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1.
Nat Chem Biol ; 14(12): 1150-1158, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30420692

RESUMO

Human muscarinic receptor M2 is one of the five subtypes of muscarinic receptors belonging to the family of G-protein-coupled receptors. Muscarinic receptors are targets for multiple neurodegenerative diseases. The challenge has been designing subtype-selective ligands against one of the five muscarinic receptors. We report high-resolution structures of a thermostabilized mutant M2 receptor bound to a subtype-selective antagonist AF-DX 384 and a nonselective antagonist NMS. The thermostabilizing mutation S110R in M2 was predicted using a theoretical strategy previously developed in our group. Comparison of the crystal structures and pharmacological properties of the M2 receptor shows that the Arg in the S110R mutant mimics the stabilizing role of the sodium cation, which is known to allosterically stabilize inactive state(s) of class A GPCRs. Molecular dynamics simulations reveal that tightening of the ligand-residue contacts in M2 receptors compared to M3 receptors leads to subtype selectivity of AF-DX 384.


Assuntos
Antagonistas Muscarínicos/metabolismo , Pirenzepina/análogos & derivados , Receptor Muscarínico M2/química , Receptor Muscarínico M2/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Estabilidade Enzimática , Humanos , Simulação de Dinâmica Molecular , Antagonistas Muscarínicos/química , Mutação , N-Metilescopolamina/química , N-Metilescopolamina/metabolismo , Pirenzepina/química , Pirenzepina/metabolismo , Receptor Muscarínico M2/antagonistas & inibidores
2.
Nature ; 475(7354): 65-70, 2011 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-21697825

RESUMO

The biogenic amine histamine is an important pharmacological mediator involved in pathophysiological processes such as allergies and inflammations. Histamine H(1) receptor (H(1)R) antagonists are very effective drugs alleviating the symptoms of allergic reactions. Here we show the crystal structure of the H(1)R complex with doxepin, a first-generation H(1)R antagonist. Doxepin sits deep in the ligand-binding pocket and directly interacts with Trp 428(6.48), a highly conserved key residue in G-protein-coupled-receptor activation. This well-conserved pocket with mostly hydrophobic nature contributes to the low selectivity of the first-generation compounds. The pocket is associated with an anion-binding region occupied by a phosphate ion. Docking of various second-generation H(1)R antagonists reveals that the unique carboxyl group present in this class of compounds interacts with Lys 191(5.39) and/or Lys 179(ECL2), both of which form part of the anion-binding region. This region is not conserved in other aminergic receptors, demonstrating how minor differences in receptors lead to pronounced selectivity differences with small molecules. Our study sheds light on the molecular basis of H(1)R antagonist specificity against H(1)R.


Assuntos
Doxepina/metabolismo , Antagonistas dos Receptores Histamínicos/metabolismo , Receptores Histamínicos H1/química , Receptores Histamínicos H1/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Doxepina/química , Antagonistas dos Receptores Histamínicos/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Isomerismo , Ligantes , Modelos Moleculares , Fosfatos/química , Fosfatos/metabolismo , Ligação Proteica , Conformação Proteica , Receptores Adrenérgicos beta 2/química , Receptores de Dopamina D3/química , Especificidade por Substrato
3.
Structure ; 32(3): 352-361.e5, 2024 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-38194963

RESUMO

Orexin neuropeptides have many physiological roles in the sleep-wake cycle, feeding behavior, reward demands, and stress responses by activating cognitive receptors, the orexin receptors (OX1R and OX2R), distributed in the brain. There are only subtle differences between OX1R and OX2R in the orthosteric site, which has hindered the rational development of subtype-selective antagonists. In this study, we utilized solution-state NMR to capture the structural plasticity of OX2R labeled with 13CH3-ε-methionine in complex with antagonists. Mutations in the orthosteric site allosterically affected the intracellular tip of TM6. Ligand exchange experiments with the subtype-selective EMPA and the nonselective suvorexant identified three methionine residues that were substantially perturbed. The NMR spectra suggested that the suvorexant-bound state exhibited more structural plasticity than the EMPA-bound state, which has not been foreseen from the close similarity of their crystal structures, providing insights into dynamic features to be considered in understanding the ligand recognition mode.


Assuntos
Metionina , Humanos , Orexinas , Ligantes , Receptores de Orexina/genética , Receptores de Orexina/química , Espectroscopia de Ressonância Magnética
4.
Microb Cell Fact ; 11: 78, 2012 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-22694812

RESUMO

BACKGROUND: Recent successes in the determination of G-protein coupled receptor (GPCR) structures have relied on the ability of receptor variants to overcome difficulties in expression and purification. Therefore, the quick screening of functionally expressed stable receptor variants is vital. RESULTS: We developed a platform using Saccharomyces cerevisiae for the rapid construction and evaluation of functional GPCR variants for structural studies. This platform enables us to perform a screening cycle from construction to evaluation of variants within 6-7 days. We firstly confirmed the functional expression of 25 full-length class A GPCRs in this platform. Then, in order to improve the expression level and stability, we generated and evaluated the variants of the four GPCRs (hADRB2, hCHRM2, hHRH1 and hNTSR1). These stabilized receptor variants improved both functional activity and monodispersity. Finally, the expression level of the stabilized hHRH1 in Pichia pastoris was improved up to 65 pmol/mg from negligible expression of the functional full-length receptor in S. cerevisiae at first screening. The stabilized hHRH1 was able to be purified for use in crystallization trials. CONCLUSIONS: We demonstrated that the S. cerevisiae system should serve as an easy-to-handle and rapid platform for the construction and evaluation of GPCR variants. This platform can be a powerful prescreening method to identify a suitable GPCR variant for crystallography.


Assuntos
Receptores Acoplados a Proteínas G/metabolismo , Saccharomyces cerevisiae/metabolismo , Cristalização , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Pichia/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores Histamínicos H1/genética , Receptores Histamínicos H1/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
5.
Methods ; 55(4): 281-6, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21903167

RESUMO

G-protein coupled receptors (GPCRs) play essential roles in regulation of many physiological processes and are one of the major targets of pharmaceutical drugs. The 3D structure can provide important information for the understanding of GPCR function and the design of new drugs. However, the success of structure determination relies largely on the production of recombinant GPCRs, because the expression levels of GPCRs are very low in native tissues except rhodopsin. All non-rhodopsin GPCRs whose structures were determined so far were expressed in insect cells and the availability of other hosts was unknown. Recently, we succeeded to determine the structure of human histamine H(1) receptor (H(1)R) expressed in Pichia pastoris. Here, we report the expression and purification procedures of recombinant H(1)R used in the structural determination. The receptor was designed to possess a N-terminal 19-residue deletion and a replacement of the third cytoplasmic loop with T4-lysozyme. The receptor was verified to show similar binding activities with the receptor expressed in other hosts. The receptor was purified by the immobilized metal ion affinity chromatography and used for the crystallographic study that resulted in the successful structure determination.


Assuntos
Pichia/genética , Receptores Histamínicos H1/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Sequência de Aminoácidos , Cromatografia de Afinidade , Clonagem Molecular , Técnicas de Cultura , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Humanos , Dados de Sequência Molecular , Ligação Proteica , Biossíntese de Proteínas , Proteólise , Pirilamina/química , Receptores Histamínicos H1/química , Receptores Histamínicos H1/isolamento & purificação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Saccharomyces cerevisiae
6.
Cell Rep ; 40(11): 111323, 2022 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-36103815

RESUMO

Prostaglandin receptors have been implicated in a wide range of functions, including inflammation, immune response, reproduction, and cancer. Our group has previously determined the crystal structure of the active-like EP3 bound to its endogenous agonist, prostaglandin E2. Here, we present the single-particle cryoelectron microscopy (cryo-EM) structure of the human EP3-Gi signaling complex at a resolution of 3.4 Å. The structure reveals the binding mode of Gi to EP3 and the structural changes induced in EP3 by Gi binding. In addition, we compare the structure of the EP3-Gi complex with other subtypes of prostaglandin receptors (EP2 and EP4) bound to Gs that have been previously reported and examine the differences in amino acid composition at the receptor-G protein interface. Mutational analysis reveals that the selectivity of the G protein depends on specific amino acid residues in the second intracellular loop and TM5.


Assuntos
Dinoprostona , Receptores de Prostaglandina E , Aminoácidos , Microscopia Crioeletrônica , Dinoprostona/farmacologia , Humanos , Receptores de Prostaglandina E/agonistas , Receptores de Prostaglandina E/metabolismo , Receptores de Prostaglandina E Subtipo EP3/metabolismo
7.
Microb Cell Fact ; 10: 24, 2011 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-21513509

RESUMO

BACKGROUND: Various protein expression systems, such as Escherichia coli (E. coli), Saccharomyces cerevisiae (S. cerevisiae), Pichia pastoris (P. pastoris), insect cells and mammalian cell lines, have been developed for the synthesis of G protein-coupled receptors (GPCRs) for structural studies. Recently, the crystal structures of four recombinant human GPCRs, namely ß2 adrenergic receptor, adenosine A2a receptor, CXCR4 and dopamine D3 receptor, were successfully determined using an insect cell expression system. GPCRs expressed in insect cells are believed to undergo mammalian-like posttranscriptional modifications and have similar functional properties than in mammals. Crystal structures of GPCRs have not yet been solved using yeast expression systems. In the present study, P. pastoris and insect cell expression systems for the human muscarinic acetylcholine receptor M2 subtype (CHRM2) were developed and the quantity and quality of CHRM2 synthesized by both expression systems were compared for the application in structural studies. RESULTS: The ideal conditions for the expression of CHRM2 in P. pastoris were 60 hr at 20°C in a buffer of pH 7.0. The specific activity of the expressed CHRM2 was 28.9 pmol/mg of membrane protein as determined by binding assays using [3H]-quinuclidinyl benzilate (QNB). Although the specific activity of the protein produced by P. pastoris was lower than that of Sf9 insect cells, CHRM2 yield in P. pastoris was 2-fold higher than in Sf9 insect cells because P. pastoris was cultured at high cell density. The dissociation constant (Kd) for QNB in P. pastoris was 101.14 ± 15.07 pM, which was similar to that in Sf9 insect cells (86.23 ± 8.57 pM). There were no differences in the binding affinity of CHRM2 for QNB between P. pastoris and Sf9 insect cells. CONCLUSION: Compared to insect cells, P. pastoris is easier to handle, can be grown at lower cost, and can be expressed quicker at a large scale. Yeast, P. pastoris, and insect cells are all effective expression systems for GPCRs. The results of the present study strongly suggested that protein expression in P. pastoris can be applied to the structural and biochemical studies of GPCRs.


Assuntos
Expressão Gênica , Técnicas Genéticas , Pichia/genética , Receptor Muscarínico M2/genética , Receptor Muscarínico M2/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Humanos , Dados de Sequência Molecular , Pichia/metabolismo , Processamento de Proteína Pós-Traducional , Spodoptera
8.
RSC Adv ; 11(21): 12559-12567, 2021 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-35423811

RESUMO

The M2 muscarinic acetylcholine receptor (M2R) is a prototypical G protein-coupled receptor (GPCR) that responds to acetylcholine (ACh) and mediates various cellular responses in the nervous system. We recently established Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopy for ligand binding to M2R reconstituted in lipid membranes, paving the way to understand the mechanism in atomic detail. However, the obtained difference FTIR spectra upon ligand binding contained ligand, protein, lipid, and water signals, so a vibrational assignment was needed for a thorough understanding. In the present study, we compared difference FTIR spectra between unlabeled and 2-13C labeled ACh, and assigned the bands at 1741 and 1246 cm-1 as the C[double bond, length as m-dash]O and C-O stretches of ACh, respectively. The C[double bond, length as m-dash]O stretch of ACh in M2R is close to that in aqueous solution (1736 cm-1), and much lower in frequency than the free C[double bond, length as m-dash]O stretch (1778-1794 cm-1), indicating a strong hydrogen bond, which probably formed with N4046.52. We propose that a water molecule bridges ACh and N4046.52. The other ACh terminal is positively charged, and it interacts with negatively charged D1033.32. The present study revealed that D1033.32 is deprotonated (negatively charged) in both ACh-bound and free states, a suggested mechanism to stabilize the negative charge of D1033.32 in the free M2R.

9.
Commun Biol ; 4(1): 1321, 2021 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-34815515

RESUMO

The intrinsic efficacy of ligand binding to G protein-coupled receptors (GPCRs) reflects the ability of the ligand to differentially activate its receptor to cause a physiological effect. Here we use attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy to examine the ligand-dependent conformational changes in the human M2 muscarinic acetylcholine receptor (M2R). We show that different ligands affect conformational alteration appearing at the C=O stretch of amide-I band in M2R. Notably, ATR-FTIR signals strongly correlated with G-protein activation levels in cells. Together, we propose that amide-I band serves as an infrared probe to distinguish the ligand efficacy in M2R and paves the path to rationally design ligands with varied efficacy towards the target GPCR.


Assuntos
Receptor Muscarínico M2/química , Humanos , Ligantes , Análise Espectral
10.
Nat Struct Mol Biol ; 28(8): 694-701, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34354246

RESUMO

Melatonin receptors (MT1 and MT2) transduce inhibitory signaling by melatonin (N-acetyl-5-methoxytryptamine), which is associated with sleep induction and circadian rhythm modulation. Although recently reported crystal structures of ligand-bound MT1 and MT2 elucidated the basis of ligand entry and recognition, the ligand-induced MT1 rearrangement leading to Gi-coupling remains unclear. Here we report a cryo-EM structure of the human MT1-Gi signaling complex at 3.3 Å resolution, revealing melatonin-induced conformational changes propagated to the G-protein-coupling interface during activation. In contrast to other Gi-coupled receptors, MT1 exhibits a large outward movement of TM6, which is considered a specific feature of Gs-coupled receptors. Structural comparison of Gi and Gs complexes demonstrated conformational diversity of the C-terminal entry of the Gi protein, suggesting loose and variable interactions at the end of the α5 helix of Gi protein. These notions, together with our biochemical and computational analyses, highlight variable binding modes of Gαi and provide the basis for the selectivity of G-protein signaling.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Melatonina/metabolismo , Receptor MT1 de Melatonina/metabolismo , Microscopia Crioeletrônica , Humanos , Proteínas de Membrana/metabolismo , Estrutura Quaternária de Proteína , Transdução de Sinais/fisiologia , Relação Estrutura-Atividade
11.
Biochem Biophys Res Commun ; 380(2): 271-6, 2009 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-19167344

RESUMO

N-linked glycosylation is the most common post-translational modification of G-protein-coupled receptors (GPCRs) and is correlated to the localization and function of the receptors depending on each receptor. However, heterogeneity of glycosylation can interfere with protein crystallization. The removal of N-linked glycosylation from membrane proteins improves the ability to crystallize these proteins. We screened 25 non-glycosylated GPCRs for functional receptor production in the methylotrophic yeast Pichia pastoris using specific ligand-receptor binding assays. We found that five clones were expressed at greater than 10 pmol/mg, 9 clones at 1-10 pmol/mg and 11 clones at less than 1 pmol/mg of membrane protein. Further optimization of culture parameters including culture scale, induction time, pH and temperature enabled us to achieve expression of a functional human muscarinic acetylcholine receptor subtype 2 (CHRM2) with a B(max) value of 51.2 pmol/mg of membrane protein. Approximately 1.9 mg of the human CHRM2 was produced from a 1-L culture.


Assuntos
Pichia , Receptor Muscarínico M2/biossíntese , Receptores Acoplados a Proteínas G/biossíntese , Proteínas Recombinantes/biossíntese , Glicosilação , Humanos , Biossíntese de Proteínas , Receptor Muscarínico M2/química , Receptores Acoplados a Proteínas G/química , Proteínas Recombinantes/química
12.
J Phys Chem Lett ; 10(22): 7270-7276, 2019 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-31692352

RESUMO

M2 muscarinic acetylcholine receptor (M2R) is a prototypical G protein-coupled receptor (GPCR) that responds to acetylcholine and mediates various cellular responses in the nervous system. Here, we used attenuated total reflection-Fourier transform infrared spectroscopy analyses on M2R reconstituted in a lipid membrane to understand the molecular mechanism behind the ligand binding-induced conformational changes. Upon agonist binding, M2R shows large spectral change of the amide-I band corresponding to backbone C═O stretch, which likely connects with the receptor activation in the lipid environment. These results pave the way to probe effects of different ligand binding on GPCRs using vibrational spectroscopy.


Assuntos
Receptor Muscarínico M2/química , Sítios de Ligação , Humanos , Ligantes , Conformação Proteica , Espectroscopia de Infravermelho com Transformada de Fourier
13.
Endocrinology ; 149(2): 483-91, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17991722

RESUMO

Peripheral arterial diseases are caused by arterial sclerosis and impaired collateral vessel formation, which are exacerbated by diabetes, often leading to leg amputation. We have reported that an activation of the natriuretic peptides/cGMP/cGMP-dependent protein kinase pathway accelerated vascular regeneration and blood flow recovery in murine legs, for which ischemia had been induced by a femoral arterial ligation as a model for peripheral arterial diseases. In this study, ip injection of carperitide, a human recombinant atrial natriuretic peptide, accelerated blood flow recovery with increasing capillary density in ischemic legs not only in nondiabetic mice but also in mice kept upon streptozotocin-induced hyperglycemia for 16 wk, which significantly impaired the blood flow recovery compared with nondiabetic mice. Based on these findings, we tried to apply the administration of carperitide to the treatment of peripheral arterial diseases. The study group comprised a continuous series of 13 patients with peripheral arterial diseases (Fontaine's classification I, one; II, five; III, two; and IV, five), for whom conventional therapies had not accomplished appreciable results. Carperitide was administrated continuously and intravenously for 2 wk to Fontaine's class I-III patients and for 4 weeks to class IV patients. The dose was gradually increased to the maximum, with the patient's systolic blood pressure being kept above 100 mm Hg. Carperitide administration improved the ankle-brachial pressure index, intermittent claudication, rest pain, and ulcers. In conclusion, this study showed a therapeutic potential of carperitide to treat peripheral arterial diseases refractory to conventional therapies.


Assuntos
Fator Natriurético Atrial/administração & dosagem , Neovascularização Fisiológica/efeitos dos fármacos , Doenças Vasculares Periféricas/tratamento farmacológico , Fluxo Sanguíneo Regional/efeitos dos fármacos , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Arteriosclerose Obliterante/tratamento farmacológico , Arteriosclerose Obliterante/fisiopatologia , Pressão Sanguínea/efeitos dos fármacos , Diabetes Mellitus Experimental/complicações , Angiopatias Diabéticas/tratamento farmacológico , Angiopatias Diabéticas/fisiopatologia , Feminino , Úlcera do Pé/tratamento farmacológico , Úlcera do Pé/fisiopatologia , Gangrena/tratamento farmacológico , Gangrena/fisiopatologia , Humanos , Infusões Intravenosas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Doenças Vasculares Periféricas/fisiopatologia , Tromboangiite Obliterante/tratamento farmacológico , Tromboangiite Obliterante/fisiopatologia , Resultado do Tratamento
14.
Endocrinology ; 149(8): 3764-77, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18436714

RESUMO

Mineralocorticoid receptors (MRs) are classically known to be expressed in the distal collecting duct of the kidney. Recently it was reported that MR is identified in the heart and vasculature. Although MR expression is also found in the brain, it is restricted to the hippocampus and cerebral cortex under normal condition, and the role played by MRs in brain remodeling after cerebral ischemia remains unclear. In the present study, we used the mouse 20-min middle cerebral artery occlusion model to examine the time course of MR expression and activity in the ischemic brain. We found that MR-positive cells remarkably increased in the ischemic striatum, in which MR expression is not observed under normal conditions, during the acute and, especially, subacute phases after stroke and that the majority of MR-expressing cells were astrocytes that migrated to the ischemic core. Treatment with the MR antagonist spironolactone markedly suppressed superoxide production within the infarct area during this period. Quantitative real-time RT-PCR revealed that spironolactone stimulated the expression of neuroprotective or angiogenic factors, such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), whereas immunohistochemical analysis showed astrocytes to be cells expressing bFGF and VEGF. Thereby the incidence of apoptosis was reduced. The up-regulated bFGF and VEGF expression also appeared to promote endogenous angiogenesis and blood flow within the infarct area and to increase the number of neuroblasts migrating toward the ischemic striatum. By these beneficial effects, the infarct volume was significantly reduced in spironolactone-treated mice. Spironolactone may thus provide therapeutic neuroprotective effects in the ischemic brain after stroke.


Assuntos
Encéfalo/fisiologia , Infarto Cerebral/fisiopatologia , Regeneração Nervosa/fisiologia , Receptores de Mineralocorticoides/fisiologia , Proteínas Angiogênicas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Pressão Sanguínea/efeitos dos fármacos , Encéfalo/irrigação sanguínea , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Infarto Cerebral/metabolismo , Avaliação Pré-Clínica de Medicamentos , Frequência Cardíaca/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Antagonistas de Receptores de Mineralocorticoides , Atividade Motora/efeitos dos fármacos , Regeneração Nervosa/efeitos dos fármacos , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Receptores de Mineralocorticoides/metabolismo , Fluxo Sanguíneo Regional/efeitos dos fármacos , Espironolactona/farmacologia , Fatores de Tempo
15.
J Transl Med ; 6: 54, 2008 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-18823569

RESUMO

BACKGROUND: We previously demonstrated that vascular endothelial growth factor receptor type 2 (VEGF-R2)-positive cells induced from mouse embryonic stem (ES) cells can differentiate into both endothelial cells (ECs) and mural cells (MCs) and these vascular cells construct blood vessel structures in vitro. Recently, we have also established a method for the large-scale expansion of ECs and MCs derived from human ES cells. We examined the potential of vascular cells derived from human ES cells to contribute to vascular regeneration and to provide therapeutic benefit for the ischemic brain. METHODS: Phosphate buffered saline, human peripheral blood mononuclear cells (hMNCs), ECs-, MCs-, or the mixture of ECs and MCs derived from human ES cells were intra-arterially transplanted into mice after transient middle cerebral artery occlusion (MCAo). RESULTS: Transplanted ECs were successfully incorporated into host capillaries and MCs were distributed in the areas surrounding endothelial tubes. The cerebral blood flow and the vascular density in the ischemic striatum on day 28 after MCAo had significantly improved in ECs-, MCs- and ECs+MCs-transplanted mice compared to that of mice injected with saline or transplanted with hMNCs. Moreover, compared to saline-injected or hMNC-transplanted mice, significant reduction of the infarct volume and of apoptosis as well as acceleration of neurological recovery were observed on day 28 after MCAo in the cell mixture-transplanted mice. CONCLUSION: Transplantation of ECs and MCs derived from undifferentiated human ES cells have a potential to contribute to therapeutic vascular regeneration and consequently reduction of infarct area after stroke.


Assuntos
Células Sanguíneas/transplante , Vasos Sanguíneos/fisiologia , Células-Tronco Embrionárias/transplante , Regeneração , Acidente Vascular Cerebral/fisiopatologia , Acidente Vascular Cerebral/terapia , Indutores da Angiogênese/metabolismo , Animais , Apoptose , Células Sanguíneas/citologia , Vasos Sanguíneos/patologia , Infarto Encefálico/patologia , Infarto Encefálico/fisiopatologia , Linhagem Celular , Circulação Cerebrovascular , Citoproteção , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica , Humanos , Infarto da Artéria Cerebral Média/induzido quimicamente , Infarto da Artéria Cerebral Média/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Recuperação de Função Fisiológica
16.
Structure ; 26(1): 7-19.e5, 2018 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-29225076

RESUMO

Orexin peptides in the brain regulate physiological functions such as the sleep-wake cycle, and are thus drug targets for the treatment of insomnia. Using serial femtosecond crystallography and multi-crystal data collection with a synchrotron light source, we determined structures of human orexin 2 receptor in complex with the subtype-selective antagonist EMPA (N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-pyridin-3-ylmethyl-acetamide) at 2.30-Å and 1.96-Å resolution. In comparison with the non-subtype-selective antagonist suvorexant, EMPA contacted fewer residues through hydrogen bonds at the orthosteric site, explaining the faster dissociation rate. Comparisons among these OX2R structures in complex with selective antagonists and previously determined OX1R/OX2R structures bound to non-selective antagonists revealed that the residue at positions 2.61 and 3.33 were critical for the antagonist selectivity in OX2R. The importance of these residues for binding selectivity to OX2R was also revealed by molecular dynamics simulation. These results should facilitate the development of antagonists for orexin receptors.


Assuntos
Aminopiridinas/química , Azepinas/química , Antagonistas dos Receptores de Orexina/química , Receptores de Orexina/química , Orexinas/química , Sulfonamidas/química , Triazóis/química , Aminopiridinas/metabolismo , Animais , Azepinas/metabolismo , Baculoviridae/genética , Baculoviridae/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia/métodos , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Ligação de Hidrogênio , Cinética , Simulação de Dinâmica Molecular , Antagonistas dos Receptores de Orexina/metabolismo , Receptores de Orexina/genética , Receptores de Orexina/metabolismo , Orexinas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Sulfonamidas/metabolismo , Síncrotrons , Termodinâmica , Triazóis/metabolismo
17.
Endocrinology ; 147(4): 1642-53, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16384868

RESUMO

Adrenomedullin (AM) is a vasodilating hormone secreted mainly from vascular wall, and its expression is markedly enhanced after stroke. We have revealed that AM promotes not only vasodilation but also vascular regeneration. In this study, we focused on the roles of AM in the ischemic brain and examined its therapeutic potential. We developed novel AM-transgenic (AM-Tg) mice that overproduce AM in the liver and performed middle cerebral artery occlusion for 20 min (20m-MCAO) to examine the effects of AM on degenerative or regenerative processes in ischemic brain. The infarct area and gliosis after 20m-MCAO was reduced in AM-Tg mice in association with suppression of leukocyte infiltration, oxidative stress, and apoptosis in the ischemic core. In addition, vascular regeneration and subsequent neurogenesis were enhanced in AM-Tg mice, preceded by increase in mobilization of CD34(+) mononuclear cells, which can differentiate into endothelial cells. The vasculo-neuro-regenerative actions observed in AM-Tg mice in combination with neuroprotection resulted in improved recovery of motor function. Brain edema was also significantly reduced in AM-Tg mice via suppression of vascular permeability. In vitro, AM exerted direct antiapoptotic and neurogenic actions on neuronal cells. Exogenous administration of AM in mice after 20m-MCAO also reduced the infarct area, and promoted vascular regeneration and functional recovery. In summary, this study suggests the neuroprotective and vasculo-neuro-regenerative roles of AM and provides basis for a new strategy to rescue ischemic brain through its multiple hormonal actions.


Assuntos
Isquemia Encefálica/terapia , Regeneração Nervosa , Fármacos Neuroprotetores , Peptídeos/fisiologia , Adrenomedulina , Animais , Infarto Cerebral/terapia , Corpo Estriado/irrigação sanguínea , Membro Posterior/irrigação sanguínea , Humanos , Camundongos , Camundongos Transgênicos , Neovascularização Fisiológica , Peptídeos/genética , Espécies Reativas de Oxigênio
18.
Mol Cell Biol ; 29(22): 6018-32, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19737918

RESUMO

Vascular fibrosis is a major complication of hypertension and atherosclerosis, yet it is largely untreatable. Natriuretic peptides (NPs) repress fibrogenic activation of vascular smooth muscle cells (VSMCs), but the intracellular mechanism mediating this effect remains undetermined. Here we show that inhibition of RhoA through phosphorylation at Ser188, the site targeted by the NP effector cyclic GMP (cGMP)-dependent protein kinase I (cGK I), is critical to fully exert antifibrotic potential. cGK I(+/-) mouse blood vessels exhibited an attenuated P-RhoA level and concurrently increased RhoA/ROCK signaling. Importantly, cGK I insufficiency caused dynamic recruitment of ROCK into the fibrogenic programs, thereby eliciting exaggerated vascular hypertrophy and fibrosis. Transgenic expression of cGK I-unphosphorylatable RhoA(A188) in VSMCs augmented ROCK activity, vascular hypertrophy, and fibrosis more prominently than did that of wild-type RhoA, consistent with the notion that RhoA(A188) escapes the intrinsic inhibition by cGK I. Additionally, VSMCs expressing RhoA(A188) became refractory to the antifibrotic effects of NPs. Our results identify cGK I-mediated Ser188 phosphorylation of RhoA as a converging node for pro- and antifibrotic signals and may explain how diminished cGMP signaling, commonly associated with vascular malfunction, predisposes individuals to vascular fibrosis.


Assuntos
Vasos Sanguíneos/enzimologia , Vasos Sanguíneos/patologia , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Fosfosserina/metabolismo , Transdução de Sinais , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Angiotensina II/farmacologia , Animais , Vasos Sanguíneos/efeitos dos fármacos , GMP Cíclico/farmacologia , Proteínas Quinases Dependentes de GMP Cíclico/deficiência , Ativação Enzimática/efeitos dos fármacos , Fibrose , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hipertrofia , Camundongos , Camundongos Transgênicos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/patologia , Proteínas Mutantes/metabolismo , Especificidade de Órgãos/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Elemento de Resposta Sérica/genética , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos
19.
Diabetes ; 58(12): 2880-92, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19690065

RESUMO

OBJECTIVE: Natriuretic peptides (NPs) have been characterized as vascular hormones that regulate vascular tone via guanylyl cyclase (GC), cyclic GMP (cGMP), and cGMP-dependent protein kinase (cGK). Recent clinical studies have shown that plasma NP levels were lower in subjects with the metabolic syndrome. The present study was conducted to elucidate the roles for NP/cGK cascades in energy metabolism. RESEARCH DESIGN AND METHODS: We used three types of genetically engineered mice: brain NP (BNP) transgenic (BNP-Tg), cGK-Tg, and guanylyl cyclase-A (GCA) heterozygous knockout (GCA(+/-)) mice and analyzed the metabolic consequences of chronic activation of NP/cGK cascades in vivo. We also examined the effect of NPs in cultured myocytes. RESULTS: BNP-Tg mice fed on high-fat diet were protected against diet-induced obesity and insulin resistance, and cGK-Tg mice had reduced body weight even on standard diet; surprisingly, giant mitochondria were densely packed in the skeletal muscle. Both mice showed an increase in muscle mitochondrial content and fat oxidation through upregulation of peroxisome proliferator-activated receptor (PPAR)-gamma coactivator (PGC)-1alpha and PPARdelta. The functional NP receptors, GCA and guanylyl cyclase-B, were downregulated by feeding a high-fat diet, while GCA(+/-) mice showed increases in body weight and glucose intolerance when fed a high-fat diet. NPs directly increased the expression of PGC-1alpha and PPARdelta and mitochondrial content in cultured myocytes. CONCLUSIONS: The findings together suggest that NP/cGK cascades can promote muscle mitochondrial biogenesis and fat oxidation, as to prevent obesity and glucose intolerance. The vascular hormone, NP, would contribute to coordinated regulation of oxygen supply and consumption.


Assuntos
GMP Cíclico/metabolismo , Gorduras na Dieta/metabolismo , Intolerância à Glucose/metabolismo , Resistência à Insulina , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , Peptídeos Natriuréticos/metabolismo , Obesidade/prevenção & controle , Proteínas Quinases/metabolismo , Animais , Glicemia/metabolismo , Células Cultivadas , Gorduras na Dieta/administração & dosagem , Gorduras na Dieta/efeitos adversos , Regulação para Baixo , Engenharia Genética , Intolerância à Glucose/etiologia , Peroxidação de Lipídeos , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Células Musculares/metabolismo , Peptídeo Natriurético Encefálico/metabolismo , Obesidade/etiologia , Obesidade/metabolismo , Consumo de Oxigênio , PPAR delta/metabolismo , PPAR gama/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Receptores do Fator Natriurético Atrial/metabolismo , Transativadores/metabolismo , Fatores de Transcrição , Regulação para Cima
20.
Proc Natl Acad Sci U S A ; 100(6): 3404-9, 2003 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-12621153

RESUMO

Natriuretic peptides (NPs), which consist of atrial, brain, and C-type natriuretic peptides (ANP, BNP, and CNP, respectively), are characterized as cardiac or vascular hormones that elicit their biological effects by activation of the cGMPcGMP-dependent protein kinase (cGK) pathway. We recently reported that adenoviral gene transfer of CNP into rabbit blood vessels not only suppressed neointimal formation but also accelerated reendothelialization, a required step for endothelium-dependent vasorelaxation and antithrombogenicity. Accordingly, we investigated the therapeutic potential of the NPscGMPcGK pathway for vascular regeneration. In transgenic (Tg) mice that overexpress BNP in response to hindlimb ischemia, neovascularization with appropriate mural cell coating was accelerated without edema or bleeding, and impaired angiogenesis by the suppression of nitric oxide production was effectively rescued. Furthermore, in BNP-Tg mice, inflammatory cell infiltration in ischemic tissue and vascular superoxide production were suppressed compared with control mice. Ischemia-induced angiogenesis was also significantly potentiated in cGK type I Tg mice, but attenuated in cGK type I knockout mice. NPs significantly stimulated capillary network formation of cultured endothelial cells by cGK stimulation and subsequent Erk12 activation. Furthermore, gene transfer of CNP into ischemic muscles effectively accelerated angiogenesis. These findings reveal an action of the NPscGMPcGK pathway to exert multiple vasculoprotective and regenerative actions in the absence of apparent adverse effects, and therefore suggest that NPs as the endogenous cardiovascular hormone can be used as a strategy of therapeutic angiogenesis in patients with tissue ischemia.


Assuntos
Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/fisiologia , Proteínas Quinases Dependentes de GMP Cíclico/fisiologia , GMP Cíclico/fisiologia , Peptídeo Natriurético Encefálico/fisiologia , Regeneração/efeitos dos fármacos , Regeneração/fisiologia , Animais , Fator Natriurético Atrial/genética , Fator Natriurético Atrial/fisiologia , Fator Natriurético Atrial/uso terapêutico , Células Cultivadas , Expressão Gênica , Técnicas de Transferência de Genes , Humanos , Inflamação/etiologia , Inflamação/patologia , Isquemia/terapia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Peptídeo Natriurético Encefálico/genética , Peptídeo Natriurético Encefálico/uso terapêutico , Peptídeo Natriurético Tipo C/genética , Peptídeo Natriurético Tipo C/fisiologia , Peptídeo Natriurético Tipo C/uso terapêutico , Neovascularização Fisiológica
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