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1.
Proc Natl Acad Sci U S A ; 120(30): e2216329120, 2023 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-37478163

RESUMO

To accomplish concerted physiological reactions, nature has diversified functions of a single hormone at at least two primary levels: 1) Different receptors recognize the same hormone, and 2) different cellular effectors couple to the same hormone-receptor pair [R.P. Xiao, Sci STKE 2001, re15 (2001); L. Hein, J. D. Altman, B.K. Kobilka, Nature 402, 181-184 (1999); Y. Daaka, L. M. Luttrell, R. J. Lefkowitz, Nature 390, 88-91 (1997)]. Not only these questions lie in the heart of hormone actions and receptor signaling but also dissecting mechanisms underlying these questions could offer therapeutic routes for refractory diseases, such as kidney injury (KI) or X-linked nephrogenic diabetes insipidus (NDI). Here, we identified that Gs-biased signaling, but not Gi activation downstream of EP4, showed beneficial effects for both KI and NDI treatments. Notably, by solving Cryo-electron microscope (cryo-EM) structures of EP3-Gi, EP4-Gs, and EP4-Gi in complex with endogenous prostaglandin E2 (PGE2)or two synthetic agonists and comparing with PGE2-EP2-Gs structures, we found that unique primary sequences of prostaglandin E2 receptor (EP) receptors and distinct conformational states of the EP4 ligand pocket govern the Gs/Gi transducer coupling selectivity through different structural propagation paths, especially via TM6 and TM7, to generate selective cytoplasmic structural features. In particular, the orientation of the PGE2 ω-chain and two distinct pockets encompassing agonist L902688 of EP4 were differentiated by their Gs/Gi coupling ability. Further, we identified common and distinct features of cytoplasmic side of EP receptors for Gs/Gi coupling and provide a structural basis for selective and biased agonist design of EP4 with therapeutic potential.


Assuntos
Dinoprostona , Transdução de Sinais , Dinoprostona/metabolismo , Transdução de Sinais/fisiologia , Receptores de Prostaglandina/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Hormônios , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Receptores de Prostaglandina E Subtipo EP3/metabolismo
2.
Science ; 380(6640): eadd6220, 2023 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-36862765

RESUMO

Individual free fatty acids (FAs) play important roles in metabolic homeostasis, many through engagement with more than 40G protein-coupled receptors. Searching for receptors to sense beneficial omega-3 FAs of fish oil enabled the identification of GPR120, which is involved in a spectrum of metabolic diseases. Here, we report six cryo-electron microscopy structures of GPR120 in complex with FA hormones or TUG891 and Gi or Giq trimers. Aromatic residues inside the GPR120 ligand pocket were responsible for recognizing different double-bond positions of these FAs and connect ligand recognition to distinct effector coupling. We also investigated synthetic ligand selectivity and the structural basis of missense single-nucleotide polymorphisms. We reveal how GPR120 differentiates rigid double bonds and flexible single bonds. The knowledge gleaned here may facilitate rational drug design targeting to GPR120.


Assuntos
Desenho de Fármacos , Ácidos Graxos Ômega-3 , Receptores Acoplados a Proteínas G , Microscopia Crioeletrônica , Ligantes , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Ácidos Graxos Ômega-3/química , Ácidos Graxos Ômega-3/metabolismo , Humanos , Compostos de Bifenilo/química , Compostos de Bifenilo/farmacologia , Fenilpropionatos/química , Fenilpropionatos/farmacologia , Conformação Proteica , Ácido Eicosapentaenoico/química , Ácido Eicosapentaenoico/metabolismo , Mutação de Sentido Incorreto , Polimorfismo de Nucleotídeo Único
3.
Nature ; 604(7907): 771-778, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35418677

RESUMO

Adhesion G protein-coupled receptors (aGPCRs) constitute an evolutionarily ancient family of receptors that often undergo autoproteolysis to produce α and ß subunits1-3. A tethered agonism mediated by the 'Stachel sequence' of the ß subunit has been proposed to have central roles in aGPCR activation4-6. Here we present three cryo-electron microscopy structures of aGPCRs coupled to the Gs heterotrimer. Two of these aGPCRs are activated by tethered Stachel sequences-the ADGRG2-ß-Gs complex and the ADGRG4-ß-Gs complex (in which ß indicates the ß subunit of the aGPCR)-and the other is the full-length ADGRG2 in complex with the exogenous ADGRG2 Stachel-sequence-derived peptide agonist IP15 (ADGRG2(FL)-IP15-Gs). The Stachel sequences of both ADGRG2-ß and ADGRG4-ß assume a U shape and insert deeply into the seven-transmembrane bundles. Constituting the FXφφφXφ motif (in which φ represents a hydrophobic residue), five residues of ADGRG2-ß or ADGRG4-ß extend like fingers to mediate binding to the seven-transmembrane domain and activation of the receptor. The structure of the ADGRG2(FL)-IP15-Gs complex reveals the structural basis for the improved binding affinity of IP15 compared with VPM-p15 and indicates that rational design of peptidic agonists could be achieved by exploiting aGPCR-ß structures. By converting the 'finger residues' to acidic residues, we develop a method to generate peptidic antagonists towards several aGPCRs. Collectively, our study provides structural and biochemical insights into the tethered activation mechanism of aGPCRs.


Assuntos
Peptídeos , Receptores Acoplados a Proteínas G , Microscopia Crioeletrônica , Humanos , Peptídeos/metabolismo , Domínios Proteicos , Receptores Acoplados a Proteínas G/metabolismo
4.
Nat Commun ; 12(1): 5979, 2021 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-34645801

RESUMO

The lobula giant movement detector (LGMD) is the movement-sensitive, wide-field visual neuron positioned in the third visual neuropile of lobula. LGMD neuron can anticipate collision and trigger avoidance efficiently owing to the earlier occurring firing peak before collision. Vision chips inspired by the LGMD have been successfully implemented in very-large-scale-integration (VLSI) system. However, transistor-based chips and single devices to simulate LGMD neurons make them bulky, energy-inefficient and complicated. The devices with relatively compact structure and simple operation mode to mimic the escape response of LGMD neuron have not been realized yet. Here, the artificial LGMD visual neuron is implemented using light-mediated threshold switching memristor. The non-monotonic response to light flow field originated from the formation and break of Ag conductive filaments is analogue to the escape response of LGMD neuron. Furthermore, robot navigation with obstacle avoidance capability and biomimetic compound eyes with wide field-of-view (FoV) detection capability are demonstrated.

5.
Front Pharmacol ; 12: 625233, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33959005

RESUMO

In order to develop improved anxiolytic drugs, 8-substituted analogs of triazolam were synthesized in an effort to discover compounds with selectivity for α2/α3 subunit-containing GABAA subtypes. Two compounds in this series, XLi-JY-DMH (6-(2-chlorophenyl)-8-ethynyl-1-methyl-4H-benzo [f][1,2,4]triazolo[4,3-a][1,4]diazepine) and SH-TRI-108 [(E)-8-ethynyl-1-methyl-6-(pyridin-2-yl)-4H-benzo [f][1,2,4]triazolo[4,3-a][1,4]diazepine], were evaluated for in vitro and in vivo properties associated with GABAA subtype-selective ligands. In radioligand binding assays conducted in transfected HEK cells containing rat αXß3γ2 subtypes (X = 1,2,3,5), no evidence of selectivity was obtained, although differences in potency relative to triazolam were observed overall (triazolam > XLi-JY-DMH > SH-TRI-108). In studies with rat αXß3γ2 subtypes (X = 1,2,3,5) using patch-clamp electrophysiology, no differences in maximal potentiation of GABA-mediated Cl- current was obtained across subtypes for any compound. However, SH-TRI-108 demonstrated a 25-fold difference in functional potency between α1ß3γ2 vs. α2ß3γ2 subtypes. We evaluated the extent to which this potency difference translated into behavioral pharmacological differences in monkeys. In a rhesus monkey conflict model of anxiolytic-like effects, triazolam, XLi-JY-DMH, and SH-TR-108 increased rates of responding attenuated by shock (anti-conflict effect) but also attenuated non-suppressed responding. In a squirrel monkey observation procedure, both analogs engendered a profile of sedative-motor effects similar to that reported previously for triazolam. In molecular docking studies, we found that the interactions of the 8-ethynyl triazolobenzodiazepines with the C-loop of the α1GABAA site was stronger than that of imidazodiazepines XHe-II-053 and HZ-166, which may account for the non-sedating yet anxiolytic profile of these latter compounds when evaluated in previous studies.

6.
Nat Commun ; 12(1): 2396, 2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33888704

RESUMO

Arrestins recognize different receptor phosphorylation patterns and convert this information to selective arrestin functions to expand the functional diversity of the G protein-coupled receptor (GPCR) superfamilies. However, the principles governing arrestin-phospho-receptor interactions, as well as the contribution of each single phospho-interaction to selective arrestin structural and functional states, are undefined. Here, we determined the crystal structures of arrestin2 in complex with four different phosphopeptides derived from the vasopressin receptor-2 (V2R) C-tail. A comparison of these four crystal structures with previously solved Arrestin2 structures demonstrated that a single phospho-interaction change results in measurable conformational changes at remote sites in the complex. This conformational bias introduced by specific phosphorylation patterns was further inspected by FRET and 1H NMR spectrum analysis facilitated via genetic code expansion. Moreover, an interdependent phospho-binding mechanism of phospho-receptor-arrestin interactions between different phospho-interaction sites was unexpectedly revealed. Taken together, our results provide evidence showing that phospho-interaction changes at different arrestin sites can elicit changes in affinity and structural states at remote sites, which correlate with selective arrestin functions.


Assuntos
Receptores de Vasopressinas/metabolismo , beta-Arrestina 1/metabolismo , Cristalografia por Raios X , Células HEK293 , Humanos , Mutação , Ressonância Magnética Nuclear Biomolecular , Fosfopeptídeos/química , Fosfopeptídeos/metabolismo , Fosforilação , Conformação Proteica em alfa-Hélice , Domínios Proteicos/genética , Receptores de Vasopressinas/química , Receptores de Vasopressinas/ultraestrutura , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , beta-Arrestina 1/genética , beta-Arrestina 1/isolamento & purificação , beta-Arrestina 1/ultraestrutura
7.
Nanoscale ; 12(46): 23391-23423, 2020 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-33227110

RESUMO

The substantial amount of data generated every second in the big data age creates a pressing requirement for new and advanced data storage techniques. Luminescent nanomaterials (LNMs) not only possess the same optical properties as their bulk materials but also have unique electronic and mechanical characteristics due to the strong constraints of photons and electrons at the nanoscale, enabling the development of revolutionary methods for data storage with superhigh storage capacity, ultra-long working lifetime, and ultra-low power consumption. In this review, we investigate the latest achievements in LNMs for constructing next-generation data storage systems, with a focus on optical data storage and optoelectronic data storage. We summarize the LNMs used in data storage, namely upconversion nanomaterials, long persistence luminescent nanomaterials, and downconversion nanomaterials, and their applications in optical data storage and optoelectronic data storage. We conclude by discussing the superiority of the two types of data storage and survey the prospects for the field.

8.
Nat Commun ; 11(1): 4857, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32978402

RESUMO

Characterization of the dynamic conformational changes in membrane protein signaling complexes by nuclear magnetic resonance (NMR) spectroscopy remains challenging. Here we report the site-specific incorporation of 4-trimethylsilyl phenylalanine (TMSiPhe) into proteins, through genetic code expansion. Crystallographic analysis revealed structural changes that reshaped the TMSiPhe-specific amino-acyl tRNA synthetase active site to selectively accommodate the trimethylsilyl (TMSi) group. The unique up-field 1H-NMR chemical shift and the highly efficient incorporation of TMSiPhe enabled the characterization of multiple conformational states of a phospho-ß2 adrenergic receptor/ß-arrestin-1(ß-arr1) membrane protein signaling complex, using only 5 µM protein and 20 min of spectrum accumulation time. We further showed that extracellular ligands induced conformational changes located in the polar core or ERK interaction site of ß-arr1 via direct receptor transmembrane core interactions. These observations provided direct delineation and key mechanism insights that multiple receptor ligands were able to induce distinct functionally relevant conformational changes of arrestin.


Assuntos
Arrestina/química , Arrestina/genética , Arrestina/metabolismo , Ligantes , Espectroscopia de Prótons por Ressonância Magnética/métodos , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Fenilalanina , Ligação Proteica , Conformação Proteica , Receptores Adrenérgicos beta 2/metabolismo , Transdução de Sinais , beta-Arrestina 1/química , beta-Arrestina 1/genética , beta-Arrestina 1/metabolismo
9.
Nature ; 587(7834): 499-504, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32698187

RESUMO

The G-protein-coupled bile acid receptor (GPBAR) conveys the cross-membrane signalling of a vast variety of bile acids and is a signalling hub in the liver-bile acid-microbiota-metabolism axis1-3. Here we report the cryo-electron microscopy structures of GPBAR-Gs complexes stabilized by either the high-affinity P3954 or the semisynthesized bile acid derivative INT-7771,3 at 3 Å resolution. These structures revealed a large oval pocket that contains several polar groups positioned to accommodate the amphipathic cholic core of bile acids, a fingerprint of key residues to recognize diverse bile acids in the orthosteric site, a putative second bile acid-binding site with allosteric properties and structural features that contribute to bias properties. Moreover, GPBAR undertakes an atypical mode of activation and G protein coupling that features a different set of key residues connecting the ligand-binding pocket to the Gs-coupling site, and a specific interaction motif that is localized in intracellular loop 3. Overall, our study not only reveals unique structural features of GPBAR that are involved in bile acid recognition and allosteric effects, but also suggests the presence of distinct connecting mechanisms between the ligand-binding pocket and the G-protein-binding site in the G-protein-coupled receptor superfamily.


Assuntos
Ácidos e Sais Biliares/metabolismo , Microscopia Crioeletrônica , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/ultraestrutura , Regulação Alostérica/efeitos dos fármacos , Ácidos e Sais Biliares/química , Sítios de Ligação/efeitos dos fármacos , Ácidos Cólicos/química , Ácidos Cólicos/farmacologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/ultraestrutura , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Especificidade por Substrato
11.
Anal Chem ; 91(23): 14936-14942, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31670502

RESUMO

Förster resonance energy transfer (FRET) is a well-established method for studying macromolecular interactions and conformational changes within proteins. Such a method normally uses fluorescent proteins or chemical-labeling methods which are often only accessible to surface-exposed residues and risk-disturbing target protein structures. Here, we demonstrate that the genetic incorporation of a synthetic fluorescent amino acid, L-(7-hydroxycoumarin-4-yl) ethylglycine (Cou) and natural endogenous fluorophore Tryptophan (Trp) residues of a protein could serve as an efficient FRET pair to monitor protein interactions, using the signaling transducer ß-arrestin-1 as a model system. We used this technology to record the dynamic spectra in both binding and competition experiments of ß-arrestin-1, the contribution of each specific phosphate in ternary complex formation, in a rapid and efficient manner. The determined Kd value for the association between the active arrestin and Fab30 is 0.68 µM in the three-component interaction system. Moreover, we were able to determine the contributions of the site 3 phospho-site and the site 6 phospho-site binding, each contributing to the high affinity ternary complex assembly as 2.7 fold and 15.5 fold, respectively, which were never determined before. These results thus highlighted the potential usage of this new method in measurement of the allosteric-induced enhanced affinity with small amount proteins and in a fast manner and in a complex system. Collectively, our newly developed Trp:Cou FRET system based on genetic expansion technology has extended the molecular toolboxes available for biochemical and structural biology studies.


Assuntos
Aminoácidos/química , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Engenharia de Proteínas/métodos , Sítios de Ligação , Glicina/análogos & derivados , Glicina/química , Glicina/genética , Ligação Proteica , Triptofano/química , Umbeliferonas/química , beta-Arrestina 1
12.
Redox Biol ; 17: 386-399, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29860106

RESUMO

The adaptive immune system plays a critical role in hyperhomocysteinemia (HHcy)-accelerated atherosclerosis. Recent studies suggest that HHcy aggravates atherosclerosis with elevated oxidative stress and reduced S-nitrosylation level of redox-sensitive protein residues in the vasculature. However, whether and how S-nitrosylation contributes to T-cell-driven atherosclerosis remain unclear. In the present study, we report that HHcy reduced the level of protein S-nitrosylation in T cells by inducing S-nitrosoglutathione reductase (GSNOR), the key denitrosylase that catalyzes S-nitrosoglutathione (GSNO), which is the main restored form of nitric oxide in vivo. Consequently, secretion of inflammatory cytokines [interferon-γ (IFN-γ) and interleukin-2] and proliferation of T cells were increased. GSNOR knockout or GSNO stimulation rectified HHcy-induced inflammatory cytokine secretion and T-cell proliferation. Site-directed mutagenesis of Akt at Cys224 revealed that S-nitrosylation at this site was pivotal for the reduced phosphorylation at Akt Ser473, which led to impaired Akt signaling. Furthermore, on HHcy challenge, as compared with GSNOR+/+ApoE-/- littermate controls, GSNOR-/-ApoE-/- double knockout mice showed reduced T-cell activation with concurrent reduction of atherosclerosis. Adoptive transfer of GSNOR-/- T cells to ApoE-/- mice fed homocysteine (Hcy) decreased atherosclerosis, with fewer infiltrated T cells and macrophages in plaques. In patients with HHcy and coronary artery disease, the level of plasma Hcy was positively correlated with Gsnor expression in peripheral blood mononuclear cells and IFN-γ+ T cells but inversely correlated with the S-nitrosylation level in T cells. These data reveal that T cells are activated, in part via GSNOR-dependent Akt denitrosylation during HHcy-induced atherosclerosis. Thus, suppression of GSNOR in T cells may reduce the risk of atherosclerosis.


Assuntos
Aldeído Oxirredutases/genética , Apolipoproteínas E/genética , Aterosclerose/genética , Hiper-Homocisteinemia/genética , Proteínas Proto-Oncogênicas c-akt/genética , Imunidade Adaptativa/genética , Idoso , Animais , Aterosclerose/metabolismo , Aterosclerose/patologia , Feminino , Humanos , Hiper-Homocisteinemia/metabolismo , Hiper-Homocisteinemia/patologia , Interferon gama/genética , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Óxido Nítrico/metabolismo , Oxirredução , Estresse Oxidativo/genética , Fosforilação/genética , S-Nitrosoglutationa/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo
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