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1.
Proc Natl Acad Sci U S A ; 114(10): 2562-2567, 2017 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-28223524

RESUMO

ß-Arrestins (ßarrs) interact with G protein-coupled receptors (GPCRs) to desensitize G protein signaling, to initiate signaling on their own, and to mediate receptor endocytosis. Prior structural studies have revealed two unique conformations of GPCR-ßarr complexes: the "tail" conformation, with ßarr primarily coupled to the phosphorylated GPCR C-terminal tail, and the "core" conformation, where, in addition to the phosphorylated C-terminal tail, ßarr is further engaged with the receptor transmembrane core. However, the relationship of these distinct conformations to the various functions of ßarrs is unknown. Here, we created a mutant form of ßarr lacking the "finger-loop" region, which is unable to form the core conformation but retains the ability to form the tail conformation. We find that the tail conformation preserves the ability to mediate receptor internalization and ßarr signaling but not desensitization of G protein signaling. Thus, the two GPCR-ßarr conformations can carry out distinct functions.


Assuntos
Endocitose/genética , Proteínas Mutantes/química , Receptores Acoplados a Proteínas G/química , beta-Arrestinas/química , Sequência de Aminoácidos/genética , Reguladores de Proteínas de Ligação ao GTP/genética , Células HEK293 , Humanos , Conformação Molecular , Complexos Multiproteicos , Proteínas Mutantes/genética , Receptores Acoplados a Proteínas G/genética , beta-Arrestinas/genética
2.
Proc Natl Acad Sci U S A ; 114(7): 1708-1713, 2017 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-28130548

RESUMO

The ß2-adrenergic receptor (ß2AR) has been a model system for understanding regulatory mechanisms of G-protein-coupled receptor (GPCR) actions and plays a significant role in cardiovascular and pulmonary diseases. Because all known ß-adrenergic receptor drugs target the orthosteric binding site of the receptor, we set out to isolate allosteric ligands for this receptor by panning DNA-encoded small-molecule libraries comprising 190 million distinct compounds against purified human ß2AR. Here, we report the discovery of a small-molecule negative allosteric modulator (antagonist), compound 15 [([4-((2S)-3-(((S)-3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)amino)-2-(2-cyclohexyl-2-phenylacetamido)-3-oxopropyl)benzamide], exhibiting a unique chemotype and low micromolar affinity for the ß2AR. Binding of 15 to the receptor cooperatively enhances orthosteric inverse agonist binding while negatively modulating binding of orthosteric agonists. Studies with a specific antibody that binds to an intracellular region of the ß2AR suggest that 15 binds in proximity to the G-protein binding site on the cytosolic surface of the ß2AR. In cell-signaling studies, 15 inhibits cAMP production through the ß2AR, but not that mediated by other Gs-coupled receptors. Compound 15 also similarly inhibits ß-arrestin recruitment to the activated ß2AR. This study presents an allosteric small-molecule ligand for the ß2AR and introduces a broadly applicable method for screening DNA-encoded small-molecule libraries against purified GPCR targets. Importantly, such an approach could facilitate the discovery of GPCR drugs with tailored allosteric effects.


Assuntos
Antagonistas Adrenérgicos beta/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Receptores Adrenérgicos beta 2/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Antagonistas Adrenérgicos beta/química , Antagonistas Adrenérgicos beta/metabolismo , Animais , Sítios de Ligação/genética , Ligação Competitiva/efeitos dos fármacos , DNA/genética , Humanos , Ligantes , Estrutura Molecular , Mutação , Receptores Adrenérgicos beta 2/genética , Células Sf9 , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Spodoptera
3.
J Pharmacol Exp Ther ; 358(3): 423-30, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27340210

RESUMO

Growth differentiation factor-11 (GDF11) and myostatin (MSTN) are highly related transforming growth factor-ß (TGF-ß) ligands with 89% amino acid sequence homology. They have different biologic activities and diverse tissue distribution patterns. However, the activities of these ligands are indistinguishable in in vitro assays. SMAD2/3 signaling has been identified as the canonical pathway for GDF11 and MSTN, However, it remains unclear which receptor heterodimer and which antagonists preferentially mediate and regulate signaling. In this study, we investigated the initiation and regulation of GDF11 and MSTN signaling at the receptor level using a novel receptor dimerization detection technology. We used the dimerization platform to link early receptor binding events to intracellular downstream signaling. This approach was instrumental in revealing differential receptor binding activity within the TGF-ß family. We verified the ActR2b/ALK5 heterodimer as the predominant receptor for GDF11- and MSTN-induced SMAD2/3 signaling. We also showed ALK7 specifically mediates activin-B signaling. We verified follistatin as a potent antagonist to neutralize both SMAD2/3 signaling and receptor dimerization. More remarkably, we showed that the two related antagonists, growth and differentiation factor-associated serum protein (GASP)-1 and GASP2, differentially regulate GDF11 (and MSTN) signaling. GASP1 blocks both receptor dimerization and downstream signaling. However, GASP2 blocks only downstream signaling without interference from receptor dimerization. Our data strongly suggest that physical binding of GDF11 (and MSTN) to both ActR2b and ALK5 receptors is required for initiation of signaling.


Assuntos
Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteína 2 Relacionada a Actina/química , Proteína 2 Relacionada a Actina/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Fatores de Diferenciação de Crescimento/metabolismo , Células Hep G2 , Humanos , Miostatina/metabolismo , Ligação Proteica , Multimerização Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Quaternária de Proteína , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/química , Transdução de Sinais , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Especificidade por Substrato
4.
Mutat Res ; 664(1-2): 77-83, 2009 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-19428384

RESUMO

According to a recent IARC Working Group report, alcohol consumption is causally related to an increased risk of cancer of the upper aerodigestive tract, liver, colorectum, and female breast [R. Baan, K. Straif, Y. Grosse, B. Secretan, F. El Ghissassi, V. Bouvard, A. Altieri, V. Cogliano, Carcinogenicity of alcoholic beverages, Lancet Oncol. 8 (2007) 292-293]. Several lines of evidence indicate that acetaldehyde (AA), the first product of alcohol metabolism, plays a very important role in alcohol-related carcinogenesis, particularly in the esophagus. We previously proposed a model for alcohol-related carcinogenesis in which AA, generated from alcohol metabolism, reacts in cells to generate DNA lesions that form interstrand crosslinks (ICLs) [J.A. Theruvathu, P. Jaruga, R.G. Nath, M. Dizdaroglu, P.J. Brooks, Polyamines stimulate the formation of mutagenic 1,N2-propanodeoxyguanosine adducts from acetaldehyde, Nucleic Acids Res. 33 (2005) 3513-3520]. Since the Fanconi anemia-breast cancer associated (FANC-BRCA) DNA damage response network plays a crucial role in protecting cells against ICLs, in the present work we tested this hypothesis by exposing cells to AA and monitoring activation of this network. We found that AA exposure results in a concentration-dependent increase in FANCD2 monoubiquitination, which is dependent upon the FANC core complex. AA also stimulated BRCA1 phosphorylation at Ser1524 and increased the level of gammaH2AX, with both modifications occurring in a dose-dependent manner. However, AA did not detectably increase the levels of hyperphosphorylated RPA34, a marker of single-stranded DNA exposure at replication forks. These results provide the initial description of the AA-DNA damage response, which is qualitatively similar to the cellular response to mitomycin C, a known DNA crosslinking agent. We discuss the mechanistic implications of these results, as well as their possible relationship to alcohol-related carcinogenesis in different human tissues.


Assuntos
Acetaldeído/toxicidade , Proteína BRCA1/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Histonas/metabolismo , Consumo de Bebidas Alcoólicas/efeitos adversos , Linhagem Celular , Reagentes de Ligações Cruzadas/toxicidade , Dano ao DNA/efeitos dos fármacos , Etanol/toxicidade , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Feminino , Humanos , Técnicas In Vitro , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Masculino , Mitomicina/toxicidade , Neoplasias/etiologia , Neoplasias/genética , Neoplasias/metabolismo , Fosforilação/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos
5.
Mol Cell Biol ; 23(15): 5421-30, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12861027

RESUMO

The rare hereditary disorder Fanconi anemia (FA) is characterized by progressive bone marrow failure, congenital skeletal abnormality, elevated susceptibility to cancer, and cellular hypersensitivity to DNA cross-linking chemicals and sometimes other DNA-damaging agents. Molecular cloning identified six causative genes (FANCA, -C, -D2, -E, -F, and -G) encoding a multiprotein complex whose precise biochemical function remains elusive. Recent studies implicate this complex in DNA damage responses that are linked to the breast cancer susceptibility proteins BRCA1 and BRCA2. Mutations in BRCA2, which participates in homologous recombination (HR), are the underlying cause in some FA patients. To elucidate the roles of FA genes in HR, we disrupted the FANCG/XRCC9 locus in the chicken B-cell line DT40. FANCG-deficient DT40 cells resemble mammalian fancg mutants in that they are sensitive to killing by cisplatin and mitomycin C (MMC) and exhibit increased MMC and radiation-induced chromosome breakage. We find that the repair of I-SceI-induced chromosomal double-strand breaks (DSBs) by HR is decreased approximately 9-fold in fancg cells compared with the parental and FANCG-complemented cells. In addition, the efficiency of gene targeting is mildly decreased in FANCG-deficient cells, but depends on the specific locus. We conclude that FANCG is required for efficient HR-mediated repair of at least some types of DSBs.


Assuntos
Dano ao DNA , Proteínas de Ligação a DNA/fisiologia , DNA/efeitos da radiação , Recombinação Genética , Sequência de Aminoácidos , Animais , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Western Blotting , Divisão Celular , Linhagem Celular , Núcleo Celular/metabolismo , Galinhas , Aberrações Cromossômicas , Cisplatino/farmacologia , Clonagem Molecular , Cricetinae , Reagentes de Ligações Cruzadas/farmacologia , DNA/efeitos dos fármacos , Reparo do DNA , DNA Complementar/metabolismo , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Resistência a Medicamentos , Proteína do Grupo de Complementação G da Anemia de Fanconi , Citometria de Fluxo , Teste de Complementação Genética , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/metabolismo , Camundongos , Mitomicina/farmacologia , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Raios X
6.
Chem Biol ; 21(6): 743-53, 2014 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-24856822

RESUMO

Environmental exposures to chemically heterogeneous endocrine-disrupting chemicals (EDCs) mimic or interfere with hormone actions and negatively affect human health. Despite public interest and the prevalence of EDCs in the environment, methods to mechanistically classify these diverse chemicals in a high throughput (HT) manner have not been actively explored. Here, we describe the use of multiparametric, HT microscopy-based platforms to examine how a prototypical EDC, bisphenol A (BPA), and 18 poorly studied BPA analogs (BPXs), affect estrogen receptor (ER). We show that short exposure to BPA and most BPXs induces ERα and/or ERß loading to DNA changing target gene transcription. Many BPXs exhibit higher affinity for ERß and act as ERß antagonists, while they act largely as agonists or mixed agonists and antagonists on ERα. Finally, despite binding to ERs, some BPXs exhibit lower levels of activity. Our comprehensive view of BPXs activities allows their classification and the evaluation of potential harmful effects. The strategy described here used on a large-scale basis likely offers a faster, more cost-effective way to identify safer BPA alternatives.


Assuntos
Compostos Benzidrílicos/química , Compostos Benzidrílicos/farmacologia , Receptor alfa de Estrogênio/agonistas , Receptor beta de Estrogênio/antagonistas & inibidores , Ensaios de Triagem em Larga Escala , Fenóis/química , Fenóis/farmacologia , Compostos Benzidrílicos/efeitos adversos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Células HeLa , Humanos , Células MCF-7 , Microscopia , Fenóis/efeitos adversos , Relação Estrutura-Atividade , Células Tumorais Cultivadas
7.
Methods Mol Biol ; 756: 61-73, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21870220

RESUMO

Despite rapid growth in our knowledge of potential disease targets following completion of the first drafts of the human genome over 10 years ago, the success rate of new therapeutic discovery has been frustratingly low. In addition to the widely reported costs and single-digit success rate of the entire drug discovery and development process, it has recently been estimated that even the preliminary process of transitioning new targets to preclinical development succeeds in less than 3% of attempts [Vogel (ed.) Drug Discovery and Evaluation: Pharmacological Assays. 3rd ed. Springer, Berlin (2007)]. At these early stages of development, poor understanding of therapeutic mechanisms and lack of compound selectivity are often to blame for failed compounds. It is worth noting than the emerging class of nucleic acid-based therapeutics, including miRNA and RNAi, are likely to be even more prone to unexpected system-wide and off-target activities. For all therapeutic approaches, it is clear that discovery strategies permitting the assessment of drug targets in their native context are required. At the same time, these strategies need to retain the high throughput of current reductionist approaches to enable broad assessment of chemical space for small molecule and genetic therapeutics. We describe here an integrated system based on high-content cellular analysis combined with system-wide pathway interrogation. The platform can be applied to novel therapeutic target and drug candidate identification, and for providing detailed mechanistic and selectivity information at an early stage of development.


Assuntos
Descoberta de Drogas/métodos , Proteínas/análise , Animais , Técnicas Citológicas/instrumentação , Técnicas Citológicas/métodos , Descoberta de Drogas/instrumentação , Ensaios de Triagem em Larga Escala/instrumentação , Ensaios de Triagem em Larga Escala/métodos , Humanos , Modelos Moleculares , Proteínas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Biologia de Sistemas/instrumentação , Biologia de Sistemas/métodos
8.
Mutagenesis ; 19(3): 237-44, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15123790

RESUMO

The human FANCG/XRCC9 gene, which is defective in Fanconi anemia complementation group G (FA-G) cells, was first cloned by genetic complementation of the mitomycin C (MMC) sensitivity of CHO mutant UV40. The CHO NM3 mutant was subsequently assigned to the same complementation group. The parental AA8 CHO cells are hemizygous at the FancG locus, and we identified frameshift mutations that result in N-terminal truncations of the protein in both UV40 and NM3. Hypersensitivity to DNA cross-linking agents, such as MMC, typically characterizes FA cells. By introducing the native CHO FancG gene into mutant NM3, we demonstrate that hamster FancG fully corrects the 3-fold sensitivity to methyl methanesulfonate (MMS) as well as the 10-fold sensitivity to MMC, whereas resistance to ionizing radiation did not increase appreciably. In contrast, hamster cDNA transformants showed incomplete correction for both MMC and MMS sensitivity. The constitutively expressed FancG protein is present in the cytoplasmic, nuclear and chromatin fractions. FancG protein levels and subcellular localization do not change appreciably as a function of cell cycle position. Our results are consistent with roles of FancG in both the nuclear and cytoplasmic compartments to maintain genomic stability in response to various genotoxic agents.


Assuntos
Proteínas de Ligação a DNA/genética , Mutação , Alquilantes/farmacologia , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Anemia de Fanconi/genética , Proteína do Grupo de Complementação G da Anemia de Fanconi , Metanossulfonato de Metila/farmacologia , Mitomicina/farmacologia , Dados de Sequência Molecular , Mutagênicos/farmacologia
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