Protocol for analyzing TRAIL- and Fas-induced signaling complexes by immunoprecipitation from human cells.

Engagement of TRAIL or Fas death receptors can trigger the assembly of cytoplasmic caspase-8/FADD/RIPK1 (FADDosome) signaling complexes that promote nuclear factor κB (NF-κB) activation. Here, we present a protocol for immunoprecipitation of TRAIL- or Fas-induced FADDosomes from human cell lines. We describe steps for stimulating human cells with TRAIL or Fas ligand, followed by preparation of membrane death receptor-associated, as well as cytoplasmic FADDosome, signaling complexes. This protocol has application in the analysis of death receptor-induced signaling complex formation. For complete details on the use and execution of this protocol, please refer to Davidovich et al.1.

Molluscan pleiotropic FADD involved in innate immune signaling and induces apoptosis.

Fas-associated protein with death domain (FADD) was initially identified as a crucial adaptor protein in the apoptotic pathway mediated by death receptor (DR). Subsequently, many studies have confirmed that FADD plays a vital role in innate immunity and inflammatory responses in animals. However, the function of this pleiotropic molecule in mollusk species has not been well explored. In this study, we successfully verified the gene sequence of FADD in the Zhikong scallop (Chlamys farreri) and designated it as CfFADD. The CfFADD protein contains a conserved death effector and death domains. Phylogenetic analysis showed that CfFADD is a novel addition to the molluscan FADD family with a close evolutionary relationship with molluscan FADD subfamily proteins. CfFADD mRNA expression in various scallop tissues was significantly induced by challenge with pathogen-associated molecular patterns (lipopolysaccharide, peptidoglycan, and poly(I:C)), suggesting its role in innate immunity in scallops. Co-immunoprecipitation showed that CfFADD interacted with the scallop DR (tumor necrosis factor receptor) and a signaling molecule involved in the Toll-like receptor pathway (interleukin-1 receptor-associated kinase), confirming that CfFADD may be involved in DR-mediated apoptosis and innate immune signaling pathways. Further studies showed that CfFADD interacted with CfCaspase-8 and activated caspase-3. HEK293T cells exhibited distinct apoptotic features after transfection with a CfFADD-expression plasmid, suggesting a functional DR-FADD-caspase apoptotic pathway in scallops. Overexpression of CfFADD led to a significant dose-dependent activation of interferon ß and nuclear factor-κB reporter genes, demonstrating the key role of CfFADD in innate immunity. In summary, our research has confirmed the critical roles of CfFADD in innate immunity and apoptosis and provides valuable information for developing comparative immunology theories.

ZBP1 causes inflammation by inducing RIPK3-mediated necroptosis and RIPK1 kinase activity-independent apoptosis.

Z-DNA binding protein 1 (ZBP1) has important functions in anti-viral immunity and in the regulation of inflammatory responses. ZBP1 induces necroptosis by directly engaging and activating RIPK3, however, the mechanisms by which ZBP1 induces inflammation and in particular the role of RIPK1 and the contribution of cell death-independent signaling remain elusive. Here we show that ZBP1 causes skin inflammation by inducing RIPK3-mediated necroptosis and RIPK1-caspase-8-mediated apoptosis in keratinocytes. ZBP1 induced TNFR1-independent skin inflammation in mice with epidermis-specific ablation of FADD by triggering keratinocyte necroptosis. Moreover, transgenic expression of C-terminally truncated constitutively active ZBP1 (ZBP1ca) in mouse epidermis caused skin inflammation that was only partially inhibited by abrogation of RIPK3-MLKL-dependent necroptosis and fully prevented by combined deficiency in MLKL and caspase-8. Importantly, ZBP1ca induced caspase-8-mediated skin inflammation by RHIM-dependent but kinase activity-independent RIPK1 signaling. Furthermore, ZBP1ca-induced inflammatory cytokine production in the skin was completely prevented by combined inhibition of apoptosis and necroptosis arguing against a cell death-independent pro-inflammatory function of ZBP1. Collectively, these results showed that ZBP1 induces inflammation by activating necroptosis and RIPK1 kinase activity-independent apoptosis.

Expression of RIPK1 and FADD are associated with chemosensitivity and survival in head and heck squamous cell carcinoma via tanshinone IIA-mediated modulation of the RIPK1-FADD-Caspase 8 complex.

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.

The RIPK1 death domain restrains ZBP1- and TRIF-mediated cell death and inflammation.

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.

Achillea fragrantissima (Forssk.) Sch.Bip instigates the ROS/FADD/c-PARP expression that triggers apoptosis in breast cancer cell (MCF-7).

Achillea fragrantissima is a shrub plant that belongs to the Asteraceae family in Arabia and Egypt. It is used as folk medicine and is a good source of phenolic acids, flavonoids, and some active compounds. To investigate the anti-cancer effect of A.fragrantissima on breast cancer MCF-7 cells and find the critical mechanism involved in apoptosis. The toxicity and pharmacokinetic studies of ethanolic extract of A.fragrantissima was examined for anti-breast cancer properties. In turn, cytotoxicity and cell viability were achieved by the MTT method. Furthermore, the trypan blue exclusion and microscopy examination proved the presence of apoptotic cells. Again, fluorescent staining such as AO/EtBr, DCFH-DA, Rho-123, and Hoechst-33342 reveals the cellular cytoplasmic disciplines upon A. fragrantissima effect. Moreover, cellular functioning tests like wound healing, colony formation, and Transwell invasion assay were demonstrated. In addition, the qRT-PCR technique authenticates the A. fragrantissima -induced apoptotic network genes (Caspase-3, Caspase-8, Caspase-9, Cytochrome c, BCL-2, BID, BAX, PARP, PTEN, PI3K, and Akt) expression were evaluated. Mainly, the Immunoblot technique proved the expressed level of apoptotic proteins such as cleaved PARP, CYCS, and FADD. This study confirmed that the A. fragrantissima exerts cytotoxicity at 20 µg/mL for 24 hrs in MCF-7 cells. Also, decreases cellular viability, producing apoptotic cells and damaged cellular surfaces with dead matter. Consequently, it creates ROS species accumulation, loss of mitochondrial membrane potential, and fragmentation of DNA in MCF-7 cells. Furthermore, it arrests cell migration, induces colony-forming ability loss, and suppresses cell invasion. In addition, A. fragrantissima significantly upregulates genes such as caspase-3, 9, cytochrome c, BID, BAX, and PTEN while downregulating the Pi3K/ Akt signaling. Nonetheless, A.fragrantissima induced cleaved PARP, CYCS, and FADD proteins in MCF-7 cells to avail apoptosis.

Identification of PANoptosis-related biomarkers and analysis of prognostic values in head and neck squamous cell carcinoma.

PANoptosis plays a crucial role in cancer initiation and progression. However, the roles of PANoptosis-related genes (PARGs) in the prognosis and immune landscape of head and neck squamous cell carcinoma (HNSCC) remain unclear. Integrated bioinformatics analyses based on the data of HNSCC patients in the TCGA database were conducted. We extracted 48 PARGs expression profile and then conducted differentially expressed analysis, following building a Cox model to predict the survival of HNSCC patients. Subsequently, the relationships between the risk score, immune landscape, chemo-, and immune-therapy responses were analyzed, respectively. Moreover, we investigated the prognostic value, and further predicted the pathways influenced by PARGs. Finally, we identified the biological function of crucial PARGs. A total of 18 differentially expressed PARGs were identified in HNSCC, and a Cox model including CASP8, FADD, NLRP1, TNF, and ZBP1 was constructed, which showed that the risk score was associated with the prognosis as well as immune infiltration of HNSCC patients, and the risk score could be regarded as an independent biomarker. Additionally, patients with high-risk score might be an indicator of lymph node metastasis and advanced clinical stage. High-risk scores also contributed to the chemotherapy resistance and immune escape of HNSCC patients. In addition, FADD and ZBP1 played a crucial role in various cancer-related pathways, such as the MAPK, WNT, and MTOR signaling pathways. On the other hand, we suggested that FADD facilitated the progression and 5-fluorouracil (5-FU) resistance of HNSCC cells. A signature based on PANoptosis showed great predictive power for lymph node metastasis and advanced stage, suggesting that the risk score might be an independent prognostic biomarker for HNSCC. Meanwhile, FADD, identified as a prognostic biomarker, may represent an effective therapeutic target for HNSCC.

Cellular Dynamics of Fas-Associated Death Domain in the Regulation of Cancer and Inflammation.

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.

Combined germline and somatic human FADD mutations cause autoimmune lymphoproliferative syndrome.

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.

cFLIPL acts as a suppressor of TRAIL- and Fas-initiated inflammation by inhibiting assembly of caspase-8/FADD/RIPK1 NF-κB-activating complexes.

TRAIL and FasL are potent inducers of apoptosis but can also promote inflammation through assembly of cytoplasmic caspase-8/FADD/RIPK1 (FADDosome) complexes, wherein caspase-8 acts as a scaffold to drive FADD/RIPK1-mediated nuclear factor κB (NF-κB) activation. cFLIP is also recruited to FADDosomes and restricts caspase-8 activity and apoptosis, but whether cFLIP also regulates death receptor-initiated inflammation is unclear. Here, we show that silencing or deletion of cFLIP leads to robustly enhanced Fas-, TRAIL-, or TLR3-induced inflammatory cytokine production, which can be uncoupled from the effects of cFLIP on caspase-8 activation and apoptosis. Mechanistically, cFLIPL suppresses Fas- or TRAIL-initiated NF-κB activation through inhibiting the assembly of caspase-8/FADD/RIPK1 FADDosome complexes, due to the low affinity of cFLIPL for FADD. Consequently, increased cFLIPL occupancy of FADDosomes diminishes recruitment of FADD/RIPK1 to caspase-8, thereby suppressing NF-κB activation and inflammatory cytokine production downstream. Thus, cFLIP acts as a dual suppressor of apoptosis and inflammation via distinct modes of action.

Ginsenoside Rb1 promotes the activation of PPARα pathway via inhibiting FADD to ameliorate heart failure.

PURPOSE: Ginsenoside Rb1 (GRb1), a dammarane-type triterpene saponin compound mainly distributed in ginseng (Panax ginseng), has been demonstrated to ameliorate cardiovascular diseases. However, it remains unclear whether GRb1 alleviates heart failure (HF) by maintaining cardiac energy metabolism balance. Therefore, this work aimed to investigate the cardiac benefits of GRb1 against cardiac energy deficit and explore its mechanism of action. METHODS AND RESULTS: Isoproterenol (ISO) induced HF Sprague-Dawley rats were administrated with GRb1 or fenofibrate for 6 weeks. ISO-induced primary neonatal rat cardiomyocytes (NRCMs) were used as the in vitro model. In vivo, GRb1 significantly improved the structural and metabolic disorder, as demonstrated by the restoration of cardiac function, inhibition of cardiac hypertrophy and fibrosis, and increased adenosine triphosphate (ATP) generation. In vitro, GRb1 effectively protected mitochondrial function and scavenged excessive reactive oxygen species. Moreover, in ISO-induced NRCMs, GRb1 significantly inhibited the abnormal upregulation of Fas-associated death domain (FADD), promoted transcriptional activation of peroxisome proliferator-activated receptor-alpha (PPARα), improved the aberrant expression of cardiac energy metabolism-related enzymes and cardiac fatty acid oxidation, and subsequently increased the synthesis of ATP. Noticeably, GRb1 could inhibit the increased binding between FADD and PPARα, which contributed to the activation of PPARα. Furthermore, GRb1 strengthened the thermal stabilization of FADD and might bind to FADD directly. CONCLUSIONS: Collectively, it's part of the in-depth mechanism of GRb1's cardio-protection that GRb1 could directly bind to FADD and counteract its negative role in the transcription of PPARα thus ameliorating cardiac energy derangement and HF.

PW06 Triggered Fas-FADD to Induce Apoptotic Cell Death In Human Pancreatic Carcinoma MIA PaCa-2 Cells through the Activation of the Caspase-Mediated Pathway.

Pancreatic cancer has higher incidence and mortality rates worldwide. PW06 [(E)-3-(9-ethyl-9H-carbazol-3-yl)-1-(2,5-dimethoxyphenyl) prop-2-en-1-one] is a carbazole derivative containing chalcone moiety which was designed for inhibiting tumorigenesis in human pancreatic cancer. This study is aimed at investigating PW06-induced anticancer effects in human pancreatic cancer MIA PaCa-2 cells in vitro. The results showed PW06 potent antiproliferative/cytotoxic activities and induced cell morphological changes in a human pancreatic cancer cell line (MIA PaCa-2), and these effects are concentration-dependent (IC50 is 0.43 µM). Annexin V and DAPI staining assays indicated that PW06 induced apoptotic cell death and DNA condensation. Western blotting indicated that PW06 increased the proapoptotic proteins such as Bak and Bad but decreased the antiapoptotic protein such as Bcl-2 and Bcl-xL. Moreover, PW06 increased the active form of caspase-8, caspase-9, and caspase-3, PARP, releasing cytochrome c, AIF, and Endo G from mitochondria in MIA PaCa-2 cells. Confocal laser microscopy assay also confirmed that PW06 increased Bak and decreased Bcl-xL. Also, the cells were pretreated with inhibitors of caspase-3, caspase-8, and caspase-9 and then were treated with PW06, resulting in increased viable cell number compared to PW06 treated only. Furthermore, PW06 showed a potent binding ability with hydrophobic interactions in the core site of the Fas-Fas death domains (FADD). In conclusion, PW06 can potent binding ability to the Fas-FADD which led to antiproliferative, cytotoxic activities, and apoptosis induction accompanied by the caspase-dependent and mitochondria-dependent pathways in human pancreatic cancer MIA PaCa-2 cells.

Bioluminescent RIPoptosome Assay for FADD/RIPK1 Interaction Based on Split Luciferase Assay in a Human Neuroblastoma Cell Line SH-SY5Y.

Different programed cell death (PCD) modalities involve protein-protein interactions in large complexes. Tumor necrosis factor α (TNFα) stimulated assembly of receptor-interacting protein kinase 1 (RIPK1)/Fas-associated death domain (FADD) interaction forms Ripoptosome complex that may cause either apoptosis or necroptosis. The present study addresses the interaction of RIPK1 and FADD in TNFα signaling by fusion of C-terminal (CLuc) and N-terminal (NLuc) luciferase fragments to RIPK1-CLuc (R1C) or FADD-NLuc (FN) in a caspase 8 negative neuroblastic SH-SY5Y cell line, respectively. In addition, based on our findings, an RIPK1 mutant (R1C K612R) had less interaction with FN, resulting in increasing cell viability. Moreover, presence of a caspase inhibitor (zVAD.fmk) increases luciferase activity compared to Smac mimetic BV6 (B), TNFα -induced (T) and non-induced cell. Furthermore, etoposide decreased luciferase activity, but dexamethasone was not effective in SH-SY5Y. This reporter assay might be used to evaluate basic aspects of this interaction as well as for screening of necroptosis and apoptosis targeting drugs with potential therapeutic application.

A Dual Role for FADD in Human Precursor T-Cell Neoplasms.

A reduction in FADD levels has been reported in precursor T-cell neoplasms and other tumor types. Such reduction would impact on the ability of tumor cells to undergo apoptosis and has been associated with poor clinical outcomes. However, FADD is also known to participate in non-apoptotic functions, but these mechanisms are not well-understood. Linking FADD expression to the severity of precursor T-cell neoplasms could indicate its use as a prognostic marker and may open new avenues for targeted therapeutic strategies. Using transcriptomic and clinical data from patients with precursor T-cell neoplasms, complemented by in vitro analysis of cellular functions and by high-throughput interactomics, our results allow us to propose a dual role for FADD in precursor T-cell neoplasms, whereby resisting cell death and chemotherapy would be a canonical consequence of FADD deficiency in these tumors, whereas deregulation of the cellular metabolism would be a relevant non-canonical function in patients expressing FADD. These results reveal that evaluation of FADD expression in precursor T-cell neoplasms may aid in the understanding of the biological processes that are affected in the tumor cells. The altered biological processes can be of different natures depending on the availability of FADD influencing its ability to exert its canonical or non-canonical functions. Accordingly, specific therapeutic interventions would be needed in each case.

FADD as a key molecular player in cancer progression.

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.

Super-Resolution Imaging of Fas/CD95 Reorganization Induced by Membrane-Bound Fas Ligand Reveals Nanoscale Clustering Upstream of FADD Recruitment.

Signaling through the TNF-family receptor Fas/CD95 can trigger apoptosis or non-apoptotic cellular responses and is essential for protection from autoimmunity. Receptor clustering has been observed following interaction with Fas ligand (FasL), but the stoichiometry of Fas, particularly when triggered by membrane-bound FasL, the only form of FasL competent at inducing programmed cell death, is not known. Here we used super-resolution microscopy to study the behavior of single molecules of Fas/CD95 on the plasma membrane after interaction of Fas with FasL on planar lipid bilayers. We observed rapid formation of Fas protein superclusters containing more than 20 receptors after interactions with membrane-bound FasL. Fluorescence correlation imaging demonstrated recruitment of FADD dependent on an intact Fas death domain, with lipid raft association playing a secondary role. Flow-cytometric FRET analysis confirmed these results, and also showed that some Fas clustering can occur in the absence of FADD and caspase-8. Point mutations in the Fas death domain associated with autoimmune lymphoproliferative syndrome (ALPS) completely disrupted Fas reorganization and FADD recruitment, confirming structure-based predictions of the critical role that these residues play in Fas-Fas and Fas-FADD interactions. Finally, we showed that induction of apoptosis correlated with the ability to form superclusters and recruit FADD.

The Classical Apoptotic Adaptor FADD Regulates Glycolytic Capacity in Acute Lymphoblastic Leukemia.

The Fas-associated death domain (FADD) has long been regarded as a crucial adaptor protein in the extrinsic apoptotic pathway. Despite the non-apoptotic function of FADD is gradually being discovered and confirmed, its corresponding physiological and pathological significance is still unclear. Based on the database of GWAS catalog and GTEx Portal, 17 SNPs associated with leukemia susceptibility were found to be linked to FADD expression. We then investigated a regulatory role of FADD in T-acute lymphoblastic leukemia (T-ALL) using Jurkat cells as a model. Jurkat cells stably depleted of FADD (FADD-/- Jurkat) expression exhibited dampened proliferation, hypersensitivity to Etoposide-induced intrinsic apoptosis whereas near total resistance to TRAIL-induced extrinsic apoptosis. Comparison between wild type and FADD-/- Jurkat cells using iTRAQ-based proteomics revealed considerably altered expression spectrum of genes, and led us to focus on metabolic pathways. Investigation of glycolytic and mitochondrial pathways and relevant enzymes revealed that FADD knockout triggered a metabolic shift from glycolysis to mitochondrial respiration in Jurkat cells. Re-expression of FADD in FADD-/- Jurkat cells partially rescued glycolytic capacity. FADD loss triggers global metabolic reprogramming in Jurkat cells and therefore remains as a potential druggable target for ALL treatment.

The role of Fas-FasL-FADD signaling pathway in arsenic-mediated neuronal apoptosis in vivo and in vitro.

The molecular mechanisms underlying arsenic-induced neurotoxicity have not been completely elucidated. Our study aimed to determine the role of the Fas-FasL-FADD signaling pathway in arsenic-mediated neuronal apoptosis. Pathological and molecular biological tests were performed on the cerebral cortex of arsenic-exposed rats and SH-SY5Y neuroblastoma cells. Arsenic induced apoptosis in the cortical neurons, which corresponded to abnormal ultrastructural changes. Mechanistically, arsenic activated the Fas-FasL-FADD signaling pathway and the downstream caspases both in vivo and in vitro. ZB4 treatment reversed the apoptotic effects of arsenic on the SHSY5Y cells. Taken together, arsenic induces neurotoxicity by activating the Fas-FasL-FADD signaling pathway.

ANKRD13a controls early cell-death checkpoint by interacting with RIP1 independent of NF-κB.

In TNF signaling, ubiquitination of RIP1 functions as an early cell-death checkpoint, which prevents the spatial transition of the signaling complex from complex-I to death-inducing complex-II. Here, we report that ankyrin repeat domain 13a (ANKRD13a) acts as a novel component of complex-II to set a higher signal threshold for the cytotoxic potential of TNF. ANKRD13a deficiency is sufficient to turn the response to TNF from survival to death by promoting the formation of complex-II without affecting NF-κB activation. ANKRD13a binds to ubiquitinated-RIP1 via its UIM, and subsequently limits the association of FADD and caspase-8 with RIP1. Moreover, high ANKRD13a expression is inversely correlated with apoptotic phenotypes in ovarian cancer tissues and is associated with poor prognosis. Our work identifies ANKRD13a as a novel gatekeeper of the early cell-death checkpoint, which may function as part of an escape mechanism from cell death in some cancers.