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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.

The anti-inflammatory interleukin-1 receptor associated kinase-M (IRAK-M) is a negative regulator of MyD88/IRAK-4/IRAK-1 signaling. However, IRAK-M has also been reported to activate NF-κB through the MyD88/IRAK-4/IRAK-M myddosome in a MEKK-3 dependent manner. Here we provide support that IRAK-M uses three surfaces of its Death Domain (DD) to activate NF-κB downstream of MyD88/IRAK-4/IRAK-M. Surface 1, with central residue Trp74, binds to MyD88/IRAK-4. Surface 2, with central Lys60, associates with other IRAK-M DDs to form an IRAK-M homotetramer under the MyD88/IRAK-4 scaffold. Surface 3; with central residue Arg97 is located on the opposite side of Trp74 in the IRAK-M DD tetramer, lacks any interaction points with the MyD88/IRAK-4 complex. Although the IRAK-M DD residue Arg97 is not directly involved in the association with MyD88/IRAK-4, Arg97 was responsible for 50% of the NF-κB activation though the MyD88/IRAK-4/IRAK-M myddosome. Arg97 was also found to be pivotal for IRAK-M’s interaction with IRAK-1, and important for IRAK-M’s interaction with TRAF6. Residue Arg97 was responsible for 50% of the NF-κB generated by MyD88/IRAK-4/IRAK-M myddosome in IRAK-1/MEKK3 double knockout cells. By structural modeling we found that the IRAK-M tetramer surface around Arg97 has excellent properties that allow formation of an IRAK-M homo-octamer. This model explains why mutation of Arg97 results in an IRAK-M molecule with increased inhibitory properties: it still binds to myddosome, competing with myddosome IRAK-1 binding, while resulting in less NF-κB formation. The findings further identify the structure-function properties of IRAK-M, which is a potential therapeutic target in inflammatory disease.

Hypohidrotic ectodermal dysplasia is a rare condition characterized by hypohidrosis, hypodontia, and hypotrichosis. The disease can show X‐linked recessive, autosomal dominant or autosomal recessive inheritance trait. Of these, the autosomal forms are caused by mutations in either EDAR or EDARADD. To date, the underlying pathomechanisms or genotype–phenotype correlations for autosomal forms have not completely been disclosed. In this study, we performed a series of in vitro studies for four missense mutations in the death domain of EDAR protein: p.R358Q, p.G382S, p.I388T, and p.T403M. The results revealed that p.R358Q‐ and p.T403M‐mutant EDAR showed different expression patterns from wild‐type EDAR in both western blots and immunostainings. NF‐κB reporter assays demonstrated that all the mutant EDAR showed reduced activation of NF‐κB, but the reduction by p.G382S‐ and p.I388T‐mutant EDAR was moderate. Co‐immunoprecipitation assays showed that p.R358Q‐ and p.T403M‐mutant EDAR did not bind with EDARADD at all, whereas p.G382S‐ and p.I388T‐mutant EDAR maintained the affinity to some extent. Furthermore, we demonstrated that all the mutant EDAR proteins analyzed aberrantly bound with TRAF6. Sum of the data suggest that the degree of loss‐of‐function is different among the mutant EDAR proteins, which may be associated with the severity of the disease. [ABSTRACT FROM AUTHOR]

PIDDosome formation followed by caspase-2 activation is critical for genotoxic stress-induced apoptotic cell death. Failure of proper caspase-2 activation causes a neurodevelopmental disorder and intellectual disability. R815W, R862W, and Q863stop mutations in p53-induced protein with a death domain (PIDD), a component of the PIDDosome, also lead to this disorder. However, the molecular mechanisms underlying this pathogenesis remain elusive. In this study, we analyzed the molecular mechanisms underlying the pathogenesis of the PIDD DD pathogenic variants R815W, R862W, and Q863stop. We determined that these mutations prevented the interaction between PIDD and RIP-associated Ich-1/Ced-3 homologous protein with a death domain (RAIDD), a molecule that mediates PIDDosome formation. The disruption of this interaction affects PIDDosome formation and caspase-2 activation. [Display omitted] • Three PIDD pathogenic variants (R815W, R862W, and Q863stop) causing a neurodevelopmental disorder was analyzed. • Mapping study showed that three pathogenetic mutations are located in the PIDD DD. • Q863stop mutant was aggregated by misfolding, indicating that helix 6 is critical for the proper folding of PIDD DD. • R815W and R862W mutants lost its capability of binding to RAIDD. • Failure of binding of PIDD pathogenic variants to RAIDD cause to failure of PIDDosome formation. [ABSTRACT FROM AUTHOR]

Cell surface death receptors are members of the tumor necrosis factor receptor (TNFR) superfamily and mediate signals leading to the induction of apoptosis or necroptosis, as well as NF-κB-mediated cell survival. These biochemical processes play key roles in cell growth, development, tissue homeostasis, and immune responses. The downstream signaling complexes activated by different death receptors can differ significantly and are subject to multiple, distinct regulatory mechanisms. Dysregulation of signaling by the TNFR superfamily contributes to a variety of pathologic conditions, including defective immune responses and cancer. Caspase-8 signaling is important for mediating death receptor signals leading to either apoptosis or NF-κB activation. By contrast, inactivation of caspase-8 or loss of caspase-8 expression shifts death receptor signaling to the necroptosis pathway. Notably, the gene encoding caspase-8 is mutated in roughly ten percent of head and neck cancers. These findings support the hypothesis that alterations in the biochemical pathways mediated by death receptors have important consequences for the development of head and neck, and possibly other, cancers.

Cells communication in response to extracellular or biophysical stimulus relies on elaborated systems of signal transduction. In the course of most signal pathway, the cascades involve signal protein complexes, which are often assembled by adaptor proteins. Tumor necrosis factor receptor type 1-associated death domain protein (TRADD) is an adaptor molecule involved in various signal pathways and mediating multiple biological activities, including cell survival, cell proliferation, cell differentiation, apoptosis, necroptosis and inflammation. TRADD contains an N terminal tumor necrosis factor receptor-associated factor 2 (TRAF2) binding domain and a C terminal death domain (DD) for interacting with multiple DD-containing proteins. Following activation of specific receptors, such as tumor necrosis factor receptor 1 (TNFR1), death receptor 3 (DR3), tumor necrosis factor-related apoptosis-inducing ligand receptor 1 (TRAILR1, DR4), TRAILR1 (DR5), DR6 and p75 neurotrophin receptor (p75NTR)，TRADD can bind to the receptors, serving as a platform for the recruitment of the downstream molecules for signal propagating and thus mediating various physiological and pathological processes. In this review, we provide a brief overview of the current knowledge on TRADD and discuss the roles of TRADD in infectious and inflammatory diseases, cardiovascular diseases, central nervous system diseases, cancer, endometriosis, hepatocyte proliferation, preterm birth and perinatal development.

The CARMA-BCL10-MALT1 (CBM) signalosome functions as a pivotal supramolecular module, integrating diverse receptor-induced signaling pathways to regulate BCL10-dependent NF-kB activation in innate and adaptive immunity. Conversely, the API2-MALT1 fusion protein in t(11; 18)(q21; q21) MALT lymphoma constitutively induces BCL10-independent NF-kB activation. MALT1 dimer formation is indispensable for the requisite proteolytic activity and is critical for NF-kB activation regulation in both scenarios. However, the molecular assembly of MALT1 individual domains in CBM activation remains elusive. Here we report the crystal structure of the MALT1 death domain (DD) at a resolution of 2.1 Å, incorporating reconstructed residues in previously disordered loops 1 and 2. Additionally, we observe a conformational regulation element (CRE) regulating stem-helix formation in NLRPs pyrin (PYD) within the MALT1 DD structure. The structure reveals a stem-helix-mediated dimer further corroborated in solution. To elucidate how the BCL10 filament facilitates MALT1 dimerization, we reconstitute a BCL10-CARD-MALT1-DD-IG1-IG2 complex model. We propose a N+7 rule for BCL10-dependent MALT1 dimerization via the IG1-IG2 domain and for MALT1-dependent cleavage in trans. Biochemical data further indicates concentration-dependent dimerization of the MALT1 IG1-IG2 domain, facilitating MALT1 dimerization in BCL10-independent manner. Our findings provide a structural and biochemical foundation for understanding MALT1 dimeric mechanisms, shedding light on potential BCL10-independent MALT1 dimer formation and high-order BCL10-MALT1 assembly. • A high-resolution crystal structure of MALT1 death domain was determined. • MALT1 death domain forms a stem-helix dimer. • A pH-sensitive system may structurally regulate the conformation of MALT1 DD helix α6. • BCL10-filament-mediated MALT1 dimerization is via a N+7 rule. • MALT1 IG1-IG2 behaves differently in BCL10-depedent and BCL10-independent assembly. [ABSTRACT FROM AUTHOR]

As a member of the tumor necrosis factor receptor superfamily (TNFRSF), death receptor 6 (DR6) has a similar structural architecture to other family members. The extracellular region of DR6 contains four cysteine-rich domains, followed by a single-pass transmembrane domain and an intracellular region. Since its discovery, DR6 has become an orphan receptor ubiquitously expressed to transduce unique signaling pathways. Although the free ectodomains of β-amyloid precursor protein (APP) can bind to DR6 to induce apoptotic signals, the natural ligands of DR6 still remain largely unknown. In this review, we focus on recent research progress of structural and functional studies on DR6 for better understanding DR6-mediated signaling and the treatment of DR6-related diseases. [ABSTRACT FROM AUTHOR]

As a member of the tumor necrosis factor receptor superfamily (TNFRSF), death receptor 6 (DR6) has a similar structural architecture to other family members. The extracellular region of DR6 contains four cysteine-rich domains, followed by a single-pass transmembrane domain and an intracellular region. Since its discovery, DR6 has become an orphan receptor ubiquitously expressed to transduce unique signaling pathways. Although the free ectodomains of β-amyloid precursor protein (APP) can bind to DR6 to induce apoptotic signals, the natural ligands of DR6 still remain largely unknown. In this review, we focus on recent research progress of structural and functional studies on DR6 for better understanding DR6-mediated signaling and the treatment of DR6-related diseases.

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.

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus that causes acute enteric disease in piglets and severely threatens the pig industry all over the world. Death domain-associated protein (DAXX) is a classical chaperone protein involved in multiple biological processes, such as cell apoptosis, transcriptional regulation, DNA damage repair, and host innate immunity. However, whether DAXX functions in the anti-PEDV innate immune responses remains unclear. In this study, we found that PEDV infection upregulated DAXX expression and induced its nucleocytoplasmic translocation in IPEC-J2 cells. Furthermore, we found that DAXX overexpression was inhibitory to PEDV replication, while downregulation of DAXX by RNA interference facilitated PEDV replication. The antiviral activity of DAXX was due to its positive effect on IFN-λ3-STAT1 signaling, as DAXX positively regulated STAT1 activation through their interaction in cytoplasm and enhancing the downstream ISG15 expression. Mutation of tryptophan at 621 to alanine in DAXX increased its abundance in the cytoplasm, leading to the upregulation of STAT1 phosphorylation and ISG15 expression. It indicated that cytoplasmic fraction of DAXX was advantageous for the STAT1-ISG15 signaling axis and PEDV inhibition. In summary, these results show that DAXX inhibits PEDV infection by increasing IFN-λ3-induced STAT1 phosphorylation and the downstream ISG15 expression., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Caspases are a family of cysteine proteases that play an essential role in inflammation, apoptosis, cell death, and development. Here we delve into the effects caused by heterologous expression of human caspase-1 in the yeast Saccharomyces cerevisiae and compare them to those of caspase-8. Overexpression of both caspases in the heterologous model led to their activation and caused mitochondrial hyperpolarization, damage to different organelles, and cell death. All these effects were dependent on their protease activity, and caspase-8 was more aggressive than caspase-1. Growth arrest could be at least partially explained by dysfunction of the actin cytoskeleton as a consequence of the processing of the yeast Bni1 formin, which we identify here as a likely direct substrate of both caspases. Through the modulation of the GAL1 promoter by using different galactose:glucose ratios in the culture medium, we have established a scenario in which caspase-1 is sufficiently expressed to become activated while yeast growth is not impaired. Finally, we used the yeast model to explore the role of death-fold domains (DD) of both caspases in their activity. Peculiarly, the DDs of either caspase showed an opposite involvement in its intrinsic activity, as the deletion of the caspase activation and recruitment domain (CARD) of caspase-1 enhanced its activity, whereas the deletion of the death effector domain (DED) of caspase-8 diminished it. We show that caspase-1 is able to efficiently process its target gasdermin D (GSDMD) when co-expressed in yeast. In sum, we propose that S. cerevisiae provides a manageable tool to explore caspase-1 activity and structure–function relationships.

Caspases are a family of cysteine proteases that play an essential role in inflammation, apoptosis, cell death, and development. Here we delve into the effects caused by heterologous expression of human caspase-1 in the yeast Saccharomyces cerevisiae and compare them to those of caspase-8. Overexpression of both caspases in the heterologous model led to their activation and caused mitochondrial hyperpolarization, damage to different organelles, and cell death. All these effects were dependent on their protease activity, and caspase-8 was more aggressive than caspase-1. Growth arrest could be at least partially explained by dysfunction of the actin cytoskeleton as a consequence of the processing of the yeast Bni1 formin, which we identify here as a likely direct substrate of both caspases. Through the modulation of the GAL1 promoter by using different galactose:glucose ratios in the culture medium, we have established a scenario in which caspase-1 is sufficiently expressed to become activated while yeast growth is not impaired. Finally, we used the yeast model to explore the role of death-fold domains (DD) of both caspases in their activity. Peculiarly, the DDs of either caspase showed an opposite involvement in its intrinsic activity, as the deletion of the caspase activation and recruitment domain (CARD) of caspase-1 enhanced its activity, whereas the deletion of the death effector domain (DED) of caspase-8 diminished it. We show that caspase-1 is able to efficiently process its target gasdermin D (GSDMD) when co-expressed in yeast. In sum, we propose that S. cerevisiae provides a manageable tool to explore caspase-1 activity and structure–function relationships. [ABSTRACT FROM AUTHOR]

Little is known regarding molecular markers in head and neck squamous cell carcinoma (HNSCC) that predict responsiveness to different therapeutic regimens or predict HNSCC progression. Mutations in procaspase-8 occur in 9% of HNSCC primary tumors, but the functional consequences of these mutations are poorly understood. In this study, we examined the impact of four, representative, HNSCC-associated procaspase-8 mutations on activation of the extrinsic apoptosis pathway, as well as cellular migration and invasion, and in vivo tumor growth. All four mutant proteins acted to potently inhibit activation of apoptosis following treatment with TRAIL or agonistic anti-Fas. In contrast to wild-type procaspase-8, the mutant proteins were not recruited to FADD following treatment with TRAIL or anti-Fas, but may be constitutively bound by FADD. Three of the four procaspase-8 mutants promoted enhanced cellular migration and invasion through matrigel, relative to that seen with the wild-type procaspase-8 protein. Procaspase-8 mutation also stimulated the growth of HNSCC xenograft tumors. These findings indicate that HNSCC-associated procaspase-8 mutations inhibit activation of the extrinsic apoptosis pathway and are likely to represent markers for resistance to therapeutic regimens incorporating death receptor activators. Moreover, procaspase-8 mutations may serve as markers of HNSCC tumor progression, as exemplified by enhanced migration, invasion, and tumor growth.

The Precambrian explosion led to the rapid appearance of most major animal phyla alive today. It has been argued that the complexity of life has steadily increased since that event. Here we challenge this hypothesis through the characterization of apoptosis in reef-building corals, representatives of some of the earliest animals. Bioinformatic analysis reveals that all of the major components of the death receptor pathway are present in coral with high-predicted structural conservation with Homo sapiens. The TNF receptor-ligand superfamilies (TNFRSF/TNFSF) are central mediators of the death receptor pathway, and the predicted proteome of Acropora digitifera contains more putative coral TNFRSF members than any organism described thus far, including humans. This high abundance of TNFRSF members, as well as the predicted structural conservation of other death receptor signaling proteins, led us to wonder what would happen if corals were exposed to a member of the human TNFSF (HuTNFα). HuTNFα was found to bind directly to coral cells, increase caspase activity, cause apoptotic blebbing and cell death, and finally induce coral bleaching. Next, immortalized human T cells (Jurkats) expressing a functional death receptor pathway (WT) and a corresponding Fas-associated death domain protein (FADD) KO cell line were exposed to a coral TNFSF member (AdTNF1) identified and purified here. AdTNF1 treatment resulted in significantly higher cell death (P < 0.0001) in WT Jurkats compared with the corresponding FADD KO, demonstrating that coral AdTNF1 activates the H. sapiens death receptor pathway. Taken together, these data show remarkable conservation of the TNF-induced apoptotic response representing 550 My of functional conservation.

The Map kinase Activating Death Domain containing protein (MADD) isoform of the IG20 gene is over-expressed in different types of cancer tissues and cell lines and it functions as a negative regulator of apoptosis. Therefore, we speculated that MADD might be over-expressed in human breast cancer tissues and that MADD knock-down might synergize with chemotherapeutic or TRAIL-induced apoptosis of breast cancer cells. Analyses of breast tissue microarrays revealed over-expression of MADD in ductal and invasive carcinomas relative to benign tissues. MADD knockdown resulted in enhanced spontaneous apoptosis in human breast cancer cell lines. Moreover, MADD knockdown followed by treatment with TRAIL or doxorubicin resulted in increased cell death compared to either treatment alone. Enhanced cell death was found to be secondary to increased caspase-8 activation. These data indicate that strategies to decrease MADD expression or function in breast cancer may be utilized to increase tumor cell sensitivity to TRAIL and doxorubicin induced apoptosis.

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.

Insulin resistance (IR) is the key pathophysiological cause of type 2 diabetes, and inflammation has been implicated in it. The death domain (DD) of the adaptor protein, MyD88 plays a crucial role in the transduction of TLR4-associated inflammatory signal. Herein, we have identified a 10-residue peptide (M10), from the DD of MyD88 which seems to be involved in Myddosome formation. We hypothesized that M10 could inhibit MyD88-dependent TLR4-signaling and might have effects on inflammation-associated IR. Intriguingly, 10-mer M10 showed oligomeric nature and reversible self-assembly property indicating the peptide's ability to recognize its own amino acid sequence. M10 inhibited LPS-induced nuclear translocation of NF-κB in L6 myotubes and also reduced LPS-induced IL-6 and TNF-α production in peritoneal macrophages of BALB/c mice. Remarkably, M10 inhibited IL-6 and TNF-α secretion in diabetic, db/db mice. Notably, M10 abrogated IR in insulin-resistant L6 myotubes, which was associated with an increase in glucose uptake and a decrease in Ser307-phosphorylation of IRS1, TNF-α-induced JNK activation and nuclear translocation of NF-κB in these cells. Alternate day dosing with M10 (10 and 20 mg/kg) for 30 days in db/db mice significantly lowered blood glucose and improved glucose intolerance after loading, 3.0 g/kg glucose orally. Furthermore, M10 increased insulin and adiponectin secretion in db/db mice. M10-induced glucose uptake in L6 myotubes involved the activation of PI3K/AKT/GLUT4 pathways. A scrambled M10-analog was mostly inactive. Overall, the results show the identification of a 10-mer peptide from the DD of MyD88 with anti-inflammatory and anti-diabetic properties, suggesting that targeting of TLR4-inflammatory pathway, could lead to the discovery of molecules against IR and diabetes., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Stereotactic radiation approaches are gaining more popularity for the treatment of intracranial as well as extracranial tumors in organs such as the liver and lung. Technology, rather than biology, is driving the rapid adoption of stereotactic body radiation therapy (SBRT), also known as stereotactic ablative radiotherapy (SABR), in the clinic due to advances in precise positioning and targeting. Dramatic improvements in tumor control have been demonstrated; however, our knowledge of normal tissue biology response mechanisms to large fraction sizes is lacking. Herein, we will discuss how SABR can induce cellular expression of MHC I, adhesion molecules, costimulatory molecules, heat shock proteins, inflammatory mediators, immunomodulatory cytokines, and death receptors to enhance antitumor immune responses.

Social cellular aggregation or multicellular organization pose increased risk of transmission of infections through the system upon infection of a single cell. The generality of the evolutionary responses to this outside of Metazoa remains unclear. We report the discovery of several thematically unified, remarkable biological conflict systems preponderantly present in multicellular prokaryotes. These combine thresholding mechanisms utilizing NTPase chaperones (the MoxR-vWA couple), GTPases and proteolytic cascades with hypervariable effectors, which vary either by using a reverse transcriptase-dependent diversity-generating system or through a system of acquisition of diverse protein modules, typically in inactive form, from various cellular subsystems. Conciliant lines of evidence indicate their deployment against invasive entities, like viruses, to limit their spread in multicellular/social contexts via physical containment, dominant-negative interactions or apoptosis. These findings argue for both a similar operational ‘grammar’ and shared protein domains in the sensing and limiting of infections during the multiple emergences of multicellularity.

Signaling activation is a tightly regulated process involving myriad posttranslational modifications such as phosphorylation/dephosphorylation, ubiquitylation/deubiquitylation, proteolytical cleavage events as well as translocation of proteins to new compartments within the cell. In addition to each of these events potentially regulating individual proteins, the assembly of very large supramolecular complexes has emerged as a common theme in signal transduction and is now known to regulate many signaling events. This is particularly evident in pathways regulating both inflammation and cell death/survival. Regulation of the assembly and silencing of these complexes plays important roles in immune signaling and inflammation and the fate of cells to either die or survive. Here we will give a summary of some of the better studied supramolecular complexes involved in inflammation and cell death, particularly with a focus on diseases caused by their autoactivation and the role autophagy either plays or may be playing in their regulation.

The anti-inflammatory interleukin-1 receptor associated kinase-M (IRAK-M) is a negative regulator of MyD88/IRAK-4/IRAK-1 signaling. However, IRAK-M has also been reported to activate NF-κB through the MyD88/IRAK-4/IRAK-M myddosome in a MEKK-3 dependent manner. Here we provide support that IRAK-M uses three surfaces of its Death Domain (DD) to activate NF-κB downstream of MyD88/IRAK-4/IRAK-M. Surface 1, with central residue Trp74, binds to MyD88/IRAK-4. Surface 2, with central Lys60, associates with other IRAK-M DDs to form an IRAK-M homotetramer under the MyD88/IRAK-4 scaffold. Surface 3; with central residue Arg97 is located on the opposite side of Trp74 in the IRAK-M DD tetramer, lacks any interaction points with the MyD88/IRAK-4 complex. Although the IRAK-M DD residue Arg97 is not directly involved in the association with MyD88/IRAK-4, Arg97 was responsible for 50% of the NF-κB activation though the MyD88/IRAK-4/IRAK-M myddosome. Arg97 was also found to be pivotal for IRAK-M's interaction with IRAK-1, and important for IRAK-M's interaction with TRAF6. Residue Arg97 was responsible for 50% of the NF-κB generated by MyD88/IRAK-4/IRAK-M myddosome in IRAK-1/MEKK3 double knockout cells. By structural modeling we found that the IRAK-M tetramer surface around Arg97 has excellent properties that allow formation of an IRAK-M homo-octamer. This model explains why mutation of Arg97 results in an IRAK-M molecule with increased inhibitory properties: it still binds to myddosome, competing with myddosome IRAK-1 binding, while resulting in less NF-κB formation. The findings further identify the structure-function properties of IRAK-M, which is a potential therapeutic target in inflammatory disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor TR declared a past collaboration with the author WW., (Copyright © 2024 Gürkan, Poelman, Pereverzeva, Kruijswijk, de Vos, Groenen, Nollet, Wichapong, Lutgens, van der Poll, Du, Wiersinga, Nicolaes and van ‘t Veer.)

Innate immune adaptor proteins are critical components of the innate immune system that propagate pro-inflammatory responses from their upstream receptors, and lead to pathogen clearance from the host. Bacterial pathogens have developed strategies to survive inside host cells without triggering the innate immune surveillance in ways that are still not fully understood. Here, it is reported that Pseudomonas aeruginosa induces its quorum sensing mechanism after macrophage engulfment. Further investigation of its secretome identified a quorum sensing regulated product, LasB, is responsible for innate immune suppression depending on the MyD88-mediated signaling. Moreover, it is showed that this specific type of pathogen-mediated innate immune suppression is due to the enzymatic digestion of the death domains of the innate immune adaptors, mainly MyD88, and attributed to LasB's large substrate binding groove. Lastly, it is demonstrated that the secretion of LasB from P. aeruginosa directly contributed to MyD88 degradation within macrophages. Hence, it is discovered an example of bacterial quorum sensing-regulated cellular innate immune suppression by direct cleavage of immune adaptors., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

The death domain (DD) is a globular protein motif with a signature feature of an all‐helical Greek‐key motif. It is a primary mediator of a variety of biological activities, including apoptosis, cell survival and cytoskeletal changes, which are related to many neurodegenerative diseases, neurotrauma, and cancers. DDs exist in a wide range of signalling proteins including p75 neurotrophin receptor (p75NTR), a member of the tumour necrosis factor receptor superfamily. The specific signalling mediated by p75NTR in a given cell depends on the type of ligand engaging the extracellular domain and the recruitment of cytosolic interactors to the intracellular domain, especially the DD, of the receptor. In solution, the p75NTR‐DDs mainly form a symmetric non‐covalent homodimer. In response to extracellular signals, conformational changes in the p75NTR extracellular domain (ECD) propagate to the p75NTR‐DD through the disulfide‐bonded transmembrane domain (TMD) and destabilize the p75NTR‐DD homodimer, leading to protomer separation and exposure of binding sites on the DD surface. In this review, we focus on recent advances in the study of the structural mechanism of p75NTR‐DD signalling through recruitment of diverse intracellular interactors for the regulation and control of diverse functional outputs. [ABSTRACT FROM AUTHOR]

The inflammasome is a multi-protein platform that assembles upon the presence of cues derived from infection or tissue damage, and triggers the inflammatory response. Inflammasome components include sensor proteins that detect danger signals, procaspase 1 and the adapter ASC (apoptosis-associated speck-like protein containing a CARD) tethering these molecules together. Upon inflammasome assembly, procaspase 1 self-activates and renders functional cytokines to arbitrate in the defense mechanism. This assembly is mediated by self-association and protein interactions via Death Domains. The inflammasome plays a critical role in innate immunity and its dysregulation is the culprit of many autoimmune disorders. An in-depth understanding of the factors involved in inflammasome assembly could help fight these conditions. This review describes our current knowledge on the biophysical aspects of inflammasome formation from the perspective of ASC. The specific characteristics of the three-dimensional solution structure and interdomain dynamics of ASC are explained in relation to its function in inflammasome assembly. Additionally, the review elaborates on the identification of ASC interacting surfaces at the amino acid level using NMR techniques. Finally, the macrostructures formed by full-length ASC and its two Death Domains studied with Transmission Electron Microscopy are compared in the context of a directional model for inflammasome assembly. [ABSTRACT FROM AUTHOR]

Death receptor 3 (DR3) (a.k.a. tumor necrosis factor receptor superfamily 25) plays a key role in the immune system by activating nuclear factor kappa‐light‐chain‐enhancer of activated B cells signaling pathway. Here we present the crystal structures of human and mouse DR3 intracellular death domain (DD) at 2.7 and 2.5 Å resolutions, respectively. The mouse DR3 DD adopts a classical six‐helix bundle structure while human DR3 DD displays an extended fold. Though there is one‐amino‐acid difference in the linker between maltose‐binding protein (MBP) tag and DR3 DD, according to our self‐interaction analysis, the hydrophobic interface discovered in MBP–hDR3 DD crystal structure is responsible for both hDR3 DD and mDR3 DD homotypic interaction. Furthermore, our biochemical analysis indicates that the sequence variation between human and mouse DR3 DD does not affect its structure and function. Small‐angle X‐ray scattering analysis shows the averaged solution structures of both human and mouse MBP‐DR3 DD are the combination of different conformations with different proportion. Through switching to the open conformation, DR3 DD could improve the interaction with downstream element TNFR‐associated death domain (TRADD). Here we propose an activation‐dependent structural rearrangement model: the DD region is folded as the six‐helix bundles in the resting state, while upon extracellular ligand engagement, it switches to the open conformation, which facilitates its self‐association and the recruitment of TRADD. Our results provide detailed insights into the architecture of DR3 DD and the molecular mechanism of activation. Databases: All refined structure coordinates as well as the corresponding structure factors have been deposited in the PDB under the accession codes 5YGS, 5YEV, 5YGP, 5ZNY, 5ZNZ. [ABSTRACT FROM AUTHOR]

p75 neurotrophin receptor (p75NTR) contains a C-terminal globular protein module known as the death domain (DD), which plays a central role in apoptotic and inflammatory signaling through the formation of oligomeric protein complexes. A monomeric state of the p75NTR-DD also exists depending on its chemical environment in vitro. However, studies on the oligomeric states of the p75NTR-DD have produced conflicting findings and sparked great controversy. Here we present new evidence from biophysical and biochemical studies to demonstrate the coexistence of symmetric and asymmetric dimers of the p75NTR-DD, which may equilibrate with the monomeric form in solution and in the absence of any other protein. The reversible close-open solution behavior may be important for the p75NTR-DD to serve as an intracellular signaling hub. This result supports an intrinsic ability of the p75NTR-DD to self-associate, in congruence with the oligomerization properties of all members of the DD superfamily. [ABSTRACT FROM AUTHOR]

Ectodermal dysplasia (ED), which exhibits a wide range of clinical symptoms, may be classified into three major types: hypohidrotic, anhidrotic, and hidrotic. A male child (proband) showing anhidrotic dysplasia was used as the subject of this study. The biopsy of the big toe revealed that the male child had no sweat glands. Genetic analysis of the patient revealed a mutation caused by a homozygous nucleotide substitution in the EDAR-associated death domain (EDARADD) (rs114632254) gene c.439G>A (p.Gly147Arg). Phenotypically, his teeth were sharp, but eight teeth were missing (oligodontia). The patient had normal nails with dry skin, sparse hair, everted lower lip vermilion, hyperpigmented eyelids, and abnormal nasal bridge morphology around the eyes. There is also a homozygous dominant (healthy) female and a heterozygous male in this family, who are cousins (aunt children) to the heterozygous parents. The daughter of the patient was also heterozygous. This mutation represents homozygous recessive inheritance, which we describe for the first time. Furthermore, we demonstrated that this genetic disorder can be readily diagnosed using the restriction fragment length polymorphism (RFLP) method after digestion with MnII restriction endonuclease.

The current research is based on computational study of the Death Domain (DD) superfamily of proteins involved in the cytoplasmic inflammasome complexes associated with apoptosis and inflammation in response to the nanoparticle treatment. The PYD domains of the DD superfamily proteins, NALP3 and ASC, were chosen for investigation as they are found to be primarily involved in the regulation of innate immunity and are associated with apoptosis and inflammation. The in-vitro studies of these proteins have proven to be a challenge as the proteins have a tendency to aggregate under laboratory conditions. The interactions between PYD-PYD domains of NALP3 & ASC proteins as well as PYD-PYD domains of NALP3 and ASC2 proteins were studied using the computational tools. In our study, the protein structures were taken from Protein Data Bank, and molecular dynamics simulation was performed using NAMD software followed by molecular docking studies using HADDOCK. The generated protein models were validated using PROCHECK and then the protein-protein interactions were analyzed using the molecular visualization tool CHIMERA. We noticed that the affinity between PYD of NALP3-ASC complex is better when compared to the NALP3-ASC2 complex based on their binding energies and docking scores. In the case of NALP3-ASC complex, seven key amino acids of ASC-PYD protein interface interact with four key amino acids of NALP3-PYD protein interface. However, in the case of NALP3-ASC2 complex, six key amino acids of ASC-PYD protein interact with four key amino acids of NALP3-PYD protein. Although, there is not much difference in the number of interacting amino acid residues between the two protein complexes, we understand that the NALP3-ASC exhibits better binding interaction and stability than the NALP3-ASC2 protein complex because the number of hydrogen bonding between them is more when compared to the latter. The hydrogen bonds between the interacting amino acids of the NALP3-ASC protein interface are 12, whereas it is 6 in the case of NALP3-ASC2 protein interaction. The protein-protein interaction seems to be dominated by the energetically significant hydrogen bonding followed by the electrostatic interaction in the NALP3-ASC protein interface, whereas both hydrogen bonding and the interaction between charged residues appears to play a significant role at the NALP3-ASC2 protein interfaces. [ABSTRACT FROM AUTHOR]

Context: Targeted cancer therapy has lately been the preferred cancer treatment modality. It is principally based on silencing or downregulating of specific genes responsible for cancer. Extensive exploration in this field has revealed the fact that CD95/CD95 ligand (CD95L), apart from their apoptotic role in killing cancer cells, also plays a non-apoptotic role in tumor advancement. Scientists have discovered that by eliminating CD95/CD95L, there is considerable decrease in tumor growth and invasiveness. Objective: Elimination of CD95/CD95L may offer a novel approach to targeted cancer therapy by suppressing the multiplication of tumor cells and thereby ensuring their death. Materials and methods: Data were obtained and analyzed from electronic database searches of relevant published literatures from PubMed and Google Scholar. Conclusion: Targeting the multiple tumor-promoting activity of CD95/CD95L is a potent treatment modality that can better the prognosis and increase the survival rate of cancer patients. [ABSTRACT FROM AUTHOR]

RIPK1 is a critical mediator of cell death and inflammation downstream of TNFR1 upon stimulation by TNFα, a potent proinflammatory cytokine involved in a multitude of human inflammatory and degenerative diseases. RIPK1 contains an N-terminal kinase domain, an intermediate domain, and a C-terminal death domain (DD). The kinase activity of RIPK1 promotes cell death and inflammation. Here, we investigated the involvement of RIPK1-DD in the regulation of RIPK1 kinase activity. We show that a charge-conserved mutation of a lysine located on the surface of DD (K599R in human RIPK1 or K584R in murine RIPK1) blocks RIPK1 activation in necroptosis and RIPK1-dependent apoptosis and the formation of complex II. Ripk1K584R/K584R knockin mutant cells are resistant to RIPK1 kinasedependent apoptosis and necroptosis. The resistance of K584R cells, however, can be overcome by forced dimerization of RIPK1. Finally, we show that the K584R RIPK1 knockin mutation protects mice against TNFα-induced systematic inflammatory response syndrome. Our study demonstrates the role of RIPK1-DD in mediating RIPK1 dimerization and activation of its kinase activity during necroptosis and RIPK1-dependent apoptosis. [ABSTRACT FROM AUTHOR]