RESUMEN
G protein-coupled receptors (GPCRs) mediate most cellular responses to hormones, neurotransmitters, and environmental stimulants. However, whether GPCRs participate in tissue homeostasis through ferroptosis remains unclear. Here we identify that GPR56/ADGRG1 renders cells resistant to ferroptosis and deficiency of GPR56 exacerbates ferroptosis-mediated liver injury induced by doxorubicin (DOX) or ischemia-reperfusion (IR). Mechanistically, GPR56 decreases the abundance of phospholipids containing free polyunsaturated fatty acids (PUFAs) by promoting endocytosis-lysosomal degradation of CD36. By screening a panel of steroid hormones, we identified that 17α-hydroxypregnenolone (17-OH PREG) acts as an agonist of GPR56 to antagonize ferroptosis and efficiently attenuates liver injury before or after insult. Moreover, disease-associated GPR56 mutants were unresponsive to 17-OH PREG activation and insufficient to defend against ferroptosis. Together, our findings uncover that 17-OH PREG-GPR56 axis-mediated signal transduction works as a new anti-ferroptotic pathway to maintain liver homeostasis, providing novel insights into the potential therapy for liver injury.
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Histamine is a biogenic amine that is critical in various physiological and pathophysiological processes, including but not limited to allergic reactions, wakefulness, gastric acid secretion and neurotransmission. Here, we determine 9 cryo-electron microscopy (cryo-EM) structures of the 4 histamine receptors in complex with four different G protein subtypes, with endogenous or synthetic agonists bound. Inside the ligand pocket, we identify key motifs for the recognition of histamine, the distinct binding orientations of histamine and three subpockets that facilitate the design of specific ligands. In addition, we also identify key residues responsible for the selectivity of immethridine. Moreover, we reveal distinct structural features as determinants of Gq vs. Gs or Gs vs. Gi coupling differences among the histamine receptors. Our study provides a structural framework for understanding the ligand recognition and G protein coupling of all 4 histamine receptors, which may facilitate the rational design of ligands targeting these receptors.
Asunto(s)
Microscopía por Crioelectrón , Histamina , Receptores Histamínicos , Ligandos , Humanos , Histamina/metabolismo , Histamina/química , Receptores Histamínicos/metabolismo , Receptores Histamínicos/química , Células HEK293 , Unión Proteica , Sitios de Unión , Agonistas de los Receptores Histamínicos/química , Agonistas de los Receptores Histamínicos/metabolismo , Agonistas de los Receptores Histamínicos/farmacología , Modelos MolecularesRESUMEN
Omega-3 polyunsaturated fatty acids (ω-3 PUFAs) have been associated with potential cardiovascular benefits, partly attributed to their bioactive metabolites. However, the underlying mechanisms responsible for these advantages are not fully understood. We previously reported that metabolites of the cytochrome P450 pathway derived from eicosapentaenoic acid (EPA) mediated the atheroprotective effect of ω-3 PUFAs. Here, we show that 17,18-epoxyeicosatetraenoic acid (17,18-EEQ) and its receptor, sphingosine-1-phosphate receptor 1 (S1PR1), in endothelial cells (ECs) can inhibit oscillatory shear stress- or tumor necrosis factor-α-induced endothelial activation in cultured human ECs. Notably, the atheroprotective effect of 17,18-EEQ and purified EPA is circumvented in male mice with endothelial S1PR1 deficiency. Mechanistically, the anti-inflammatory effect of 17,18-EEQ relies on calcium release-mediated endothelial nitric oxide synthase (eNOS) activation, which is abolished upon inhibition of S1PR1 or Gq signaling. Furthermore, 17,18-EEQ allosterically regulates the conformation of S1PR1 through a polar interaction with Lys34Nter. Finally, we show that Vascepa, a prescription drug containing highly purified and stable EPA ethyl ester, exerts its cardiovascular protective effect through the 17,18-EEQ-S1PR1 pathway in male and female mice. Collectively, our findings indicate that the anti-inflammatory effect of 17,18-EEQ involves the activation of the S1PR1-Gq-Ca2+-eNOS axis in ECs, offering a potential therapeutic target against atherosclerosis.
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Ácido Eicosapentaenoico , Receptores de Esfingosina-1-Fosfato , Animales , Ácido Eicosapentaenoico/farmacología , Ácido Eicosapentaenoico/análogos & derivados , Ácido Eicosapentaenoico/metabolismo , Humanos , Ratones , Receptores de Esfingosina-1-Fosfato/metabolismo , Masculino , Óxido Nítrico Sintasa de Tipo III/metabolismo , Aterosclerosis/metabolismo , Aterosclerosis/prevención & control , Receptores de Lisoesfingolípidos/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/efectos de los fármacos , Ácidos AraquidónicosRESUMEN
G-protein-coupled receptors (GPCRs) transmit downstream signals predominantly via G-protein pathways. However, the conformational basis of selective coupling of primary G-protein remains elusive. Histamine receptors H2R and H3R couple with Gs- or Gi-proteins respectively. Here, three cryo-EM structures of H2R-Gs and H3R-Gi complexes are presented at a global resolution of 2.6-2.7 Å. These structures reveal the unique binding pose for endogenous histamine in H3R, wherein the amino group interacts with E2065.46 of H3R instead of the conserved D1143.32 of other aminergic receptors. Furthermore, comparative analysis of the H2R-Gs and H3R-Gi complexes reveals that the structural geometry of TM5/TM6 determines the primary G-protein selectivity in histamine receptors. Machine learning (ML)-based structuromic profiling and functional analysis of class A GPCR-G-protein complexes illustrate that TM5 length, TM5 tilt, and TM6 outward movement are key determinants of the Gs and Gi/o selectivity among the whole Class A family. Collectively, the findings uncover the common structural geometry within class A GPCRs that determines the primary Gs- and Gi/o-coupling selectivity.
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Microscopía por Crioelectrón , Receptores Acoplados a Proteínas G , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Humanos , Microscopía por Crioelectrón/métodos , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/genética , Histamina/metabolismo , Histamina/química , Receptores Histamínicos H2/metabolismo , Receptores Histamínicos H2/genética , Receptores Histamínicos H2/química , Receptores Histamínicos H3/metabolismo , Receptores Histamínicos H3/química , Receptores Histamínicos H3/genética , Transducción de SeñalRESUMEN
BACKGROUND AND PURPOSE: Pancreatic islets are modulated by cross-talk among different cell types and paracrine signalling plays important roles in maintaining glucose homeostasis. Urocortin 3 (UCN3) secreted by pancreatic ß cells activates the CRF2 receptor (CRF2R) and downstream pathways mediated by different G protein or arrestin subtypes in δ cells to cause somatostatin (SST) secretion, and constitutes an important feedback circuit for glucose homeostasis. EXPERIMENTAL APPROACH: Here, we used Arrb1-/-, Arrb2-/-, Gsfl/fl and Gqfl/fl knockout mice, the G11-shRNA-GFPfl/fl lentivirus, as well as functional assays and pharmacological characterization to study how the coupling of Gs, G11 and ß-arrestin1 to CRF2R contributed to UCN3-induced SST secretion in pancreatic δ cells. KEY RESULTS: Our study showed that CRF2R coupled to a panel of G protein and arrestin subtypes in response to UCN3 engagement. While RyR3 phosphorylation by PKA at the S156, S2706 and S4697 sites may underlie the Gs-mediated UCN3- CRF2R axis for SST secretion, the interaction of SYT1 with ß-arrestin1 is also essential for efficient SST secretion downstream of CRF2R. The specific expression of the transcription factor Stat6 may contribute to G11 expression in pancreatic δ cells. Furthermore, we found that different UCN3 concentrations may have distinct effects on glucose homeostasis, and these effects may depend on different CRF2R downstream effectors. CONCLUSIONS AND IMPLICATIONS: Collectively, our results provide a landscape view of signalling mediated by different G protein or arrestin subtypes downstream of paracrine UCN3- CRF2R signalling in pancreatic ß-δ-cell circuits, which may facilitate the understanding of fine-tuned glucose homeostasis networks.
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Receptores de Hormona Liberadora de Corticotropina , Transducción de Señal , Somatostatina , Urocortinas , Animales , Masculino , Ratones , Proteínas de Unión al GTP/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Somatostatina/metabolismo , Células Secretoras de Somatostatina/metabolismo , Urocortinas/metabolismoRESUMEN
Prostaglandin F2α (PGF2α) and thromboxane A2 (TXA2) are endogenous arachidonic acid metabolites, modulating diverse physiological processes including inflammation and cardiovascular homeostasis through activating PGF2α receptor (FP) and TXA2 receptor (TP). Ligands targeting FP and TP have demonstrated efficacy in treating conditions like glaucoma and cardiovascular diseases in humans, as well as reproductive-related diseases in animals. Here, we present five cryoelectron microscopy structures illustrating FP and TP in complex with Gq and bound to PGF2α (endogenous ligand), latanoprost acid (a clinical drug), and two other synthetic agonists. Combined with mutational and functional studies, these structures reveal not only structural features for the specific recognition of endogenous ligands and attainment of receptor selectivity of FP and TP but also the common mechanisms of receptor activation and Gq protein coupling. The findings may enrich our knowledge of ligand recognition and signal transduction of the prostanoid receptor family and facilitate rational ligand design toward these two receptors.
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Receptores de Prostaglandina , Transducción de Señal , Humanos , Animales , Ligandos , Microscopía por Crioelectrón , Receptores de Prostaglandina/metabolismo , ProstaglandinasRESUMEN
The existing available antipsychotics have failed to manage the cognitive impairment of schizophrenia and induced a number of seriously undesirable effects. Trace amine-associated receptor 1 (TAAR1) has emerged as an ideal target for the design of antischizophrenia drugs, with the ability to mediate multiple psychological functions by sensing endogenous amine-containing metabolites without the side effects of catalepsy. In this work, a series of novel TAAR1 agonists were designed based on the structural analysis of the TAAR1 activation pocket. Among them, 6e displayed a potent TAAR1-Gs/Gq dual-pathway activation property, being different from that of the clinical drug candidate SEP-363856 with only TAAR1-Gs pathway activation. In rodent models, 6e significantly alleviated MK-801-induced schizophrenia-like cognitive phenotypes without inducing catalepsy. Furthermore, 6e·HCl exhibited favorable pharmacokinetic (T1/2 = 2.31 h, F = 39%) and safety properties. All these demonstrated that 6e·HCl may be used as a novel drug candidate for schizophrenia treatment.
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Antipsicóticos , Esquizofrenia , Humanos , Antipsicóticos/farmacología , Antipsicóticos/uso terapéutico , Catalepsia , Receptores Acoplados a Proteínas G/metabolismo , Esquizofrenia/tratamiento farmacológicoRESUMEN
Adhesion G protein-coupled receptors (aGPCRs) are evolutionarily ancient receptors involved in a variety of physiological and pathophysiological processes. Modulators of aGPCR, particularly antagonists, hold therapeutic promise for diseases like cancer and immune and neurological disorders. Hindered by the inactive state structural information, our understanding of antagonist development and aGPCR activation faces challenges. Here, we report the cryo-electron microscopy structures of human CD97, a prototypical aGPCR that plays crucial roles in immune system, in its inactive apo and G13-bound fully active states. Compared with other family GPCRs, CD97 adopts a compact inactive conformation with a constrained ligand pocket. Activation induces significant conformational changes for both extracellular and intracellular sides, creating larger cavities for Stachel sequence binding and G13 engagement. Integrated with functional and metadynamics analyses, our study provides significant mechanistic insights into the activation and signaling of aGPCRs, paving the way for future drug discovery efforts.
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Antígenos CD , Receptores Acoplados a Proteínas G , Transducción de Señal , Humanos , Adhesión Celular , Microscopía por Crioelectrón , Complejo GPIb-IX de Glicoproteína Plaquetaria , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Antígenos CD/química , Antígenos CD/metabolismoRESUMEN
GPR101 is an orphan G protein-coupled receptor actively participating in energy homeostasis. Here we report the cryo-electron microscopy structure of GPR101 constitutively coupled to Gs heterotrimer, which reveals unique features of GPR101, including the interaction of extracellular loop 2 within the 7TM bundle, a hydrophobic chain packing-mediated activation mechanism and the structural basis of disease-related mutants. Importantly, a side pocket is identified in GPR101 that facilitates in silico screening to identify four small-molecule agonists, including AA-14. The structure of AA-14-GPR101-Gs provides direct evidence of the AA-14 binding at the side pocket. Functionally, AA-14 partially restores the functions of GH/IGF-1 axis and exhibits several rejuvenating effects in wild-type mice, which are abrogated in Gpr101-deficient mice. In summary, we provide a structural basis for the constitutive activity of GPR101. The structure-facilitated identification of GPR101 agonists and functional analysis suggest that targeting this orphan receptor has rejuvenating potential.
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Receptores Acoplados a Proteínas G , Ratones , Animales , Microscopía por Crioelectrón , Receptores Acoplados a Proteínas G/metabolismo , LigandosRESUMEN
Trace-amine-associated receptors (TAARs), a group of biogenic amine receptors, have essential roles in neurological and metabolic homeostasis1. They recognize diverse endogenous trace amines and subsequently activate a range of G-protein-subtype signalling pathways2,3. Notably, TAAR1 has emerged as a promising therapeutic target for treating psychiatric disorders4,5. However, the molecular mechanisms underlying its ability to recognize different ligands remain largely unclear. Here we present nine cryo-electron microscopy structures, with eight showing human and mouse TAAR1 in a complex with an array of ligands, including the endogenous 3-iodothyronamine, two antipsychotic agents, the psychoactive drug amphetamine and two identified catecholamine agonists, and one showing 5-HT1AR in a complex with an antipsychotic agent. These structures reveal a rigid consensus binding motif in TAAR1 that binds to endogenous trace amine stimuli and two extended binding pockets that accommodate diverse chemotypes. Combined with mutational analysis, functional assays and molecular dynamic simulations, we elucidate the structural basis of drug polypharmacology and identify the species-specific differences between human and mouse TAAR1. Our study provides insights into the mechanism of ligand recognition and G-protein selectivity by TAAR1, which may help in the discovery of ligands or therapeutic strategies for neurological and metabolic disorders.
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Proteínas de Unión al GTP , Receptores Acoplados a Proteínas G , Animales , Humanos , Ratones , Aminas/metabolismo , Anfetamina/metabolismo , Antipsicóticos/química , Antipsicóticos/metabolismo , Sitios de Unión , Catecolaminas/agonistas , Catecolaminas/química , Catecolaminas/metabolismo , Microscopía por Crioelectrón , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/ultraestructura , Ligandos , Simulación de Dinámica Molecular , Mutación , Polifarmacología , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/ultraestructura , Especificidad de la Especie , Especificidad por SustratoRESUMEN
Trace amine-associated receptor 1 (TAAR1) senses a spectrum of endogenous amine-containing metabolites (EAMs) to mediate diverse psychological functions and is useful for schizophrenia treatment without the side effects of catalepsy. Here, we systematically profiled the signaling properties of TAAR1 activation and present nine structures of TAAR1-Gs/Gq in complex with EAMs, clinical drugs, and synthetic compounds. These structures not only revealed the primary amine recognition pocket (PARP) harboring the conserved acidic D3.32 for conserved amine recognition and "twin" toggle switch for receptor activation but also elucidated that targeting specific residues in the second binding pocket (SBP) allowed modulation of signaling preference. In addition to traditional drug-induced Gs signaling, Gq activation by EAM or synthetic compounds is beneficial to schizophrenia treatment. Our results provided a structural and signaling framework for molecular recognition by TAAR1, which afforded structural templates and signal clues for TAAR1-targeted candidate compounds design.
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Receptores Acoplados a Proteínas G , Transducción de Señal , Humanos , Aminas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Esquizofrenia/metabolismoRESUMEN
Chronic inflammation due to islet-residing macrophages plays key roles in the development of type 2 diabetes mellitus. By systematically profiling intra-islet lipid-transmembrane receptor signalling in islet-resident macrophages, we identified endogenous 9(S)-hydroxy-10,12-octadecadienoic acid-G-protein-coupled receptor 132 (GPR132)-Gi signalling as a significant contributor to islet macrophage reprogramming and found that GPR132 deficiency in macrophages reversed metabolic disorders in mice fed a high-fat diet. The cryo-electron microscopy structures of GPR132 bound with two endogenous agonists, N-palmitoylglycine and 9(S)-hydroxy-10,12-octadecadienoic acid, enabled us to rationally design both GPR132 agonists and antagonists with high potency and selectivity through stepwise translational approaches. We ultimately identified a selective GPR132 antagonist, NOX-6-18, that modulates macrophage reprogramming within pancreatic islets, decreases weight gain and enhances glucose metabolism in mice fed a high-fat diet. Our study not only illustrates that intra-islet lipid signalling contributes to islet macrophage reprogramming but also provides a broadly applicable strategy for the identification of important G-protein-coupled receptor targets in pathophysiological processes, followed by the rational design of therapeutic leads for refractory diseases such as diabetes.
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Diabetes Mellitus Tipo 2 , Islotes Pancreáticos , Ratones , Animales , Diabetes Mellitus Tipo 2/metabolismo , Microscopía por Crioelectrón , Islotes Pancreáticos/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transducción de SeñalRESUMEN
MRGPRX1, a Mas-related GPCR (MRGPR), is a key receptor for itch perception and targeting MRGPRX1 may have potential to treat both chronic itch and pain. Here we report cryo-EM structures of the MRGPRX1-Gi1 and MRGPRX1-Gq trimers in complex with two peptide ligands, BAM8-22 and CNF-Tx2. These structures reveal a shallow orthosteric pocket and its conformational plasticity for sensing multiple different peptidic itch allergens. Distinct from MRGPRX2, MRGPRX1 contains a unique pocket feature at the extracellular ends of TM3 and TM4 to accommodate the peptide C-terminal "RF/RY" motif, which could serve as key mechanisms for peptidic allergen recognition. Below the ligand binding pocket, the G6.48XP6.50F6.51G6.52X(2)F/W6.55 motif is essential for the inward tilting of the upper end of TM6 to induce receptor activation. Moreover, structural features inside the ligand pocket and on the cytoplasmic side of MRGPRX1 are identified as key elements for both Gi and Gq signaling. Collectively, our studies provide structural insights into understanding itch sensation, MRGPRX1 activation, and downstream G protein signaling.
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Receptores Acoplados a Proteínas G , Transducción de Señal , Humanos , Citoplasma , Citosol , Ligandos , PruritoRESUMEN
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.
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Dinoprostona , Transducción de Señal , Dinoprostona/metabolismo , Transducción de Señal/fisiología , Receptores de Prostaglandina/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Hormonas , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Subtipo EP3 de Receptores de Prostaglandina E/metabolismoRESUMEN
The serotonin receptor 5-HT6R is an important G-protein-coupled receptor (GPCR) that involved in essential functions within the central and peripheral nervous systems and is linked to various psychiatric disorders. Selective activation of 5-HT6R promotes neural stem cell regeneration activity. As a 5-HT6R selective agonist, 2-(5 chloro-2-methyl-1H-indol-3-yl)-N, N-dimethylethanolamine (ST1936) has been widely used to investigate the functions of the 5-HT6R. The molecular mechanism of how ST1936 is recognized by 5-HT6R and how it effectively couples with Gs remain unclear. Here, we reconstituted the ST1936-5-HT6R-Gs complex in vitro and solved its cryo-electron microscopy structure at 3.1 Å resolution. Further structural analysis and mutational studies facilitated us to identify the residues of the Y3107.43 and "toggle switch" W2816.48 of the 5-HT6R contributed to the higher efficacy of ST1936 compared with 5-HT. By uncovering the structural foundation of how 5-HT6R specifically recognizes agonists and elucidating the molecular process of G protein activation, our discoveries offer valuable insights and pave the way for the development of promising 5-HT6R agonists.
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Receptores de Serotonina , Serotonina , Humanos , Microscopía por Crioelectrón , Receptores de Serotonina/metabolismo , IndolesRESUMEN
Odorants are detected as smell in the nasal epithelium of mammals by two G-protein-coupled receptor families, the odorant receptors and the trace amine-associated receptors1,2 (TAARs). TAARs emerged following the divergence of jawed and jawless fish, and comprise a large monophyletic family of receptors that recognize volatile amine odorants to elicit both intraspecific and interspecific innate behaviours such as attraction and aversion3-5. Here we report cryo-electron microscopy structures of mouse TAAR9 (mTAAR9) and mTAAR9-Gs or mTAAR9-Golf trimers in complex with ß-phenylethylamine, N,N-dimethylcyclohexylamine or spermidine. The mTAAR9 structures contain a deep and tight ligand-binding pocket decorated with a conserved D3.32W6.48Y7.43 motif, which is essential for amine odorant recognition. In the mTAAR9 structure, a unique disulfide bond connecting the N terminus to ECL2 is required for agonist-induced receptor activation. We identify key structural motifs of TAAR family members for detecting monoamines and polyamines and the shared sequence of different TAAR members that are responsible for recognition of the same odour chemical. We elucidate the molecular basis of mTAAR9 coupling to Gs and Golf by structural characterization and mutational analysis. Collectively, our results provide a structural basis for odorant detection, receptor activation and Golf coupling of an amine olfactory receptor.
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Aminas Biogénicas , Odorantes , Percepción Olfatoria , Poliaminas , Receptores Odorantes , Animales , Ratones , Aminas Biogénicas/análisis , Aminas Biogénicas/química , Aminas Biogénicas/metabolismo , Microscopía por Crioelectrón , Subunidades alfa de la Proteína de Unión al GTP Gs/química , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/ultraestructura , Odorantes/análisis , Percepción Olfatoria/fisiología , Poliaminas/análisis , Poliaminas/química , Poliaminas/metabolismo , Receptores de Amina Biogénica/química , Receptores de Amina Biogénica/genética , Receptores de Amina Biogénica/metabolismo , Receptores de Amina Biogénica/ultraestructura , Receptores Odorantes/química , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Receptores Odorantes/ultraestructura , Olfato/fisiología , Espermidina/análisis , Espermidina/química , Espermidina/metabolismoRESUMEN
In response to spinal surgery, neurons secrete a large amount of substance P into the epidural area. Substance P is involved in macrophage differentiation and fibrotic disease. However, the specific roles and mechanisms of substance P in epidural fibrosis remain unclear. In this study, we established a mouse model of L1-L3 laminectomy and found that dorsal root ganglion neurons and the macrophages infiltrating into the wound area released sphingolipids. In vitro experiments revealed that type 1 macrophages secreted substance P, which promoted differentiation of type 1 macrophages towards a type 2 phenotype. High-throughput mRNA-seq analysis revealed that the sphingolipid metabolic pathway may be involved in the regulation of type 2 macrophages by substance P. Specifically, sphingomyelin synthase 2, a component of the sphingolipid metabolic pathway, promoted M2 differentiation in substance P-treated macrophages, while treating the macrophages with LY93, a sphingomyelin synthase 2 inhibitor, suppressed M2 differentiation. In addition, substance P promoted the formation of neutrophil extracellular traps, which further boosted M2 differentiation. Blocking substance P with the neurokinin receptor 1 inhibitor RP67580 decreased the number of M2 macrophages in the wound area after spinal surgery and alleviated epidural fibrosis, as evidenced by decreased fibronectin, α-smooth muscle actin, and collagen I in the scar tissue. These results demonstrated that substance P promotes M2 macrophage differentiation in epidural fibrosis via sphingomyelin synthase 2 and neutrophil extracellular traps. These findings provide a novel strategy for the treatment of epidural fibrosis.
RESUMEN
The ankle-link complex (ALC) consists of USH2A, WHRN, PDZD7, and ADGRV1 and plays an important role in hair cell development. At present, its architectural organization and signaling role remain unclear. By establishing Adgrv1 Y6236fsX1 mutant mice as a model of the deafness-associated human Y6244fsX1 mutation, the authors show here that the Y6236fsX1 mutation disrupts the interaction between adhesion G protein-coupled receptor V subfamily member 1 (ADGRV1) and other ALC components, resulting in stereocilia disorganization and mechanoelectrical transduction (MET) deficits. Importantly, ADGRV1 inhibits WHRN phosphorylation through regional cAMP-PKA signaling, which in turn regulates the ubiquitination and stability of USH2A via local signaling compartmentalization, whereas ADGRV1 Y6236fsX1 does not. Yeast two-hybrid screening identified the E3 ligase WDSUB1 that binds to WHRN and regulates the ubiquitination of USH2A in a WHRN phosphorylation-dependent manner. Further FlAsH-BRET assay, NMR spectrometry, and mutagenesis analysis provided insights into the architectural organization of ALC and interaction motifs at single-residue resolution. In conclusion, the present data suggest that ALC organization and accompanying local signal transduction play important roles in regulating the stability of the ALC.
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Sordera , Animales , Humanos , Ratones , Proteínas Portadoras/genética , Sordera/genética , Sordera/metabolismo , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Mutación/genética , FosforilaciónRESUMEN
Natural astaxanthin is a high-value ketone carotenoid mainly derived from Haematococcus pluvialis, which is an excellent antioxidant for human consumption. To study the role of lipids in accumulation of astaxanthin, the H. pluvialis-derived astaxanthin synthesis pathway genes (ß-carotene ketolase gene, BKT and ß-carotene hydroxylase gene, BCH) and fatty acid elongation gene (mitochondrial trans-2-enoyl-coa reductase gene, MECR) were heterologously co-expressed in C. reinhardtii. Zeaxanthin, the precursor of astaxanthin synthesis, was significantly increased after BKT and BCH were expressed. In contrast, the α-carotene that competes with astaxanthin synthesis for lycopene decreased significantly. This redistribution of carbon flow was conducive to the synthesis of astaxanthin. In addition, the transformant only expressed astaxanthin metabolism related genes (BKT, BCH) would lead to an increase in total lipid, a decrease in monounsaturated fatty acids and an increase in polyunsaturated fatty acids. On this basis, the expression of MECR gene further increased the total lipid, and the relative content of different fatty acids also changed. The astaxanthin content of algal strains transformed with BKT and BCH genes was nearly 50% higher than that of the wild type. On this basis, the astaxanthin content of transformants expressing MECR gene related to long-chain fatty acid synthesis was increased by 227.5%. In this study, an astaxanthin production model similar to H. pluvialis by combining carotenoid metabolism and fatty acid metabolism was constructed in C. reinhardtii. The results suggest that the increase in astaxanthin is indeed linked to the regulation of fatty acid metabolism, and this link may involve the type of fatty acids and the dynamics of astaxanthin ester in cells. The strategy of promoting the synthesis of fatty acids has potential to achieve efficient production of astaxanthin in C. reinhardtii.