RESUMO
Ultraviolet irradiation induces melanin accumulation, which can be reduced by the use of chemical whitening products. However, the associated safety concerns of such products have prompted the search for natural and harmless alternatives. This study aimed to identify a natural acidic formulation to reduce skin pigmentation. The metabolite propionic acid (CH3CH2COOH, PA) was the most abundant fatty acid in the filtrate from Pluronic F68 (PF68) fermentation of Cutibacterium acnes (C. acnes) and reduced the DOPA-positive melanocytes by significantly inhibiting cellular tyrosinase activity via binding to the free fatty acid receptor 2 (FFAR2). Moreover, 4 mM PA treatment did not alter melanocyte proliferation, indicating that it is an effective solution for hyperpigmentation, causing no cellular damage. The reduced DOPA-positive melanocytes and tyrosinase activity were also observed in mice ear skin tissue injected with a mixture of C. acnes and PF68, supporting that the inhibition of melanogenesis is likely to be mediated through fermentation metabolites from C. acnes fermentation using PF68 as a carbon source. Additionally, PA did not affect the growth of its parent bacteria C. acnes, hence is a potent fermentation metabolite that does not disrupt the balance of the skin microbiome.
Assuntos
Melaninas/síntese química , Propionatos/metabolismo , Propionibacterium acnes/metabolismo , Animais , Proliferação de Células , Orelha , Feminino , Fermentação , Humanos , Hiperpigmentação , Melanócitos/citologia , Melanócitos/metabolismo , Metaboloma , Camundongos Endogâmicos ICR , Processos Fotoquímicos , Propionatos/química , Receptores Acoplados a Proteínas G/efeitos da radiação , Pele , Pigmentação da Pele , Raios UltravioletaRESUMO
Animals have evolved light-sensitive G protein-coupled receptors, known as opsins, to detect coherent and ambient light for visual and nonvisual functions. These opsins have evolved to satisfy the particular lighting niches of the organisms that express them. While many unique patterns of evolution have been identified in mammals for rod and cone opsins, far less is known about the atypical mammalian opsins. Using genomic data from over 400 mammalian species from 22 orders, unique patterns of evolution for each mammalian opsins were identified, including photoisomerases, RGR-opsin (RGR) and peropsin (RRH), as well as atypical opsins, encephalopsin (OPN3), melanopsin (OPN4), and neuropsin (OPN5). The results demonstrate that OPN5 and rhodopsin show extreme conservation across all mammalian lineages. The cone opsins, SWS1 and LWS, and the nonvisual opsins, OPN3 and RRH, demonstrate a moderate degree of sequence conservation relative to other opsins, with some instances of lineage-specific gene loss. Finally, the photoisomerase, RGR, and the best-studied atypical opsin, OPN4, have high sequence diversity within mammals. These conservation patterns are maintained in human populations. Importantly, all mammalian opsins retain key amino acid residues important for conjugation to retinal-based chromophores, permitting light sensitivity. These patterns of evolution are discussed along with known functions of each atypical opsin, such as in circadian or metabolic physiology, to provide insight into the observed patterns of evolutionary constraint.
Assuntos
Evolução Molecular , Mamíferos/metabolismo , Opsinas/metabolismo , Opsinas/efeitos da radiação , Animais , Ritmo Circadiano/efeitos da radiação , Sequência Conservada , Humanos , Camundongos , Opsinas/química , Opsinas/genética , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/efeitos da radiação , Retina/metabolismo , Retina/efeitos da radiação , Rodopsina/química , Rodopsina/genética , Rodopsina/metabolismo , Rodopsina/efeitos da radiaçãoRESUMO
Precise manipulation of protein activity in living systems has broad applications in biomedical sciences. However, it is challenging to use light to manipulate protein activity in living systems without genetic modification. Here, we report a technique to optically switch off protein activity in living cells with high spatiotemporal resolution, referred to as molecular hyperthermia (MH). MH is based on the nanoscale-confined heating of plasmonic gold nanoparticles by short laser pulses to unfold and photoinactivate targeted proteins of interest. First, we show that protease-activated receptor 2 (PAR2), a G-protein-coupled receptor and an important pathway that leads to pain sensitization, can be photoinactivated in situ by MH without compromising cell proliferation. PAR2 activity can be switched off in laser-targeted cells without affecting surrounding cells. Furthermore, we demonstrate the molecular specificity of MH by inactivating PAR2 while leaving other receptors intact. Second, we demonstrate that the photoinactivation of a tight junction protein in brain endothelial monolayers leads to a reversible blood-brain barrier opening in vitro. Lastly, the protein inactivation by MH is below the nanobubble generation threshold and thus is predominantly due to the nanoscale heating. MH is distinct from traditional hyperthermia (that induces global tissue heating) in both its time and length scales: nanoseconds versus seconds, nanometers versus millimeters. Our results demonstrate that MH enables selective and remote manipulation of protein activity and cellular behavior without genetic modification.
Assuntos
Temperatura Alta , Proteínas de Membrana , Nanopartículas Metálicas/química , Óptica e Fotônica/métodos , Barreira Hematoencefálica/química , Linhagem Celular , Ouro/química , Humanos , Lasers , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas de Membrana/efeitos da radiação , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/efeitos da radiaçãoRESUMO
While rods in the mammalian retina regenerate rhodopsin through a well-characterized pathway in cells of the retinal pigment epithelium (RPE), cone visual pigments are thought to regenerate in part through an additional pathway in Müller cells of the neural retina. The proteins comprising this intrinsic retinal visual cycle are unknown. Here, we show that RGR opsin and retinol dehydrogenase-10 (Rdh10) convert all-trans-retinol to 11-cis-retinol during exposure to visible light. Isolated retinas from Rgr+/+ and Rgr-/- mice were exposed to continuous light, and cone photoresponses were recorded. Cones in Rgr-/- retinas lost sensitivity at a faster rate than cones in Rgr+/+ retinas. A similar effect was seen in Rgr+/+ retinas following treatment with the glial cell toxin, α-aminoadipic acid. These results show that RGR opsin is a critical component of the Müller cell visual cycle and that regeneration of cone visual pigment can be driven by light.
Assuntos
Células Ependimogliais/metabolismo , Proteínas do Olho/genética , Receptores Acoplados a Proteínas G/genética , Células Fotorreceptoras Retinianas Cones/metabolismo , Pigmentos da Retina/metabolismo , Ácido 2-Aminoadípico/farmacologia , Oxirredutases do Álcool/metabolismo , Oxirredutases do Álcool/efeitos da radiação , Animais , Células Ependimogliais/efeitos dos fármacos , Células Ependimogliais/efeitos da radiação , Antagonistas de Aminoácidos Excitatórios/farmacologia , Proteínas do Olho/metabolismo , Proteínas do Olho/efeitos da radiação , Luz , Camundongos , Camundongos Knockout , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/efeitos da radiação , Células Fotorreceptoras Retinianas Cones/efeitos da radiação , Pigmentos da Retina/efeitos da radiação , Vitamina A/metabolismoRESUMO
G protein-coupled receptors (GPCRs) are key biological switches that transmit both internal and external stimuli into the cell interior. Among the GPCRs, the "light receptor" rhodopsin has been shown to activate with a rearrangement of the transmembrane (TM) helix bundle within â¼1 ms, while all other receptors are thought to become activated within â¼50 ms to seconds at saturating concentrations. Here, we investigate synchronous stimulation of a dimeric GPCR, the metabotropic glutamate receptor type 1 (mGluR1), by two entirely different methods: (i) UV light-triggered uncaging of glutamate in intact cells or (ii) piezo-driven solution exchange in outside-out patches. Submillisecond FRET recordings between labels at intracellular receptor sites were used to record conformational changes in the mGluR1. At millimolar ligand concentrations, the initial rearrangement between the mGluR1 subunits occurs at a speed of τ1 â¼ 1-2 ms and requires the occupancy of both binding sites in the mGluR1 dimer. These rapid changes were followed by significantly slower conformational changes in the TM domain (τ2 â¼ 20 ms). Receptor deactivation occurred with time constants of â¼40 and â¼900 ms for the inter- and intrasubunit conformational changes, respectively. Together, these data show that, at high glutamate concentrations, the initial intersubunit activation of mGluR1 proceeds with millisecond speed, that there is loose coupling between this initial step and activation of the TM domain, and that activation and deactivation follow a cyclic pathway, including-in addition to the inactive and active states-at least two metastable intermediate states.
Assuntos
Receptores Acoplados a Proteínas G/metabolismo , Dimerização , Células HEK293 , Humanos , Cinética , Receptores Acoplados a Proteínas G/efeitos da radiaçãoRESUMO
An innocuous sensory stimulus that reliably signals an upcoming aversive event can be conditioned to elicit locomotion to a safe location before the aversive outcome ensues. The neural circuits that mediate the expression of this signaled locomotor action, known as signaled active avoidance, have not been identified. While exploring sensorimotor midbrain circuits in mice of either sex, we found that excitation of GABAergic cells in the substantia nigra pars reticulata blocks signaled active avoidance by inhibiting cells in the pedunculopontine tegmental nucleus (PPT), not by inhibiting cells in the superior colliculus or thalamus. Direct inhibition of putative-glutamatergic PPT cells, excitation of GABAergic PPT cells, or excitation of GABAergic afferents in PPT, abolish signaled active avoidance. Conversely, excitation of putative-glutamatergic PPT cells, or inhibition of GABAergic PPT cells, can be tuned to drive avoidance responses. The PPT is an essential junction for the expression of signaled active avoidance gated by nigral and other synaptic afferents.SIGNIFICANCE STATEMENT When a harmful situation is signaled by a sensory stimulus (e.g., street light), subjects typically learn to respond with active or passive avoidance responses that circumvent the threat. During signaled active avoidance behavior, subjects move away to avoid a threat signaled by a preceding innocuous stimulus. We identified a part of the midbrain essential to process the signal and avoid the threat. Inhibition of neurons in this area eliminates avoidance responses to the signal but preserves escape responses caused by presentation of the threat. The results highlight an essential part of the neural circuits that mediate signaled active avoidance behavior.
Assuntos
Aprendizagem da Esquiva/fisiologia , Reação de Fuga/fisiologia , Neurônios GABAérgicos/fisiologia , Rede Nervosa/fisiologia , Parte Reticular da Substância Negra/fisiologia , Núcleo Tegmental Pedunculopontino/fisiologia , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Aprendizagem da Esquiva/efeitos da radiação , Mapeamento Encefálico , Proteínas de Transporte/genética , Proteínas de Transporte/efeitos da radiação , Clozapina/análogos & derivados , Clozapina/farmacologia , Condicionamento Clássico , Dependovirus/genética , Comportamento de Ingestão de Líquido , Eletrochoque , Reação de Fuga/efeitos dos fármacos , Reação de Fuga/efeitos da radiação , Mutação com Ganho de Função , Genes Reporter , Vetores Genéticos/administração & dosagem , Luz , Camundongos , Ruído/efeitos adversos , Optogenética , Parte Reticular da Substância Negra/citologia , Tempo de Reação , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/efeitos da radiação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/efeitos da radiação , Colículos Superiores/citologia , Colículos Superiores/fisiologia , Tálamo/citologia , Tálamo/fisiologiaRESUMO
Remote and selective spatiotemporal control of the activity of neurons to regulate behavior and physiological functions has been a long-sought goal in system neuroscience. Identification and subsequent bioengineering of light-sensitive ion channels (e.g., channelrhodopsins, halorhodopsin, and archaerhodopsins) from the bacteria have made it possible to use light to artificially modulate neuronal activity, namely optogenetics. Recent advance in genetics has also allowed development of novel pharmacological tools to selectively and remotely control neuronal activity using engineered G protein-coupled receptors, which can be activated by otherwise inert drug-like small molecules such as the designer receptors exclusively activated by designer drug, a form of chemogenetics. The cutting-edge optogenetics and pharmacogenetics are powerful tools in neuroscience that allow selective and bidirectional modulation of the activity of defined populations of neurons with unprecedented specificity. These novel toolboxes are enabling significant advances in deciphering how the nervous system works and its influence on various physiological processes in health and disease. Here, we discuss the fundamental elements of optogenetics and chemogenetics approaches and some of the applications that yielded significant advances in various areas of neuroscience and beyond.
Assuntos
Canais Iônicos , Neurônios , Neurociências/métodos , Optogenética/métodos , Farmacogenética/métodos , Receptores Acoplados a Proteínas G , Transdução de Sinais , Animais , Humanos , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Canais Iônicos/efeitos da radiação , Luz , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/efeitos da radiação , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/efeitos da radiação , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/efeitos da radiaçãoRESUMO
Opsins comprise the protein component of light sensitive G protein-coupled receptors (GPCRs) in the retina of the eye that are responsible for the transduction of light into a biochemical signal. Here, we used hydrogen/deuterium (H/D) exchange coupled with mass spectrometry to map conformational changes in green cone opsin upon light activation. We then compared these findings with those reported for rhodopsin. The extent of H/D exchange in green cone opsin was greater than in rhodopsin in the dark and bleached states, suggesting a higher structural heterogeneity for green cone opsin. Further analysis revealed that green cone opsin exists as a dimer in both dark (inactive) and bleached (active) states, and that the predicted glycosylation sites at N32 and N34 are indeed glycosylated. Comparison of deuterium uptake between inactive and active states of green cone opsin also disclosed a reduced solvent accessibility of the extracellular N-terminal region and an increased accessibility of the chromophore binding site. Increased H/D exchange at the extracellular side of transmembrane helix four (TM4) combined with an analysis of sequence alignments revealed a conserved Pro-Pro motif in extracellular loop 2 (EL2) of monostable visual GPCRs. These data present new insights into the locus of chromophore release at the extracellular side of TM4 and TM5 and provide a foundation for future functional evaluation.
Assuntos
Opsinas dos Cones/química , Receptores Acoplados a Proteínas G/química , Opsinas de Bastonetes/química , Motivos de Aminoácidos , Substituição de Aminoácidos , Asparagina/metabolismo , Sítios de Ligação , Biologia Computacional , Opsinas dos Cones/genética , Opsinas dos Cones/metabolismo , Opsinas dos Cones/efeitos da radiação , Sequência Conservada , Medição da Troca de Deutério , Glicosilação , Humanos , Ligantes , Luz , Mutação Puntual , Prolina/química , Conformação Proteica , Redobramento de Proteína/efeitos da radiação , Estrutura Terciária de Proteína , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/efeitos da radiação , Proteínas Recombinantes , Opsinas de Bastonetes/genética , Opsinas de Bastonetes/metabolismo , Opsinas de Bastonetes/efeitos da radiação , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
The activation of a G protein-coupled receptor is generally triggered by binding of an agonist to the receptor's binding pocket, or, in the case of rhodopsin, by light-induced changes of the pre-bound retinal. This is followed by a series of a conformational changes towards an active receptor conformation, which is capable of signalling to G proteins and other downstream proteins. In the past few years, a number of new techniques have been employed to analyze the kinetics of this activation process, including X-ray crystallographic three-dimensional structures of receptors in the inactive and the active states, NMR studies of labelled receptors, molecular simulations, and optical analyses with fluorescence resonance energy transfer (FRET). Here we review our current understanding of the activation process of GPCRs as well as open questions in the sequence of events ranging from (sub-)microsecond activation by light or agonist binding to millisecond activation of receptors by soluble ligands and the subsequent generation of an intracellular signal.
Assuntos
Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Animais , Cristalografia por Raios X , Transferência Ressonante de Energia de Fluorescência , Humanos , Cinética , Ligantes , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/isolamento & purificação , Receptores Acoplados a Proteínas G/efeitos da radiação , Rodopsina/química , Rodopsina/metabolismo , Rodopsina/efeitos da radiação , Transdução de Sinais/efeitos da radiaçãoRESUMO
X-ray crystallography of G protein-coupled receptors and other membrane proteins is hampered by difficulties associated with growing sufficiently large crystals that withstand radiation damage and yield high-resolution data at synchrotron sources. We used an x-ray free-electron laser (XFEL) with individual 50-femtosecond-duration x-ray pulses to minimize radiation damage and obtained a high-resolution room-temperature structure of a human serotonin receptor using sub-10-micrometer microcrystals grown in a membrane mimetic matrix known as lipidic cubic phase. Compared with the structure solved by using traditional microcrystallography from cryo-cooled crystals of about two orders of magnitude larger volume, the room-temperature XFEL structure displays a distinct distribution of thermal motions and conformations of residues that likely more accurately represent the receptor structure and dynamics in a cellular environment.
Assuntos
Cristalografia por Raios X/instrumentação , Cristalografia por Raios X/métodos , Receptores Acoplados a Proteínas G/química , Humanos , Lasers , Conformação Proteica , Receptor 5-HT2B de Serotonina/química , Receptor 5-HT2B de Serotonina/efeitos da radiação , Receptores Acoplados a Proteínas G/efeitos da radiação , Fatores de TempoRESUMO
Dimerization and oligomerization of G protein-coupled receptors (GPCRs), proposed almost 30 years ago, have crucial relevance for drug design. Indeed, formation of GPCR oligomers may affect the diversity and performance by which extracellular signals are transferred to G proteins in the process of receptor transduction. Thus, the control of oligomer assembly/disassembly and signaling will be a powerful pharmacological tool. This, however, requires (i) the determination that oligomerization takes place between particular receptors, (ii) the confirmation that the oligomer has pharmacological importance and (iii) the availability of the oligomer 3D structure. This review aims at presenting experimental methods which unveil the complexity of GPCR dimerization/oligomerization focusing on biochemical and biophysical approaches. In total, we review 22 methods, including biochemical methods (radiation inactivation technique, receptor co-expression and trans-complementation studies, cross-linking experiments, co-immunoprecipitation and immunoblotting studies and analysis of receptor mutants and chimeras) and biophysical methods (Fluorescence Resonance Energy Transfer, (FRET), including photobleaching FRET (pb-FRET) and Time-Resolved FRET (TR-FRET), Luminescence Resonance Energy Transfer (LRET), Bioluminescence Resonance Energy Transfer (BRET), Bimolecular Fluorescence Complementation (BiFC), Luminescence Fluorescence Complementation (BiLC), Fluorescence Recovery after Photobleaching (FRAP), Confocal Microscopy, Immunofluorescence Microscopy, Single Fluorescent-Molecule Imaging, Transmission Electron Microscopy, Immunoelectron Microscopy, Atomic Force Microscopy, Total Internal Reflectance Fluorescence Microscopy (TIRFM) and X-ray Crystallography). For each method the scientific basis of the approach is shortly described followed by the extensive description of its application for studying GPCR oligomers presented according to their classes and families. Based on the wealth of experimental evidence, there is no doubt about the existence of GPCR dimers, oligomers and receptor mosaics which constitute a new and highly promising group of novel drug targets for more selective and safer drugs.
Assuntos
Multimerização Proteica , Receptores Acoplados a Proteínas G/química , Reagentes de Ligações Cruzadas/química , Cristalografia por Raios X , Desenho de Fármacos , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Ligação ao GTP/química , Imunoprecipitação , Luminescência , Medições Luminescentes , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência/métodos , Mutação , Fotodegradação , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/efeitos da radiação , Proteínas Recombinantes de FusãoRESUMO
Recent studies have implicated the lipid mediator platelet-activating factor (PAF) in UVB-mediated systemic immunosuppression known to be a major cause for skin cancers. Previously, our group has demonstrated that UVB irradiation triggers the production of PAF and oxidized glycerophosphocholines that act as PAF-receptor (PAF-R) agonists. The present studies explored the mechanisms by which UVB generates PAF-R agonists. UVB irradiation of human epidermal KB cells resulted in both increased levels of reactive oxygen species (ROS) and PAF-R agonistic activity. Pretreatment of KB cells with antioxidants vitamin C and N-acetylcysteine or the pharmacological inhibitor PD168393 specific for the epidermal growth factor receptor all inhibited UVB-induced ROS as well as PAF-R agonists, yet had no effect on fMLP-mediated PAF-R agonist production. In addition, in vivo production of PAF-R agonists from UVB-irradiated mouse skin was blocked by both systemic vitamin C administration and topical PD168393 application. Moreover, both vitamin C and PD168393 abolished UVB-mediated but not the PAF-R agonist 1-hexadecyl-2-N-methylcarbamoyl glycerophosphocholine-mediated immunosuppression as measured by the inhibition of delayed type contact hypersensitivity to the chemical dinitrofluorobenzene. These studies suggest that UVB-induced systemic immunosuppression is due to epidermal growth factor receptor-mediated ROS which results in PAF-R agonist formation.
Assuntos
Receptores ErbB/fisiologia , Fator de Ativação de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/agonistas , Glicoproteínas da Membrana de Plaquetas/efeitos da radiação , Espécies Reativas de Oxigênio/metabolismo , Espécies Reativas de Oxigênio/efeitos da radiação , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/efeitos da radiação , Raios Ultravioleta , Animais , Dermatite de Contato/etiologia , Dermatite de Contato/imunologia , Dermatite de Contato/metabolismo , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/efeitos da radiação , Receptores ErbB/efeitos da radiação , Humanos , Terapia de Imunossupressão , Células KB , Camundongos , Camundongos Pelados , Camundongos Endogâmicos C57BL , Fator de Ativação de Plaquetas/efeitos da radiação , Glicoproteínas da Membrana de Plaquetas/biossíntese , Espécies Reativas de Oxigênio/farmacologia , Receptores Acoplados a Proteínas G/biossíntese , Pele/imunologia , Pele/metabolismo , Pele/efeitos da radiaçãoRESUMO
Conformational possibilities of flexible loops in rhodopsin, a prototypical G-protein-coupled receptor, were studied by modeling both in the dark-adapted (R) and activated (R*) states. Loop structures were built onto templates representing the R and R* states of the TM region of rhodopsin developed previously (G. V. Nikiforovich and G. R. Marshall. 2003. Biochemistry. 42:9110). Geometrical sampling and energy calculations were performed for each individual loop, as well as for the interacting intracellular loops IC1, IC2, and IC3 and the extracellular loops EC1, EC2, and EC3 mounted on the R and R* templates. Calculations revealed that the intra- and extracellular loops of rhodopsin possess low-energy structures corresponding to large conformational movements both in the R and R* states. Results of these calculations are in good agreement with the x-ray data available for the dark-adapted rhodopsin as well as with the available experimental biophysical data on the disulfide-linked mutants of rhodopsin. The calculated results are used to exemplify how the combined application of the results of independent calculations with emerging experimental data can be used to select plausible three-dimensional structures of the loops in rhodopsin.
Assuntos
Membrana Celular/química , Luz , Modelos Químicos , Modelos Moleculares , Rodopsina/química , Rodopsina/efeitos da radiação , Adaptação Fisiológica/efeitos da radiação , Simulação por Computador , Adaptação à Escuridão , Conformação Proteica/efeitos da radiação , Estrutura Secundária de Proteína/efeitos da radiação , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/efeitos da radiaçãoRESUMO
An overview of the rhodopsin crystal structure provides a structural basis for understanding the structures and functions of other G-protein coupled receptors (GPCRs). All of the structural details observed to date for rhodopsin will not necessarily carry over to other GPCRs, but major features such as the arrangement of the seven transmembrane helices, the retinal/ligand binding site, the D(E)RY and NPXXY sequence and structural motifs, and the bent helices are likely characteristics of the GPCRs most closely related to rhodopsin. A general view of these structural features is presented here.
Assuntos
Receptores Acoplados a Proteínas G/fisiologia , Rodopsina/química , Rodopsina/fisiologia , Animais , Humanos , Modelos Biológicos , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/efeitos da radiação , Rodopsina/efeitos dos fármacosRESUMO
Rhodopsin is the best-understood member of the large G protein-coupled receptor (GPCR) superfamily. The G-protein amplification cascade is triggered by poorly understood light-induced conformational changes in rhodopsin that are homologous to changes caused by agonists in other GPCRs. We have applied the "antibody imprint" method to light-activated rhodopsin in native membranes by using nine monoclonal antibodies (mAbs) against aqueous faces of rhodopsin. Epitopes recognized by these mAbs were found by selection from random peptide libraries displayed on phage. A new computer algorithm, FINDMAP, was used to map the epitopes to discontinuous segments of rhodopsin that are distant in the primary sequence but are in close spatial proximity in the structure. The proximity of a segment of the N-terminal and the loop between helices VI and VIII found by FINDMAP is consistent with the X-ray structure of the dark-adapted rhodopsin. Epitopes to the cytoplasmic face segregated into two classes with different predicted spatial proximities of protein segments that correlate with different preferences of the antibodies for stabilizing the metarhodopsin I or metarhodopsin II conformations of light-excited rhodopsin. Epitopes of antibodies that stabilize metarhodopsin II indicate conformational changes from dark-adapted rhodopsin, including rearrangements of the C-terminal tail and altered exposure of the cytoplasmic end of helix VI, a portion of the C-3 loop, and helix VIII. As additional antibodies are subjected to antibody imprinting, this approach should provide increasingly detailed information on the conformation of light-excited rhodopsin and be applicable to structural studies of other challenging protein targets.