Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 2.354
Filtrar
1.
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
2.
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
3.
Proc Natl Acad Sci U S A ; 120(50): e2314698120, 2023 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-38064509

RESUMO

Mutations in many visual cycle enzymes in photoreceptors and retinal pigment epithelium (RPE) cells can lead to the chronic accumulation of toxic retinoid byproducts, which poison photoreceptors and the underlying RPE if left unchecked. Without a functional ATP-binding cassette, sub-family A, member 4 (ABCA4), there is an elevation of all-trans-retinal and prolonged buildup of all-trans-retinal adducts, resulting in a retinal degenerative disease known as Stargardt-1 disease. Even in this monogenic disorder, there is significant heterogeneity in the time to onset of symptoms among patients. Using a combination of molecular techniques, we studied Abca4 knockout (simulating human noncoding disease variants) and Abca4 knock-in mice (simulating human misfolded, catalytically inactive protein variants), which serve as models for Stargardt-1 disease. We compared the two strains to ascertain whether they exhibit differential responses to agents that affect cytokine signaling and/or ceramide metabolism, as alterations in either of these pathways can exacerbate retinal degenerative phenotypes. We found different degrees of responsiveness to maraviroc, a known immunomodulatory CCR5 antagonist, and to the ceramide-lowering agent AdipoRon, an agonist of the ADIPOR1 and ADIPOR2 receptors. The two strains also display different degrees of transcriptional deviation from matched WT controls. Our phenotypic comparison of the two distinct Abca4 mutant-mouse models sheds light on potential therapeutic avenues previously unexplored in the treatment of Stargardt disease and provides a surrogate assay for assessing the effectiveness for genome editing.


Assuntos
Degeneração Macular , Degeneração Retiniana , Humanos , Camundongos , Animais , Doença de Stargardt/metabolismo , Degeneração Macular/tratamento farmacológico , Degeneração Macular/genética , Degeneração Macular/metabolismo , Retinaldeído/metabolismo , Retina/metabolismo , Degeneração Retiniana/tratamento farmacológico , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Modelos Animais de Doenças , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo
4.
J Phys Chem B ; 127(46): 9873-9886, 2023 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-37940604

RESUMO

Photoisomerization of an all-trans-retinal chromophore triggers ion transport in microbial ion-pumping rhodopsins. Understanding chromophore structures in the electronically excited (S1) state provides insights into the structural evolution on the potential energy surface of the photoexcited state. In this study, we examined the structure of the S1-state chromophore in Natronomonas pharaonis halorhodopsin (NpHR), a chloride ion-pumping rhodopsin, using time-resolved resonance Raman spectroscopy. The spectral patterns of the S1-state chromophore were completely different from those of the ground-state chromophore, resulting from unique vibrational characteristics and the structure of the S1 state. Mode assignments were based on a combination of deuteration shifts of the Raman bands and hybrid quantum mechanics-molecular mechanics calculations. The present observations suggest a weakened bond alternation in the π conjugation system. A strong hydrogen-out-of-plane bending band was observed in the Raman spectra of the S1-state chromophore in NpHR, indicating a twisted polyene structure. Similar frequency shifts for the C═N/C═C and C-C stretching modes of the S1-state chromophore in NpHR were observed in the Raman spectra of sodium ion-pumping and proton-pumping rhodopsins, suggesting that these unique features are common to the S1 states of ion-pumping rhodopsins.


Assuntos
Rodopsina , Rodopsinas Microbianas , Rodopsina/química , Retinaldeído/química , Halorrodopsinas/química
5.
Curr Biol ; 33(21): 4733-4740.e4, 2023 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-37776863

RESUMO

Animals with enhanced dim-light sensitivity are at higher risk of light-induced retinal degeneration when exposed to bright light conditions.1,2,3,4 This trade-off is mediated by the rod photoreceptor sensory protein, rhodopsin (RHO), and its toxic vitamin A chromophore by-product, all-trans retinal.5,6,7,8 Rod arrestin (Arr-1) binds to RHO and promotes sequestration of excess all-trans retinal,9,10 which has recently been suggested as a protective mechanism against photoreceptor cell death.2,11 We investigated Arr-1 evolution in animals at high risk of retinal damage due to periodic bright-light exposure of rod-dominated retinas. Here, we find the convergent evolution of enhanced Arr-1/RHO all-trans-retinal sequestration in owls and deep-diving whales. Statistical analyses reveal a parallel acceleration of Arr-1 evolutionary rates in these lineages, which is associated with the introduction of a rare Arr-1 mutation (Q69R) into the RHO-Arr-1 binding interface. Using in vitro assays, we find that this single mutation significantly enhances RHO-all-trans-retinal sequestration by ∼30%. This functional convergence across 300 million years of evolutionary divergence suggests that Arr-1 and RHO may play an underappreciated role in the photoprotection of the eye, with potentially vast clinical significance.


Assuntos
Degeneração Retiniana , Estrigiformes , Animais , Estrigiformes/metabolismo , Retinaldeído/metabolismo , Baleias , Células Fotorreceptoras Retinianas Bastonetes , Retina/metabolismo , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Rodopsina/metabolismo
6.
J Transl Med ; 21(1): 546, 2023 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-37587475

RESUMO

BACKGROUND: ABCA4, the gene implicated in Stargardt disease (STGD1), contains 50 exons, of which 17 contain multiples of three nucleotides. The impact of in-frame exon skipping is yet to be determined. Antisense oligonucleotides (AONs) have been investigated in Usher syndrome-associated genes to induce skipping of in-frame exons carrying severe variants and mitigate their disease-linked effect. Upon the identification of a STGD1 proband carrying a novel exon 17 canonical splice site variant, the activity of ABCA4 lacking 22 amino acids encoded by exon 17 was examined, followed by design of AONs able to induce exon 17 skipping. METHODS: A STGD1 proband was compound heterozygous for the splice variant c.2653+1G>A, that was predicted to result in in-frame skipping of exon 17, and a null variant [c.735T>G, p.(Tyr245*)]. Clinical characteristics of this proband were studied using multi-modal imaging and complete ophthalmological examination. The aberrant splicing of c.2653+1G>A was investigated in vitro in HEK293T cells with wild-type and mutant midigenes. The residual activity of the mutant ABCA4 protein lacking Asp864-Gly885 encoded by exon 17 was analyzed with all-trans-retinal-activated ATPase activity assay, along with its subcellular localization. To induce exon 17 skipping, the effect of 40 AONs was examined in vitro in WT WERI-Rb-1 cells and 3D human retinal organoids. RESULTS: Late onset STGD1 in the proband suggests that c.2653+1G>A does not have a fully deleterious effect. The in vitro splice assay confirmed that this variant leads to ABCA4 transcripts without exon 17. ABCA4 Asp864_Gly863del was stable and retained 58% all-trans-retinal-activated ATPase activity compared to WT ABCA4. This sequence is located in an unstructured linker region between transmembrane domain 6 and nucleotide-binding domain-1 of ABCA4. AONs were designed to possibly reduce pathogenicity of severe variants harbored in exon 17. The best AON achieved 59% of exon 17 skipping in retinal organoids. CONCLUSIONS: Exon 17 deletion in ABCA4 does not result in the absence of protein activity and does not cause a severe STGD1 phenotype when in trans with a null allele. By applying AONs, the effect of severe variants in exon 17 can potentially be ameliorated by exon skipping, thus generating partial ABCA4 activity in STGD1 patients.


Assuntos
Adenosina Trifosfatases , Retinaldeído , Humanos , Doença de Stargardt/genética , Células HEK293 , Éxons/genética , Proteínas Mutantes , Transportadores de Cassetes de Ligação de ATP/genética
7.
Cell Rep ; 42(8): 112982, 2023 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-37585292

RESUMO

In daylight, demand for visual chromophore (11-cis-retinal) exceeds supply by the classical visual cycle. This shortfall is compensated, in part, by the retinal G-protein-coupled receptor (RGR) photoisomerase, which is expressed in both the retinal pigment epithelium (RPE) and in Müller cells. The relative contributions of these two cellular pools of RGR to the maintenance of photoreceptor light responses are not known. Here, we use a cell-specific gene reactivation approach to elucidate the kinetics of RGR-mediated recovery of photoreceptor responses following light exposure. Electroretinographic measurements in mice with RGR expression limited to either cell type reveal that the RPE and a specialized subset of Müller glia contribute both to scotopic and photopic function. We demonstrate that 11-cis-retinal formed through photoisomerization is rapidly hydrolyzed, consistent with its role in a rapid visual pigment regeneration process. Our study shows that RGR provides a pan-retinal sink for all-trans-retinal released under sustained light conditions and supports rapid chromophore regeneration through the photic visual cycle.


Assuntos
Epitélio Pigmentado da Retina , Retinaldeído , Animais , Camundongos , Epitélio Pigmentado da Retina/metabolismo , Retinaldeído/metabolismo , Pigmentos da Retina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Neuroglia/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo
8.
Tissue Eng Regen Med ; 20(6): 951-964, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37440108

RESUMO

BACKGROUND: Retinal degenerative disease (RDD), one of the most common causes of blindness, is predominantly caused by the gradual death of retinal pigment epithelial cells (RPEs) and photoreceptors due to various causes. Cell-based therapies, such as stem cell implantation, have been developed for the treatment of RDD, but potential risks, including teratogenicity and immune reactions, have hampered their clinical application. Stem cell-derived extracellular vesicles (EVs) have recently emerged as a cell-free alternative therapeutic strategy; however, additional invasiveness and low yield of the stem cell extraction process is problematic. METHODS: To overcome these limitations, we developed therapeutic EVs for the treatment of RDD which were extracted from tonsil-derived mesenchymal stem cells obtained from human tonsil tissue discarded as medical waste following tonsillectomy (T-MSC EVs). To verify the biocompatibility and cytoprotective effect of T-MSC EVs, we measured cell viability by co-culture with human RPE without or with toxic all-trans-retinal. To elucidate the cytoprotective mechanism of T-MSC EVs, we performed transcriptome sequencing using RNA extracted from RPEs. The in vivo protective effect of T-MSC EVs was evaluated using Pde6b gene knockout rats as an animal model of retinitis pigmentosa. RESULTS: T-MSC EVs showed high biocompatibility and the human pigment epithelial cells were significantly protected in the presence of T-MSC EVs from the toxic effect of all-trans-retinal. In addition, T-MSC EVs showed a dose-dependent cell death-delaying effect in real-time quantification of cell death. Transcriptome sequencing analysis revealed that the efficient ability of T-MSC EVs to regulate intracellular oxidative stress may be one of the reasons explaining their excellent cytoprotective effect. Additionally, intravitreally injected T-MSC EVs had an inhibitory effect on the destruction of the outer nuclear layer in the Pde6b gene knockout rat. CONCLUSIONS: Together, the results of this study indicate the preventive and therapeutic effects of T-MSC EVs during the initiation and development of retinal degeneration, which may be a beneficial alternative for the treatment of RDD.


Assuntos
Vesículas Extracelulares , Células-Tronco Mesenquimais , Degeneração Retiniana , Humanos , Ratos , Animais , Degeneração Retiniana/terapia , Degeneração Retiniana/metabolismo , Tonsila Palatina , Retinaldeído/metabolismo , Vesículas Extracelulares/metabolismo , Células-Tronco Mesenquimais/metabolismo
9.
Bioessays ; 45(9): e2300068, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37454357

RESUMO

The photocycle of visual opsins is essential to maintain the light sensitivity of the retina. The early physical observations of the rhodopsin photocycle by Böll and Kühne in the 1870s inspired over a century's worth of investigations on rhodopsin biochemistry. A single photon isomerizes the Schiff-base linked 11-cis-retinylidene chromophore of rhodopsin, converting it to the all-trans agonist to elicit phototransduction through photoactivated rhodopsin (Rho*). Schiff base hydrolysis of the agonist is a key step in the photocycle, not only diminishing ongoing phototransduction but also allowing for entry and binding of fresh 11-cis chromophore to regenerate the rhodopsin pigment and maintain light sensitivity. Many challenges have been encountered in measuring the rate of this hydrolysis, but recent advancements have facilitated studies of the hydrolysis within the native membrane environment of rhodopsin. These techniques can now be applied to study hydrolysis of agonist in other opsin proteins that mediate phototransduction or chromophore turnover. In this review, we discuss the progress that has been made in characterizing the rhodopsin photocycle and the journey to characterize the hydrolysis of its all-trans-retinylidene agonist.


Assuntos
Fotofobia , Rodopsina , Humanos , Rodopsina/metabolismo , Retinaldeído/química , Retinaldeído/metabolismo , Retina
10.
Photochem Photobiol Sci ; 22(11): 2499-2517, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37498510

RESUMO

Water is usually indispensable for protein function. For ion-pumping rhodopsins, water molecules inside the proteins play an important role in ion transportation. In addition to amino acid residues, water molecules regulate the colors of retinal proteins. It was reported that a sodium-pumping rhodopsin, Krokinobacter eikastus rhodopsin 2 (KR2), showed a color change from red to purple upon dehydration under crystalline conditions. Here, we applied comprehensive visible and IR absorption spectroscopy and resonance Raman spectroscopy to KR2 in liposomes under hydration-controlled conditions. A large increase in the hydrogen-out-of-plane (HOOP) vibration at 947 (H-C11=C12-H Au mode) and moderate increases at 893 (C7-H and C10-H) and 808 (C14-H) cm-1 were observed under dehydrated conditions, which were assigned by using systematically deuterated retinal. Moreover, the Asn variant at Asp116, which functions as a counter ion for the protonated retinal Schiff base (PRSB), caused a large redshift in the absorption maximum and constitutive increase in the HOOP modes under hydrated and dehydrated conditions. The protonation of a counter ion at Asp116 clearly causes a redshift in the absorption maximum as the all-trans retinal chromophore twists upon dehydration. Namely, the results strongly suggested that water molecules are important for maintaining the hydrogen-bonding network at the PRSB and deprotonation state of Asp116 in KR2.


Assuntos
Retinaldeído , Rodopsina , Humanos , Retinaldeído/química , Desidratação , Hidrogênio , Água
11.
J Med Chem ; 66(12): 8140-8158, 2023 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-37279401

RESUMO

In the eye, the isomerization of all-trans-retinal to 11-cis-retinal is accomplished by a metabolic pathway termed the visual cycle that is critical for vision. RPE65 is the essential trans-cis isomerase of this pathway. Emixustat, a retinoid-mimetic RPE65 inhibitor, was developed as a therapeutic visual cycle modulator and used for the treatment of retinopathies. However, pharmacokinetic liabilities limit its further development including: (1) metabolic deamination of the γ-amino-α-aryl alcohol, which mediates targeted RPE65 inhibition, and (2) unwanted long-lasting RPE65 inhibition. We sought to address these issues by more broadly defining the structure-activity relationships of the RPE65 recognition motif via the synthesis of a family of novel derivatives, which were tested in vitro and in vivo for RPE65 inhibition. We identified a potent secondary amine derivative with resistance to deamination and preserved RPE65 inhibitory activity. Our data provide insights into activity-preserving modifications of the emixustat molecule that can be employed to tune its pharmacological properties.


Assuntos
Propanolaminas , Retinoides , Retinoides/farmacologia , Retinoides/metabolismo , Éteres Fenílicos/farmacologia , Visão Ocular , Retinaldeído/metabolismo , Proteínas do Olho
12.
Phys Chem Chem Phys ; 25(18): 12833-12840, 2023 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-37165904

RESUMO

Heliorhodopsins (HeRs) are a new category of rhodopsins. They exist as a dimer and exhibit a characteristic inverted topology. HeRs bind all-trans-retinal as a chromophore in the dark, and its isomerization to the 13-cis form by light illumination leads to a photocyclic reaction involving several photo-intermediates: K, L, M, and O. In this study, the kinetics of conformational changes of HeR from Thermoplasmatales archaeon SG8-52-1 (TaHeR) were studied by the transient grating (TG) and circular dichroism (CD) methods. The TG method reveals that the diffusion coefficient (D) does not change until the O formation suggesting no significant conformation change at the surface of the protein during the early steps of the reaction. Subsequently, D decreases upon the O formation. Although two time constants (202 µs and 2.6 ms) are observed for the conversion from the M to O by the absorption detection, D decreases only at the first step (202 µs). Light-induced unfolding of helical structure is detected by the CD method. To examine the contribution of a characteristic helix in the intracellular loop 1 (ICL1 helix), Tyr93 on the ICL1 helix was replaced by Gly (Y93G), and the reaction of this mutant was also investigated. It was found that this replacement partially suppresses the D-change, although the CD-change is almost the same as that of the wild type. These results are interpreted in terms of different sensitivities of TG and CD methods, that is, D is sensitive to the structure of the solvent-exposed surface and selectively observes the conformational change in the ICL1 region. It is suggested that the structure of hydrophilic residues in the ICL1 helix is changed during this process.


Assuntos
Rodopsina , Rodopsinas Microbianas , Rodopsinas Microbianas/química , Dicroísmo Circular , Retinaldeído/química , Conformação Proteica
13.
Biomed Pharmacother ; 164: 114930, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37236031

RESUMO

Vitamin A (retinol) is a lipid-soluble vitamin that acts as a precursor for several bioactive compounds, such as retinaldehyde (retinal) and isomers of retinoic acid. Retinol and all-trans-retinoic acid (atRA) penetrate the blood-brain barrier and are reported to be neuroprotective in several animal models. We characterised the impact of retinol and its metabolites, all-trans-retinal (atRAL) and atRA, on ferroptosis-a programmed cell death caused by iron-dependent phospholipid peroxidation. Ferroptosis was induced by erastin, buthionine sulfoximine or RSL3 in neuronal and non-neuronal cell lines. We found that retinol, atRAL and atRA inhibited ferroptosis with a potency superior to α-tocopherol, the canonical anti-ferroptotic vitamin. In contrast, we found that antagonism of endogenous retinol with anhydroretinol sensitises ferroptosis induced in neuronal and non-neuronal cell lines. Retinol and its metabolites atRAL and atRA directly interdict lipid radicals in ferroptosis since these compounds displayed radical trapping properties in a cell-free assay. Vitamin A, therefore, complements other anti-ferroptotic vitamins, E and K; metabolites of vitamin A, or agents that alter their levels, may be potential therapeutics for diseases where ferroptosis is implicated.


Assuntos
Ferroptose , Vitamina A , Animais , Vitamina A/farmacologia , Peroxidação de Lipídeos/fisiologia , Tretinoína/farmacologia , Vitaminas , Retinaldeído , Lipídeos
14.
Photochem Photobiol Sci ; 22(8): 1809-1823, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37036621

RESUMO

A putative xanthorhodopsin-encoding gene, XR34, was found in the genome of the moderately halophilic gammaproteobacterium Salinivibrio socompensis S34, isolated from modern stromatolites found on the shore of Laguna Socompa (3570 m), Argentina Puna. XR-encoding genes were clustered together with genes encoding X-carotene, retinal (vitamin-A aldehyde), and carotenoid biosynthesis enzymes while the carotene ketolase gene critical for the salinixanthin antenna compound was absent. To identify its functional behavior, we herein overexpressed and characterized this intriguing microbial rhodopsin. Recombinant XR34 showed all the salient features of canonical microbial rhodopsin and covalently bound retinal as a functional chromophore with λmax = 561 nm (εmax ca. 60,000 M-1 cm-1). Two canonical counterions with pK values of around 6 and 3 were identified by pH titration of the recombinant protein. With a recovery time of approximately half an hour in the dark, XR34 shows light-dark adaptation shifting the absorption maximum from 551 to 561 nm. Laser-flash induced photochemistry at pH 9 (deprotonated primary counterion) identified a photocycle starting with a K-like intermediate, followed by an M-state (λmax ca. 400 nm, deprotonated Schiff base), and a final long wavelength-absorbing N- or O-like intermediate before returning to the parental 561 nm-state. Initiating the photocycle at pH 5 (protonated counterion) yields only bathochromic intermediates, due to the lacking capacity of the counterion to accept the Schiff base proton. Illumination of the membrane-embedded protein yielded a capacitive transport current. The presence of the M-intermediate under these conditions was demonstrated by a blue light-induced shunt process.


Assuntos
Bacteriorodopsinas , Bases de Schiff , Bases de Schiff/química , Carotenoides/metabolismo , Retinaldeído/química , Rodopsinas Microbianas/genética , Rodopsinas Microbianas/química , Rodopsinas Microbianas/metabolismo , Concentração de Íons de Hidrogênio
15.
J Biol Chem ; 299(5): 104686, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37031820

RESUMO

Dry age-related macular degeneration (AMD) and recessive Stargardt's disease (STGD1) lead to irreversible blindness in humans. The accumulation of all-trans-retinal (atRAL) induced by chaos in visual cycle is closely associated with retinal atrophy in dry AMD and STGD1 but its critical downstream signaling molecules remain ambiguous. Here, we reported that activation of eukaryotic translation initiation factor 2α (eIF2α) by atRAL promoted retinal degeneration and photoreceptor loss through activating c-Jun N-terminal kinase (JNK) signaling-dependent apoptosis and gasdermin E (GSDME)-mediated pyroptosis. We determined that eIF2α activation by atRAL in photoreceptor cells resulted from endoplasmic reticulum homeostasis disruption caused at least in part by reactive oxygen species production, and it activated JNK signaling independent of and dependent on activating transcription factor 4 and the activating transcription factor 4/transcription factor C/EBP homologous protein (CHOP) axis. CHOP overexpression induced apoptosis of atRAL-loaded photoreceptor cells through activating JNK signaling rather than inhibiting the expression of antiapoptotic gene Bcl2. JNK activation by eIF2α facilitated photoreceptor cell apoptosis caused by atRAL via caspase-3 activation and DNA damage. Additionally, we demonstrated that eIF2α was activated in neural retina of light-exposed Abca4-/-Rdh8-/- mice, a model that shows severe defects in atRAL clearance and displays primary features of human dry AMD and STGD1. Of note, inhibition of eIF2α activation by salubrinal effectively ameliorated retinal degeneration and photoreceptor apoptosis in Abca4-/-Rdh8-/- mice upon light exposure. The results of this study suggest that eIF2α is an important target to develop drug therapies for the treatment of dry AMD and STGD1.


Assuntos
Fator de Iniciação 2 em Eucariotos , Degeneração Retiniana , Retinaldeído , Doença de Stargardt , Animais , Humanos , Camundongos , Fator 4 Ativador da Transcrição/metabolismo , Apoptose , Transportadores de Cassetes de Ligação de ATP/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/metabolismo , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Retinaldeído/metabolismo , Doença de Stargardt/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo
16.
Biochemistry ; 62(9): 1429-1432, 2023 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-37057907

RESUMO

Retinal G-protein-coupled receptor (RGR) plays a crucial role in the visual system of vertebrates as a retinal photoisomerase, which isomerizes all-trans-retinal to 11-cis-retinal to maintain the photosensitivity of visual rhodopsins. Despite the previous characterization of bovine RGR, little is known about the spectral properties of RGR from other species. In addition, photoreactivity of the 11-cis-retinal-binding form remains unclear. In this study, we revealed that human and chicken RGRs form blue-absorbing pigments similar to bovine RGR. Furthermore, the spectroscopic and biochemical analyses revealed that bovine and chicken RGRs are bistable rhodopsins displaying a reversible photoreaction. These findings provide insight into the behavior of RGR as a retinal photoisomerase and aid in understanding its role in the visual system.


Assuntos
Retina , Retinaldeído , Animais , Bovinos , Humanos , Retinaldeído/química , Receptores Acoplados a Proteínas G , cis-trans-Isomerases , Proteínas do Olho/química , Rodopsina
17.
Nature ; 615(7954): 939-944, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36949205

RESUMO

Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs)1. A photon is absorbed by the 11-cis retinal chromophore of rhodopsin, which isomerizes within 200 femtoseconds to the all-trans conformation2, thereby initiating the cellular signal transduction processes that ultimately lead to vision. However, the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains experimentally unclear. Here we use ultrafast time-resolved crystallography at room temperature3 to determine how an isomerized twisted all-trans retinal stores the photon energy that is required to initiate the protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at a 1-ps time delay after photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Notably, the very early structural motions in the protein side chains of rhodopsin appear in regions that are involved in later stages of the conserved class A GPCR activation mechanism. Our study sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated GPCR activation.


Assuntos
Rodopsina , Visão Ocular , Animais , Sítios de Ligação/efeitos da radiação , Cristalografia , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Isomerismo , Fótons , Ligação Proteica/efeitos da radiação , Conformação Proteica/efeitos da radiação , Retinaldeído/química , Retinaldeído/metabolismo , Retinaldeído/efeitos da radiação , Rodopsina/química , Rodopsina/metabolismo , Rodopsina/efeitos da radiação , Fatores de Tempo , Visão Ocular/fisiologia , Visão Ocular/efeitos da radiação
18.
Biophys Chem ; 296: 106991, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36905840

RESUMO

Heliorhodopsin (HeR) is a seven-helical transmembrane protein with a retinal chromophore that corresponds to a new rhodopsin family. HeR from the archaebacterium Thermoplasmatales archaeon (TaHeR) exhibits unique features, such as the inverted protein orientation in the membrane compared to other rhodopsins and a long photocycle. Here, we used solid-state nuclear magnetic resonance (NMR) spectroscopy to investigate the 13C and 15N NMR signals of the retinal chromophore and protonated Schiff base (RPSB) in TaHeR embedded in POPE/POPG membrane. Although the 14- and 20-13C retinal signals indicated 13-trans/15-anti (all-trans) configurations, the 20-13C chemical shift value was different from that of other microbial rhodopsins, indicating weakly steric hinderance between Phe203 and the C20 methyl group. 15N RPSB/λmax plot deviated from the linear correlation based on retinylidene-halide model compounds. Furthermore, 15N chemical shift anisotropy (CSA) suggested that Ser112 and Ser234 polar residues distinguish the electronic environment tendencies of RPSB from those of other microbial rhodopsins. Our NMR results revealed that the retinal chromophore and the RPSB in TaHeR exhibit unique electronic environments.


Assuntos
Retinaldeído , Thermoplasmales , Retinaldeído/química , Retinaldeído/metabolismo , Bases de Schiff/química , Rodopsina/química , Rodopsina/metabolismo , Rodopsinas Microbianas/química , Espectroscopia de Ressonância Magnética/métodos , Thermoplasmales/metabolismo , Archaea/metabolismo
19.
J Phys Chem B ; 127(10): 2169-2176, 2023 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-36857774

RESUMO

Opsins are photosensitive G protein-coupled receptor proteins and are classified into visual and nonvisual receptors. Opn5L1 is a nonvisual opsin that binds all-trans retinal as a chromophore. A unique feature of Opn5L1 is that the protein exhibits a photocyclic reaction upon photoexcitation. Determining the chromophore structures of intermediates in the photocycle is essential for understanding the functional mechanism of Opn5L1. A previous study revealed that a long-lived intermediate in the photocycle cannot activate the G protein and forms a covalent bond between the retinal chromophore and a nearby cysteine residue. However, the position of this covalent bond in the chromophore remains undetermined. Here, we report a resonance Raman study on isotopically labeled samples in combination with density functional theory calculations and reveal that the 11th carbon atom of the chromophore of the intermediate forms a covalent linkage to the cysteine residue. Furthermore, vibrational assignments based on the isotopic substitutions and density functional theory calculations suggested that the Schiff base of the intermediate is deprotonated. The chromophore structure determined in the present study well explains the mechanism of the photocyclic reaction, which is crucial to the photobiological function of Opn5L1.


Assuntos
Carbono , Cisteína , Retinaldeído/química , Opsinas , Proteínas de Ligação ao GTP/metabolismo
20.
Ecotoxicol Environ Saf ; 252: 114602, 2023 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-36773439

RESUMO

Over the last decade, fluctuations of retinoids (RETs), also known as vitamin A and derivatives, have proved to be useful biomarkers to assess the environmental chemical pressure on a wide variety of non-target vertebrates. This use of RET-based biomarkers is of particular interest in the non-target sentinel species Gammarus fossarum in which RETs were shown to influence crucial physiological functions. To study and probe this metabolism in this crustacean model, a UHPLC-MS/MS method was developed to 1) identify and 2) monitor several endogenous RETs in unexposed females throughout their reproductive cycle. Then, females were exposed in controlled conditions to exogenous all-trans retinoic acid (atRA) and citral (CIT), a RA synthesis inhibitor, to simulate an excess or deficiency in RA. Perturbation of vitamin A metabolism by pesticides was further studied in response to methoprene (MET), a juvenile hormone analog as well as glyphosate (GLY). The developed method allowed, for the first time in this model, the identification of RA metabolites (all-trans 4-oxo and 13-cis 4-oxo RA), RA isomers (all-trans and 13-cis RA) as well as retinaldehyde (RALD) isomers (all-trans, 11-cis, and 13-cis RALD) and showed two distinct phases in the reproductive cycle. Retinoic acid successfully increased the tissular concentration of both RA isomers and CIT proved to be efficient at perturbating the conversion from RALD to RA. Methoprene perturbed the ratios between RA isomers whereas GLY had no observed effects on the RET system of G. fossarum females. We were able to discriminate different dynamics of RET perturbations by morphogens (atRA or CIT) or MET which highlights the plausible mediation of RETs in MET-induced disorders. Ultimately, our study shows that RETs are influenced by exposure to MET and strengthen their potential to assess aquatic ecosystem chemical status.


Assuntos
Metoprene , Vitamina A , Animais , Feminino , Ecossistema , Espectrometria de Massas em Tandem , Tretinoína , Retinoides , Isotretinoína , Retinaldeído/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...