Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 47
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Toxicol Lett ; 389: 26-33, 2023 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-37871705

RESUMO

Early-life ozone exposure disrupts normal patterns of lung development, but the molecular determinants underlying these changes are not well understood. This study aimed to elucidate changes in gene expression following episodic ozone exposure to identify potential mechanisms of ozone-mediated impairments in lung development. Rat pups were exposed to either filtered air or ozone (0.5 ppm, 6 hr./day, 5 days/week) from postnatal day (PND) 7-28 (16 dams total with 8 pups each, 4 M & 4 F) and sacrificed at either PND 30-31 or PND 80-84. Lung microdissection isolated major regions for RNA-Seq analysis. Ozone modified inherent differences in gene expression between lung regions in both male and female rat pups, whereas statistically significant changes in gene expression directly attributed to ozone were only identified in females. The greatest number of differentially expressed genes was observed between the distal airways and the parenchyma of ozone-exposed juvenile female rats, with 355 genes being differentially expressed. Genes modulating epithelial-to-mesenchymal transition, cell growth, and adhesion were differentially expressed in the parenchyma of ozone exposed juvenile females, suggesting that episodic ozone exposure may affect branching morphogenesis and lung cell growth. Importantly, our study provides novel targets for future experiments investigating the impact of ozone on lung development.


Assuntos
Pulmão , Ozônio , Ratos , Animais , Masculino , Feminino , Pulmão/metabolismo , Ozônio/toxicidade , Expressão Gênica
2.
bioRxiv ; 2023 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-37461561

RESUMO

There are two main families of G protein-coupled receptors that detect odours in humans, the odorant receptors (ORs) and the trace amine-associated receptors (TAARs). Their amino acid sequences are distinct, with the TAARs being most similar to the aminergic receptors such as those activated by adrenaline, serotonin and histamine. To elucidate the structural determinants of ligand recognition by TAARs, we have determined the cryo-EM structure of a murine receptor, mTAAR7f, coupled to the heterotrimeric G protein Gs and bound to the odorant N,N-dimethylcyclohexylamine (DMCH) to an overall resolution of 2.9 Å. DMCH is bound in a hydrophobic orthosteric binding site primarily through van der Waals interactions and a strong charge-charge interaction between the tertiary amine of the ligand and an aspartic acid residue. This site is distinct and non-overlapping with the binding site for the odorant propionate in the odorant receptor OR51E2. The structure, in combination with mutagenesis data and molecular dynamics simulations suggests that the activation of the receptor follows a similar pathway to that of the ß-adrenoceptors, with the significant difference that DMCH interacts directly with one of the main activation microswitch residues.

3.
Toxicol Sci ; 191(1): 79-89, 2023 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-36331340

RESUMO

Asthma is a common chronic respiratory disease exacerbated by multiple environmental factors. Acute ozone exposure has previously been implicated in airway inflammation, airway hyperreactivity, and other characteristics of asthma, which may be attributable to altered sphingolipid metabolism. This study tested the hypothesis that acute ozone exposure alters sphingolipid metabolism within the lung, which contributes to exacerbations in characteristics of asthma in allergen-sensitized mice. Adult male and female BALB/c mice were sensitized intranasally to house dust mite (HDM) allergen on days 1, 3, and 5 and challenged on days 12-14. Mice were exposed to ozone following each HDM challenge for 6 h/day. Bronchoalveolar lavage, lung lobes, and microdissected lung airways were collected for metabolomics analysis (N = 8/sex/group). Another subset of mice underwent methacholine challenge using a forced oscillation technique to measure airway resistance (N = 6/sex/group). Combined HDM and ozone exposure in male mice synergistically increased airway hyperreactivity that was not observed in females and was accompanied by increased airway inflammation and eosinophilia relative to control mice. Importantly, glycosphingolipids were significantly increased following combined HDM and ozone exposure relative to controls in both male and female airways, which was also associated with both airway resistance and eosinophilia. However, 15 glycosphingolipid species were increased in females compared with only 6 in males, which was concomitant with significant associations between glycosphingolipids and airway resistance that ranged from R2 = 0.33-0.51 for females and R2 = 0.20-0.34 in male mice. These observed sex differences demonstrate that glycosphingolipids potentially serve to mitigate exacerbations in characteristics of allergic asthma.


Assuntos
Asma , Eosinofilia , Ozônio , Feminino , Masculino , Animais , Camundongos , Ozônio/toxicidade , Líquido da Lavagem Broncoalveolar , Asma/induzido quimicamente , Pulmão , Inflamação , Alérgenos/toxicidade , Esfingolipídeos , Modelos Animais de Doenças , Camundongos Endogâmicos BALB C
4.
NanoImpact ; 26: 100404, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35560287

RESUMO

Two-dimensional (2D) engineered nanomaterials are widely used in consumer and industrial goods due to their unique chemical and physical characteristics. Engineered nanomaterials are incredibly small and capable of being aerosolized during manufacturing, with the potential for biological interaction at first-contact sites such as the eye and lung. The unique properties of 2D nanomaterials that make them of interest to many industries may also cause toxicity towards epithelial cells. Using murine and human respiratory epithelial cell culture models, we tested the cytotoxicity of eight 2D engineered nanomaterials: graphene (110 nm), graphene oxide (2 um), graphene oxide (400 nm), reduced graphene oxide (2 um), reduced graphene oxide (400 nm), partially reduced graphene oxide (400 nm), molybdenum disulfide (400 nm), and hexagonal boron nitride (150 nm). Non-graphene nanomaterials were also tested in human corneal epithelial cells for ocular epithelial cytotoxicity. Hexagonal boron nitride was found to be cytotoxic in mouse tracheal, human alveolar, and human corneal epithelial cells. Hexagonal boron nitride was also tested for inhibition of wound healing in alveolar epithelial cells; no inhibition was seen at sub-cytotoxic doses. Nanomaterials should be considered with care before use, due to specific regional cytotoxicity that also varies by cell type. Supported by U01ES027288 and T32HL007013 and T32ES007059.


Assuntos
Epitélio Corneano , Nanoestruturas , Células Epiteliais Alveolares , Animais , Células Epiteliais , Camundongos , Nanoestruturas/toxicidade , Tórax
5.
Toxicol Sci ; 184(2): 214-222, 2021 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-34498071

RESUMO

Naphthalene is a ubiquitous environmental contaminant produced by combustion of fossil fuels and is a primary constituent of both mainstream and side stream tobacco smoke. Naphthalene elicits region-specific toxicity in airway club cells through cytochrome P450 (P450)-mediated bioactivation, resulting in depletion of glutathione and subsequent cytotoxicity. Although effects of naphthalene in mice have been extensively studied, few experiments have characterized global metabolomic changes in the lung. In individual lung regions, we found metabolomic changes in microdissected mouse lung conducting airways and parenchyma obtained from animals sacrificed at 3 timepoints following naphthalene treatment. Data on 577 unique identified metabolites were acquired by accurate mass spectrometry-based assays focusing on lipidomics and nontargeted metabolomics of hydrophilic compounds. Statistical analyses revealed distinct metabolite profiles between the 2 lung regions. Additionally, the number and magnitude of statistically significant exposure-induced changes in metabolite abundance were different between airways and parenchyma for unsaturated lysophosphatidylcholines, dipeptides, purines, pyrimidines, and amino acids. Importantly, temporal changes were found to be highly distinct for male and female mice with males exhibiting predominant treatment-specific changes only at 2 h postexposure. In females, metabolomic changes persisted until 6 h postnaphthalene treatment, which may explain the previously characterized higher susceptibility of female mice to naphthalene toxicity. In both males and females, treatment-specific changes corresponding to lung remodeling, oxidative stress response, and DNA damage were observed. Overall, this study provides insights into potential mechanisms contributing to naphthalene toxicity and presents a novel approach for lung metabolomic analysis that distinguishes responses of major lung regions.


Assuntos
Pulmão , Microdissecção , Naftalenos/toxicidade , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Feminino , Pulmão/patologia , Masculino , Metabolômica/métodos , Camundongos , Fatores Sexuais
6.
Nanoscale ; 13(31): 13519-13528, 2021 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-34477756

RESUMO

Membrane proteins are of fundamental importance to cellular processes and nano-encapsulation strategies that preserve their native lipid bilayer environment are particularly attractive for studying and exploiting these proteins. Poly(styrene-co-maleic acid) (SMA) and related polymers poly(styrene-co-(N-(3-N',N'-dimethylaminopropyl)maleimide)) (SMI) and poly(diisobutylene-alt-maleic acid) (DIBMA) have revolutionised the study of membrane proteins by spontaneously solubilising membrane proteins direct from cell membranes within nanoscale discs of native bilayer called SMA lipid particles (SMALPs), SMILPs and DIBMALPs respectively. This systematic study shows for the first time, that conformational changes of the encapsulated protein are dictated by the solubilising polymer. The photoactivation pathway of rhodopsin (Rho), a G-protein-coupled receptor (GPCR), comprises structurally-defined intermediates with characteristic absorbance spectra that revealed conformational restrictions with styrene-containing SMA and SMI, so that photoactivation proceeded only as far as metarhodopsin-I, absorbing at 478 nm, in a SMALP or SMILP. In contrast, full attainment of metarhodopsin-II, absorbing at 382 nm, was observed in a DIBMALP. Consequently, different intermediate states of Rho could be generated readily by simply employing different SMA-like polymers. Dynamic light-scattering and analytical ultracentrifugation revealed differences in size and thermostability between SMALP, SMILP and DIBMALP. Moreover, encapsulated Rho exhibited different stability in a SMALP, SMILP or DIBMALP. Overall, we establish that SMA, SMI and DIBMA constitute a 'toolkit' of solubilising polymers, so that selection of the appropriate solubilising polymer provides a spectrum of useful attributes for studying membrane proteins.


Assuntos
Proteínas de Membrana , Polímeros , Bicamadas Lipídicas , Maleatos , Poliestirenos , Rodopsina , Estireno
7.
Proc Natl Acad Sci U S A ; 118(31)2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34326250

RESUMO

G protein-coupled receptors (GPCRs) are important pharmaceutical targets for the treatment of a broad spectrum of diseases. Although there are structures of GPCRs in their active conformation with bound ligands and G proteins, the detailed molecular interplay between the receptors and their signaling partners remains challenging to decipher. To address this, we developed a high-sensitivity, high-throughput matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) method to interrogate the first stage of signal transduction. GPCR-G protein complex formation is detected as a proxy for the effect of ligands on GPCR conformation and on coupling selectivity. Over 70 ligand-GPCR-partner protein combinations were studied using as little as 1.25 pmol protein per sample. We determined the selectivity profile and binding affinities of three GPCRs (rhodopsin, beta-1 adrenergic receptor [ß1AR], and angiotensin II type 1 receptor) to engineered Gα-proteins (mGs, mGo, mGi, and mGq) and nanobody 80 (Nb80). We found that GPCRs in the absence of ligand can bind mGo, and that the role of the G protein C terminus in GPCR recognition is receptor-specific. We exemplified our quantification method using ß1AR and demonstrated the allosteric effect of Nb80 binding in assisting displacement of nadolol to isoprenaline. We also quantified complex formation with wild-type heterotrimeric Gαißγ and ß-arrestin-1 and showed that carvedilol induces an increase in coupling of ß-arrestin-1 and Gαißγ to ß1AR. A normalization strategy allows us to quantitatively measure the binding affinities of GPCRs to partner proteins. We anticipate that this methodology will find broad use in screening and characterization of GPCR-targeting drugs.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Receptores Opioides/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Arrestina/genética , Arrestina/metabolismo , Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica , Células HEK293 , Humanos , Ligantes , Camundongos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Receptores Opioides/química , Anticorpos de Cadeia Única , Perus , beta-Arrestina 1/genética , beta-Arrestina 1/metabolismo
8.
Regul Toxicol Pharmacol ; 116: 104761, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32768664

RESUMO

4-Methylimidazole (4MEI) is a contaminant in food and consumer products. Pulmonary toxicity and carcinogenicity following chronic dietary exposures to 4MEI is a regulatory concern based on previous rodent studies. This study examined acute pulmonary toxicity in B6C3F1 mice from 6 h to 5 days after oral gavage with a single dose of 150 mg/kg 4MEI, a double dose delivered 6 h apart, or vehicle controls. Oral gavage of 150 mg/kg naphthalene, a prototypical Club cell toxicant, was used as a positive control. Intrapulmonary conducting airway cytotoxicity was assessed in fixed-pressure inflated lungs using qualitative histopathology scoring, quantitative morphometric measurement of vacuolated and exfoliating epithelial cells, and immunohistochemistry. 4MEI treatment did not change markers of cytotoxicity including the mass of vacuolated epithelium, the thickness of the epithelium, or the distributions of epithelial proteins: secretoglobin 1A1, proliferating cell nuclear antigen, calcitonin gene-related peptide, and myeloperoxidase. 4MEI and vehicle controls caused slight cytotoxicity with rare vacuolization of the epithelium relative to the severe bronchiolar epithelial cell toxicity found in the naphthalene exposed mice at terminal bronchioles, intrapulmonary airways, or airway bifurcations. In summary, 4MEI caused minimal airway epithelial toxicity without characteristic Club Cell toxicity when compared to naphthalene, a canonical Club Cell toxicant.


Assuntos
Poluentes Ambientais/toxicidade , Imidazóis/toxicidade , Naftalenos/toxicidade , Mucosa Respiratória/efeitos dos fármacos , Administração Oral , Animais , Feminino , Masculino , Camundongos , Mucosa Respiratória/patologia
9.
Nature ; 583(7818): 862-866, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32555462

RESUMO

The ß1-adrenoceptor (ß1AR) is a G-protein-coupled receptor (GPCR) that couples1 to the heterotrimeric G protein Gs. G-protein-mediated signalling is terminated by phosphorylation of the C terminus of the receptor by GPCR kinases (GRKs) and by coupling of ß-arrestin 1 (ßarr1, also known as arrestin 2), which displaces Gs and induces signalling through the MAP kinase pathway2. The ability of synthetic agonists to induce signalling preferentially through either G proteins or arrestins-known as biased agonism3-is important in drug development, because the therapeutic effect may arise from only one signalling cascade, whereas the other pathway may mediate undesirable side effects4. To understand the molecular basis for arrestin coupling, here we determined the cryo-electron microscopy structure of the ß1AR-ßarr1 complex in lipid nanodiscs bound to the biased agonist formoterol5, and the crystal structure of formoterol-bound ß1AR coupled to the G-protein-mimetic nanobody6 Nb80. ßarr1 couples to ß1AR in a manner distinct to that7 of Gs coupling to ß2AR-the finger loop of ßarr1 occupies a narrower cleft on the intracellular surface, and is closer to transmembrane helix H7 of the receptor when compared with the C-terminal α5 helix of Gs. The conformation of the finger loop in ßarr1 is different from that adopted by the finger loop of visual arrestin when it couples to rhodopsin8. ß1AR coupled to ßarr1 shows considerable differences in structure compared with ß1AR coupled to Nb80, including an inward movement of extracellular loop 3 and the cytoplasmic ends of H5 and H6. We observe weakened interactions between formoterol and two serine residues in H5 at the orthosteric binding site of ß1AR, and find that formoterol has a lower affinity for the ß1AR-ßarr1 complex than for the ß1AR-Gs complex. The structural differences between these complexes of ß1AR provide a foundation for the design of small molecules that could bias signalling in the ß-adrenoceptors.


Assuntos
Microscopia Crioeletrônica , Fumarato de Formoterol/química , Fumarato de Formoterol/metabolismo , Receptores Adrenérgicos beta 1/química , Receptores Adrenérgicos beta 1/ultraestrutura , beta-Arrestina 1/química , beta-Arrestina 1/ultraestrutura , Sequência de Aminoácidos , Animais , Sítios de Ligação , 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 , Células HEK293 , Humanos , Modelos Moleculares , Complexos Multiproteicos , Receptores Adrenérgicos beta 1/metabolismo , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/metabolismo , Anticorpos de Cadeia Única/ultraestrutura , Peixe-Zebra , beta-Arrestina 1/metabolismo
10.
J Vis Exp ; (157)2020 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-32225143

RESUMO

The key to determining crystal structures of membrane protein complexes is the quality of the sample prior to crystallization. In particular, the choice of detergent is critical, because it affects both the stability and monodispersity of the complex. We recently determined the crystal structure of an active state of bovine rhodopsin coupled to an engineered G protein, mini-Go, at 3.1 Å resolution. Here, we detail the procedure for optimizing the preparation of the rhodopsin-mini-Go complex. Dark-state rhodopsin was prepared in classical and neopentyl glycol (NPG) detergents, followed by complex formation with mini-Go under light exposure. The stability of the rhodopsin was assessed by ultraviolet-visible (UV-VIS) spectroscopy, which monitors the reconstitution into rhodopsin of the light-sensitive ligand, 9-cis retinal. Automated size-exclusion chromatography (SEC) was used to characterize the monodispersity of rhodopsin and the rhodopsin-mini-Go complex. SDS-polyacrylamide electrophoresis (SDS-PAGE) confirmed the formation of the complex by identifying a 1:1 molar ratio between rhodopsin and mini-Go after staining the gel with Coomassie blue. After cross-validating all this analytical data, we eliminated unsuitable detergents and continued with the best candidate detergent for large-scale preparation and crystallization. An additional problem arose from the heterogeneity of N-glycosylation. Heterologously-expressed rhodopsin was observed on SDS-PAGE to have two different N-glycosylated populations, which would probably have hindered crystallogenesis. Therefore, different deglycosylation enzymes were tested, and endoglycosidase F1 (EndoF1) produced rhodopsin with a single species of N-glycosylation. With this strategic pipeline for characterizing protein quality, preparation of the rhodopsin-mini-Go complex was optimized to deliver the crystal structure. This was only the third crystal structure of a GPCR-G protein signaling complex. This approach can also be generalized for other membrane proteins and their complexes to facilitate sample preparation and structure determination.


Assuntos
Cristalização/métodos , Proteínas de Ligação ao GTP/metabolismo , Transdução de Sinais
11.
Science ; 364(6442): 775-778, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31072904

RESUMO

G protein-coupled receptors (GPCRs) in the G protein-coupled active state have higher affinity for agonists as compared with when they are in the inactive state, but the molecular basis for this is unclear. We have determined four active-state structures of the ß1-adrenoceptor (ß1AR) bound to conformation-specific nanobodies in the presence of agonists of varying efficacy. Comparison with inactive-state structures of ß1AR bound to the identical ligands showed a 24 to 42% reduction in the volume of the orthosteric binding site. Potential hydrogen bonds were also shorter, and there was up to a 30% increase in the number of atomic contacts between the receptor and ligand. This explains the increase in agonist affinity of GPCRs in the active state for a wide range of structurally distinct agonists.


Assuntos
Agonistas de Receptores Adrenérgicos beta 1/química , Desenho de Fármacos , Receptores Acoplados a Proteínas G/agonistas , Agonistas de Receptores Adrenérgicos beta 1/farmacologia , Sítio Alostérico/imunologia , Domínio Catalítico/imunologia , Ligação de Hidrogênio , Ligantes , Ligação Proteica , Estrutura Secundária de Proteína , Receptores Adrenérgicos beta 1/química , Receptores Adrenérgicos beta 1/imunologia , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/imunologia , Anticorpos de Domínio Único/imunologia
12.
Sci Adv ; 4(9): eaat7052, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30255144

RESUMO

Selective coupling of G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors (GPCRs) to specific Gα-protein subtypes is critical to transform extracellular signals, carried by natural ligands and clinical drugs, into cellular responses. At the center of this transduction event lies the formation of a signaling complex between the receptor and G protein. We report the crystal structure of light-sensitive GPCR rhodopsin bound to an engineered mini-Go protein. The conformation of the receptor is identical to all previous structures of active rhodopsin, including the complex with arrestin. Thus, rhodopsin seems to adopt predominantly one thermodynamically stable active conformation, effectively acting like a "structural switch," allowing for maximum efficiency in the visual system. Furthermore, our analysis of the well-defined GPCR-G protein interface suggests that the precise position of the carboxyl-terminal "hook-like" element of the G protein (its four last residues) relative to the TM7/helix 8 (H8) joint of the receptor is a significant determinant in selective G protein activation.


Assuntos
Receptores Acoplados a Proteínas G/química , Rodopsina/química , Rodopsina/metabolismo , Animais , Sítios de Ligação , Bovinos , Cristalografia por Raios X , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Mutação , Conformação Proteica , Receptores Acoplados a Proteínas G/metabolismo , Rodopsina/genética
13.
Nature ; 558(7711): 620-623, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29925951

RESUMO

G-protein-coupled receptors (GPCRs) form the largest family of receptors encoded by the human genome (around 800 genes). They transduce signals by coupling to a small number of heterotrimeric G proteins (16 genes encoding different α-subunits). Each human cell contains several GPCRs and G proteins. The structural determinants of coupling of Gs to four different GPCRs have been elucidated1-4, but the molecular details of how the other G-protein classes couple to GPCRs are unknown. Here we present the cryo-electron microscopy structure of the serotonin 5-HT1B receptor (5-HT1BR) bound to the agonist donitriptan and coupled to an engineered Go heterotrimer. In this complex, 5-HT1BR is in an active state; the intracellular domain of the receptor is in a similar conformation to that observed for the ß2-adrenoceptor (ß2AR) 3 or the adenosine A2A receptor (A2AR) 1 in complex with Gs. In contrast to the complexes with Gs, the gap between the receptor and the Gß-subunit in the Go-5-HT1BR complex precludes molecular contacts, and the interface between the Gα-subunit of Go and the receptor is considerably smaller. These differences are likely to be caused by the differences in the interactions with the C terminus of the Go α-subunit. The molecular variations between the interfaces of Go and Gs in complex with GPCRs may contribute substantially to both the specificity of coupling and the kinetics of signalling.


Assuntos
Microscopia Crioeletrônica , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/ultraestrutura , Receptor 5-HT1B de Serotonina/metabolismo , Receptor 5-HT1B de Serotonina/ultraestrutura , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Humanos , Modelos Moleculares , Nitrilas/química , Nitrilas/metabolismo , Piperazinas/química , Piperazinas/metabolismo , Conformação Proteica , Receptor 5-HT1B de Serotonina/química , Agonistas do Receptor 5-HT1 de Serotonina/química , Agonistas do Receptor 5-HT1 de Serotonina/metabolismo , Triptaminas/química , Triptaminas/metabolismo
14.
Methods Enzymol ; 594: 243-264, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28779842

RESUMO

Structure determination of G protein-coupled receptors (GPCRs) in the inactive state bound to high-affinity antagonists has been very successful through the implementation of a number of protein engineering and crystallization strategies. However, the structure determination of GPCRs in their fully active state coupled to a G protein is still very challenging. Recently, mini-G proteins were developed, which recapitulate the coupling of a full heterotrimeric G protein to a GPCR despite being less than one-third of the size. This allowed the structure determination of the agonist-bound adenosine A2A receptor (A2AR) coupled to mini-Gs. Although this is extremely encouraging, A2AR is very stable compared with many other GPCRs, particularly when an agonist is bound. In contrast, the agonist-bound conformation of the human corticotropin-releasing factor receptor is considerably less stable, impeding the formation of good quality crystals for structure determination. We have therefore developed a novel strategy for the thermostabilization of a GPCR-mini-G protein complex. In this chapter, we will describe the theoretical and practical principles of the thermostability assay for stabilizing this complex, discuss its strengths and weaknesses, and show some typical results from the thermostabilization process.


Assuntos
Bioquímica/métodos , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/agonistas , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas de Anfíbios/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Radioisótopos do Iodo/química , Hormônios Peptídicos/química , Estabilidade Proteica , Receptores de Hormônio Liberador da Corticotropina/agonistas , Receptores de Hormônio Liberador da Corticotropina/química , Receptores de Hormônio Liberador da Corticotropina/metabolismo
15.
PLoS One ; 12(4): e0175642, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28426733

RESUMO

Mini-G proteins are the engineered GTPase domains of Gα subunits. They couple to GPCRs and recapitulate the increase in agonist affinity observed upon coupling of a native heterotrimeric G protein. Given the small size and stability of mini-G proteins, and their ease of expression and purification, they are ideal for biophysical studies of GPCRs in their fully active state. The first mini-G protein developed was mini-Gs. Here we extend the family of mini-G proteins to include mini-Golf, mini-Gi1, mini-Go1 and the chimeras mini-Gs/q and mini-Gs/i. The mini-G proteins were shown to couple to relevant GPCRs and to form stable complexes with purified receptors that could be purified by size exclusion chromatography. Agonist-bound GPCRs coupled to a mini-G protein showed higher thermal stability compared to the agonist-bound receptor alone. Fusion of GFP at the N-terminus of mini-G proteins allowed receptor coupling to be monitored by fluorescence-detection size exclusion chromatography (FSEC) and, in a separate assay, the affinity of mini-G protein binding to detergent-solubilised receptors was determined. This work provides the foundation for the development of any mini-G protein and, ultimately, for the structure determination of GPCRs in a fully active state.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sequência de Aminoácidos , Cromatografia em Gel , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/classificação , Humanos , Ligantes , Filogenia , Conformação Proteica , Receptores Acoplados a Proteínas G/química , Homologia de Sequência de Aminoácidos , Espectrometria de Fluorescência
16.
Methods Mol Biol ; 1432: 63-78, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27485330

RESUMO

Overexpression of mammalian membrane proteins in mammalian cells is an effective strategy to produce sufficient protein for biophysical analyses and structural studies, because the cells generally express proteins in a correctly folded state. However, obtaining high levels of expression suitable for protein purification on a milligram scale can be challenging. As membrane protein overexpression often has a negative impact on cell viability, it is usual to make stable cell lines where the protein of interest is expressed from an inducible promoter. Here we describe a methodology for optimizing the inducible production of any membrane protein fused to GFP through the isolation of clonal cell lines. Flow cytometry is used to sort uninduced cells and the most fluorescent 5 % of the cell population are used to make clonal cell lines.


Assuntos
Técnicas de Cultura de Células/métodos , Proteínas de Membrana/metabolismo , Tetraciclina/farmacologia , Animais , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Células Clonais/citologia , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/genética , Regiões Promotoras Genéticas , Engenharia de Proteínas
17.
Toxicol Pathol ; 44(5): 673-86, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27025955

RESUMO

Silver nanoparticle (Ag NP) production methods are being developed and refined to produce more uniform Ag NPs through chemical reactions involving silver salt solutions, solvents, and capping agents to control particle formation. These chemical reactants are often present as contaminants and/or coatings on the Ag NPs, which could alter their interactions in vivo. To determine pulmonary effects of citrate-coated Ag NPs, Sprague-Dawley rats were exposed once nose-only to aerosolized Ag NPs (20 nm [C20] or 110 nm [C110] Ag NPs) for 6 hr. Bronchoalveolar lavage fluid (BALF) and lung tissues were obtained at 1, 7, 21, and 56 days postexposure for analyses. Inhalation of Ag NPs, versus citrate buffer control, produced significant inflammatory and cytotoxic responses that were measured in BALF cells and supernatant. At day 7, total cells, protein, and lactate dehydrogenase were significantly elevated in BALF, and peak histopathology was noted after C20 or C110 exposure versus control. At day 21, BALF polymorphonuclear cells and tissue inflammation were significantly greater after C20 versus C110 exposure. By day 56, inflammation was resolved in Ag NP-exposed animals. Overall, results suggest delayed, short-lived inflammatory and cytotoxic effects following C20 or C110 inhalation and potential for greater responses following C20 exposure.


Assuntos
Pulmão/patologia , Nanopartículas Metálicas/toxicidade , Prata/toxicidade , Administração por Inalação , Animais , Líquido da Lavagem Broncoalveolar , Pulmão/efeitos dos fármacos , Masculino , Nanopartículas Metálicas/administração & dosagem , Tamanho da Partícula , Ratos , Ratos Sprague-Dawley , Prata/administração & dosagem
18.
PLoS One ; 11(3): e0151582, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26982879

RESUMO

The factors defining the correct folding and stability of integral membrane proteins are poorly understood. Folding of only a few select membrane proteins has been scrutinised, leaving considerable deficiencies in knowledge for large protein families, such as G protein coupled receptors (GPCRs). Complete reversible folding, which is problematic for any membrane protein, has eluded this dominant receptor family. Moreover, attempts to recover receptors from denatured states are inefficient, yielding at best 40-70% functional protein. We present a method for the reversible unfolding of an archetypal family member, the ß1-adrenergic receptor, and attain 100% recovery of the folded, functional state, in terms of ligand binding, compared to receptor which has not been subject to any unfolding and retains its original, folded structure. We exploit refolding on a solid support, which could avoid unwanted interactions and aggregation that occur in bulk solution. We determine the changes in structure and function upon unfolding and refolding. Additionally, we employ a method that is relatively new to membrane protein folding; pulse proteolysis. Complete refolding of ß1-adrenergic receptor occurs in n-decyl-ß-D-maltoside (DM) micelles from a urea-denatured state, as shown by regain of its original helical structure, ligand binding and protein fluorescence. The successful refolding strategy on a solid support offers a defined method for the controlled refolding and recovery of functional GPCRs and other membrane proteins that suffer from instability and irreversible denaturation once isolated from their native membranes.


Assuntos
Receptores Acoplados a Proteínas G/metabolismo , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Ligantes , Dobramento de Proteína , Desdobramento de Proteína , Proteólise , Receptores Acoplados a Proteínas G/química , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Ureia/química
19.
Mol Pharmacol ; 87(6): 907-15, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25762024

RESUMO

The adenosine A2A receptor (A(2A)R) plays a key role in transmembrane signaling mediated by the endogenous agonist adenosine. Here, we describe the crystal structure of human A2AR thermostabilized in an active-like conformation bound to the selective agonist 2-[p-(2-carboxyethyl)phenylethyl-amino]-5'-N-ethylcarboxamido adenosine (CGS21680) at a resolution of 2.6 Å. Comparison of A(2A)R structures bound to either CGS21680, 5'-N-ethylcarboxamido adenosine (NECA), UK432097 [6-(2,2-diphenylethylamino)-9-[(2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxy-tetrahydrofuran-2-yl]-N-[2-[[1-(2-pyridyl)-4-piperidyl]carbamoylamino]ethyl]purine-2-carboxamide], or adenosine shows that the adenosine moiety of the ligands binds to the receptor in an identical fashion. However, an extension in CGS21680 compared with adenosine, the (2-carboxyethyl)phenylethylamino group, binds in an extended vestibule formed from transmembrane regions 2 and 7 (TM2 and TM7) and extracellular loops 2 and 3 (EL2 and EL3). The (2-carboxyethyl)phenylethylamino group makes van der Waals contacts with side chains of amino acid residues Glu169(EL2), His264(EL3), Leu267(7.32), and Ile274(7.39), and the amine group forms a hydrogen bond with the side chain of Ser67(2.65). Of these residues, only Ile274(7.39) is absolutely conserved across the human adenosine receptor subfamily. The major difference between the structures of A(2A)R bound to either adenosine or CGS21680 is that the binding pocket narrows at the extracellular surface when CGS21680 is bound, due to an inward tilt of TM2 in that region. This conformation is stabilized by hydrogen bonds formed by the side chain of Ser67(2.65) to CGS21680, either directly or via an ordered water molecule. Mutation of amino acid residues Ser67(2.65), Glu169(EL2), and His264(EL3), and analysis of receptor activation either in the presence or absence of ligands implicates this region in modulating the level of basal activity of A(2A)R.


Assuntos
Agonistas do Receptor A2 de Adenosina/química , Adenosina/análogos & derivados , Fenetilaminas/química , Receptor A2A de Adenosina/química , Adenosina/química , Adenosina/farmacologia , Agonistas do Receptor A2 de Adenosina/farmacologia , Animais , Células CHO , Cricetulus , Cristalografia por Raios X , AMP Cíclico/biossíntese , Humanos , Modelos Moleculares , Fenetilaminas/farmacologia , Conformação Proteica , Receptor A2A de Adenosina/metabolismo
20.
Toxicol Sci ; 144(1): 151-62, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25628415

RESUMO

Silver nanoparticles (Ag NPs) can be found in myriad consumer products, medical equipment/supplies, and public spaces. However, questions remain regarding the risks associated with Ag NP exposure. As part of a consortium-based effort to better understand these nanomaterials, this study examined how Ag NPs with varying sizes and coatings affect pulmonary responses at different time-points. Four types of Ag NPs were tested: 20 nm (C20) and 110 nm (C110) citrate-stabilized NPs, and 20 nm (P20) and 110 nm (P110) PVP-stabilized NPs. Male, Sprague Dawley rats were intratracheally instilled with Ag NPs (0, 0.1, 0.5, or 1.0 mg/kg bodyweight [BW]), and bronchoalveolar lavage fluid (BALF) and lung tissues were obtained at 1, 7, and 21 days post-exposure for analysis of BAL cells and histopathology. All Ag NP types produced significantly elevated polymorphonuclear cells (PMNs) in BALF on Days 1, 7, and/or 21 at the 0.5 and/or 1.0 mg/kg BW dose(s). Histology of animals exposed to 1.0 mg/kg BW Ag NPs showed patchy, focal, centriacinar inflammation for all time-points; though neutrophils, macrophages, and/or monocytes were also found in the airway submucosa and perivascular regions at Days 1 and 7. Confocal microscopy of ethidium homodimer-stained lungs at Day 1 showed dead/dying cells at branch points along the main airway. By Day 21, only animals exposed to the high dose of C110 or P110 exhibited significant BALF neutrophilia and marked cellular debris in alveolar airspaces. Findings suggest that 110 nm Ag NPs may produce lasting effects past Day 21 post instillation.


Assuntos
Exposição por Inalação , Pulmão/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Pneumonia/induzido quimicamente , Prata/toxicidade , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Ácido Cítrico/química , Ácido Cítrico/toxicidade , Relação Dose-Resposta a Droga , Pulmão/metabolismo , Pulmão/patologia , Masculino , Nanopartículas Metálicas/química , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/patologia , Tamanho da Partícula , Pneumonia/metabolismo , Pneumonia/patologia , Povidona/química , Povidona/toxicidade , Ratos Sprague-Dawley , Medição de Risco , Prata/química , Propriedades de Superfície , Fatores de Tempo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...