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Hypohidrotic ectodermal dysplasia (HED) is a genetic disorder characterized by hypohidrosis, hypodontia, and hypotrichosis. Autosomal forms of the disease are caused by mutations in either EDAR or EDARADD. To date, the underlying pathomechanisms for HED resulting from EDARADD mutations have not fully been disclosed. In this study, we performed detailed in vitro analyses in order to characterize three dominantly inherited missense mutations, p.D120Y, p.L122R, and p.D123N, and one recessively inherited missense mutation, p.E152K, in the EDARADD gene. Nuclear factor (NF)-κB reporter assays demonstrated that all the mutant EDARADD showed reduction in activation of NF-κB. Importantly, p.D120Y-, p.L122R-, and p.D123N-mutant EDARADD slightly reduced the NF-κB activity induced by wild-type EDARADD in a dominant negative manner. Co-immunoprecipitation assays showed that all of the mutant EDARADD were capable of binding to EDAR and wild-type EDARADD. Additional co-immunoprecipitation assays revealed that p.D120Y-, p.L122R-, and p.D123N-mutant EDARADD markedly prevented the interaction between EDAR and wild-type EDARADD, which further indicated a dominant negative effect by these mutations. Finally, we found that p.D120Y-, p.L122R-, and p.D123N-mutant EDARADD completely lost the ability to bind with TRAF6, while p.E152K-mutant EDARADD showed a mild reduction in the affinity. Our findings will provide crucial information toward unraveling the molecular mechanisms how EDARADD gene mutations cause the disease.

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.

BACKGROUND: Non-small cell lung cancer (NSCLC) is one of the most widespread cancer with increasing morbidity and mortality. FAS-associated protein with death domain (FADD) is considered as an essential instrument in cell death, whereas Bcl-XS promotes apoptosis through inhibiting the activity of Bcl-2 and Bcl-XL. OBJECTIVE AND METHODS: We detected the expression of FADD and Bcl-XS in resected NSCLC tissues by immunohistochemistry, and investigated their association with clinicopathological characteristics and prognostic significance of NSCLC patients. RESULTS: Bcl-XS expression was significantly increased in well and moderate differentiated lung SCC (P= 0.004). Lung ADC patients with overexpression of FADD and lung SCC patients with low expression of Bcl-XS had importantly lower overall survival rates by Kaplan-Meier analysis (P= 0.033, P= 0.02, respectively). Multivariate analysis confirmed that elevated expression of FADD was an independent poor prognostic factor for patients with surgically resected lung ADC (P= 0.027) and increased expression of Bcl-XS was an independent good prognostic factor for patients with surgically resected lung SCC (P= 0.016) CONCLUSION: Elevated expression of FADD was identified as independent poor prognostic factor for patients with surgically resected lung ADC, however, increased expression of Bcl-XS was an independent good prognostic biomarker for patients with surgically resected lung SCC.

NLRP3-PYD inflammasome activates an inflammatory pathway in response to a wide variety of cell damage or infections. Dysregulated NLRP3 inflammatory signaling has many chronic inflammatory and autoimmune disorders. NLRP3 and ASC have a PYD, a superfamily member of the Death Domain, which plays a key role in inflammatory assembly. The ASC interacts with NLRP3 through a homotypic PYD and recruits the procaspase-1 through a homotypic caspase recruitment domain interaction. Here, we used several computational approaches to reveal the interactions of the NLRP3 and ASC PYD domains that lead to the activation of the inflammasome complex. We have characterized ASC and NLRP3-PYD intermolecular interactions by protein-protein docking, and further molecular dynamics (MD) simulations were conducted to evaluate the stability of NLRP3/ASC-PYD complex. Subsequently, we have identified several residues that stabilize the NLRP3/ASC-PYD complex in different faces (i.e., Face-1 to Face-4). The research framework offers new insights into the molecular mechanisms of inflammasome and apoptosis signaling as well as the ease of the drug discovery process.

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.