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1.
Genes Cells ; 22(6): 535-551, 2017 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-28493531

RESUMEN

Retinoid-related orphan receptor gamma (RORγ) directly controls the differentiation of Th17 cell and the production of interleukin-17, which plays an integral role in autoimmune diseases. To obtain insight into RORγ, we have determined the first crystal structure of a ternary complex containing RORγ ligand-binding domain (LBD) bound with a novel synthetic inhibitor and a repressor peptide, 22-mer peptide from silencing mediator of retinoic acid and thyroid hormone receptor (SMRT). Comparison of a binary complex of nonliganded (apo) RORγ-LBD with a nuclear receptor co-activator (NCoA-1) peptide has shown that our inhibitor displays a unique mechanism different from those caused by natural inhibitor, ursolic acid (UA). The compound unprecedentedly induces indirect disruption of a hydrogen bond between His479 on helix 11 (H11) and Tyr502 on H12, which is crucial for active conformation. This crystallographic study will allow us to develop novel synthetic compounds for autoimmune disease therapy.


Asunto(s)
Co-Represor 2 de Receptor Nuclear/metabolismo , Coactivador 1 de Receptor Nuclear/metabolismo , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Sitios de Unión , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Mutación , Co-Represor 2 de Receptor Nuclear/agonistas , Co-Represor 2 de Receptor Nuclear/química , Co-Represor 2 de Receptor Nuclear/genética , Coactivador 1 de Receptor Nuclear/química , Coactivador 1 de Receptor Nuclear/genética , Fragmentos de Péptidos , Unión Proteica , Conformación Proteica , Triterpenos/farmacología , Ácido Ursólico
2.
J Biol Chem ; 291(31): 16240-8, 2016 07 29.
Artículo en Inglés | MEDLINE | ID: mdl-27235396

RESUMEN

The HIV-1 transactivator protein Tat is a critical regulator of HIV transcription primarily enabling efficient elongation of viral transcripts. Its interactions with RNA and various host factors are regulated by ordered, transient post-translational modifications. Here, we report a novel Tat modification, monomethylation at lysine 71 (K71). We found that Lys-71 monomethylation (K71me) is catalyzed by KMT7, a methyltransferase that also targets lysine 51 (K51) in Tat. Using mass spectrometry, in vitro enzymology, and modification-specific antibodies, we found that KMT7 monomethylates both Lys-71 and Lys-51 in Tat. K71me is important for full Tat transactivation, as KMT7 knockdown impaired the transcriptional activity of wild type (WT) Tat but not a Tat K71R mutant. These findings underscore the role of KMT7 as an important monomethyltransferase regulating HIV transcription through Tat.


Asunto(s)
VIH-1/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Activación Transcripcional , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , VIH-1/genética , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Células Jurkat , Lisina/genética , Lisina/metabolismo , Metilación , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética
3.
PLoS Pathog ; 7(8): e1002184, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21876670

RESUMEN

The essential transactivator function of the HIV Tat protein is regulated by multiple posttranslational modifications. Although individual modifications are well characterized, their crosstalk and dynamics of occurrence during the HIV transcription cycle remain unclear.We examine interactions between two critical modifications within the RNA-binding domain of Tat: monomethylation of lysine 51 (K51) mediated by Set7/9/KMT7, an early event in the Tat transactivation cycle that strengthens the interaction of Tat with TAR RNA, and acetylation of lysine 50 (K50) mediated by p300/KAT3B, a later process that dissociates the complex formed by Tat, TAR RNA and the cyclin T1 subunit of the positive transcription elongation factor b (P-TEFb). We find K51 monomethylation inhibited in synthetic Tat peptides carrying an acetyl group at K50 while acetylation can occur in methylated peptides, albeit at a reduced rate. To examine whether Tat is subject to sequential monomethylation and acetylation in cells, we performed mass spectrometry on immunoprecipitated Tat proteins and generated new modification-specific Tat antibodies against monomethylated/acetylated Tat. No bimodified Tat protein was detected in cells pointing to a demethylation step during the Tat transactivation cycle. We identify lysine-specific demethylase 1 (LSD1/KDM1) as a Tat K51-specific demethylase, which is required for the activation of HIV transcription in latently infected T cells. LSD1/KDM1 and its cofactor CoREST associates with the HIV promoter in vivo and activate Tat transcriptional activity in a K51-dependent manner. In addition, small hairpin RNAs directed against LSD1/KDM1 or inhibition of its activity with the monoamine oxidase inhibitor phenelzine suppresses the activation of HIV transcription in latently infected T cells.Our data support the model that a LSD1/KDM1/CoREST complex, normally known as a transcriptional suppressor, acts as a novel activator of HIV transcription through demethylation of K51 in Tat. Small molecule inhibitors of LSD1/KDM1 show therapeutic promise by enforcing HIV latency in infected T cells.


Asunto(s)
Histona Demetilasas/metabolismo , Transcripción Genética/efectos de los fármacos , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Acetilación , Animales , Epigénesis Genética/fisiología , Genes Virales/efectos de los fármacos , Histona Demetilasas/antagonistas & inhibidores , Metilación , Fenelzina/farmacología , Factor B de Elongación Transcripcional Positiva/metabolismo , Conejos , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/inmunología
4.
Antimicrob Agents Chemother ; 56(8): 4250-6, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22615294

RESUMEN

JTK-853 is a novel piperazine derivative nonnucleoside inhibitor of hepatitis C virus (HCV) RNA-dependent RNA polymerase. JTK-853 showed potent inhibitory activity against genotype 1 HCV polymerase, with a 50% inhibitory concentration in the nanomolar range, and showed potent antiviral activity against the genotype 1b replicon, with a 50% effective concentration of 0.035 µM. The presence of human serum at up to 40% had little effect on the antiviral activity of JTK-853. Structure analysis of HCV polymerase with JTK-853 revealed that JTK-853 associates with the palm site and ß-hairpin region of HCV polymerase, and JTK-853 showed decreased antiviral activity against HCV replicons bearing the resistance mutations C316Y, M414T, Y452H, and L466V in the palm site region of HCV polymerase. JTK-853 showed an additive combination effect with other DAAs (direct antiviral agents), such as nucleoside polymerase inhibitor, thumb pocket-binding nonnucleoside polymerase inhibitor, NS5A inhibitor, and protease inhibitor. Collectively, these data demonstrate that JTK-853 is a potent and novel nonnucleoside palm site-binding HCV polymerase inhibitor, suggesting JTK-853 as a potentially useful agent in combination with other DAAs for treatment of HCV infections.


Asunto(s)
Hepacivirus/efectos de los fármacos , Piperazinas/farmacología , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Antivirales/química , Antivirales/farmacología , Sitios de Unión , Línea Celular , Farmacorresistencia Viral/genética , Genotipo , Hepacivirus/enzimología , Hepacivirus/genética , Humanos , Pruebas de Sensibilidad Microbiana , Mutación , Piperazinas/química , Estructura Cuaternaria de Proteína , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/genética , Proteínas no Estructurales Virales/antagonistas & inhibidores
5.
Shinrigaku Kenkyu ; 81(3): 240-6, 2010 Aug.
Artículo en Japonés | MEDLINE | ID: mdl-20845730

RESUMEN

This study examines how children's social power cognitions about themselves and the collective efficacy of their classrooms affect their selection and use of influence tactics toward their classmates. The effect of daily usage of influence tactics by children on agents' perception of social power is also examined. The results of linear regression analysis indicate that children who see themselves as friendly holders of power deriving from the perception of affable friendships use both collaborative and coercive influence tactics. While the use of collaborative tactics encourages agents' friendly social power cognition, the use of coercive tactics only reinforces their coercive power cognition. In addition, it is suggested that the effect of social power over agents' use of influence tactics is moderated by the collective efficacy of their classroom.


Asunto(s)
Cognición , Poder Psicológico , Psicología Infantil , Niño , Femenino , Humanos , Masculino , Conducta Social , Percepción Social
6.
Shinrigaku Kenkyu ; 78(1): 70-6, 2007 Apr.
Artículo en Japonés | MEDLINE | ID: mdl-17511250

RESUMEN

This study examines how the use of influence tactics between peers affects the likelihood of eliciting social support. The participants (261 college students) were asked to imagine themselves in one of six scenarios, which described a hypothetical peer agent who usually uses specific influence tactics and the strength of the friendship. Then the participants rated the likelihood of supporting the agent, when the agent confronted a stressful situation. The result shows that the participants gave less spontaneous support to an agent who used hard influence tactics, such as restricting their freedom in choosing a course of action. Regarding emotional support, when they are in a close relationship with the agent, the participants gave the most spontaneous support to the agent using soft influence tactics, such as ingratiation. Participants gave steady socio-emotional support to agents using rational influence tactics, regardless of their relationship. This study also examined how influence tactics affect the participants' impression of the agent.


Asunto(s)
Emociones , Relaciones Interpersonales , Grupo Paritario , Apoyo Social , Adulto , Femenino , Humanos , Masculino
7.
ACS Med Chem Lett ; 7(1): 23-7, 2016 Jan 14.
Artículo en Inglés | MEDLINE | ID: mdl-26819660

RESUMEN

A novel series of RORγ inhibitors was identified starting with the HTS hit 1. After SAR investigation based on a prospective consideration of two drug-likeness metrics, ligand efficiency (LE) and fraction of sp(3) carbon atoms (Fsp(3)), significant improvement of metabolic stability as well as reduction of CYP inhibition was observed, which finally led to discovery of a selective and orally efficacious RORγ inhibitor 3z.

8.
Cardiovasc Res ; 59(1): 95-104, 2003 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-12829180

RESUMEN

OBJECTIVE: Inducible costimulator (ICOS) is a member of the CD28 family. Although inflammation is an essential pathological feature of myocarditis, the role of ICOS in myocarditis remains unclear. METHODS AND RESULTS: Lewis rats were immunized on day 0 with purified porcine cardiac myosin to establish experimental autoimmune myocarditis (EAM). Flow cytometry was used to examine expression of ICOS on myocardial infiltrating cells. Anti-ICOS antibody or ICOS-immunoglobulin (ICOSIg) was administered intravenously, and rats were killed on day 14 or 21 to study effects of ICOS/ICOS-ligand (ICOSL) pathway blockade during the antigen priming phase (days 0-14) or immune response phase (days 14-21), respectively. The heart weight to body weight ratio was determined, and histological examination and echocardiogram were performed to evaluate the severity of the disease. Cytokine expression in the heart and T cell proliferation against cardiac myosin were analyzed. Flow cytometry revealed that the majority of infiltrating cells, especially CD4-positive cells, expressed ICOS. Blockade of the ICOS/ICOSL pathway during the immune response phase attenuated EAM development. However, blockade of the ICOS/ICOSL pathway during the antigen priming phase did not attenuate and exacerbate EAM. Blockade of T cell activation through ICOS suppressed expression of cytokines including INF-gamma, IL-4, IL-6, IL-10, IL-1 beta, and TNF-alpha and inhibited T cell proliferation in vitro. CONCLUSIONS: Blockade of T cell activation through ICOS during the immune response phase regulates development of EAM, and therefore, ICOS may be an effective target for treating myocarditis.


Asunto(s)
Anticuerpos Bloqueadores/farmacología , Antígenos de Diferenciación de Linfocitos T/inmunología , Enfermedades Autoinmunes/inmunología , Miocarditis/inmunología , Linfocitos T/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , División Celular , Citocinas/metabolismo , Ecocardiografía , Citometría de Flujo , Proteína Coestimuladora de Linfocitos T Inducibles , Activación de Linfocitos , Masculino , Ratas , Ratas Endogámicas Lew , Linfocitos T/química
9.
Transplantation ; 78(11): 1590-6, 2004 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-15591946

RESUMEN

BACKGROUND: Costimulatory signals have been reported to play an important role in islet-xenograft rejection, although the precise mechanisms remain unknown. The aim of the present study was to determine a role of a novel costimulatory molecule, inducible costimulator (ICOS), in rat islet-xenograft rejection in conjunction with CTLA4Ig with respect to cellular as well as humoral immune responses. METHODS: Isolated rat islets were transplanted into the liver of streptozotocin (180 mg/kg) induced diabetic mice. Cellular immune responses to islet xenografts, and productions of anti-rat antibody in mice were examined by flow cytometry (FACS) after transplantation. RESULTS: Intrahepatic rat islet xenografts were rejected in mice within 8 days after transplantation. FACS analysis revealed an expansion of CD8(+) T cells in the liver as well as a production of anti-rat antibody in recipient mice in association with rejection. The treatment with anti-ICOS antibody in conjunction with CTLA4Ig produced a marked prolongation of islet-xenograft survival with neither expansion of CD8(+) T cells nor production of anti-rat antibody, whereas, in contrast, those treated with anti-ICOS antibody or CTLA4Ig alone did not have prolonged survival, and CD8(+) T cells were expanded. CONCLUSION: These findings demonstrate that cellular rather than humoral immune responses are considered responsible for islet-xenograft rejection from rat to mouse and that the blockade of costimulatory signals with anti-ICOS antibody in conjunction with CTLA4Ig has a favorable effect on prevention of islet xenograft rejection.


Asunto(s)
Anticuerpos/uso terapéutico , Antígenos de Diferenciación de Linfocitos T/fisiología , Rechazo de Injerto/prevención & control , Inmunoconjugados/uso terapéutico , Trasplante de Islotes Pancreáticos/inmunología , Trasplante Heterólogo/inmunología , Abatacept , Animales , Linfocitos T CD4-Positivos/inmunología , Supervivencia de Injerto , Proteína Coestimuladora de Linfocitos T Inducibles , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Endogámicas Lew
10.
Transplantation ; 75(8): 1115-8, 2003 Apr 27.
Artículo en Inglés | MEDLINE | ID: mdl-12717187

RESUMEN

BACKGROUND: An inducible costimulator (ICOS) has been found to be a novel costimulator for T-cell activation, although its precise role in transplant immunobiology remains unclear. This study determined whether ICOS plays an essential role in rejection of intrahepatic islet allografts in streptozotocin-induced diabetic mice. METHODS: Mononuclear cells in the liver of mice were isolated and examined by flow cytometry with respect to expression of ICOS in association with rejection, and the effects of in vivo treatment with an anti-ICOS antibody on survival of intrahepatic islet allografts were determined. RESULTSFlow cytometric analysis of mononuclear cells in the liver of normal untreated mice revealed that ICOS is expressed on CD4+CD3int natural killer T cells. The expression of ICOS was up-regulated on CD4+CD3bright T cells and expanded CD8 T cells in the liver in association with rejection. Posttransplant short-term administration of anti-ICOS antibody alone produced a significant prolongation of islet allograft survival. Administration of the antibody in conjunction with a subtherapeutic regimen of FK506 prevented rejection, leading to the acceptance of islet allografts. CONCLUSION: ICOS has an essential role in rejection of intrahepatic islet allografts and the blockade of ICOS interaction might be a novel approach for preventing islet allograft rejection.


Asunto(s)
Anticuerpos/farmacología , Antígenos de Diferenciación de Linfocitos T/inmunología , Supervivencia de Injerto/efectos de los fármacos , Trasplante de Islotes Pancreáticos , Animales , Antígenos de Diferenciación de Linfocitos T/metabolismo , Complejo CD3/análisis , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Sinergismo Farmacológico , Rechazo de Injerto/inmunología , Rechazo de Injerto/patología , Proteína Coestimuladora de Linfocitos T Inducibles , Trasplante de Islotes Pancreáticos/inmunología , Células Asesinas Naturales/inmunología , Hígado/citología , Hígado/lesiones , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Tacrolimus/farmacología , Regulación hacia Arriba
11.
Vaccine ; 23(29): 3850-6, 2005 May 31.
Artículo en Inglés | MEDLINE | ID: mdl-15893624

RESUMEN

Porphyromonas gingivalis has been implicated as an important pathogen in the development of adult periodontitis, and its colonization of subgingival sites is critical in the pathogenic process. One potential virulence factor, hemagglutinin, may mediate bacteria attachment onto and penetration into host cells, as well as agglutinate and lyses erythrocytes to intake heme, an absolute requirement for growth. Toward the development of passive immunotherapy, the construction of a human type monoclonal antibody, which is capable of inhibiting the hemagglutinating ability, will be significant and important. The human mAbs, both exhibiting a high degree of specificity and affinity against the recombinant 130 kDa hemagglutinin domain protein have been prepared using XenoMouse technology. The constructed Xeno-mAbs, IgG2 subclass, significantly inhibited hemagglutination of P. gingivalis and its vesicles. The newly constructed Xeno-mAbs may prove to be useful for the development of passive immunization against periodontal diseases caused by P. gingivalis infection, pending the results of fertility study in disease mode.


Asunto(s)
Anticuerpos Antibacterianos/biosíntesis , Anticuerpos Monoclonales/biosíntesis , Proteínas Bacterianas/inmunología , Hemaglutininas/inmunología , Porphyromonas gingivalis/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Proteínas Bacterianas/genética , Pruebas de Hemaglutinación , Hemaglutininas/genética , Humanos , Hibridomas , Ratones , Ratones Transgénicos , Pruebas de Neutralización , Periodontitis/microbiología , Periodontitis/prevención & control
12.
Biochem Biophys Res Commun ; 299(5): 755-61, 2002 Dec 20.
Artículo en Inglés | MEDLINE | ID: mdl-12470643

RESUMEN

Connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) displays multiple functions in several types of mesenchymal cells, including the promotion of proliferation and differentiation of chondrocytes. Recently, the internalization and intracellular function of CTGF/Hcs24 were indicated as well. In this study, a binding protein for this factor was purified from the cytosolic fraction of human chondrosarcoma-derived chondrocytic cell line (HCS-2/8) by CTGF/Hcs24-affinity chromatography. The apparent molecular weight of the protein was 42kDa and determination of the internal amino acid sequence revealed this protein to be beta- or gamma-actin. An in vitro competitive binding assay of 125I-labeled recombinant CTGF/Hcs24 with cold-rCTGF/Hcs24 showed that the binding between actin and 125I-CTGF/Hcs24 was specific. Immunoprecipitation analysis also showed that CTGF/Hcs24 bound to actin in HCS-2/8 cells. However, rCTGF/Hcs24 had no effects on the expression level of gamma-actin mRNA or total actin protein. These findings suggest that a significant portion of intracellular CTGF/Hcs24 may regulate certain cell biological events in chondrocytes through the interaction with this particular cytoskeletal protein.


Asunto(s)
Actinas/metabolismo , Condrocitos/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Secuencia de Aminoácidos , Unión Competitiva , Factor de Crecimiento del Tejido Conjuntivo , Proteínas del Citoesqueleto/aislamiento & purificación , Humanos , Datos de Secuencia Molecular , Células Tumorales Cultivadas
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