RESUMO
Phenotypic screening in drug discovery has been revived with the expectation of providing promising lead compounds and drug targets and improving the success rate of drug approval. However, target identification remains a major bottleneck in phenotype-based drug discovery. We identified the lead compounds K542 and K405 with a selective inhibition of cell viability against sphingosine-1-phosphate lyase 1 (SGPL1)-transduced ES-2 cells by phenotypic screening. We therefore performed an in vivo pharmacological examination and observed the antitumor activity of K542 in an HT-1080 tumor-bearing mouse xenograft model. SGPL1 was expected to be a therapeutic target in some cancers, suggesting that these lead molecules might be promising candidates; however, their mechanisms of action still remain unexplained. We therefore synthesized the affinity probe Ind-tag derived from K542 and identified the proteins binding to Ind-tag via a pull-down experiment. Proteomics and biochemical analyses revealed that the target molecule of these lead compounds was Nicotinamide phosphoribosyltransferase (NAMPT). We established K542-resistant DLD-1 and HT-1080 cells, and genetic analyses of these cells identified a missense mutation in the NAMPT-encoding gene. This enzymatic experiment clearly showed that K393 exerts enzymatic inhibition against NAMPT. These proteomics, genetics and biochemical analyses clarified that compounds K542 and K405 were NAMPT inhibitors.
Assuntos
Ensaios de Seleção de Medicamentos Antitumorais/métodos , Nicotinamida Fosforribosiltransferase/efeitos dos fármacos , Nicotinamida Fosforribosiltransferase/metabolismo , Aldeído Liases/efeitos dos fármacos , Aldeído Liases/metabolismo , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Citocinas/efeitos dos fármacos , Citocinas/metabolismo , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Moleculares , Neoplasias/tratamento farmacológico , Fenótipo , Relação Estrutura-AtividadeRESUMO
Amino-terminal enhancer of split (Aes) is a member of Groucho/Transducin-like enhancer (TLE) family. Aes is a recently found metastasis suppressor of colorectal cancer (CRC) that inhibits Notch signalling, and forms nuclear foci together with TLE1. Although some Notch-associated proteins are known to form subnuclear bodies, little is known regarding the dynamics or functions of these structures. Here, we show that Aes nuclear foci in CRC observed under an electron microscope are in a rather amorphous structure, lacking surrounding membrane. Investigation of their behaviour during the cell cycle by time-lapse cinematography showed that Aes nuclear foci dissolve during mitosis and reassemble after completion of cytokinesis. We have also found that heat shock cognate 70 (HSC70) is an essential component of Aes foci. Pharmacological inhibition of the HSC70 ATPase activity with VER155008 reduces Aes focus formation. These results provide insight into the understanding of Aes-mediated inhibition of Notch signalling.
Assuntos
Adenosina Trifosfatases/metabolismo , Núcleo Celular/metabolismo , Neoplasias Colorretais/metabolismo , Proteínas de Choque Térmico HSC70/metabolismo , Proteínas Repressoras/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Animais , Núcleo Celular/ultraestrutura , Proteínas Correpressoras , Citocinese , Células HCT116 , Células HEK293 , Proteínas de Choque Térmico HSC70/antagonistas & inibidores , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Mitose , Nucleosídeos de Purina/farmacologia , Receptores Notch/metabolismo , Proteínas Repressoras/genética , Transdução de Sinais , Imagem com Lapso de TempoRESUMO
Intestinal epithelial cells contribute to regulation of intestinal immunity in mammals, but the detailed molecular mechanisms of such regulation have remained largely unknown. Stomach-cancer-associated protein tyrosine phosphatase 1 (SAP-1, also known as PTPRH) is a receptor-type protein tyrosine phosphatase that is localized specifically at microvilli of the brush border in gastrointestinal epithelial cells. Here we show that SAP-1 ablation in interleukin (IL)-10-deficient mice, a model of inflammatory bowel disease, resulted in a marked increase in the severity of colitis in association with up-regulation of mRNAs for various cytokines and chemokines in the colon. Tyrosine phosphorylation of carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 20, an intestinal microvillus-specific transmembrane protein of the Ig superfamily, was greatly increased in the intestinal epithelium of the SAP-1-deficient animals, suggesting that this protein is a substrate for SAP-1. Tyrosine phosphorylation of CEACAM20 by the protein tyrosine kinase c-Src and the consequent association of CEACAM20 with spleen tyrosine kinase (Syk) promoted the production of IL-8 in cultured cells through the activation of nuclear factor-κB (NF-κB). In addition, SAP-1 and CEACAM20 were found to form a complex through interaction of their ectodomains. SAP-1 and CEACAM20 thus constitute a regulatory system through which the intestinal epithelium contributes to intestinal immunity.
Assuntos
Moléculas de Adesão Celular/metabolismo , Colite/enzimologia , Colite/prevenção & controle , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/metabolismo , Animais , Contagem de Células , Quimiocinas/genética , Quimiocinas/metabolismo , Colite/patologia , Colo/patologia , Feminino , Células Caliciformes/metabolismo , Células Caliciformes/patologia , Células HEK293 , Humanos , Interleucina-10/deficiência , Interleucina-10/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos , NF-kappa B/metabolismo , Fosforilação , Fosfotirosina/metabolismo , Ligação Proteica , Transporte Proteico , Proteínas Tirosina Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/deficiência , Quinase Syk , Domínios de Homologia de src , Quinases da Família src/metabolismoRESUMO
DNA double-strand breaks (DSB) occur frequently during replication in sister chromatids and are dramatically increased when cells are exposed to chemotherapeutic agents including camptothecin. Such DSBs are efficiently repaired specifically by homologous recombination (HR) with the intact sister chromatid. HR, therefore, plays pivotal roles in cellular proliferation and cellular tolerance to camptothecin. Mammalian cells carry several structure-specific endonucleases, such as Xpf-Ercc1 and Mus81-Eme1, in which Xpf and Mus81 are the essential subunits for enzymatic activity. Here, we show the functional overlap between Xpf and Mus81 by conditionally inactivating Xpf in the chicken DT40 cell line, which has no Mus81 ortholog. Although mammalian cells deficient in either Xpf or Mus81 are viable, Xpf inactivation in DT40 cells was lethal, resulting in a marked increase in the number of spontaneous chromosome breaks. Similarly, inactivation of both Xpf and Mus81 in human HeLa cells and murine embryonic stem cells caused numerous spontaneous chromosome breaks. Furthermore, the phenotype of Xpf-deficient DT40 cells was reversed by ectopic expression of human Mus81-Eme1 or human Xpf-Ercc1 heterodimers. These observations indicate the functional overlap of Xpf-Ercc1 and Mus81-Eme1 in the maintenance of genomic DNA. Both Mus81-Eme1 and Xpf-Ercc1 contribute to the completion of HR, as evidenced by the data that the expression of Mus81-Eme1 or Xpf-Ercc1 diminished the number of camptothecin-induced chromosome breaks in Xpf-deficient DT40 cells, and to preventing early steps in HR by deleting XRCC3 suppressed the nonviability of Xpf-deficient DT40 cells. In summary, Xpf and Mus81 have a substantially overlapping function in completion of HR.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Recombinação Homóloga , Animais , Morte Celular/genética , Linhagem Celular Tumoral , Galinhas , Aberrações Cromossômicas , Quebras de DNA de Cadeia Dupla , Células HeLa , Humanos , CamundongosRESUMO
The elucidation of drug resistance mechanisms is important in the development of clinical therapies for the treatment of leukemia. To study the drug resistance mechanisms, protein expression profiles of 1-ß-D-arabinofuranosylcytosine (AraC)-sensitive K562 (K562S) cells and AraC-resistant K562 (K562AC) cells were compared using two-dimensional fluorescence difference gel electrophoresis. In a comparison of protein expression profiles, 2073 protein spots were found to be altered, and 15 proteins of them were remarkably altered. These proteins were identified by mass spectrometry. The most differently expressed proteins were aldehyde dehydrogenase 1 family member A2 (ALDH1A2) and vimentin. Both proteins were verified using reverse transcriptase polymerase chain reaction and Western blot analysis. ALDH1A2 protein was found to be effective in AraC resistance. ALDH1A2 knock-down induced sensitivity to AraC treatment in K562AC cells, and ALDH1A2 overexpressed K562S cells acquired the AraC resistance. Furthermore, the findings also suggest that ALDH1A2 expression is increased after the appearance of AraC resistance in clinical cases. These results will be helpful in understanding the mechanism of AraC resistance.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Citarabina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Leucêmica da Expressão Gênica , Leucemia/enzimologia , Retinal Desidrogenase/fisiologia , Família Aldeído Desidrogenase 1 , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Citarabina/administração & dosagem , Doxorrubicina/farmacologia , Eletroforese em Gel Bidimensional/métodos , Indução Enzimática , Humanos , Idarubicina/administração & dosagem , Células K562/efeitos dos fármacos , Células K562/metabolismo , Leucemia/sangue , Leucemia/tratamento farmacológico , Leucemia/genética , Leucemia Eritroblástica Aguda/enzimologia , Leucemia Eritroblástica Aguda/patologia , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Pró-Fármacos/farmacologia , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Retinal Desidrogenase/biossíntese , Retinal Desidrogenase/genética , Transfecção , Regulação para CimaRESUMO
AIMS: Accumulating evidence indicates that oxidative stress is associated with inflammation, and the cellular redox status can determine the sensitivity and the final outcome in response to inflammatory stimuli. To control the redox balance, mammalian cells contain a variety of oxidoreductases belonging to the thioredoxin superfamily. The large number of these enzymes suggests a complex mechanism of redox regulation in mammals, but the precise function of each family member awaits further investigations. RESULTS: We generated mice deficient in transmembrane thioredoxin-related protein (TMX), a transmembrane oxidoreductase in the endoplasmic reticulum (ER). When exposed to lipopolysaccharide (LPS) and d-(+)-galactosamine (GalN) to induce inflammatory liver injury, mutant mice were highly susceptible to the toxicants and developed severe liver damage. LPS-induced production of inflammatory mediators was equivalent in both wild-type and TMX(-/-) mice, whereas neutralization of the proinflammatory cytokine tumor necrosis factor-α suppressed the toxic effects of LPS/GalN in the mutant mice. Liver transcriptional profiles revealed enhanced activation of the p53-signaling pathway in the TMX(-/-) mice after LPS/GalN treatment. Furthermore, TMX deficiency also caused increased sensitivity to thioacetamide, which exerts its hepatotoxicity through the generation of reactive oxygen species. INNOVATION: The present study is the first to address the role of the oxidoreductase TMX in inflammatory liver injury. The phenotype of mice deficient in TMX suggests a functional link between redox regulation in the ER and susceptibility to oxidative tissue damage. CONCLUSION: We conclude that TMX plays a major role in host defense under the type of inflammatory conditions associated with oxidative stress.
Assuntos
Hepatite/genética , Proteínas de Membrana/genética , Oxirredutases/genética , Tiorredoxinas/genética , Animais , Apoptose/genética , Apoptose/imunologia , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/genética , Feminino , Galactosamina/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Ordem dos Genes , Marcação de Genes , Predisposição Genética para Doença , Hepatite/imunologia , Homozigoto , Lipopolissacarídeos/imunologia , Fígado/efeitos dos fármacos , Fígado/imunologia , Fígado/patologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Estresse Oxidativo , Oxirredutases/metabolismo , Tiorredoxinas/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/farmacologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
FADD is an adaptor protein that transmits apoptotic signals from death receptors. Additionally, FADD has been shown to play a role in various functions including cell proliferation. However, the physiological role of FADD during embryonic development remains to be delineated. Here, we show the novel roles FADD plays in development and the molecular mechanisms of these roles in Xenopus embryos. By whole-mount in situ hybridization and RT-PCR analysis, we observed that fadd is constantly expressed in early embryos. The upregulation or downregulation of FADD proteins by embryonic manipulation resulted in induction of apoptosis or size changes in the heart during development. Expression of a truncated form of FADD, FADDdd, which lacks pro-apoptotic activity, caused growth retardation of embryos associated with dramatic expressional fluctuations of genes that are regulated by NF-κB. Moreover, we isolated a homolog of mammalian cullin-4 (Cul4), a component of the ubiquitin E3 ligase family, as a FADDdd-interacting molecule in Xenopus embryos. Thus, our study shows that FADD has multiple functions in embryos; it plays a part in the regulation of NF-κB activation and heart formation, in addition to apoptosis. Furthermore, our findings provide new insights into how Cul4-based ligase is related to FADD signaling in embryogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Antígenos de Diferenciação/fisiologia , Apoptose , Proteína de Domínio de Morte Associada a Fas/fisiologia , Coração/embriologia , NF-kappa B/metabolismo , Receptores Imunológicos/fisiologia , Proteínas de Xenopus/fisiologia , Xenopus/embriologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Blastômeros/enzimologia , Blastômeros/metabolismo , Proteínas Culina/química , Proteínas Culina/genética , Proteínas Culina/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Proteína de Domínio de Morte Associada a Fas/genética , Proteína de Domínio de Morte Associada a Fas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Dados de Sequência Molecular , Morfolinos/genética , NF-kappa B/fisiologia , Fragmentos de Peptídeos/química , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Análise de Sequência de DNA , Deleção de Sequência , Transdução de Sinais , Ativação Transcricional , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMO
Asbestos is a potent carcinogen associated with increased risks of malignant mesothelioma and lung cancer in humans. Although the mechanism of carcinogenesis remains elusive, the physicochemical characteristics of asbestos play a role in the progression of asbestos-induced diseases. Among these characteristics, a high capacity to adsorb and accommodate biomolecules on its abundant surface area has been linked to cellular and genetic toxicity. Several previous studies identified asbestos-interacting proteins. Here, with the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry, we systematically identified proteins from various lysates that adsorbed to the surface of commercially used asbestos and classified them into the following groups: chromatin/nucleotide/RNA-binding proteins, ribosomal proteins, cytoprotective proteins, cytoskeleton-associated proteins, histones and hemoglobin. The surfaces of crocidolite and amosite, two iron-rich types of asbestos, caused more protein scissions and oxidative modifications than that of chrysotile by in situ-generated 4-hydroxy-2-nonenal. In contrast, we confirmed the intense hemolytic activity of chrysotile and found that hemoglobin attached to chrysotile, but not silica, can work as a catalyst to induce oxidative DNA damage. This process generates 8-hydroxy-2'-deoxyguanosine and thus corroborates the involvement of iron in the carcinogenicity of chrysotile. This evidence demonstrates that all three types of asbestos adsorb DNA and specific proteins, providing a niche for oxidative modification via catalytic iron. Therefore, considering the affinity of asbestos for histones/DNA and the internalization of asbestos into mesothelial cells, our results suggest a novel hypothetical mechanism causing genetic alterations during asbestos-induced carcinogenesis.
Assuntos
Amianto Amosita/química , Asbesto Crocidolita/química , Asbestos Serpentinas/química , Dano ao DNA , Proteínas/metabolismo , 8-Hidroxi-2'-Desoxiguanosina , Aldeídos/metabolismo , Animais , Amianto Amosita/metabolismo , Amianto Amosita/toxicidade , Asbesto Crocidolita/toxicidade , Asbestos Serpentinas/metabolismo , Cromatina/metabolismo , Citoesqueleto/metabolismo , DNA/química , DNA/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/biossíntese , Hemoglobinas/metabolismo , Histonas/metabolismo , Ferro/metabolismo , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/patologia , Mesotelioma/etiologia , Mesotelioma/patologia , Camundongos , Oxirredução , Proteínas/química , Proteínas de Ligação a RNA/metabolismo , Ratos , Proteínas Ribossômicas/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Propriedades de SuperfícieRESUMO
The effects of the application of the jasmonic acid derivative n-propyl dihydrojasmonate (PDJ) on ethylene biosynthesis, volatile compounds, and endogenous jasmonic acid (JA) and methyl jasmonate (MeJA) were examined in Japanese apricot (Prunus mume Sieb.) infected by a pathogen (Colletotrichum gloeosporioides). The fruit were dipped into 0.4 mM PDJ solution before inoculation with the pathogen and stored at 25 °C for 6 days. The inoculation induced an increase in 1-aminocyclopropane-1-carboxylic acid (ACC), ethylene, JA, and MeJA. In contrast, PDJ application reduced the endogenous JA, MeJA, and ethylene production and expression of the ACC oxidase gene (PmACO1) caused by the pathogen infection. The lesion diameter with C. gloeosporioides decreased upon PDJ application. The alcohol, ester, ketone, and lactone concentrations and alcohol acyltransferase (AAT) activity increased in the pathogen-infected fruit, but were decreased by PDJ application. These results suggest that PDJ application might influence ethylene production through PmACO1 and that aroma volatile emissions affected by pathogen infection can be correlated with the ethylene production, which is mediated by the levels of jasmonates.
Assuntos
Colletotrichum/fisiologia , Ciclopentanos/farmacologia , Etilenos/biossíntese , Oxilipinas/farmacologia , Doenças das Plantas/microbiologia , Prunus/metabolismo , Compostos Orgânicos Voláteis/metabolismo , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Ciclopentanos/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Oxilipinas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Prunus/enzimologia , Prunus/genética , Prunus/microbiologiaRESUMO
The spatiotemporal regulation of E-cadherin expression is important during body plan development and carcinogenesis. We found that Tara (Trio-associated repeat on actin) is enriched in cadherin-based adherens junctions (AJs), and its knockdown in MDCK cells (Tara-KD cells) significantly decreases the expression of E-cadherin. Tara-KD activates Rac1 through the Trio RhoGEF, which binds to E-cadherin and subsequently increases the phosphorylation of p38 and Tbx3, a transcriptional E-cadherin repressor. Accordingly, the decrease in E-cadherin expression is abrogated by ITX3 and SB203580 (specific inhibitors of Trio RhoGEF and p38MAPK, respectively), and by dephosphomimetic Tbx3. Despite the decreased E-cadherin expression, the Tara-KD cells do not undergo an epithelial-mesenchymal transition and remain as an epithelial cell sheet, presumably due to the concomitant up-regulation of cadherin-6. Tara-KD reduces the actin-belt density in the circumferential ring, and the cells form flattened cysts, suggesting that Tara functions to modulate epithelial cell sheet formation and integrity by up-regulating E-cadherin transcription.
Assuntos
Caderinas/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas dos Microfilamentos/metabolismo , Transcrição Gênica , Proteínas rac1 de Ligação ao GTP/metabolismo , Junções Aderentes/metabolismo , Animais , Benzimidazóis/farmacologia , Linhagem Celular , Cães , Transição Epitelial-Mesenquimal , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Humanos , Imidazóis/farmacologia , Camundongos , Proteínas dos Microfilamentos/genética , Fosforilação , Piridinas/farmacologia , Fatores de Troca de Nucleotídeo Guanina Rho , Transdução de Sinais , Proteínas com Domínio T/metabolismo , Tiazóis/farmacologia , Regulação para Cima , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Integration, an indispensable step for retrovirus replication, is executed by integrase (IN), which is expressed as a part of a Gag-Pol precursor. Although mechanistic detail of the IN-catalyzed integration reaction is well defined, numerous evidence have demonstrated that IN is involved in multiple steps of retrovirus replication other than integration. In this study, Huwe1, a HECT-type E3 ubiquitin ligase, was identified as a new cellular interactor of human immunodeficiency virus type 1 (HIV-1) IN. The interaction was mediated through the catalytic core domain of IN and a wide-range region of Huwe1. Interestingly, although depletion of Huwe1 in target cells did not affect the early phase of HIV-1 infection in a human T cell line, we found that infectivity of HIV-1 released from the Huwe1 knockdown cells was significantly augmented more than that of virus produced from control cells. The increase in infectivity occurred in proviral DNA synthesis. Further analysis revealed that Huwe1 interacted with HIV-1 Gag-Pol precursor protein through an IN domain. Our results suggest that Huwe1 in HIV-1 producer cells has a negative impact on early post-entry events during the next round of virus infection via association with an IN region of Gag-Pol.
Assuntos
Proteínas de Fusão gag-pol/metabolismo , Infecções por HIV/metabolismo , HIV-1/metabolismo , HIV-1/patogenicidade , Integrases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular , Técnicas de Silenciamento de Genes , Células HEK293 , HIV-1/genética , HIV-1/ultraestrutura , Células HeLa , Humanos , Camundongos , Vírus da Leucemia Murina de Moloney/genética , Vírus da Leucemia Murina de Moloney/metabolismo , Células NIH 3T3 , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases/genéticaRESUMO
Type 2 diabetes mellitus (T2DM) is characterized by defects in both insulin sensitivity and glucose-stimulated insulin secretion (GSIS) and is often accompanied by obesity. In this study, we show that disruption of thioredoxin binding protein-2 (TBP-2, also called Txnip) in obese mice (ob/ob) dramatically improves hyperglycaemia and glucose intolerance, without affecting obesity or adipocytokine concentrations. TBP-2-deficient ob/ob mice exhibited enhanced insulin sensitivity with activated insulin receptor substrate-1/Akt signalling in skeletal muscle and GSIS in islets compared with ob/ob mice. The elevation of uncoupling protein-2 (UCP-2) expression in ob/ob islets was downregulated by TBP-2 deficiency. TBP-2 overexpression suppressed glucose-induced adenosine triphosphate production, Ca(2+) influx and GSIS. In ß-cells, TBP-2 enhanced the expression level and transcriptional activity of UCP-2 by recruitment of peroxisome proliferator-activated receptor-γ co-activator-1α to the UCP-2 promoter. Thus, TBP-2 is a key regulatory molecule of both insulin sensitivity and GSIS in diabetes, raising the possibility that inhibition of TBP-2 may be a novel therapeutic approach for T2DM.
RESUMO
BACKGROUND: Serum proteins that non-specifically bind with human S100A8/A9 (h-S100A8/A9) have been proposed. Our aim was to isolate and identify these proteins, and verify their clinical significance for monitoring the postoperative condition of liver recipients, and further to discuss the transportation of human fibronectin (h-FN) with h-S100A8/A9 and its functional role in vivo. METHODS: To isolate the serum proteins, recombinant human S100A8, S100A9 and S100A8/A9 affinity columns were used. Proteins were identified by mass spectrometry. Two enzyme-linked immunosorbent assays (ELISA) were used to measure h-S100A8/A9 and h-FN in the sera of liver recipients. Flow cytometry was employed to detect h-S100A8/A9 and h-FN on immunological cells. Western blotting was used to confirm serum constituents using antibodies specific to each constituent. RESULTS: One of the proteins was identified with h-FN, and its fluctuation pattern in the serum of the recipient was in contrast to that of CRP. Flow cytometry showed a positive reaction for h-S100A8/A9 and h-FN on neutrophils and monocytes, indicating that both proteins exist on these cells. CONCLUSIONS: The h-FN could be transported with S100A8/A9 in blood and/or on immunological cells, and effectively prevent further attack by various internal oxidants or repair damaged liver tissue in vivo.
Assuntos
Proteínas Sanguíneas/metabolismo , Calgranulina A/metabolismo , Calgranulina B/metabolismo , Transplante de Fígado/imunologia , Monitorização Imunológica/métodos , Proteínas Sanguíneas/isolamento & purificação , Fibronectinas/metabolismo , Humanos , Hepatopatias/diagnóstico , Monócitos/química , Neutrófilos/química , Cuidados Pós-Operatórios , Ligação ProteicaRESUMO
To identify a new diagnostic marker for the immune pathophysiology of aplastic anemia (AA), we screened sera of immune-mediated AA patients for the presence of antibodies (Abs) specific to proteins derived from a leukemia cell line UT-7 using two-dimensional electrophoresis followed by immunoblotting. The target proteins were identified by peptide mass fingerprinting. Heterogeneous nuclear ribonucleoprotein (hnRNP) K was identified as a novel autoantigen. An enzyme-linked immunosorbent assay revealed high titers of anti-hnRNP K Abs in 85 (31%) of 273 patients with AA. Sixty-four patients received antithymocyte globulin and cyclosporine after undergoing screening for anti-hnRNP K Ab, anti-DRS-1 Ab, anti-moesin Ab, and paroxysmal nocturnal hemoglobinuria (PNH)-type cells. Twenty (87%) of 23 patients with the presence of anti-hnRNP K Abs responded to the immunosuppressive therapy (IST), while 19 (46%) of 41 patients without the presence of anti-hnRNP K Abs responded. A multivariate analysis showed only PNH-type cells and anti-hnRNP K Abs to be significant factors for the prediction of a good response to IST. The detection of anti-hnRNP K Abs as well as PNH-type cells may therefore be useful for diagnosing the immune pathophysiology of AA.
Assuntos
Anemia Aplástica/diagnóstico , Autoanticorpos/sangue , Biomarcadores/sangue , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anemia Aplástica/imunologia , Anemia Aplástica/fisiopatologia , Autoanticorpos/imunologia , Western Blotting , Linhagem Celular Tumoral , Eletroforese em Gel Bidimensional , Ensaio de Imunoadsorção Enzimática , Feminino , Células HL-60 , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Humanos , Células K562 , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
The serine/threonine protein kinase LKB1 is a tumor suppressor gene mutated in Peutz-Jeghers syndrome patients. The mutations are found also in several types of sporadic cancer. Although LKB1 is implicated in suppression of cell growth and metastasis, the detailed mechanisms have not yet been elucidated. In this study, we investigated the effect of LKB1 on cell motility, whose acquisition occurs in early metastasis. The knockdown of LKB1 enhanced cell migration and PAK1 activity in human colon cancer HCT116 cells, whereas forced expression of LKB1 in Lkb1-null mouse embryonic fibroblasts suppressed PAK1 activity and PAK1-mediated cell migration simultaneously. Notably, LKB1 directly phosphorylated PAK1 at Thr(109) in the p21-binding domain in vitro. The phosphomimetic T109E mutant showed significantly lower protein kinase activity than wild-type PAK1, suggesting that the phosphorylation at Thr(109) by LKB1 was responsible for suppression of PAK1. Consistently, the nonphosphorylatable T109A mutant was resistant to suppression by LKB1. Furthermore, we found that PAK1 was activated in the hepatocellular carcinomas and the precancerous liver lesions of Lkb1(+/-) mice. Taken together, these results suggest that PAK1 is a direct downstream target of LKB1 and plays an essential role in LKB1-induced suppression of cell migration.
Assuntos
Proteínas Serina-Treonina Quinases/química , Treonina/química , Quinases Ativadas por p21/química , Quinases Proteína-Quinases Ativadas por AMP , Animais , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Neoplasias/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Quinases Ativadas por p21/genéticaRESUMO
OBJECTIVES: Various autoantibodies are detected in the sera of PM/DM patients. Some of them are specific to PM/DM patients and closely associated with clinical manifestations of the diseases. Recently, the anti-CADM-140 antibody was reported to be found specifically in clinically amyopathic DM (C-ADM) patients and to be associated with acute interstitial lung disease (ILD). We assessed the clinical significance of the anti-CADM-140 antibody and then investigated the autoantigen recognized by the anti-CADM-140 antibody. METHODS: Autoantibodies were screened in 192 patients with various CTDs and 21 healthy controls using immunoprecipitation with [(35)S]methionine-labelled HeLa cells. Immunoabsorbent column chromatography was used to purify an autoantigen that was subsequently subjected to peptide mass fingerprinting. RESULTS: The anti-CADM-140 antibody was revealed to be specific to DM. Most of the anti-CADM-140-positive patients were C-ADM although some of them showed apparent myositis. The anti-CADM-140-positive patients frequently showed hyperferritinaemia and acute progressive ILD with poor prognosis. The anti-CADM-140 antibody was shown to recognize IFN induced with helicase C domain protein 1 (IFIH1), also known as the melanoma differentiation-associated gene 5 (MDA5), which is one of the RIG-I-like receptors and plays a role in innate immune responses. CONCLUSION: The anti-CADM-140 antibody was a marker of DM and intractable ILD and recognized IFIH1/MDA5, which is involved in innate immunity. These findings may give a new insight into the pathogenesis of DM.
Assuntos
Autoanticorpos/imunologia , Autoantígenos/sangue , RNA Helicases DEAD-box/imunologia , Dermatomiosite/imunologia , Peptídeos/imunologia , Adulto , Sequência de Aminoácidos , Biomarcadores/sangue , RNA Helicases DEAD-box/genética , Células HeLa , Humanos , Imunidade Inata , Peptídeos e Proteínas de Sinalização Intercelular , Helicase IFIH1 Induzida por Interferon , Dados de Sequência MolecularRESUMO
Short-term synaptic facilitation plays an important role in information processing in the central nervous system. Although the crucial requirement of presynaptic Ca(2+) in the expression of this plasticity has been known for decades, the molecular mechanisms underlying the plasticity remain controversial. Here, we show that presynaptic metabotropic glutamate receptors (mGluRs) bind and release Munc18-1 (also known as rbSec1/nSec1), an essential protein for synaptic transmission, in a Ca(2+)-dependent manner, whose actions decrease and increase synaptic vesicle release, respectively. We found that mGluR4 bound Munc18-1 with an EC(50) for Ca(2+) of 168 nM, close to the resting Ca(2+) concentration, and that the interaction was disrupted by Ca(2+)-activated calmodulin (CaM) at higher concentrations of Ca(2+). Consistently, the Munc18-1-interacting domain of mGluR4 suppressed both dense-core vesicle secretion from permeabilized PC12 cells and synaptic transmission in neuronal cells. Furthermore, this domain was sufficient to induce paired-pulse facilitation. Obviously, the role of mGluR4 in these processes was independent of its classical function of activation by glutamate. On the basis of these experimental data, we propose the following model: When neurons are not active, Munc18-1 is sequestered by mGluR4, and therefore the basal synaptic transmission is kept low. After the action potential, the increase in the Ca(2+) level activates CaM, which in turn liberates Munc18-1 from mGluR4, causing short-term synaptic facilitation. Our findings unite and provide a new insight into receptor signaling and vesicular transport, which are pivotal activities involved in a variety of cellular processes.
Assuntos
Cálcio/metabolismo , Estriol/análogos & derivados , Proteínas Munc18/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Transmissão Sináptica , Animais , Estriol/metabolismo , Camundongos , Proteínas Munc18/genética , Células PC12 , Ligação Proteica , RatosRESUMO
The constitutive centromere-associated network (CCAN) proteins are central to kinetochore assembly. To define the molecular architecture of this critical kinetochore network, we sought to determine the full complement of CCAN components and to define their relationships. This work identified a centromere protein S (CENP-S)-containing subcomplex that includes the new constitutive kinetochore protein CENP-X. Both CENP-S- and CENP-X-deficient chicken DT40 cells are viable but show abnormal mitotic behavior based on live cell analysis. Human HeLa cells depleted for CENP-X also showed mitotic errors. The kinetochore localization of CENP-S and -X is abolished in CENP-T- or CENP-K-deficient cells, but reciprocal experiments using CENP-S-deficient cells did not reveal defects in the localization of CCAN components. However, CENP-S- and CENP-X-deficient cells show a significant reduction in the size of the kinetochore outer plate. In addition, we found that intrakinetochore distance was increased in CENP-S- and CENP-X-deficient cells. These results suggest that the CENP-S complex is essential for the stable assembly of the outer kinetochore.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Ciclo Celular/fisiologia , Linhagem Celular , Galinhas , Proteínas Cromossômicas não Histona/genética , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Cinetocoros/metabolismo , Complexos Multiproteicos/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fuso Acromático/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
The small GTPases RalA and RalB are multifunctional proteins regulating a variety of cellular processes. Like other GTPases, the activity of Ral is regulated by the opposing effects of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Although several RalGEFs have been identified and characterized, the molecular identity of RalGAP remains unknown. Here, we report the first molecular identification of RalGAPs, which we have named RalGAP1 and RalGAP2. They are large heterodimeric complexes, each consisting of a catalytic alpha1 or alpha2 subunit and a common beta subunit. These RalGAP complexes share structural and catalytic similarities with the tuberous sclerosis tumor suppressor complex, which acts as a GAP for Rheb. In vitro GTPase assays revealed that recombinant RalGAP1 accelerates the GTP hydrolysis rate of RalA by 280,000-fold. Heterodimerization was required for this GAP activity. In PC12 cells, knockdown of the beta subunit led to sustained Ral activation upon epidermal growth factor stimulation, indicating that the RalGAPs identified here are critical for efficient termination of Ral activation induced by extracellular stimuli. Our identification of RalGAPs will enable further understanding of Ral signaling in many biological and pathological processes.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Esclerose Tuberosa/metabolismo , Proteínas ral de Ligação ao GTP/metabolismo , Animais , Encéfalo/metabolismo , Catálise , Citosol/metabolismo , Hidrólise , Modelos Biológicos , Dados de Sequência Molecular , Células PC12 , Ratos , Suínos , Distribuição TecidualRESUMO
The membrane-anchored protease regulator RECK plays important roles in mammalian development and tumor suppression. The biochemical bases of these bioactivities, however, remain poorly understood. Here we report on the properties of a recombinant RECK protein expressed in mouse fibroblasts and purified to near homogeneity. Multiple lines of evidence indicate that RECK forms dimers. Single particle reconstruction using transmission electron microscopy revealed a unique cowbell-like shaped RECK dimer. RECK is cleaved by MMP-2 and MMP-7 and competitively inhibits MMP-7-catalyzed cleavage of fibronectin. Forced RECK expression in HT1080 cells, whose endogenous RECK expression is minimal, leads to an increase in the amount of fibronectin associated with the cell. Our data demonstrate the ability of RECK to protect fibronectin from MMP-mediated degradation.