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1.
Cell ; 185(10): 1676-1693.e23, 2022 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-35489334

RESUMO

Epidemiological studies reveal that marijuana increases the risk of cardiovascular disease (CVD); however, little is known about the mechanism. Δ9-tetrahydrocannabinol (Δ9-THC), the psychoactive component of marijuana, binds to cannabinoid receptor 1 (CB1/CNR1) in the vasculature and is implicated in CVD. A UK Biobank analysis found that cannabis was an risk factor for CVD. We found that marijuana smoking activated inflammatory cytokines implicated in CVD. In silico virtual screening identified genistein, a soybean isoflavone, as a putative CB1 antagonist. Human-induced pluripotent stem cell-derived endothelial cells were used to model Δ9-THC-induced inflammation and oxidative stress via NF-κB signaling. Knockdown of the CB1 receptor with siRNA, CRISPR interference, and genistein attenuated the effects of Δ9-THC. In mice, genistein blocked Δ9-THC-induced endothelial dysfunction in wire myograph, reduced atherosclerotic plaque, and had minimal penetration of the central nervous system. Genistein is a CB1 antagonist that attenuates Δ9-THC-induced atherosclerosis.


Assuntos
Cannabis , Doenças Cardiovasculares , Alucinógenos , Analgésicos , Animais , Agonistas de Receptores de Canabinoides/farmacologia , Dronabinol/farmacologia , Células Endoteliais , Genisteína/farmacologia , Genisteína/uso terapêutico , Inflamação/tratamento farmacológico , Camundongos , Receptor CB1 de Canabinoide , Receptores de Canabinoides
3.
Proc Natl Acad Sci U S A ; 117(50): 31824-31831, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33257561

RESUMO

The ß2 adrenergic receptor (ß2AR) is an archetypal G protein coupled receptor (GPCR). One structural signature of GPCR activation is a large-scale movement (ca. 6 to 14 Å) of transmembrane helix 6 (TM6) to a conformation which binds and activates a cognate G protein. The ß2AR exhibits a low level of agonist-independent G protein activation. The structural origin of this basal activity and its suppression by inverse agonists is unknown but could involve a unique receptor conformation that promotes G protein activation. Alternatively, a conformational selection model proposes that a minor population of the canonical active receptor conformation exists in equilibrium with inactive forms, thus giving rise to basal activity of the ligand-free receptor. Previous spin-labeling and fluorescence resonance energy transfer experiments designed to monitor the positional distribution of TM6 did not detect the presence of the active conformation of ligand-free ß2AR. Here we employ spin-labeling and pressure-resolved double electron-electron resonance spectroscopy to reveal the presence of a minor population of unliganded receptor, with the signature outward TM6 displacement, in equilibrium with inactive conformations. Binding of inverse agonists suppresses this population. These results provide direct structural evidence in favor of a conformational selection model for basal activity in ß2AR and provide a mechanism for inverse agonism. In addition, they emphasize 1) the importance of minor populations in GPCR catalytic function; 2) the use of spin-labeling and variable-pressure electron paramagnetic resonance to reveal them in a membrane protein; and 3) the quantitative evaluation of their thermodynamic properties relative to the inactive forms, including free energy, partial molar volume, and compressibility.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Receptores Adrenérgicos beta 2/ultraestrutura , Modelos Moleculares , Pressão , Conformação Proteica em alfa-Hélice , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Termodinâmica
4.
J Biol Chem ; 289(10): 6728-6738, 2014 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-24425866

RESUMO

Staphylococcus aureus causes life-threatening disease in humans. The S. aureus surface protein iron-regulated surface determinant H (IsdH) binds to mammalian hemoglobin (Hb) and extracts heme as a source of iron, which is an essential nutrient for the bacteria. However, the process of heme transfer from Hb is poorly understood. We have determined the structure of IsdH bound to human Hb by x-ray crystallography at 4.2 Å resolution, revealing the structural basis for heme transfer. One IsdH molecule is bound to each α and ß Hb subunit, suggesting that the receptor acquires iron from both chains by a similar mechanism. Remarkably, two near iron transporter (NEAT) domains in IsdH perform very different functions. An N-terminal NEAT domain binds α/ß globin through a site distant from the globin heme pocket and, via an intervening structural domain, positions the C-terminal heme-binding NEAT domain perfectly for heme transfer. These data, together with a 2.3 Å resolution crystal structure of the isolated N-terminal domain bound to Hb and small-angle x-ray scattering of free IsdH, reveal how multiple domains of IsdH cooperate to strip heme from Hb. Many bacterial pathogens obtain iron from human hemoglobin using proteins that contain multiple NEAT domains and other domains whose functions are poorly understood. Our results suggest that, rather than acting as isolated units, NEAT domains may be integrated into higher order architectures that employ multiple interaction interfaces to efficiently extract heme from host proteins.


Assuntos
Antígenos de Bactérias/química , Heme/química , Hemoglobinas/química , Ferro/metabolismo , Receptores de Superfície Celular/química , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/metabolismo , Cristalografia por Raios X , Humanos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Infecções Estafilocócicas/sangue
5.
bioRxiv ; 2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38328157

RESUMO

Large library docking can reveal unexpected chemotypes that complement the structures of biological targets. Seeking new agonists for the cannabinoid-1 receptor (CB1R), we docked 74 million tangible molecules, prioritizing 46 high ranking ones for de novo synthesis and testing. Nine were active by radioligand competition, a 20% hit-rate. Structure-based optimization of one of the most potent of these (Ki = 0.7 uM) led to '4042, a 1.9 nM ligand and a full CB1R agonist. A cryo-EM structure of the purified enantiomer of '4042 ('1350) in complex with CB1R-Gi1 confirmed its docked pose. The new agonist was strongly analgesic, with generally a 5-10-fold therapeutic window over sedation and catalepsy and no observable conditioned place preference. These findings suggest that new cannabinoid chemotypes may disentangle characteristic cannabinoid side-effects from their analgesia, supporting the further development of cannabinoids as pain therapeutics.

6.
bioRxiv ; 2023 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-37693614

RESUMO

Metabotropic glutamate receptors belong to a family of G protein-coupled receptors that are obligate dimers and possess a large extracellular ligand-binding domain (ECD) that is linked via a cysteine-rich domain (CRDs) to their 7-transmembrane (TM) domain. Upon activation, these receptors undergo a large conformational change to transmit the ligand binding signal from the ECD to the G protein-coupling TM. In this manuscript, we propose a model for a sequential, multistep activation mechanism of metabotropic glutamate receptor subtype 5. We present a series of structures in lipid nanodiscs, from inactive to fully active, including agonist-bound intermediate states. Further, using bulk and single-molecule fluorescence imaging we reveal distinct receptor conformations upon allosteric modulator and G protein binding.

7.
Science ; 369(6503)2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32732395

RESUMO

Family B heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) play important roles in carbohydrate metabolism. Recent structures of family B GPCR-Gs protein complexes reveal a disruption in the α-helix of transmembrane segment 6 (TM6) not observed in family A GPCRs. To investigate the functional impact of this structural difference, we compared the structure and function of the glucagon receptor (GCGR; family B) with the ß2 adrenergic receptor (ß2AR; family A). We determined the structure of the GCGR-Gs complex by means of cryo-electron microscopy at 3.1-angstrom resolution. This structure shows the distinct break in TM6. Guanosine triphosphate (GTP) turnover, guanosine diphosphate release, GTP binding, and G protein dissociation studies revealed much slower rates for G protein activation by the GCGR compared with the ß2AR. Fluorescence and double electron-electron resonance studies suggest that this difference is due to the inability of agonist alone to induce a detectable outward movement of the cytoplasmic end of TM6.


Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Receptores Adrenérgicos beta 2/química , Receptores de Glucagon/química , Microscopia Crioeletrônica , Ativação Enzimática , Humanos , Estrutura Secundária de Proteína
8.
Protein Sci ; 23(5): 551-65, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24677446

RESUMO

G-protein coupled receptors (GPCRs) are an important class of membrane protein that transmit extracellular signals invoked by sensing molecules such as hormones and neurotransmitters. GPCR dysfunction is implicated in many diseases and hence these proteins are of great interest to academia and the pharmaceutical industry. Leucine-rich repeat-containing GPCRs contain a characteristic extracellular domain that is an important modulator of intracellular signaling. One member of this class is the leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), a stem cell marker in intestinal crypts, and mammary glands. LGR5 modulates Wnt signaling in the presence of the ligand R-spondin (RSPO). The mechanism of activation of LGR5 by RSPO is not understood, nor is the intracellular signaling mechanism known. Recently reported structures of the extracellular domain of LGR5 bound to RSPO reveal a horseshoe-shaped architecture made up of consecutive leucine-rich repeats, with RSPO bound on the concave surface. This review discusses the discovery of LGR5 and the impact it is having on our understanding of stem cell and cancer biology of the colon. In addition, it covers functional relationships suggested by sequence homology and structural analyses, as well as some intriguing conundrums with respect to the involvement of LGR5 in Wnt signaling.


Assuntos
Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Células-Tronco/metabolismo , Sequência de Aminoácidos , Animais , Neoplasias do Colo/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Transdução de Sinais , Células-Tronco/química , Trombospondinas/metabolismo , Via de Sinalização Wnt
9.
Acta Crystallogr F Struct Biol Commun ; 70(Pt 8): 1032-7, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25084376

RESUMO

Adult haemoglobin (Hb) is made up of two α and two ß subunits. Mutations that reduce expression of the α- or ß-globin genes lead to the conditions α- or ß-thalassaemia, respectively. Whilst both conditions are characterized by anaemia of variable severity, other details of their pathophysiology are different, in part owing to the greater stability of the ß chains that is conferred through ß self-association. In contrast, α subunits interact weakly, and in the absence of stabilizing quaternary interactions the α chain (α) is prone to haem loss and denaturation. The molecular contacts that confer weak self-association of α have not been determined previously. Here, the first structure of an α2 homodimer is reported in complex with one domain of the Hb receptor from Staphylococcus aureus. The α2 dimer interface has a highly unusual, approximately linear, arrangement of four His side chains within hydrogen-bonding distance of each other. Some interactions present in the α1ß1 dimer interface of native Hb are preserved in the α2 dimer. However, a marked asymmetry is observed in the α2 interface, suggesting that steric factors limit the number of stabilizing interactions that can form simultaneously across the interface.


Assuntos
Hemoglobinas/química , Staphylococcus aureus/metabolismo , Cristalização , Cristalografia por Raios X , Dimerização , Hemoglobinas/metabolismo , Ligação Proteica , Conformação Proteica
10.
Hybridoma (Larchmt) ; 26(5): 311-5, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17979547

RESUMO

Despite the potential uses of polyclonal antisera, monoclonal antibodies (MAbs) are preferred for target-specific applications. In vitro immunizations toward the development of hybridomas have become more advantageous in situations of limited antigen availability, small molecular size, and the duration required for the production of MAbs. Cells in the mitotic stage are distinct in their protein profiles compared to cells in the other stages of the cell cycle, and studying these proteins can give various insights into the mechanisms of cell cycles and the interventional scenario, such as mitotic inhibition. Murine splenocytes were immunized in vitro with protein extracts of Chinese Hamster Ovary mitotic cells, and healthy, secretory hybridomas were generated. A 10 day incubation post-immunization in serum-free conditions, 10:1 ratio of fusion partners, and limiting dilutions in the presence of serum, conditioning medium, and syngenic feeder cells resulted in the stable hybridoma clones secreting IgG antibodies. While cell ELISA assay indicated B cells in a population of murine splenocytes and the final antigen-specific secretory cells, double diffusion and ELISA resulted in the fusion and specific efficiencies of the protocols adopted. An antigenic concentration of 2.775 microg produced the maximum fusion efficiency while 3.7 microg of the antigen produced the best specific efficiency.


Assuntos
Citosol/metabolismo , Hibridomas/citologia , Imunização , Mitose/imunologia , Baço/imunologia , Animais , Células CHO , Cricetinae , Cricetulus , Citosol/imunologia , Feminino , Hibridomas/imunologia , Hibridomas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Baço/citologia , Baço/metabolismo
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