RESUMEN
RIPK1 is a multi-functional kinase that regulates cell death and inflammation and has been implicated in the pathogenesis of inflammatory diseases. RIPK1 acts in a kinase-dependent and kinase-independent manner to promote or suppress apoptosis and necroptosis, but the underlying mechanisms remain poorly understood. Here, we show that a mutation (R588E) disrupting the RIPK1 death domain (DD) caused perinatal lethality induced by ZBP1-mediated necroptosis. Additionally, these mice developed postnatal inflammatory pathology, which was mediated by necroptosis-independent TNFR1, TRADD, and TRIF signaling, partially requiring RIPK3. Our biochemical mechanistic studies revealed that ZBP1- and TRIF-mediated activation of RIPK3 required RIPK1 kinase activity in wild-type cells but not in Ripk1R588E/R588E cells, suggesting that DD-dependent oligomerization of RIPK1 and its interaction with FADD determine the mechanisms of RIPK3 activation by ZBP1 and TRIF. Collectively, these findings revealed a critical physiological role of DD-dependent RIPK1 signaling that is important for the regulation of tissue homeostasis and inflammation.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular , Inflamación , Necroptosis , Proteínas de Unión al ARN , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Transducción de Señal , Animales , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Ratones , Inflamación/metabolismo , Inflamación/inmunología , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Proteína de Dominio de Muerte Asociada a Fas/genética , Muerte Celular , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Dominios Proteicos , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Apoptosis , Mutación , Proteína de Dominio de Muerte Asociada a Receptor de TNFRESUMEN
Cell death pathways regulate various homeostatic processes. Autoimmune lymphoproliferative syndrome (ALPS) in humans and lymphoproliferative (LPR) disease in mice result from abrogated CD95-induced apoptosis. Because caspase-8 mediates CD95 signaling, we applied genetic approaches to dissect the roles of caspase-8 in cell death and inflammation. Here, we describe oligomerization-deficient Caspase-8F122GL123G/F122GL123G and non-cleavable Caspase-8D387A/D387A mutant mice with defective caspase-8-mediated apoptosis. Although neither mouse developed LPR disease, removal of the necroptosis effector Mlkl from Caspase-8D387A/D387A mice revealed an inflammatory role of caspase-8. Ablation of one allele of Fasl, Fadd, or Ripk1 prevented the pathology of Casp8D387A/D387AMlkl-/- animals. Removing both Fadd alleles from these mice resulted in early lethality prior to post-natal day 15 (P15), which was prevented by co-ablation of either Ripk1 or Caspase-1. Our results suggest an in vivo role of the inflammatory RIPK1-caspase-8-FADD (FADDosome) complex and reveal a FADD-independent inflammatory role of caspase-8 that involves activation of an inflammasome.
Asunto(s)
Caspasa 8/genética , Susceptibilidad a Enfermedades , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Inflamación/etiología , Inflamación/metabolismo , Necroptosis/genética , Animales , Apoptosis/genética , Biomarcadores , Caspasa 8/química , Caspasa 8/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica , Inflamasomas/metabolismo , Inflamación/mortalidad , Inflamación/patología , Lipopolisacáridos/efectos adversos , Lipopolisacáridos/inmunología , Ratones , Ratones Noqueados , Mortalidad , Fenotipo , Multimerización de ProteínaRESUMEN
Pathways controlling intestinal epithelial cell (IEC) death regulate gut immune homeostasis and contribute to the pathogenesis of inflammatory bowel diseases. Here, we show that caspase-8 and its adapter FADD act in IECs to regulate intestinal inflammation downstream of Z-DNA binding protein 1 (ZBP1)- and tumor necrosis factor receptor-1 (TNFR1)-mediated receptor interacting protein kinase 1 (RIPK1) and RIPK3 signaling. Mice with IEC-specific FADD or caspase-8 deficiency developed colitis dependent on mixed lineage kinase-like (MLKL)-mediated epithelial cell necroptosis. However, MLKL deficiency fully prevented ileitis caused by epithelial caspase-8 ablation, but only partially ameliorated ileitis in mice lacking FADD in IECs. Our genetic studies revealed that caspase-8 and gasdermin-D (GSDMD) were both required for the development of MLKL-independent ileitis in mice with epithelial FADD deficiency. Therefore, FADD prevents intestinal inflammation downstream of ZBP1 and TNFR1 by inhibiting both MLKL-induced necroptosis and caspase-8-GSDMD-dependent pyroptosis-like death of epithelial cells.
Asunto(s)
Caspasa 8/genética , Proteína de Dominio de Muerte Asociada a Fas/genética , Enfermedades Inflamatorias del Intestino/etiología , Enfermedades Inflamatorias del Intestino/metabolismo , Mucosa Intestinal/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Unión a Fosfato/metabolismo , Proteínas Quinasas/metabolismo , Animales , Apoptosis/genética , Caspasa 8/metabolismo , Muerte Celular/genética , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Células Epiteliales/metabolismo , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Perfilación de la Expresión Génica , Homeostasis/genética , Inmunohistoquímica , Enfermedades Inflamatorias del Intestino/patología , Mucosa Intestinal/patología , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Noqueados , Proteínas de Unión a Fosfato/genética , Proteínas Quinasas/genéticaRESUMEN
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known for specifically killing cancer cells, whereas in resistant cancers, TRAIL/TRAIL-R can promote metastasis via Rac1 and PI3K. It remains unknown, however, whether and to what extent TRAIL/TRAIL-R signaling in cancer cells can affect the immune microenvironment. Here we show that TRAIL-triggered cytokine secretion from TRAIL-resistant cancer cells is FADD dependent and identify the TRAIL-induced secretome to drive monocyte polarization to myeloid-derived suppressor cells (MDSCs) and M2-like macrophages. TRAIL-R suppression in tumor cells impaired CCL2 production and diminished both lung MDSC presence and tumor growth. In accordance, the receptor of CCL2, CCR2, is required to facilitate increased MDSC presence and tumor growth. Finally, TRAIL and CCL2 are co-regulated with MDSC/M2 markers in lung adenocarcinoma patients. Collectively, endogenous TRAIL/TRAIL-R-mediated CCL2 secretion promotes accumulation of tumor-supportive immune cells in the cancer microenvironment, thereby revealing a tumor-supportive immune-modulatory role of the TRAIL/TRAIL-R system in cancer biology.
Asunto(s)
Adenocarcinoma/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Citocinas/metabolismo , Neoplasias Pulmonares/metabolismo , Macrófagos/metabolismo , Receptores CCR2/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Microambiente Tumoral , Células A549 , Adenocarcinoma/genética , Adenocarcinoma/inmunología , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Animales , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Pulmón de Células no Pequeñas/patología , Caspasa 8/genética , Caspasa 8/metabolismo , Proliferación Celular , Quimiocina CCL2/metabolismo , Proteína de Dominio de Muerte Asociada a Fas/genética , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Femenino , Células HCT116 , Células HeLa , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Macrófagos/inmunología , Macrófagos/patología , Ratones Endogámicos C57BL , Ratones SCID , Fenotipo , Interferencia de ARN , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Transducción de Señal , Factores de Tiempo , Transfección , Carga TumoralRESUMEN
BACKGROUND: The autoimmune lymphoproliferative syndrome (ALPS) is a noninfectious and nonmalignant lymphoproliferative disease frequently associated with autoimmune cytopenia resulting from defective FAS signaling. We previously described germline monoallelic FAS (TNFRSF6) haploinsufficient mutations associated with somatic events, such as loss of heterozygosity on the second allele of FAS, as a cause of ALPS-FAS. These somatic events were identified by sequencing FAS in DNA from double-negative (DN) T cells, the pathognomonic T-cell subset in ALPS, in which the somatic events accumulated. OBJECTIVE: We sought to identify whether a somatic event affecting the FAS-associated death domain (FADD) gene could be related to the disease onset in 4 unrelated patients with ALPS carrying a germline monoallelic mutation of the FADD protein inherited from a healthy parent. METHODS: We sequenced FADD and performed array-based comparative genomic hybridization using DNA from sorted CD4+ or DN T cells. RESULTS: We found homozygous FADD mutations in the DN T cells from all 4 patients, which resulted from uniparental disomy. FADD deficiency caused by germline heterozygous FADD mutations associated with a somatic loss of heterozygosity was a phenocopy of ALPS-FAS without the more complex symptoms reported in patients with germline biallelic FADD mutations. CONCLUSIONS: The association of germline and somatic events affecting the FADD gene is a new genetic cause of ALPS.
Asunto(s)
Síndrome Linfoproliferativo Autoinmune , Proteína de Dominio de Muerte Asociada a Fas , Humanos , Apoptosis/genética , Enfermedades Autoinmunes/genética , Síndrome Linfoproliferativo Autoinmune/genética , Hibridación Genómica Comparativa , ADN , Receptor fas/genética , Proteína de Dominio de Muerte Asociada a Fas/genética , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Células Germinativas/patología , MutaciónRESUMEN
BACKGROUND: Elevated TCRαß+CD4-CD8- double-negative T cells (DNT) and serum biomarkers help identify FAS mutant patients with autoimmune lymphoproliferative syndrome (ALPS). However, in some patients with clinical features and biomarkers consistent with ALPS, germline or somatic FAS mutations cannot be identified on standard exon sequencing (ALPS-undetermined: ALPS-U). OBJECTIVE: We sought to explore whether complex genetic alterations in the FAS gene escaping standard sequencing or mutations in other FAS pathway-related genes could explain these cases. METHODS: Genetic analysis included whole FAS gene sequencing, copy number variation analysis, and sequencing of FAS cDNA and other FAS pathway-related genes. It was guided by FAS expression analysis on CD57+DNT, which can predict somatic loss of heterozygosity (sLOH). RESULTS: Nine of 16 patients with ALPS-U lacked FAS expression on CD57+DNT predicting heterozygous "loss-of-expression" FAS mutations plus acquired somatic second hits in the FAS gene, enriched in DNT. Indeed, 7 of 9 analyzed patients carried deep intronic mutations or large deletions in the FAS gene combined with sLOH detectable in DNT; 1 patient showed a FAS exon duplication. Three patients had reduced FAS expression, and 2 of them harbored mutations in the FAS promoter, which reduced FAS expression in reporter assays. Three of the 4 ALPS-U patients with normal FAS expression carried heterozygous FADD mutations with sLOH. CONCLUSION: A combination of serum biomarkers and DNT phenotyping is an accurate means to identify patients with ALPS who are missed by routine exome sequencing.
Asunto(s)
Síndrome Linfoproliferativo Autoinmune , Receptor fas , Humanos , Síndrome Linfoproliferativo Autoinmune/diagnóstico , Síndrome Linfoproliferativo Autoinmune/genética , Biomarcadores , Variaciones en el Número de Copia de ADN , Secuenciación del Exoma , Receptor fas/genética , Proteína de Dominio de Muerte Asociada a Fas/genética , MutaciónRESUMEN
The E3 ubiquitin ligase APC/C-Cdh1 maintains the G0/G1 state, and its inactivation is required for cell cycle entry. We reveal a novel role for Fas-associated protein with death domain (FADD) in the cell cycle through its function as an inhibitor of APC/C-Cdh1. Using real-time, single-cell imaging of live cells combined with biochemical analysis, we demonstrate that APC/C-Cdh1 hyperactivity in FADD-deficient cells leads to a G1 arrest despite persistent mitogenic signaling through oncogenic EGFR/KRAS. We further show that FADDWT interacts with Cdh1, while a mutant lacking a consensus KEN-box motif (FADDKEN) fails to interact with Cdh1 and results in a G1 arrest due to its inability to inhibit APC/C-Cdh1. Additionally, enhanced expression of FADDWT but not FADDKEN, in cells arrested in G1 upon CDK4/6 inhibition, leads to APC/C-Cdh1 inactivation and entry into the cell cycle in the absence of retinoblastoma protein phosphorylation. FADD's function in the cell cycle requires its phosphorylation by CK1α at Ser-194 which promotes its nuclear translocation. Overall, FADD provides a CDK4/6-Rb-E2F-independent "bypass" mechanism for cell cycle entry and thus a therapeutic opportunity for CDK4/6 inhibitor resistance.
Asunto(s)
Proteínas de Ciclo Celular , Ubiquitina-Proteína Ligasas , Humanos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , División Celular , Expresión Génica , Células HEK293 , Mutación , Dominios Proteicos , Transporte de Proteínas/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Tanshinone IIA (Tan IIA), a main active ingredient of salvia miltiorrhiza, has a wide range of antitumor effects, while its specific role and mechanism in head and neck squamous cell carcinomas (HNSCC) is not fully understood. Totally 59 primary HNSCC patients underwent two courses of induction chemotherapy before surgery. The association between expression of Fas-Associated Death Domain (FADD) and receptor interacting protein kinase 1 (RIPK1) and chemotherapy resistance and survival were evaluated. The cell counting kit-8 was used to detect the effect of Tan IIA on the activity of cisplatin in chemoresistant HNSCC cells through a series of in vitro experiments. The quantitative real-time reverse-transcription polymerase chain reaction, Western blot analysis and flow cytometry were used. FADD and RIPK1 expressions were differentially expressed in Chemosensitive and drug-resistant patients. Furthermore, patients with tumors exhibiting high expression of FADD and RIPK1 had significantly greater risk for chemoresistance and mortality than patients with tumors that had low levels of these proteins. Moreover, Tan IIA reduced the expression of RIPK1 and FADD in HNSCC chemoresistant cell lines, which could increase the chemosensitivity of cisplatin and promote apoptosis. Overexpression of RIPK1 led to attenuation of therapeutic effects of Tan IIA, which were mainly realized through regulation of the RIPK1-FADD-Caspase 8 complex. This study is the first to demonstrate the clinical value and role of FADD and RIPK1 in the treatment of HNSCC. This work establishes the proapoptotic effects of Tan IIA and its potential to enhance chemosensitivity in HNSCC by modulating the RIPK1-FADD-Caspase 8 complex.
Asunto(s)
Abietanos , Caspasa 8 , Cisplatino , Resistencia a Antineoplásicos , Proteína de Dominio de Muerte Asociada a Fas , Neoplasias de Cabeza y Cuello , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Carcinoma de Células Escamosas de Cabeza y Cuello , Humanos , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Proteína de Dominio de Muerte Asociada a Fas/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Abietanos/farmacología , Masculino , Femenino , Caspasa 8/metabolismo , Caspasa 8/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Persona de Mediana Edad , Cisplatino/farmacología , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Neoplasias de Cabeza y Cuello/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Anciano , Apoptosis/efectos de los fármacos , Adulto , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/genéticaRESUMEN
BACKGROUND: Prion diseases are transmissible and fatal neurodegenerative diseases characterized by accumulation of misfolded prion protein isoform (PrPSc), astrocytosis, microgliosis, spongiosis, and neurodegeneration. Elevated levels of cell membrane associated PrPSc protein and inflammatory cytokines hint towards the activation of death receptor (DR) pathway/s in prion diseases. Activation of DRs regulate, either cell survival or apoptosis, autophagy and necroptosis based on the adaptors they interact. Very little is known about the DR pathways activation in prion disease. DR3 and DR5 that are expressed in normal mouse brain were never studied in prion disease, so also their ligands and any DR adaptors. This research gap is notable and investigated in the present study. METHODS: C57BL/6J mice were infected with Rocky Mountain Laboratory scrapie mouse prion strain. The progression of prion disease was examined by observing morphological and behavioural abnormalities. The levels of PrP isoforms and GFAP were measured as the marker of PrPSc accumulation and astrocytosis respectively using antibody-based techniques that detect proteins on blot and brain section. The levels of DRs, their glycosylation and ectodomain shedding, and associated factors warrant their examination at protein level, hence western blot analysis was employed in this study. RESULTS: Prion-infected mice developed motor deficits and neuropathology like PrPSc accumulation and astrocytosis similar to other prion diseases. Results from this research show higher expression of all DR ligands, TNFR1, Fas and p75NTR but decreased levels DR3 and DR5. The levels of DR adaptor proteins like TRADD and TRAF2 (primarily regulate pro-survival pathways) are reduced. FADD, which primarily regulate cell death, its level remains unchanged. RIPK1, which regulate pro-survival, apoptosis and necroptosis, its expression and proteolysis (inhibits necroptosis but activates apoptosis) are increased. CONCLUSIONS: The findings from the present study provide evidence towards the involvement of DR3, DR5, DR6, TL1A, TRAIL, TRADD, TRAF2, FADD and RIPK1 for the first time in prion diseases. The knowledge obtained from this research discuss the possible impacts of these 16 differentially expressed DR factors on our understanding towards the multifaceted neuropathology of prion diseases and towards future explorations into potential targeted therapeutic interventions for prion disease specific neuropathology.
Asunto(s)
Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Enfermedades por Prión , Animales , Enfermedades por Prión/metabolismo , Enfermedades por Prión/patología , Receptores de Muerte Celular/metabolismo , Transducción de Señal , Encéfalo/metabolismo , Encéfalo/patología , Ratones , Proteínas PrPSc/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismoRESUMEN
FIRES and NORSE are clinical presentations of disease processes that, to date, remain unexplained without an established etiology in many cases. Neuroinflammation is thought to have paramount importance in the genesis of these conditions. We hereby report the clinical, EEG, brain MRI, and genetic findings of a nuclear family with recurrent febrile-related encephalopathy with refractory de novo Status Epilepticus. Whole-exome sequencing (WES) revealed a homozygous p.C105W pathogenic variant of FADD gene (FAS-associated protein with death domain, FADD), known to cause ultrarare forms of autosomal recessive immunodeficiency that could be associated with variable degrees of lymphoproliferation, cerebral atrophy, and cardiac abnormalities. The FADD-related conditions disrupt FAS-mediated apoptosis and can cause a clinical picture with the characteristics of FIRES. This observation is important because, on one hand, it increases the number of reported patients with FADD deficiency, showing that this disorder may present variable expressivity, and on the other hand, it demonstrates a genetic cause of FIRES involving a cell-mediated inflammation regulatory pathway. This finding supports early treatment with immunomodulatory therapy and could represent a new avenue of research in the field of new onset refractory status epilepticus and related conditions.
Asunto(s)
Proteína de Dominio de Muerte Asociada a Fas , Humanos , Proteína de Dominio de Muerte Asociada a Fas/genética , Femenino , Masculino , Convulsiones Febriles/genética , Estado Epiléptico/genética , Estado Epiléptico/etiología , Linaje , Secuenciación del Exoma , Fiebre/genética , Fiebre/complicaciones , Síndromes Epilépticos/genética , ElectroencefalografíaRESUMEN
Caspase-8 activation can be triggered by death receptor-mediated formation of the death-inducing signaling complex (DISC) and by the inflammasome adaptor ASC. Caspase-8 assembles with FADD at the DISC and with ASC at the inflammasome through its tandem death effector domain (tDED), which is regulated by the tDED-containing cellular inhibitor cFLIP and the viral inhibitor MC159. Here we present the caspase-8 tDED filament structure determined by cryoelectron microscopy. Extensive assembly interfaces not predicted by the previously proposed linear DED chain model were uncovered, and were further confirmed by structure-based mutagenesis in filament formation in vitro and Fas-induced apoptosis and ASC-mediated caspase-8 recruitment in cells. Structurally, the two DEDs in caspase-8 use quasi-equivalent contacts to enable assembly. Using the tDED filament structure as a template, structural analyses reveal the interaction surfaces between FADD and caspase-8 and the distinct mechanisms of regulation by cFLIP and MC159 through comingling and capping, respectively.
Asunto(s)
Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/química , Caspasa 8/química , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/química , Proteína de Dominio de Muerte Asociada a Fas/química , Proteínas Virales/química , Secuencia de Aminoácidos , Apoptosis/efectos de los fármacos , Sitios de Unión , Proteínas Adaptadoras de Señalización CARD , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Caspasa 8/genética , Caspasa 8/metabolismo , Microscopía por Crioelectrón , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/genética , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Dominio Efector de Muerte , Proteína de Dominio de Muerte Asociada a Fas/genética , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Expresión Génica , Humanos , Células Jurkat , Plásmidos/química , Plásmidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transfección , Proteínas Virales/genética , Proteínas Virales/metabolismo , Receptor fas/farmacologíaRESUMEN
OBJECTIVE: This study aimed to clarify the relationship between FADD amplification and overexpression and the tumor immune microenvironment. METHODS: Immunohistochemical staining and bioanalysis were used to analyze the association between FADD expression in tumor cells and cells in tumor microenvironment. RNA-seq analysis was used to detect the differences in gene expression upon FADD overexpression. Flow cytometry and multicolor immunofluorescence staining (mIHC) were used to detect the differences in CD8+ T-cell infiltration in FADD-overexpressed cells or tumor tissues. RESULTS: Overexpression of FADD significantly promoted tumor growth. Cells with high FADD expression presented high expression of CD276 and FGFBP1 and low expression of proinflammatory factors (such as IFIT1-3 and CXCL8), which reduced the percentage of CD8+ T cells and created a "cold tumor" immune microenvironment, thus promoting tumor progression. In vivo and in vitro experiment confirmed that tumor tissues with excessive FADD expression exhibited CD8+ T-cell exclusion in the microenvironment. CONCLUSION: Our preliminary investigation has discovered the association between FADD expression and the immunosuppressive microenvironment in HNSCC. Due to the high frequent amplification of the chromosomal region 11q13.3, where FADD is located, targeting FADD holds promise for improving the immune-inactive state of tumors, subsequently inhibiting HNSCC tumor progression.
RESUMEN
Fas-associated death domain (FADD) is an adaptor protein that predominantly transduces the apoptosis signal from the death receptor (DR) to activate caspases, leading to the initiation of apoptotic signaling and the coordinated removal of damaged, infected, or unwanted cells. In addition to its apoptotic functions, FADD is involved in signaling pathways related to autophagy, cell proliferation, necroptosis, and cellular senescence, indicating its versatile role in cell survival and proliferation. The subcellular localization and intracellular expression of FADD play a crucial role in determining its functional outcomes, thereby highlighting the importance of spatiotemporal mechanisms and regulation. Furthermore, FADD has emerged as a key regulator of inflammatory signaling, contributing to immune responses and cellular homeostasis. This review provides a comprehensive summary and analysis of the cellular dynamics of FADD in regulating programmed cell death and inflammation through distinct molecular mechanisms associated with various signaling pathways.
Asunto(s)
Apoptosis , Neoplasias , Humanos , Dominio de Muerte , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Apoptosis/fisiología , Receptor fas/metabolismo , Inflamación , Caspasa 8/metabolismoRESUMEN
Tumour necrosis factor receptors (TNF-Rs) and their ligands, tumour necrosis factors, are highly conserved proteins described in all metazoan phyla. They function as inducers of extrinsic apoptotic signalling and facilitate inflammation, differentiation and cell survival. TNF-Rs use distinct adaptor molecules to activate signalling cascades. Fas-associated protein with death domain (FADD) family adaptors often mediate apoptosis, and TNF-R-associated factor (TRAF) family adaptors mediate cell differentiation and inflammation. Most of these pathway components are conserved in cnidarians, and, here, we investigated the Hydra TNF-R. We report that it is related to the ectodysplasin receptor, which is involved in epithelial cell differentiation in mammals. In Hydra, it is localised in epithelial cells with incorporated nematocytes in tentacles and body column, indicating a similar function. Further experiments suggest that it interacts with the Hydra homologue of a TRAF adaptor, but not with FADD proteins. Hydra FADD proteins colocalised with Hydra caspases in death effector filaments and recruited caspases, suggesting that they are part of an apoptotic signalling pathway. Regulating epithelial cell differentiation via TRAF adaptors therefore seems to be an ancient function of TNF-Rs, whereas FADD-caspase interactions may be part of a separate apoptotic pathway.
Asunto(s)
Hydra , Animales , Apoptosis , Caspasa 8 , Caspasas/metabolismo , Diferenciación Celular , Proteína de Dominio de Muerte Asociada a Fas/genética , Hydra/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
In mycobacteria, lipids are important components of the cell wall and play a critical role for pathogenic activities. Lipids need to be activated before participating in many biological pathways. FadD proteins are members of the adenylate-forming superfamily, catalyzing activation of fatty acids. FadD8 is one of the 34 Mycobacterium tuberculosis FadD proteins, which was reported to be a putative medium-long chain fatty acyl-CoA ligase. Previous studies showed FadD8 from Mycobacterium smegmatis exhibited higher activity with oxidized cholesterol than fatty acids. However, the catalytic mechanism of the FadD8 is still exclusive. Here, we reported the crystal structure of FadD8 from Mycobacterium tuberculosis, which forms homodimer. Structural analysis revealed presence of a relatively narrow pocket compared to other FadD proteins and a novel alternative pocket, implying distinct substrate binding preference. We propose that FadD8 plays a vital role in cholesterol utilization and metabolism by catalyzing activation of cholesterol. Collectively, our findings provide novel information for the further studies of the inhibitor and drug development.
Asunto(s)
Mycobacterium tuberculosis , Mycobacterium tuberculosis/metabolismo , Ligasas/metabolismo , Acilcoenzima A/metabolismo , Mycobacterium smegmatis/metabolismo , Ácidos Grasos/metabolismoRESUMEN
Human breast milk (HBM) effectively prevents and cures neonatal bronchopulmonary dysplasia (BPD). Exosomes are abundant in breast milk, but the function of HBM-derived exosomes (HBM-Exo) in BPD is still unclear. This study was to investigate the role and mechanism of HBM-Exo in BPD. Overall lung tissue photography and H&E staining showed that HBM-Exo improved the lung tissue structure collapse, alveolar structure disorder, alveolar septum width, alveolar number reduction and other injuries caused by high oxygen exposure. Immunohistochemical results showed that HBM-Exo improved the inhibition of cell proliferation and increased apoptosis caused by hyperoxia. qPCR and Western blot results also showed that HBM-Exo improved the expression of Type II alveolar epithelium (AT II) surface marker SPC. In vivo study, CCK8 and flow cytometry showed that HBM-Exo improved the proliferation inhibition and apoptosis of AT II cells induced by hyperoxia, qPCR and immunofluorescence also showed that HBM-Exo improved the down-regulation of SPC. Further RNA-Seq results in AT II cells showed that a total of 88 genes were significantly different between the hyperoxia and HBM-Exo with hyperoxia groups, including 24 up-regulated genes and 64 down-regulated genes. KEGG pathway analysis showed the enrichment of IL-17 signalling pathway was the most significant. Further rescue experiments showed that HBM-Exo improved AT II cell damage induced by hyperoxia through inhibiting downstream of IL-17 signalling pathway (FADD), which may be an important mechanism of HBM-Exo in the prevention and treatment of BPD. This study may provide new approach in the treatment of BPD.
Asunto(s)
Displasia Broncopulmonar , Exosomas , Hiperoxia , Animales , Animales Recién Nacidos , Apoptosis , Displasia Broncopulmonar/etiología , Modelos Animales de Enfermedad , Exosomas/metabolismo , Femenino , Humanos , Hiperoxia/genética , Recién Nacido , Interleucina-17/metabolismo , Pulmón/metabolismo , Leche Humana/metabolismo , RatasRESUMEN
Intestinal intraepithelial lymphocytes (IELs) play a crucial role in host defence against pathogens in the intestinal mucosa. The development of intestinal IELs is distinct from peripheral T lymphocytes and remains elusive. Fas-associated protein with death domain (FADD) is important for T cell development in the thymus. Here we describe a novel function of FADD in the IEL development. FADD (S191A), a mouse FADD mutant at Ser191 to Ala mimicking constitutively unphosphorylated FADD, promoted a rapid expansion of TCRαß+ IELs, not TCRγδ+ IELs. Mechanism investigation indicated that the dephosphorylation of FADD was required for cell activation mainly in TCRαß+ CD8+ T cells. Consistently, FADD (S191A) as dephosphorylated FADD led to a high NF-κB activation in the TCR-dependent cell expansion. In addition, The FADD (S191A)-induced abnormal IEL populations resulted in the increased incidence and severity of colitis in mice. In summary, FADD signalling is involved in the intestinal IEL development and might be a regulator for intestinal mucosal homeostasis.
Asunto(s)
Linfocitos Intraepiteliales , Receptores de Antígenos de Linfocitos T alfa-beta , Animales , Linfocitos T CD8-positivos/metabolismo , Proteína de Dominio de Muerte Asociada a Fas/genética , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Mucosa Intestinal/metabolismo , Linfocitos Intraepiteliales/metabolismo , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismoRESUMEN
Cancer is a leading disease-related cause of death worldwide. Despite advances in therapeutic interventions, cancer remains a major global public health problem. Cancer pathogenesis is extremely intricate and largely unknown. Fas-associated protein with death domain (FADD) was initially identified as an adaptor protein for death receptor-mediated extrinsic apoptosis. Recent evidence suggests that FADD plays a vital role in non-apoptotic cellular processes, such as proliferation, autophagy, and necroptosis. FADD expression and activity of are modulated by a complicated network of processes, such as DNA methylation, non-coding RNA, and post-translational modification. FADD dysregulation has been shown to be closely associated with the pathogenesis of numerous types of cancer. However, the detailed mechanisms of FADD dysregulation involved in cancer progression are still not fully understood. This review mainly summarizes recent findings on the structure, functions, and regulatory mechanisms of FADD and focuses on its role in cancer progression. The clinical implications of FADD as a biomarker and therapeutic target for cancer patients are also discussed. The information reviewed herein may expand researchers' understanding of FADD and contribute to the development of FADD-based therapeutic strategies for cancer patients.
Asunto(s)
Apoptosis , Neoplasias , Humanos , Proteína de Dominio de Muerte Asociada a Fas/genética , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Apoptosis/genética , Procesos Neoplásicos , Neoplasias/genética , Transducción de SeñalRESUMEN
Mycolic acids (MAs) are unique components of cell envelope of Mycobacterium or Corynebacterium and are key factors of their virulence to human. In order to develop new anti-Tuberculosis (TB) drugs, many efforts have paid on investigation of structures and functions of proteins involved in the biosynthesis pathway of MAs. FadD32 and polyketide synthase 13 (pks13) catalyze the last step of MAs synthesis. Here we present the crystal structures of FadD32 with substrates and holo-form of ACP-domain from Corynebacterium diphtheriae. The crystal structures and in vitro biochemical assays provide new insights into the assembly of FadD32 and pks13.
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Proteínas Bacterianas/química , Corynebacterium diphtheriae/metabolismo , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Ligandos , Modelos Moleculares , Unión Proteica , Dominios ProteicosRESUMEN
Fas-associated protein with death domain (FADD) is a pivotal adaptor protein that functions in mediating cell death, cell cycle regulation, and particular in innate immunity by the main death receptors. In this study, a second FADD gene in sea cucumber Apostichopus japonicus (termed AjFADD-2) was cloned and its potential function in the innate responses was analyzed. The full-length cDNA of AjFADD-2 consists of 2405 bp and contains a 47 bp 5'-untranslated region (UTR), a 1629 bp 3'-UTR, and a 729 bp ORF encoding 242 amino acids. AjFADD-2 possesses two conserved domains of intracellular N-terminal death effector domain and an extracellular C-terminal death domain, which is different from the first cloned FADD gene in A. japonicus that only possesses the death domain. AjFADD-2 was examined in all sampled six tissues and was significantly induced in V. splendidus-challenged sea cucumbers and LPS-exposed coelomocytes. Subcellular localization detection showed that AjFADD-2 was primarily observed in the coelomocyte cytoplasm, and transferred to the nucleus post V. splendidus challenge. Consistently, AjFADD-2 knockdown significantly inhibited apoptosis in V. splendidus-challenged sea cucumbers and LPS-exposed coelomocytes. Taken together, our results provided evidence that AjFADD functioned as a positive regulator of coelomocytes apoptosis in response to pathogen V. splendidus challenge.