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BACKGROUND: Obesity and type 2 diabetes (T2D) are major risk factors for cardiovascular diseases (CVD). Dysregulated pro-apoptotic ceramide synthesis reduces ß-cell insulin secretion, thereby promoting hyperglycemic states which may manifest as T2D. Pro-apoptotic ceramides modulate insulin sensitivity and glucose tolerance while being linked to poor cardiovascular outcomes. Sirtuin-1 (SIRT1) is a NAD + - dependent deacetylase that protects against pancreatic ß-cell dysfunction; however, systemic levels are decreased in obese T2D mice and may promote pro-apoptotic ceramide synthesis and hyperglycemia. Herein, we aimed to assess the effects of restoring circulating SIRT1 levels to prevent metabolic imbalance in obese and diabetic mice. METHODS AND RESULTS: Circulating SIRT1 levels were reduced in obese diabetic mice (db/db) as compared to age-matched non-diabetic db/+ controls. Restoration of SIRT1 plasma levels with recombinant murine SIRT1 for 4-weeks prevented body weight gain, improved glucose tolerance, insulin sensitivity and vascular function in mice models of obesity and T2D. Untargeted lipidomics revealed that SIRT1 restored insulin-secretory function of ß-cells by reducing synthesis and accumulation of pro-apoptotic ceramides. Molecular mechanisms involved direct binding to and deacetylation of Toll-like receptor 4 (TLR4) by SIRT1 in ß-cells thereby decreasing the rate limiting enzymes of sphingolipid synthesis SPTLC1/2 via AKT/NF-κB. Among T2D patients, those with high baseline plasma levels of SIRT1 prior to metabolic surgery displayed restored ß-cell function (HOMA2- ß) and were more likely to have T2D remission during follow-up. CONCLUSION: Acetylation of TLR4 promotes ß-cell dysfunction via ceramide synthesis in T2D, which is blunted by systemic SIRT1 replenishment. Hence, restoration of systemic SIRT1 may provide a novel therapeutic strategy to counteract toxic ceramide synthesis and mitigate cardiovascular complications of T2D.
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OBJECTIVE: One of the suggested mechanisms of obesity-induced insulin resistance is mitochondrial dysfunction in target tissues such as skeletal muscle. In our study, we examined whether resistin, an adipokine associated with obesity-mediated insulin resistance, induced metabolic disorders by impairing mitochondrial homeostasis. METHODS: The morphology and function of mitochondria of skeletal muscle were examined in resistin-knockout and humanized resistin mice that were subjected to high-fat diet for 3 months. Morphology was examined by transmission electron microscopy. Mitochondria bioenergetics of skeletal muscle were evaluated using a Seahorse XF96 analyzer. Human skeletal myoblasts were used for in vitro studies on signaling mechanisms in responses to resistin. RESULTS: A high-fat diet in humanized resistin mice increased fragmented and shorter mitochondria in the skeletal muscle, whereas resistin-knockout mice had healthy normal mitochondria. In vitro studies showed that human resistin treatment impaired mitochondrial homeostasis by inducing mitochondrial fission, leading to a decrease in ATP production and mitochondrial dysfunction. Induction of mitochondrial fission by resistin was accompanied by increased formation of mitochondria-associated ER membranes (MAM). At the same time, resistin induced up-regulation of the protein kinase A (PKA) pathway. This activation of PKA induced phosphorylation of Drp1 at serine 616, leading to Drp1 activation and subsequent induction of mitochondrial fission. The key molecule that mediated human resistin-induced mitochondrial fission was adenylyl cyclase-associated protein 1 (CAP1), which was reported as a bona fide receptor for human resistin. Moreover, our newly developed biomimetic selective blocking peptide could repress human resistin-mediated mitochondrial dysfunction. High-fat diet-fed mice showed lower exercise capacity and higher insulin resistance, which was prevented by a novel peptide to block the binding of resistin to CAP1 or in the CAP1-knockdown mice. CONCLUSIONS: Our study demonstrated that human resistin induces mitochondrial dysfunction by inducing abnormal mitochondrial fission. This result suggests that the resistin-CAP1 complex could be a potential therapeutic target for the treatment of obesity-related metabolic diseases such as diabetes and cardiometabolic diseases.
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Resistência à Insulina , Mitocôndrias , Obesidade , Resistina , Animais , Humanos , Camundongos , Homeostase , Resistência à Insulina/fisiologia , Camundongos Knockout , Mitocôndrias/metabolismo , Obesidade/metabolismo , Resistina/genética , Resistina/metabolismoRESUMO
Although human induced pluripotent stem cells (iPSCs) can serve as a universal cell source for regenerative medicine, the use of iPSCs in clinical applications is limited by prohibitive costs and prolonged generation time. Moreover, allogeneic iPSC transplantation requires preclusion of mismatches between the donor and recipient human leukocyte antigen (HLA). We, therefore, generated universally compatible immune nonresponsive human iPSCs by gene editing. Transcription activator-like effector nucleases (TALENs) were designed for selective elimination of HLA DR expression. The engineered nucleases completely disrupted the expression of HLA DR on human dermal fibroblast cells (HDF) that did not express HLA DR even after stimulation with IFN-γ. Teratomas formed by HLA DR knockout iPSCs did not express HLA DR, and dendritic cells differentiated from HLA DR knockout iPSCs reduced CD4+ T cell activation. These engineered iPSCs might provide a novel translational approach to treat multiple recipients from a limited number of cell donors.
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AIMS: Proprotein convertase subtilisin/kexin type-9 (PCSK9), a molecular determinant of low-density lipoprotein (LDL) receptor (LDLR) fate, has emerged as a promising therapeutic target for atherosclerotic cardiovascular diseases. However, the precise mechanism by which PCSK9 regulates the internalization and lysosomal degradation of LDLR is unknown. Recently, we identified adenylyl cyclase-associated protein 1 (CAP1) as a receptor for human resistin whose globular C-terminus is structurally similar to the C-terminal cysteine-rich domain (CRD) of PCSK9. Herein, we investigated the role of CAP1 in PCSK9-mediated lysosomal degradation of LDLR and plasma LDL cholesterol (LDL-C) levels. METHODS AND RESULTS: The direct binding between PCSK9 and CAP1 was confirmed by immunoprecipitation assay, far-western blot, biomolecular fluorescence complementation, and surface plasmon resonance assay. Fine mapping revealed that the CRD of PCSK9 binds with the Src homology 3 binding domain (SH3BD) of CAP1. Two loss-of-function polymorphisms found in human PCSK9 (S668R and G670E in CRD) were attributed to a defective interaction with CAP1. siRNA against CAP1 reduced the PCSK9-mediated degradation of LDLR in vitro. We generated CAP1 knock-out mice and found that the viable heterozygous CAP1 knock-out mice had higher protein levels of LDLR and lower LDL-C levels in the liver and plasma, respectively, than the control mice. Mechanistic analysis revealed that PCSK9-induced endocytosis and lysosomal degradation of LDLR were mediated by caveolin but not by clathrin, and they were dependent on binding between CAP1 and caveolin-1. CONCLUSION: We identified CAP1 as a new binding partner of PCSK9 and a key mediator of caveolae-dependent endocytosis and lysosomal degradation of LDLR.
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Aterosclerose/genética , Proteínas de Transporte/genética , LDL-Colesterol/sangue , Mutação , Pró-Proteína Convertase 9/genética , Receptores de LDL/sangue , Animais , Aterosclerose/metabolismo , Proteínas de Transporte/metabolismo , DNA/genética , Análise Mutacional de DNA , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Knockout , Pró-Proteína Convertase 9/metabolismoRESUMO
Toll-like receptor (TLR)/myeloid differentiation primary response protein (MYD88) signaling aggravates sepsis by impairing neutrophil migration to infection sites. However, the role of intracellular fatty acids in TLR/MYD88 signaling is unclear. Here, inhibition of fatty acid synthase by C75 improved neutrophil chemotaxis and increased the survival of mice with sepsis in cecal ligation puncture and lipopolysaccharide-induced septic shock models. C75 specifically blocked TLR/MYD88 signaling in neutrophils. Treatment with GSK2194069 that targets a different domain of fatty acid synthase, did not block TLR signaling or MYD88 palmitoylation. De novo fatty acid synthesis and CD36-mediated exogenous fatty acid incorporation contributed to MYD88 palmitoylation. The binding of IRAK4 to the MYD88 intermediate domain and downstream signal activation required MYD88 palmitoylation at cysteine 113. MYD88 was palmitoylated by ZDHHC6, and ZDHHC6 knockdown decreased MYD88 palmitoylation and TLR/MYD88 activation upon lipopolysaccharide stimulus. Thus, intracellular saturated fatty acid-dependent palmitoylation of MYD88 by ZDHHC6 is a therapeutic target of sepsis.
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Receptores Toll-Like/metabolismo , Animais , Linhagem Celular , Ácido Graxo Sintase Tipo I , Humanos , Inflamação , Lipoilação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide , NF-kappa B/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: In its RING domain, tumor necrosis factor receptor-associated factor 6 (TRAF6) has ubiquitin E3 ligase activity that facilitates the formation of lysine 63-linked polyubiquitin chains. This activity is required to activate nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and plays an important role in the IκB kinase (IKK) complex. METHODS: An in vitro ubiquitination assay was used to establish whether c-Cbl could promote TRAF6 ubiquitination. We assessed direct binding and performed fine mapping between c-Cbl and TRAF6 based on the results of an immunoprecipitation assay with cultured 293 T cells. The luciferase reporter assay was applied to establish if c-Cbl-mediated ubiquitination affected NF-κB activation after stimulus from various TRAF-mediated signals: tumor necrosis factor-α (TNF-α), receptor activator of NF-κB ligand (RANKL), and interleukin-1ß (IL-1ß). An in vivo ubiquitination assay was performed using endogenous immunoprecipitation of TRAF6 in bone marrow macrophages (BMMs) and osteoclasts. RESULTS: Here, we report on a form of TRAF6 ubiquitination that is mediated by c-Cbl, leading to the formation of lysine 48-linked polyubiquitin chains. The NF-κB activity induced by RANKL and IL-1ß treatment is inhibited when c-Cbl is overexpressed, while the NF-κB activity induced by TNFα treatment is not. c-Cbl inhibits NF-κB activity mediated by TRAF6, but not by TRAF2. These findings show that c-Cbl ubiquitin ligase activity is essential for TRAF6 ubiquitination and negative regulation of NF-κB activity. Fine mapping revealed that the proline-rich domain of c-Cbl is critical for interaction with TRAF6. Stimulation with RANKL or interferon-γ (IFN-γ) caused c-Cbl to bind to polyubiquitinated TRAF6. CONCLUSIONS: These findings indicate that the interaction of TRAF6 with c-Cbl causes lysine 48-linked polyubiquitination for both negative feedback regulation and signaling cross-talk between RANKL and IFN-γ.
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Lisina/metabolismo , NF-kappa B/metabolismo , Poliubiquitina/metabolismo , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Ubiquitinação , Células HEK293 , Humanos , Interferon gama/farmacologia , Ligação Proteica , Proteínas Proto-Oncogênicas c-cbl/química , Ligante RANK/farmacologia , Domínios RING Finger , Fator 6 Associado a Receptor de TNF/química , Ubiquitinação/efeitos dos fármacosRESUMO
Phosphatase and tensin homolog (PTEN) negatively regulates phosphoinositide 3-kinase (PI3K)/Akt signaling as a lipid phosphatase for the second messenger phosphatidylinositol 3,4,5-triphosphate. We discovered recently that inactivating glycogen synthase kinase-3ß (GSK-3ß) via Akt plays an important role in receptor activator of nuclear factor κb ligand (RANKL)-induced osteoclastogenesis. However, the signaling link between GSK-3ß and PTEN in RANKL signaling has not been revealed. Downregulating PTEN by RNA interference increases Akt and GSK-3ß phosphorylation levels by RANKL, thereby promoting the formation of osteoclasts. PTEN phosphorylation at threonine 366 (T366) decreased gradually during RANKL-induced osteoclastogenesis, whereas PTEN protein levels were unaffected. Interestingly, the PTEN phosphorylation defective mutant (T366A) showed increased osteoclastogenesis, which is consistent with its lower phosphatase activity, compared to that of wild-type PTEN. Moreover, treatment with the GSK-3 inhibitor SB216763 suppressed PTEN phosphorylation levels and phosphatase activity and enhanced Akt phosphorylation. These data suggest that inhibiting GSK-3ß during RANKL-induced osteoclastogenesis decreases PTEN phosphorylation, leading to enhanced osteoclast differentiation through Akt activation.
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Quinase 3 da Glicogênio Sintase/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ligante RANK/metabolismo , Transdução de Sinais , Animais , Diferenciação Celular , Regulação para Baixo , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Proteínas Mutantes/metabolismo , Osteoclastos/citologia , Osteoclastos/enzimologia , Osteogênese , PTEN Fosfo-Hidrolase/deficiência , FosforilaçãoRESUMO
Vascular retinopathy is the consequence of vascular disease, and the retina is the only place where the arteries can be visualized directly. The purpose of this study was to evaluate the predictive value of retinal vascular findings for carotid artery atherosclerosis. From December 2009 to January 2011, the carotid intima-media thickness (IMT) and total plaque area (TPA) were measured in 179 consecutive patients, who received a fundoscopic examination. The patients were divided into groups as follows: normal retinal artery (normal; n = 44), diabetic retinopathy (DR; n = 25), retinal artery occlusion (RAO; n = 17), retinal vein occlusion (RVO; n = 67), and hypertensive retinopathy (HTN-R; n = 26). The subjects were classified according to the presence of an increased (≥ 1 mm) IMT and plaque. The values of the mean carotid IMT in the patients with vascular retinopathy (DR, 0.87 ± 0.14 mm; RAO, 1.18 ± 0.47 mm; RVO, 0.84 ± 0.14 mm; HTN-R, 0.90 ± 0.20 mm) were significantly increased compared with those in the normal subjects (0.77 ± 0.13 mm). A total 77 of 135 vascular retinopathy patients demonstrated an increased IMT (57 %), and 97 vascular retinopathy patients had carotid artery plaque (72 %). The relative risk of vascular retinopathy in the prediction of an increased IMT and the presence of plaque was 2.79 and 3.95, respectively. Although The TPA was significantly increased in the patients with RAO (1.87 ± 2.67 cm(2)) and RVO (0.27 ± 0.23 cm(2)) compared with the normal subjects (0.18 ± 0.23 cm(2), all Ps < 0.05), there was no significant difference in the ipsilateral carotid IMT and TPA of the affected eye compared with that of the contralateral eye. In conclusion, vascular retinopathy demonstrated a good predictive value in identifying asymptomatic carotid artery atherosclerosis, and this was not confined to the ipsilateral carotid artery of the affected eye. Further recommendations with regard to carotid atherosclerosis screening in patients with vascular retinopathy should be considered.
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Artérias Carótidas/diagnóstico por imagem , Doenças das Artérias Carótidas/diagnóstico , Espessura Intima-Media Carotídea , Programas de Rastreamento/métodos , Artéria Retiniana/patologia , Doenças Retinianas/diagnóstico , Veia Retiniana/patologia , Idoso , Análise de Variância , Doenças Assintomáticas , Doenças das Artérias Carótidas/complicações , Doenças das Artérias Carótidas/diagnóstico por imagem , Estudos Transversais , Retinopatia Diabética/diagnóstico , Retinopatia Diabética/patologia , Humanos , Retinopatia Hipertensiva/diagnóstico , Retinopatia Hipertensiva/patologia , Programas de Rastreamento/normas , Pessoa de Meia-Idade , Placa Aterosclerótica , Guias de Prática Clínica como Assunto , Valor Preditivo dos Testes , Oclusão da Artéria Retiniana/diagnóstico , Oclusão da Artéria Retiniana/patologia , Doenças Retinianas/complicações , Doenças Retinianas/patologia , Oclusão da Veia Retiniana/diagnóstico , Oclusão da Veia Retiniana/patologia , Índice de Gravidade de DoençaRESUMO
Glycogen synthase kinase-3ß (GSK-3ß) is a serine/threonine kinase originally identified as a regulator of glycogen deposition. Although the role of GSK-3ß in osteoblasts is well characterized as a negative regulator of ß-catenin, its effect on osteoclast formation remains largely unidentified. Here, we show that the GSK-3ß inactivation upon receptor activator of NF-κB ligand (RANKL) stimulation is crucial for osteoclast differentiation. Regulation of GSK-3ß activity in bone marrow macrophages by retroviral expression of the constitutively active GSK-3ß (GSK3ß-S9A) mutant inhibits RANKL-induced osteoclastogenesis, whereas expression of the catalytically inactive GSK-3ß (GSK3ß-K85R) or small interfering RNA (siRNA)-mediated GSK-3ß silencing enhances osteoclast formation. Pharmacological inhibition of GSK-3ß further confirmed the negative role of GSK-3ß in osteoclast formation. We also show that overexpression of the GSK3ß-S9A mutant in bone marrow macrophages inhibits RANKL-mediated NFATc1 induction and Ca(2+) oscillations. Remarkably, transgenic mice expressing the GSK3ß-S9A mutant show an osteopetrotic phenotype due to impaired osteoclast differentiation. Further, osteoclast precursor cells from the transgenic mice show defects in expression and nuclear localization of NFATc1. These findings demonstrate a novel role for GSK-3ß in the regulation of bone remodeling through modulation of NFATc1 in RANKL signaling.
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Células da Medula Óssea/enzimologia , Remodelação Óssea/fisiologia , Diferenciação Celular/fisiologia , Núcleo Celular/enzimologia , Quinase 3 da Glicogênio Sintase/metabolismo , Osteoclastos/enzimologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/fisiologia , Substituição de Aminoácidos , Animais , Relógios Biológicos/efeitos dos fármacos , Relógios Biológicos/fisiologia , Células da Medula Óssea/citologia , Remodelação Óssea/efeitos dos fármacos , Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Núcleo Celular/genética , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Camundongos , Camundongos Transgênicos , Mutação de Sentido Incorreto , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/citologia , Ligante RANK/genética , Ligante RANK/metabolismo , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
TRANCE/TRAF6 signalling governs osteoclastogenesis in vivo. Only the TRANCE receptor (TRANCE-R) has been shown to induce osteoclastogenesis, even though other immune receptors, including CD40 and IL-1R/Toll-like receptor, use TRAF6 to activate overlapping signalling cascades. These observations led us to question whether qualitative or quantitative differences exist between the TRAF6-mediated signals induced by TRANCE and by other ligand-receptor pairs. Here we show that stimulation by overexpressed wild-type CD40 can induce osteoclastogenesis. Stimulation through modified CD40 containing increased numbers of TRAF6-binding sites in the cytoplasmic tails showed a dose-dependent increase in the activation of p38 kinase and more pronounced osteoclastogenesis. Moreover, precursors overexpressing TRAF6 differentiate into osteoclasts in the absence of additional signals from TRANCE. Our results suggest that differences in the osteoclastogenesis-inducing capacity of TRANCE-R versus other TRAF6-associated receptors may in part stem from a quantitative difference in the TRAF6-mediated signals.
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Osteoclastos/metabolismo , Transdução de Sinais/fisiologia , Fator 6 Associado a Receptor de TNF/metabolismo , Animais , Antígenos CD40/metabolismo , Diferenciação Celular/fisiologia , Humanos , Osteoclastos/citologia , Receptor Ativador de Fator Nuclear kappa-B , Receptores do Fator de Necrose Tumoral/metabolismoRESUMO
Second mitochondria-derived activator of caspase (Smac) has been implicated in the activation of apoptosis in response to cell stress. We screened for Smac/DIABLO-binding protein for further understanding of Smac-mediated apoptosis. We identified NADE, previously known as p75NTR-associated cell death executor, as a Smac-binding protein. Smac-NADE interaction was mapped to the N-terminal region of Samc and the C-terminal region of NADE. Co-expression of NADE and Smac promotes TRAIL-induced apoptosis in MCF-7 cells. Interestingly, the co-presence of Smac and NADE inhibits XIAP-mediated Smac ubiquitination. In conclusion, our results provide the first evidence that the interaction between Smac and NADE regulates apoptosis through the inhibition of Smac ubiquitination.
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Apoptose/fisiologia , Proteínas de Transporte/metabolismo , Glicoproteínas de Membrana/fisiologia , Proteínas Mitocondriais/metabolismo , Proteínas/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Proteínas Reguladoras de Apoptose , Linhagem Celular , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana/metabolismo , Ligação Proteica , Ligante Indutor de Apoptose Relacionado a TNF , Fator de Necrose Tumoral alfa/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo XRESUMO
Nuclear factor-kappaB (NF-kappaB) is a transcription factor critical for key cellular processes, including immune response, apoptosis, and cell cycle progression. A yeast two-hybrid screening, using the Rel homology domain (RHD) of the p65 subunit (RelA) of NF-kappaB as bait, led to the isolation of PIAS3, previously identified as a specific inhibitor of STAT3. We show that PIAS3 can directly associate with p65 using an in vitro pull-down and in vivo coimmunoprecipitation assays. When overexpressed, PIAS3 inhibits NF-kappaB-dependent transcription induced by treatment with tumor necrosis factor alpha (TNF-alpha) or interleukin-1beta or by overexpression of TNF family receptors such as RANK, TNFR1, and CD30 or signal transducers of TNF receptor-associated factors (TRAFs), including TRAF2, TRAF5, and TRAF6. Downregulation of PIAS3 by RNA interference reverses its effect on TNF-alpha-mediated NF-kappaB activation. We found that an N-terminal region of PIAS3 is necessary for both the interaction with p65 and the transcriptional suppression activity. In addition, we found that an LXXLL coregulator signature motif located within the N-terminal region of PIAS3 is the minimal requirement for the interaction with p65. Furthermore, we demonstrate that PIAS3 interferes with p65 binding to the CBP coactivator, thereby resulting in a decreased NF-kappaB-dependent transcription. Taken together, these data suggest that PIAS3 may function in vivo as a modulator in suppressing the transcriptional activity of p65.
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Proteínas de Transporte/metabolismo , NF-kappa B/metabolismo , NF-kappa B/fisiologia , Transcrição Gênica , Motivos de Aminoácidos , Animais , Antígenos CD/metabolismo , Western Blotting , Ciclo Celular , Linhagem Celular , Relação Dose-Resposta a Droga , Ativação Enzimática , Glutationa Transferase/metabolismo , Glicoproteínas/metabolismo , Humanos , Interleucina-1/metabolismo , Antígeno Ki-1/metabolismo , Luciferases/metabolismo , Osteoprotegerina , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Fator 5 Associado a Receptor de TNF , Fator 6 Associado a Receptor de TNF , Fatores de Tempo , Fator de Transcrição RelA , Transfecção , Fator de Necrose Tumoral alfa/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Fas-associated factor-1 (FAF1) is a Fas-binding pro-apoptotic protein that is a component of the death-inducing signaling complex in Fas-mediated apoptosis. Here, we show that FAF1 is involved in negative regulation of NF-kappaB activation. Overexpression of FAF1 decreased the basal level of NF-kappaB activity in 293 cells. NF-kappaB activation induced by tumor necrosis factor (TNF)-alpha, interleukin-1beta, and lipopolysaccharide was also inhibited by FAF1 overexpression. Moreover, FAF1 suppressed NF-kappaB activation induced by transducers of diverse NF-kappaB-activating signals such as TNF receptor-associated factor-2 and -6, MEKK1, and IkappaB kinase-beta as well as NF-kappaB p65, one of the end point molecules in the NF-kappaB activation pathway, suggesting that NF-kappaB p65 might be a target molecule upon which FAF1 acts. Subsequent study disclosed that FAF1 physically interacts with NF-kappaB p65 and that the binding domain of FAF1 is the death effector domain (DED)-interacting domain (amino acids 181-381), where DEDs of the Fas-associated death domain protein and caspase-8 interact. The NF-kappaB activity-modulating potential of FAF1 was also mapped to the DED-interacting domain. Finally, overexpression of FAF1 prevented translocation of NF-kappaB p65 into the nucleus and decreased its DNA-binding activity upon TNFalpha treatment. This study presents a novel function of FAF1, in addition to the previously known function as a component of the Fas death-inducing signaling complex, i.e. NF-kappaB activity suppressor by cytoplasmic retention of NF-kappaB p65 via physical interaction.