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The discovery of caspase homologs in bacteria highlighted the relationship between programmed cell death (PCD) evolution and eukaryogenesis. However, the origin of PCD genes in prokaryotes themselves (bacteria and archaea) is poorly understood and a source of controversy. Whether archaea also contain C14 peptidase enzymes and other death domains is largely unknown because of a historical dearth of genomic data. Archaeal genomic databases have grown significantly in the last decade, which allowed us to perform a detailed comparative study of the evolutionary histories of PCD-related death domains in major archaeal phyla, including the deepest branching phyla of Candidatus Aenigmarchaeota, Candidatus Woesearchaeota, and Euryarchaeota. We identified death domains associated with executioners of PCD, like the caspase homologs of the C14 peptidase family, in 321 archaea sequences. Of these, 15.58% were metacaspase type I orthologues and 84.42% were orthocaspases. Maximum likelihood phylogenetic analyses revealed a scattered distribution of orthocaspases and metacaspases in deep-branching bacteria and archaea. The tree topology was incongruent with the prokaryote 16S phylogeny suggesting a common ancestry of PCD genes in prokaryotes and subsequent massive horizontal gene transfer coinciding with the divergence of archaea and bacteria. Previous arguments for the origin of PCD were philosophical in nature with two popular propositions being the "addiction" and 'original sin' hypotheses. Our data support the 'original sin' hypothesis, which argues for a pleiotropic origin of the PCD toolkit with pro-life and pro-death functions tracing back to the emergence of cellular life-the Last Universal Common Ancestor State.

Background Neuroinflammation is thought to be a cause of Alzheimer’s disease (AD), which is partly caused by inadequate mitophagy. As a receptor of mitophagy, we aimed to reveal the regulatory roles of optineurin (OPTN) on neuroinflammation in the pathogenesis of AD. Methods BV2 cells and APP/PS1 transgenic (Tg) mice were used as in vitro and in vivo experimental models to determine the regulatory roles of OPTN in neuroinflammation of AD. Sophisticated molecular technologies including quantitative (q) RT-PCR, western blot, enzyme linked immunosorbent assay (ELISA), co-immunoprecipitation (Co-IP) and immunofluorescence (IF) were employed to reveal the inherent mechanisms. Results As a consequence, key roles of OPTN in regulating neuroinflammation were identified by depressing the activity of absent in melanoma 2 (AIM2) inflammasomes and receptor interacting serine/threonine kinase 1 (RIPK1)-mediated NF-κB inflammatory mechanisms. In detail, we found that expression of OPTN was downregulated, which resulted in activation of AIM2 inflammasomes due to a deficiency in mitophagy in APP/PS1 Tg mice. By ectopic expression, OPTN blocks the effects of Aβ oligomer (Aβo) on activating AIM2 inflammasomes by inhibiting mRNA expression of AIM2 and apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), leading to a reduction in the active form of caspase-1 and interleukin (IL)-1β in microglial cells. Moreover, RIPK1 was also found to be negatively regulated by OPTN via ubiquitin protease hydrolysis, resulting in the synthesis of IL-1β by activating the transcriptional activity of NF-κB in BV2 cells. As an E3 ligase, the UBAN domain of OPTN binds to the death domain (DD) of RIPK1 to facilitate its ubiquitination. Based on these observations, ectopically expressed OPTN in APP/PS1 Tg mice deactivated microglial cells and astrocytes via the AIM2 inflammasome and RIPK-dependent NF-κB pathways, leading to reduce neuroinflammation. Conclusions These results suggest that OPTN can alleviate neuroinflammation through AIM2 and RIPK1 pathways, suggesting that OPTN deficiency may be a potential factor leading to the occurrence of AD.

Xiang He,1,2,* Lei Zhang,1,2,* Lingjuan Hu,1,3,* Shengbin Liu,1,2 Anying Xiong,1,2 Junyi Wang,1,2 Ying Xiong,4 Guoping Li1,2 1Laboratory of Allergy and Precision Medicine, Chengdu Institute of Respiratory Health, The Third People’s Hospital of Chengdu, Affiliated Hospital of Southwest Jiaotong University, Chengdu, 610031, People’s Republic of China; 2Department of Pulmonary and Critical Care Medicine, Chengdu Third People’s Hospital Branch of National Clinical Research Center for Respiratory Disease, Affiliated Hospital of ChongQing Medical University, Chengdu, 610031, People’s Republic of China; 3Department of Respiratory Disease, Renshou County People’s Hospital, Renshou, 620550, People’s Republic of China; 4Department of Pulmonary and Critical Care Medicine, Sichuan Friendship Hospital, Chengdu, 610000, People’s Republic of China*These authors contributed equally to this workCorrespondence: Guoping Li; Ying Xiong Email lzlgp@163.com; ly65110@126.comBackground: Exposure to air pollutants cause exacerbation of asthma, but the experimental evidence and the mechanisms still need to be collected and addressed.Methods: Asthma model was constructed by ovalbumin (OVA) combined with or without airborne fine particulate matter 2.5 (PM2.5) exposure. Lung sections were stained by hematoxylin-eosin staining (H&E) and Masson’s trichrome. RNA-seq and gene set enrichment analysis (GSEA) was performed to identify the key pathway. TdT mediated dUTP Nick End Labeling (TUNEL) assay, real-time qPCR, Western blot, immunofluorescence and lentivirus transfection were applied for mechanism discovery.Results: In this study, we found PM2.5 aggravated airway inflammation in OVA-induced asthmatic mice. RNA-seq analysis also showed that epithelial mesenchymal transition (EMT) was enhanced in OVA-induced mice exposed to PM2.5 compared with that in OVA-induced mice. In the meantime, we observed that apoptosis was significantly increased in asthmatic mice exposed to PM2.5 by using GSEA analysis, which was validated by TUNEL assay. By using bioinformatic analysis, Fas associated via death domain (FADD), a new actor in innate immunity and inflammation, was identified to be related to apoptosis, EMT and tight junction. Furthermore, we found that the transcript and protein levels of tight junction markers, E-cadherin, zonula occludens (ZO)-1 and Occludin, were decreased after PM2.5 exposure in vivo and in vitro by using RT-qPCR and immunofluorescence, with the increased expression of FADD. Moreover, down-regulation of FADD attenuated PM2.5-induced apoptosis and tight junction disruption in human airway epithelial cells.Conclusion: Taken together, we demonstrated that PM2.5 aggravated epithelial tight junction disruption through apoptosis mediated by up-regulation of FADD in OVA-induced model.Keywords: asthma, epithelial tight junction disruption, PM2.5, FADD, apoptosis

Regulation of epithelial cell death has emerged as a key mechanism controlling immune homeostasis in barrier surfaces. Necroptosis is a type of regulated necrotic cell death induced by receptor interacting protein kinase 3 (RIPK3) that has been shown to cause inflammatory pathologies in different tissues. The role of regulated cell death and particularly necroptosis in lung homeostasis and disease remains poorly understood. Here we show that mice with Airway Epithelial Cell (AEC)-specific deficiency of Fas-associated with death domain (FADD), an adapter essential for caspase-8 activation, developed exacerbated allergic airway inflammation in a mouse model of asthma induced by sensitization and challenge with house dust mite (HDM) extracts. Genetic inhibition of RIPK1 kinase activity by crossing to mice expressing kinase inactive RIPK1 as well as RIPK3 or MLKL deficiency prevented the development of exaggerated HDM-induced asthma pathology in FADDAEC-KO mice, suggesting that necroptosis of FADD-deficient AECs augmented the allergic immune response. These results reveal a role of AEC necroptosis in amplifying airway allergic inflammation and suggest that necroptosis could contribute to asthma exacerbations caused by respiratory virus infections inducing AEC death.

Stress‐induced apoptosis is mediated primarily through the intrinsic pathway that involves caspase‐9. We previously reported that in caspase‐9‐deficient cells, a protein complex containing ATG5 and Fas‐associated death domain (FADD) facilitated caspase‐8 activation and cell death in response to endoplasmic reticulum (ER) stress. Here, we investigated whether this complex could be activated by other forms of cell stress. We show that diverse stress stimuli, including etoposide, brefeldin A and paclitaxel, as well as heat stress and gamma‐irradiation, caused formation of a complex containing ATG5‐ATG12, FADD and caspase‐8 leading to activation of downstream caspases in caspase‐9‐deficient cells. We termed this complex the ‘stressosome’. However, in these cells, only ER stress and heat shock led to stressosome‐dependent cell death. Using in silico molecular modelling, we propose the structure of the stressosome complex, with FADD acting as an adaptor protein, interacting with pro‐caspase‐8 through their respective death effector domains (DEDs) and interacting with ATG5‐ATG12 through its death domain (DD). This suggests that the complex could be regulated by cellular FADD‐like interleukin‐1β‐converting enzyme–inhibitory protein (cFLIPL), which was confirmed experimentally. This study provides strong evidence for an alternative mechanism of caspase‐8 activation involving the stressosome complex.

Cancer cells that are resistant to Bax/Bak-dependent intrinsic apoptosis can be eliminated by proteasome inhibition. Here, we show that proteasome inhibition induces the formation of high molecular weight platforms in the cytosol that serve to activate caspase-8. The activation complexes contain Fas-associated death domain (FADD) and receptor-interacting serine/threonine-protein kinase 1 (RIPK1). Furthermore, the complexes contain TRAIL-receptor 2 (TRAIL-R2) but not TRAIL-receptor 1 (TRAIL-R1). While RIPK1 inhibition or depletion did not affect proteasome inhibitor-induced cell death, TRAIL-R2 was found essential for efficient caspase-8 activation, since the loss of TRAIL-R2 expression abrogated caspase processing, significantly reduced cell death, and promoted cell re-growth after drug washout. Overall, our study provides novel insight into the mechanisms by which proteasome inhibition eliminates otherwise apoptosis-resistant cells, and highlights the crucial role of a ligand-independent but TRAIL-R2-dependent activation mechanism for caspase-8 in this scenario., Irish Research Council, European Molecular Biology Organization (EMBO), Deutsche Forschungsgemeinschaft, Projekt DEAL

Rationale: Bak is a major proapoptotic Bcl2 family member and a required molecule for apoptotic cell death. High levels of endogenous Bak were observed in both small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cell lines. Increased Bak expression was correlated with poor prognosis of NSCLC patients, suggesting that Bak protein is an attractive target for lung cancer therapy. The BH3 domain functions as death domain and is required for Bak to initiate apoptotic cell death. Thus, the BH3 domain is attractive target for discovery of Bak agonist. Methods: The BH3 death domain binding pocket (aa75-88) of Bak was chosen as a docking site for screening of small molecule Bak activators using the UCSF DOCK 6.1 program suite and the NCI chemical library (300,000 small molecules) database. The top 500 compounds determined to have the highest affinity for the BH3 domain were obtained from the NCI and tested for cytotoxicity for further screening. We identified a small molecule Bak activator BKA-073 as the lead compound. The binding affinity of BKA-073 with Bak protein was analyzed by isothermal titration calorimetry (ITC) assay. BKA-073-mediated Bak activation via oligomerization was analyzed by a cross-linking with Bis (maleimido) hexane (BMH). Sensitivity of BKA-073 to lung cancer cells in vitro was evaluated by dynamic BH3 profiling (DBP) and apoptotic cell death assay. The potency of BKA-073 alone or in combination with radiotherapy or Bcl2 inhibitor was evaluated in animal models. Results: We found that BKA-073 binds Bak at BH3 domain with high affinity and selectivity. BKA-073/Bak binding promotes Bak oligomerization and mitochondrial priming that activates its proapoptotic function. BKA-073 potently suppresses tumor growth without significant normal tissue toxicity in small cell lung cancer (SCLC) and NSCLC xenografts, patient-derived xenografts, and genetically engineered mouse models of mutant KRAS-driven cancer. Bak accumulates in radioresistant lung cancer cells and BKA-073 reverses radioresistance. Combination of BKA-073 with Bcl-2 inhibitor venetoclax exhibits strong synergy against lung cancer in vivo. Conclusions: Development of small molecule Bak activator may provide a new class of anticancer agents to treat lung cancer.

Death domain fold (DDF) superfamily comprises of the death domain (DD), death effector domain (DED), caspase activation recruitment domain (CARD), and pyrin domain (PYD). By utilizing a conserved mode of interaction involving six distinct surfaces, a DDF serves as a building block that can densely pack into homomultimers or filaments. Studies of immune signaling components have revealed that DDF-mediated filament formation plays a central role in mediating signal transduction and amplification. The unique ability of DDFs to self-oligomerize upon external signals and induce oligomerization of partner molecules underlies key processes in many innate immune signaling pathways, as exemplified by RIG-I-like receptor signalosome and inflammasome assembly. Recent studies showed that DDFs are not only limited to immune signaling pathways, but also are involved with transcriptional regulation and other biological processes. Considering that DDF annotation still remains a challenge, the current list of DDFs and their functions may represent just the tip of the iceberg within the full spectrum of DDF biology. In this review, we discuss recent advances in our understanding of DDF functions, structures, and assembly architectures with a focus on CARD- and PYD-containing proteins. We also discuss areas of future research and the potential relationship of DDFs with biomolecular condensates formed by liquid-liquid phase separation (LLPS).

We aimed to identify a microRNA (miRNA)‐E3 ubiquitin ligase regulatory network for protein substrates enriched in cell death pathways and investigate the underlying molecular mechanisms in alcohol‐associated hepatitis (AH). An miRNA‐E3 ubiquitin ligase regulatory network for protein substrates enriched in cell death pathways was constructed using integrated bioinformatics analysis. Differentially expressed hub miRNAs (GSE59492) and their validated miRNA target genes (GSE28619) were identified in the liver of patients with AH compared with healthy controls. Liver samples from patients with AH and healthy individuals and mice exposed to Gao‐binge (acute on chronic) ethanol were used for experimental validation. Using hub miRNAs identified by weighted correlation network analysis, a miRNA‐E3 ubiquitin ligase regulatory network was established based on 17 miRNAs and 7 E3 ligase genes targeted by these miRNAs that were down‐regulated in AH. Among the miRNAs in this regulatory network, miR‐150‐5p was the only miRNA regulating the E3 ligase cytokine‐inducible SH2 containing protein (CISH), the E3 ligase that regulates the largest number of substrates among all E3 ligase family members. Therefore, the CISH regulatory pathway for ubiquitinated substrates was selected for subsequent experimental validation. Consistent with the bioinformatics analysis results, expression of miR‐150‐5p was markedly increased, while CISH was decreased, in the livers of patients with AH and mice exposed to Gao‐binge ethanol. Moreover, ubiquitination of Fas‐associated protein with death domain, a predicted CISH substrate involved in the regulation of programmed cell death, was reduced in livers from mice after Gao‐binge ethanol. Conclusion: Identification of the miRNA‐E3 ubiquitin ligase regulatory network for protein substrates enriched in the cell death pathways provides insights into the molecular mechanisms contributing to hepatocyte death in AH., This study identifies a microRNA‐E3 ubiquitin ligase regulatory network for protein substrates enriched in cell death pathways. We report that expression of miR‐150‐5p was elevated in AH and experimentally verified the changes in the key components in this miR‐150‐5p‐CISH‐FADD regulatory network in the liver from both patients with AH and a murine model of acute‐on‐chronic ethanol exposure.

PURPOSEPreclinical data suggest the combination of an anti–programmed death receptor 1 antibody plus dabrafenib and trametinib to have superior antitumor activity compared with dabrafenib plus trametinib alone. These observations are supported by translational evidence suggesting that immune checkpoint inhibitors plus targeted therapy may improve treatment outcomes in patients with BRAF V600–mutant metastatic melanoma. COMBI-i is a phase III trial evaluating spartalizumab, an anti–programmed death receptor 1 antibody, in combination with dabrafenib and trametinib (sparta-DabTram), versus placebo plus dabrafenib and trametinib (placebo-DabTram) in patients with BRAF V600–mutant unresectable or metastatic melanoma.METHODSPatients received spartalizumab 400 mg intravenously every 4 weeks plus dabrafenib 150 mg orally twice daily and trametinib 2 mg orally once daily or placebo-DabTram. Participants were age ≥ 18 years with unresectable or metastatic BRAF V600–mutant melanoma. The primary end point was investigator-assessed progression-free survival. Overall survival was a key secondary end point (ClinicalTrials.gov identifier: NCT02967692 ).RESULTSAt data cutoff (July 1, 2020), the median progression-free survival was 16.2 months (95% CI, 12.7 to 23.9 months) in the sparta-DabTram arm versus 12.0 months (95% CI, 10.2 to 15.4 months) in the placebo-DabTram arm (hazard ratio, 0.82 [95% CI, 0.66 to 1.03]; P = .042 [one-sided; nonsignificant]). The objective response rates were 69% (183 of 267 patients) versus 64% (170 of 265 patients), respectively. Grade ≥ 3 treatment-related adverse events occurred in 55% (146 of 267) of patients in the sparta-DabTram arm and 33% (88 of 264) in the placebo-DabTram arm.CONCLUSIONThe study did not meet its primary end point; broad first-line use of sparta-DabTram is not supported by these results. Further biomarker-driven investigation may identify patient subpopulations who could benefit from checkpoint inhibitor plus targeted therapy combinations.

Signaling pathways in innate and adaptive immunity play vital roles in pathogen recognition and the functions of immune cells. Higher-order assemblies have recently emerged as a central principle that governs immune signaling and, by extension, cellular communication in general. There are mainly two types of higher-order assemblies: 1) ordered, solid-like large supramolecular complexes formed by stable and rigid protein-protein interactions, and 2) liquid-like phase-separated condensates formed by weaker and more dynamic intermolecular interactions. This review covers key examples of both types of higher-order assemblies in major immune pathways. By placing emphasis on the molecular structures of the examples provided, we discuss how their structural organization enables elegant mechanisms of signaling regulation.

Death receptors, members of the tumour necrosis factor receptor (TNFR) superfamily, are characterized by the presence of a death domain in the cytosolic region. TNFR1, Fas and TNF-related apoptosis-inducing ligand receptors, which are prototypical death receptors, exert pleiotropic functions in cell death, inflammation and immune surveillance. Hence, they are involved in several human diseases. The activation of death receptors and downstream intracellular signalling is regulated by various posttranslational modifications, such as phosphorylation, ubiquitination and glycosylation. Glycosylation is one of the most abundant and versatile modifications to proteins and lipids, and it plays a critical role in the development and physiology of organisms, as well as the pathology of many human diseases. Glycans control a number of cellular events, such as receptor activation, signal transduction, endocytosis, cell recognition and cell adhesion. It has been demonstrated that oligo- and monosaccharides modify death receptors and intracellular signalling proteins and regulate their functions. Here, we review the current understanding of glycan modifications of death receptor signalling and their impact on signalling activity.

Cytosolic inflammasomes are supramolecular complexes that are formed in response to intracellular pathogens and danger signals. However, as to date, the detailed description of a homotypic caspase recruitment domain (CARD) interaction between NLRP1 and ASC has not been presented. We found the CARD–CARD interaction between purified NLRP1CARD and ASCCARD experimentally and the filamentous supramolecular complex formation in an in vitro proteins solution. Moreover, we determined a high-resolution crystal structure of the death domain fold of the human ASCCARD. Mutational and structural analysis revealed three conserved interfaces of the death domain superfamily (Type I, II, and III), which mediate the assembly of the NLRP1CARD/ASCCARD complex. In addition, we validated the role of the three major interfaces of CARDs in assembly and activation of NLRP1 inflammasome in vitro. Our findings suggest a Mosaic model of homotypic CARD interactions for the activation of NLRP1 inflammasome. The Mosaic model provides insights into the mechanisms of inflammasome assembly and signal transduction amplification.

Background & Aims TNFSF15 genetic variants leading to increased TNF superfamily member 15 (TNFSF15) expression confer risk for inflammatory bowel disease (IBD), and TNFSF15 is being explored as a therapeutic target in IBD patients. Although the focus for TNFSF15-mediated inflammatory outcomes has been predominantly on its action on T cells, TNFSF15 also promotes inflammatory outcomes in human macrophages. Given the critical role for macrophages in bacterial clearance, we hypothesized that TNFSF15 promotes antimicrobial pathways in human macrophages and that macrophages from TNFSF15 IBD risk carriers with higher TNFSF15 expression have an advantage in these antimicrobial outcomes. Methods We analyzed protein expression, signaling, bacterial uptake, and intracellular bacterial clearance in human monocyte-derived macrophages through flow cytometry, enzyme-linked immunosorbent assay, and gentamicin protection. Results Autocrine/paracrine TNFSF15 interactions with death receptor 3 (DR3) were required for optimal levels of pattern-recognition-receptor (PRR)-induced bacterial clearance in human macrophages. TNFSF15 induced pyruvate dehydrogenase kinase 1-dependent bacterial uptake and promoted intracellular bacterial clearance through reactive oxygen species, nitric oxide synthase 2, and autophagy up-regulation. The TNFSF15-initiated TNF receptor-associated factor 2/receptor-interacting protein kinase 1/RIP3 pathway was required for mitogen-activated protein kinase and nuclear factor-κB activation, and, in turn, induction of each of the antimicrobial pathways; the TNFSF15-initiated Fas-associated protein with death domain/mucosa-associated lymphoid tissue lymphoma translocation protein 1/caspase-8 pathway played a less prominent role in antimicrobial functions, despite its key role in TNFSF15-induced cytokine secretion. Complementation of signaling pathways or antimicrobial pathways restored bacterial uptake and clearance in PRR-stimulated macrophages where TNFSF15:DR3 interactions were inhibited. Monocyte-derived macrophages from high TNFSF15-expressing rs6478108 TT IBD risk carriers in the TNFSF15 region showed increased levels of the identified antimicrobial pathways. Conclusions We identify that autocrine/paracrine TNFSF15 is required for optimal PRR-enhanced antimicrobial pathways in macrophages, define mechanisms regulating TNFSF15-dependent bacterial clearance, and determine how the TNFSF15 IBD risk genotype modulates these outcomes., Graphical abstract

The intracellular domain of UNC5B contains both death domain and caspase‐3 cleavage site, and is regarded as a functional domain that mediates apoptosis. However, in our previous studies, we found that the death domain of UNC5B in bladder cancer cells could not be activated to promote apoptosis. In this study, different UNC5B truncates (residue 399‐945, residue 412‐945) were created to explore whether the caspase‐3 cleavage site (site 412), as another potential functional domain of its intracellular portion, could be activated to induce apoptosis in bladder cancer cells. Using mass spectrometry, we acquired a comprehensive and detailed identification of differentially expressed proteins by overexpressing UNC5B and its truncates. Protein‐protein‐interaction (PPI) network analysis was also applied to investigate the aggregation of related proteins and predict the functional changes. EDU assay, apoptosis, xenograft tumour implantation, migration, invasion and tumour metastasis were performed to comprehensively identify the effects of UNC5B truncates on bladder cancer cells. We demonstrate that the intracellular domain of UNC5B promotes cell proliferation in vitro and tumour formation in vivo, by binding to a large number of ribosomal proteins. The overexpression of intracellular domain also facilitates cells to migrate, invade and metastasize by interacting with fibronectin, beta‐catenin and vimentin. In addition, we reveal that overexpressing the intracellular domain of UNC5B cannot bind or activate cleaved caspase‐3 to trigger apoptosis in bladder cancer cells.

Signaling by Toll-Like Receptors and the Interleukin-1 Receptor (IL1-R) involves intracellular binding of MyD88, followed by assembly of IL1-R Associated Kinases (IRAKs) into the so-called Myddosome. Using NMR, Nechama et al. determined the structure of the IRAK-M death domain monomer (PDBid: 5UKE). With this structure, they performed a docking study to model the location of IRAK-M in the Myddosome. Based on this, they present a molecular basis for selectivity of IRAK-M towards IRAK1 over IRAK2 binding. When we attempted to use 5UKE as a homology modeling template, we noticed that our 5UKE-based models had structural issues, such as disallowed torsion angles and solvent exposed tryptophans. We therefore analyzed the NMR ensemble of 5UKE using structure validation tools and we compared 5UKE with homologous high-resolution X-ray structures. We identified several structural anomalies in 5UKE, including packing issues, frayed helices and improbable side chain conformations. We used Yasara to build a homology model, based on two high resolution death domain crystal structures, as an alternative model for the IRAK-M death domain (atomic coordinates, modeling details and validation are available at https://swift.cmbi.umcn.nl/gv/service/5uke/). Our model agrees better with known death domain structure information than 5UKE and also with the chemical shift data that was deposited for 5UKE.

In pleiotropic diseases, multiple organ systems are affected causing a variety of clinical manifestations. Here, we report a pleiotropic disorder with a unique constellation of neurological, endocrine, exocrine, and haematological findings that is caused by biallelic MADD variants. MADD, the mitogen-activated protein kinase (MAPK) activating death domain protein, regulates various cellular functions, such as vesicle trafficking, activity of the Rab3 and Rab27 small GTPases, tumour necrosis factor-α (TNF-α)-induced signalling and prevention of cell death. Through national collaboration and GeneMatcher, we collected 23 patients with 21 different pathogenic MADD variants identified by next-generation sequencing. We clinically evaluated the series of patients and categorized the phenotypes in two groups. Group 1 consists of 14 patients with severe developmental delay, endo- and exocrine dysfunction, impairment of the sensory and autonomic nervous system, and haematological anomalies. The clinical course during the first years of life can be potentially fatal. The nine patients in Group 2 have a predominant neurological phenotype comprising mild-to-severe developmental delay, hypotonia, speech impairment, and seizures. Analysis of mRNA revealed multiple aberrant MADD transcripts in two patient-derived fibroblast cell lines. Relative quantification of MADD mRNA and protein in fibroblasts of five affected individuals showed a drastic reduction or loss of MADD. We conducted functional tests to determine the impact of the variants on different pathways. Treatment of patient-derived fibroblasts with TNF-α resulted in reduced phosphorylation of the extracellular signal-regulated kinases 1 and 2, enhanced activation of the pro-apoptotic enzymes caspase-3 and -7 and increased apoptosis compared to control cells. We analysed internalization of epidermal growth factor in patient cells and identified a defect in endocytosis of epidermal growth factor. We conclude that MADD deficiency underlies multiple cellular defects that can be attributed to alterations of TNF-α-dependent signalling pathways and defects in vesicular trafficking. Our data highlight the multifaceted role of MADD as a signalling molecule in different organs and reveal its physiological role in regulating the function of the sensory and autonomic nervous system and endo- and exocrine glands.

Literatures indicate that microRNA-129-5p (miR-129-5p) or Fas-associated death domain (FADD) is related to intervertebral disc degeneration (IDD), but the effect of miR-129-5p/FADD axis on IDD is not studied. The study aimed to investigate whether miR-129-5p influenced immune privilege and nucleus pulposus (NP) cell apoptosis in rats with IDD via regulating FADD.A rat model with caudal IDD was established, and injected with miR-129-5p agomir or miR-129-5p antagomir to figure out the character of miR-129-5p in the cell apoptosis and inflammation in the nucleus pulposus (NP) tissues of IDD rats. NP cells were grouped as the same ways for determining proliferation, apoptosis, and senescence in NP cells of IDD rats. Annexin V-FITC/PI double staining detected the apoptosis of macrophages and CD8+ cells co-cultured via transfected NP cells. Expression of miR-129-5p, FADD, collagen I, collagen II, aggrecan and Sox-9 in NP tissues and cells were determined.Up-regulated miR-129-5p decreased FADD, collagen I and elevated collagen Ⅱ, aggrecan, and Sox-9 in NP tissues and repressed inflammation in serum and NP tissues in IDD rats. Up-regulated miR-129-5p facilitated proliferation, inhibited senescence, apoptosis, and decreased FADD, collagen I and increased collagen Ⅱ, aggrecan, and Sox-9 in NP cells of IDD rats. Elevated miR-129-5p promoted the apoptosis of macrophages and CD8+ cells.We pronounced that up-regulated miR-129-5p inhibited the apoptosis and facilitated the proliferation of NP cells, as well as the apoptosis of macrophages and CD8+ cells via decreased FADD in IDD, suggesting that miR-129-5p had a protective effect on IDD.

The relationship between increased neuroimmune gene expression and hippocampal degeneration in alcohol use disorder (AUD) and other mental diseases is poorly understood. We report here that tumor necrosis factor receptor superfamily death receptor 3 (TNFRSF25, DR3) and Fas receptors (Fas) that initiate caspase cell death cascades are increased in AUD hippocampus and following a rat adolescent binge drinking model. Death receptors are known inducers of apoptosis and cell death that recruit death domain (DD) proteins FADD and TRADD and caspases to form death-inducing signaling complexes (DISC). In postmortem human AUD hippocampus, mRNA and IHC protein are increased for the entire death receptor cascade. In AUD hippocampus, ligand–death receptor pairs, i.e., TL1A-DR3 and FasL–Fas, were increased, as well as FADD and TRADD, and active caspase-8, -7, -9, and caspase-3. Further, pNFκB p65, a key neuroimmune transcription factor, and IL-8, a chemokine, were significantly increased. Interestingly, across AUD patients, increases in DR3 and Fas correlated with TRADD, and TRADD with active caspase+IR and IL-8+IR, consistent with coordinated activation of neuronal DISC mediated death cascades and neuroimmune gene induction in AUD. These findings support a role for DR3 and Fas neuroimmune signaling in AUD hippocampal neurodegeneration.

Supramolecular organizing center (SMOC)-mediated signal transduction is an emerging concept in the field of signal transduction that is ushering in a new era. The formation of location-specific, higher-order SMOCs is particularly important for cell death and innate immune signaling processes. Several protein interaction domains, including the death domain (DD) superfamily and the CIDE domain, are representative mediators of SMOC assembly in cell death and innate immune signaling pathways. DD superfamily- and CIDE domain-containing proteins form SMOCs that activate various caspases and provide signaling scaffold platforms. These assemblies can lead to signal transduction and amplification during signaling events. In this review, we summarize recent findings on the molecular basis of DD superfamily- and CIDE domain-mediated SMOC formation., Nonspecific immunity: Super signaling complexes Improved understanding of large molecular signaling complexes that form during innate (nonspecific) immune responses could help develop treatments for multiple diseases including cancer. Correct cell signaling requires precise protein interactions and binding, which are mediated by specific sites on the surface of the protein molecules involved. Innate immune responses and cell death mechanisms rely on such protein interactions, and defects can cause signaling abnormalities and trigger disease. Hyun Ho Park and co-workers at Chung-Ang University in Seoul, South Korea, reviewed recent insights into the presence of supramolecular organizing centers (SMOCs), localized complexes of signaling proteins that form during immune responses. The researchers highlight existing understanding of SMOC assembly processes. A better understanding of SMOCs will help to explain enzyme activation, signal amplification and cell signaling control mechanisms.

Immunogenic cell death (ICD) occurs when a dying cell releases cytokines and damage-associated molecular patterns, acting as adjuvants, and expresses Ags that induce a specific antitumor immune response. ICD is studied mainly in the context of regulated cell death pathways, especially caspase-mediated apoptosis marked by endoplasmic reticulum stress and calreticulin exposure and, more recently, also in relation to receptor-interacting protein kinase–driven necroptosis, whereas unregulated cell death like accidental necrosis is nonimmunogenic. Importantly, the murine cancer cell lines used in ICD studies often express virally derived peptides that are recognized by the immune system as tumor-associated Ags. However, it is unknown how different cell death pathways may affect neoepitope cross-presentation and Ag recognition of cancer cells. We used a prophylactic tumor vaccination model and observed that both apoptotic and necroptotic colon carcinoma CT26 cells efficiently immunized mice against challenge with a breast cancer cell line that expresses the same immunodominant tumor Ag, AH1, but only necroptotic CT26 cells would mount an immune response against CT26-specific neoepitopes. By CRISPR/Cas9 genome editing, we knocked out AH1 and saw that only necroptotic CT26 cells were still able to protect mice against tumor challenge. Hence, in this study, we show that endogenous AH1 tumor Ag expression can mask the strength of immunogenicity induced by different cell death pathways and that upon knockout of AH1, necroptosis was more immunogenic than apoptosis in a prophylactic tumor vaccination model. This work highlights necroptosis as a possible preferred ICD form over apoptosis in the treatment of cancer.

To examine the expression of P53-induced protein with a death domain (PIDD) at retina in animal model of optic nerve crush (ONC) and to investigate the role of PIDD in retinal glial activation and NF-κB activation induced by optic nerve damage, ONC animal model was established in Sprague-Dawley rats. PIDD has three isoforms (Isof); Western blot was performed to examine the expression of PIDD (Isof-1, Isof-2, and Isof-3, respectively) in retina at different time points after ONC. Retinal glial activation is closely associated with retinal neuronal death and is monitored by the expression of GFAP+ glial cells and IBA1+ microglia, then activated microglia leads to inflammatory cytokine production. NF-kB activation in glial cells also can promote neuronal death. In our study, the role of PIDD in retinal glial activation and NF-kB activation was investigated with PIDD inhibition selectively. PIDD expression (Isof-1 and Isof-3) was dramatically increased, and peaked at 3 days after ONC, while Isof-2 did not show any difference. In the ONC animal model, the number of GFAP+ glial cells and IBA1+ microglia in retinal layers was increased significantly, inflammatory cytokine production was upregulated, and NF-κB in glial cell was also activated. Moreover, those responses induced by optic nerve damage were attenuated with PIDD inhibition, which indicated that PIDD could regulate retinal glial activation, neuro-inflammation, and NF-κB activation. These results provided the direct demonstration that the PIDD (Isof-1and Isof-3) was overexpressed in retina after ONC, and PIDD may be involved in retinal neurodegenerative diseases by regulating retinal glial activation and NF-κB activation.

Antigen receptor–dependent (AgR-dependent) stimulation of the NF-κB transcription factor in lymphocytes is a required event during adaptive immune response, but dysregulated activation of this signaling pathway can lead to lymphoma. AgR stimulation promotes assembly of the CARMA1-BCL10-MALT1 complex, wherein MALT1 acts as (a) a scaffold to recruit components of the canonical NF-κB machinery and (b) a protease to cleave and inactivate specific substrates, including negative regulators of NF-κB. In multiple lymphoma subtypes, malignant B cells hijack AgR signaling pathways to promote their own growth and survival, and inhibiting MALT1 reduces the viability and growth of these tumors. As such, MALT1 has emerged as a potential pharmaceutical target. Here, we identified G protein–coupled receptor kinase 2 (GRK2) as a new MALT1-interacting protein. We demonstrated that GRK2 binds the death domain of MALT1 and inhibits MALT1 scaffolding and proteolytic activities. We found that lower GRK2 levels in activated B cell–type diffuse large B cell lymphoma (ABC-DLBCL) are associated with reduced survival, and that GRK2 knockdown enhances ABC-DLBCL tumor growth in vitro and in vivo. Together, our findings suggest that GRK2 can function as a tumor suppressor by inhibiting MALT1 and provide a roadmap for developing new strategies to inhibit MALT1-dependent lymphomagenesis.

Background: Death domain-associated protein (DAXX) is a tumor suppressor and its loss has been found in a variety of cancer types. Dysregulation of DAXX is strongly correlated with cancer metastasis. However, the role and functions of DAXX in colorectal cancer (CRC) metastasis are not fully understood. Methods: We validated the mRNA and protein expression of DAXX in CRC specimens and CRC cell lines using real-time reverse transcription-PCR and Western blot, respectively. The overexpression plasmids of ZEB1 and E-cadherin and the siRNAs for DAXX and ZEB1 knockdown were constructed to study the impact of these factors on cells. Wound-healing assay and Transwell assay were performed to examine the cell motility and cell migration and invasion abilities, respectively. Luciferase assay was performed to assess the E-cadherin promoter activity. Immunoprecipitation assay was performed to investigate the interaction between proteins. The rescue experiment was carried out to verify whether the effect of DAXX on E-cadherin expression is depended on ZEB1. Results: DAXX expression was lower in liver metastases than in primary colon cancer tissues. Our results demonstrated that DAXX directly interacted with ZEB1 and suppressed its inhibitory effect on promoter activity of E-cadherin through a ZEB1-dependent manner, and thus suppresses the cell motility, migration, and invasion of CRC cell lines. Conclusion: In sum, these findings supported that the loss of DAXX is associated with cancer cell metastases in CRC. ZEB1-mediated transcriptional suppression of E-cadherin is a possible mechanism. DAXX/ZEB-1 pathway could be a potential therapeutic target for preventing cancer metastasis in CRC.

Graphical abstract, TRADD participates in various receptor signaling pathways and plays vital roles in many biological activities, including cell survival and apoptosis, in different cellular contexts. TRADD has two distinct functional domains, a TRAF-binding domain at the N-terminus and a death domain (DD) at the C-terminus. The TRAF binding domain of TRADD folds into an α-β plait topology and is mainly responsible for binding TRAF2, while the TRADD-DD can interact with a variety of DD-containing proteins, including receptors and intracellular signaling molecules. After activation of specific receptors such as TNFR1 and DR3, TRADD can bind to the receptor through DD-DD interaction, creating a membrane-proximal platform for the recruitment of downstream molecules to propagate cellular signals. In this review, we highlight recent advances in the studies of the structural mechanism of TRADD adaptor functions for NF-κB activation and apoptosis induction. We also provide suggestions for future structure research related to TRADD-mediated signaling pathways.

Fas-associated protein with death domain (FADD) was first identified for its role in linking death receptors to the apoptotic signaling pathway with subsequent cell death. Later studies reported non-apoptotic functions for FADD in normal cells and cancer cells. Non-apoptotic functions for FADD in osteosarcoma (OS) have not been reported. In this study, FADD protein expression was knocked down in human CCHOSD, LM7, and SaOS2 OS cell lines followed by assessment of sensitivity to TNFα- or TRAIL-induced cell death. Knock down of FADD significantly increased TNFα-induced cell death in LM7 and SaOS2 cell lines. The mode of TNFα-induced cell death was apoptosis and not necroptosis. Inhibition of nuclear factor kappa B (NFκB) in wildtype cells increased TNFα-induced cell death to similar levels observed in FADD knockdown cells, suggesting a role for FADD in NFκB pro-survival cell signaling. In addition, knock down of FADD increased SMAC mimetic-mediated TNFα-induced cell death in all cell lines studied. The results of this study indicate that FADD has a pro-survival function in OS following TNFα treatment that involves NFκB signaling. The results also indicate that the pro-survival function of FADD is associated with XIAP activity.