonstrate that both mutants aggregate, with the G188R mutant exhibiting a more severe aggregation profile compared to the P23H mutant. The potential for rhodopsin mutants to aggregate in vivo was assessed by PROTEOSTAT, a dye that labels aggregated proteins. Both mutants mislocalize in photoreceptor cells and PROTEOSTAT staining was detected surrounding the nuclei of photoreceptor cells. The G188R mutant promotes a more severe retinal degeneration phenotype and greater PROTEOSTAT staining compared to that promoted by the P23H mutant. Here, we show that the level of PROTEOSTAT positive cells mirrors the progression and level of photoreceptor cell death, which suggests a potential role for rhodopsin aggregation in retinal degeneration.


Assuntos
Degeneração Retiniana , Retinite Pigmentosa , Rodopsina , Animais , Camundongos , Modelos Animais de Doenças , Mutação , Células Fotorreceptoras/metabolismo , Degeneração Retiniana/genética , Retinite Pigmentosa/genética , Retinite Pigmentosa/metabolismo , Rodopsina/genética , Rodopsina/metabolismo , Agregados Proteicos/genética
19.
J Chem Theory Comput ; 20(2): 842-855, 2024 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-38198619

RESUMO

The tuning mechanism of pH can be extremely challenging to model computationally in complex biological systems, especially with respect to the photochemical properties. This article reports a protocol aimed at modeling pH-dependent photodynamics using a combination of constant-pH molecular dynamics and semiclassical nonadiabatic molecular dynamics simulations. With retinal photoisomerization in Anabaena sensory rhodopsin (ASR) as a testbed, we show that our protocol produces pH-dependent photochemical properties, such as the isomerization quantum yield or decay rates. We decompose our results into single-titrated residue contributions, identifying some key tuning amino acids. Additionally, we assess the validity of the single protonation state picture to represent the system at a given pH and propose the most populated protein charge state as a compromise between cost and accuracy.


Assuntos
Anabaena , Rodopsina , Fotoquímica , Rodopsina/química , Anabaena/química , Concentração de Íons de Hidrogênio
20.
Microb Cell Fact ; 23(1): 4, 2024 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-38172917

RESUMO

BACKGROUND: The supply of ATP is a limiting factor for cellular metabolism. Therefore, cell factories require a sufficient ATP supply to drive metabolism for efficient bioproduction. In the current study, a light-driven proton pump in the vacuolar membrane was constructed in yeast to reduce the ATP consumption required by V-ATPase to maintain the acidification of the vacuoles and increase the intracellular ATP supply for bioproduction. RESULTS: Delta rhodopsin (dR), a microbial light-driven proton-pumping rhodopsin from Haloterrigena turkmenica, was expressed and localized in the vacuolar membrane of Saccharomyces cerevisiae by conjugation with a vacuolar membrane-localized protein. Vacuoles with dR were isolated from S. cerevisiae, and the light-driven proton pumping activity was evaluated based on the pH change outside the vacuoles. A light-induced increase in the intracellular ATP content was observed in yeast harboring vacuoles with dR. CONCLUSIONS: Yeast harboring the light-driven proton pump in the vacuolar membrane developed in this study are a potential optoenergetic cell factory suitable for various bioproduction applications.


Assuntos
Saccharomyces cerevisiae , ATPases Vacuolares Próton-Translocadoras , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Vacúolos , Prótons , Rodopsina/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/metabolismo , Trifosfato de Adenosina/metabolismo
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