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 β 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.

BACKGROUND: Peripheral artery disease (PAD) is an ischemic disease with a rising incidence worldwide. The lncRNA H19 (H19) is enriched in endothelial progenitor cells (EPCs), and transplantation of pyroptosis-resistant H19-overexpressed EPCs (oe-H19-EPCs) may promote vasculogenesis and blood flow recovery in PAD, especially with critical limb ischemia (CLI).
METHODS: EPCs isolated from human peripheral blood was characterized using immunofluorescence and flow cytometry. Cell proliferation was determined with CCK8 and EdU assays. Cell migration was assessed by Transwell and wound healing assays. The angiogenic potential was evaluated using tube formation assay. The pyroptosis pathway-related protein in EPCs was detected by western blot. The binding sites of H19 and FADD on miR-107 were analyzed using Luciferase assays. In vivo, oe-H19-EPCs were transplanted into a mouse ischemic limb model, and blood flow was detected by laser Doppler imaging. The transcriptional landscape behind the therapeutic effects of oe-H19-EPCs on ischemic limbs were examined with whole transcriptome sequencing.
RESULTS: Overexpression of H19 in EPCs led to an increase in proliferation, migration, and tube formation abilities. These effects were mediated through pyroptosis pathway, which is regulated by the H19/miR-107/FADD axis. Transplantation of oe-H19-EPCs in a mouse ischemic limb model promoted vasculogenesis and blood flow recovery. Whole transcriptome sequencing indicated significant activation of vasculogenesis pathway in the ischemic limbs following treatment with oe-H19-EPCs.
CONCLUSIONS: Overexpression of H19 increases FADD level by competitively binding to miR-107, leading to enhanced proliferation, migration, vasculogenesis, and inhibition of pyroptosis in EPCs. These effects ultimately promote the recovery of blood flow in CLI.

Perturbation of the apoptosis and necroptosis pathways critically influences embryogenesis. Receptor-associated protein kinase-1 (RIPK1) interacts with Fas-associated via death domain (FADD)-caspase-8-cellular Flice-like inhibitory protein long (cFLIPL) to regulate both extrinsic apoptosis and necroptosis. Here, we describe Ripk1-mutant animals (Ripk1R588E [RE]) in which the interaction between FADD and RIPK1 is disrupted, leading to embryonic lethality. This lethality is not prevented by further removal of the kinase activity of Ripk1 (Ripk1R588E K45A [REKA]). Both Ripk1RE and Ripk1REKA animals survive to adulthood upon ablation of Ripk3. While embryonic lethality of Ripk1RE mice is prevented by ablation of the necroptosis effector mixed lineage kinase-like (MLKL), animals succumb to inflammation after birth. In contrast, Mlkl ablation does not prevent the death of Ripk1REKA embryos, but animals reach adulthood when both MLKL and caspase-8 are removed. Ablation of the nucleic acid sensor Zbp1 largely prevents lethality in both Ripk1RE and Ripk1REKA embryos. Thus, the RIPK1-FADD interaction prevents Z-DNA binding protein-1 (ZBP1)-induced, RIPK3-caspase-8-mediated embryonic lethality, affected by the kinase activity of RIPK1.

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.

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.

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.

BACKGROUND: Cardiomyocyte apoptosis plays a vital role in myocardial ischemia-reperfusion (MI/R) injury; however, the role of beclin1 (BECN1) remains unclear. This study aimed at revealing the function of BECN1 during cardiomyocyte apoptosis after MI/R injury.
METHODS: In vivo, TTC and Evan\'s blue double staining was applied to verify the gross morphological alteration in both wild type (WT) mice and BECN1 transgene mice (BECN1-TG), and TUNEL staining and western blot were adopted to evaluate the cardiomyocyte apoptosis. In vitro, a hypoxia/reoxygenation (H/R) model was established in H9c2 cells to simulate MI/R injury. Proteomics analysis was preformed to verify if apoptosis occurs in the H/R cellular model. And apoptosis factors, RIPK1, Caspase-1, Caspase-3, and cleaved Caspase-3, were investigated using western bolting. In addition, the mRNA level were verified using RT-PCR. To further investigate the protein interactions small interfering RNA and lentiviral transfection were used. To continue investigate the protein interactions, immunofluorescence and coimmunoprecipitation were applied.
RESULTS: Morphologically, BECN1 significantly attenuated the apoptosis from TTC-Evan\'s staining, TUNEL, and cardiac tissue western blot. After H/R, a RIPK1-induced complex (complex II) containing RIPK1, Caspase-8, and FADD was formed. Thereafter, cleaved Caspase-3 was activated, and myocyte apoptosis occurred. However, BECN1 decreased the expression of RIPK1, Caspase-8, and FADD. Nevertheless, BECN1 overexpression increased RIPK1 ubiquitination before apoptosis by inhibiting OTUD1.
CONCLUSIONS: BECN1 regulates FADD/RIPK1/Caspase-8 complex formation via RIPK1 ubiquitination by downregulating OTUD1 in C-Caspase-3-induced myocyte apoptosis after MI/R injury. Therefore, BECN1 can function as a cardioprotective candidate.

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.

Mucosal epithelial death is an essential pathological characteristic of portal hypertensive gastropathy (PHG). FADDosome can regulate mucosal homeostasis by controlling mitochondrial status and cell death. However, it remains ill-defined whether and how the FADDosome is involved in the epithelial death of PHG. The FADDosome formation, mitochondrial dysfunction, glycolysis process and NLRP3 inflammasome activation in PHG from both human sections and mouse models were investigated. NLRP3 wild-type (NLRP3-WT) and NLRP3 knockout (NLRP3-KO) littermate models, critical element inhibitors and cell experiments were utilized. The mechanism underlying FADDosome-regulated mitochondrial dysfunction and epithelial death in PHG was explored. Here, we found that FADD recruited caspase-8 and receptor-interacting serine/threonine-protein kinase 1 (RIPK1) to form the FADDosome to promote Drp1-dependent mitochondrial fission and dysfunction in PHG. Also, FADDosome modulated NOX2 signaling to strengthen Drp1-dependent mitochondrial fission and alter glycolysis as well as enhance mitochondrial reactive oxygen species (mtROS) production. Moreover, due to the dysfunction of electron transport chain (ETC) and alteration of antioxidant enzymes activity, this altered glycolysis also contributed to mtROS production. Subsequently, the enhanced mtROS production induced NLRP3 inflammasome activation to result in the epithelial pyroptosis and mucosal injury in PHG. Thus, the FADDosome-regulated pathways may provide a potential therapeutic target for PHG.

Fas-associated protein with death domain (FADD), procaspase-8, and cellular FLICE-inhibitory proteins (cFLIP) assemble through death-effector domains (DEDs), directing death receptor signaling towards cell survival or apoptosis. Understanding their three-dimensional regulatory mechanism has been limited by the absence of atomic coordinates for their ternary DED complex. By employing X-ray crystallography and cryogenic electron microscopy (cryo-EM), we present the atomic coordinates of human FADD-procaspase-8-cFLIP complexes, revealing structural insights into these critical interactions. These structures illustrate how FADD and cFLIP orchestrate the assembly of caspase-8-containing complexes and offer mechanistic explanations for their role in promoting or inhibiting apoptotic and necroptotic signaling. A helical procaspase-8-cFLIP hetero-double layer in the complex appears to promote limited caspase-8 activation for cell survival. Our structure-guided mutagenesis supports the role of the triple-FADD complex in caspase-8 activation and in regulating receptor-interacting protein kinase 1 (RIPK1). These results propose a unified mechanism for DED assembly and procaspase-8 activation in the regulation of apoptotic and necroptotic signaling across various cellular pathways involved in development, innate immunity, and disease.